B CELL DEVELOPMENT: THE IMPACT OF THE …...B cell development: the impact of the microenvironment...
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B CELL DEVELOPMENT: THE IMPACT OF THE MICROENVIRONMENT by Nathalie Simard A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate department of Immunology University of Toronto © Copyright by Nathalie Simard 2013
Transcript of B CELL DEVELOPMENT: THE IMPACT OF THE …...B cell development: the impact of the microenvironment...
B CELL DEVELOPMENT: THE IMPACT OF THE MICROENVIRONMENT
by
Nathalie Simard
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Graduate department of Immunology University of Toronto
© Copyright by Nathalie Simard 2013
ABSTRACT
B cell development: the impact of the microenvironment
Nathalie Simard
Doctor of Philosophy
Graduate department of Immunology
University of Toronto
2013
B lymphocytes develop from pluripotent stem cells, and differentiate to plasma cells (PCs) in
reaction to signals from the supportive microenvironment. Different sets of signals, which are
derived from multiple sources such as soluble cytokines and cell-cell contacts, are required at
different stages of development. For instance, murine B cell progenitors require the action of
interleukin-7 (IL-7) in the early phase of their development in the bone marrow (BM). The
necessity for IL-7 decreases as the cell matures, and this event is correlated with the appearance
of CD22. The first two chapters of this thesis focus on the early stages of B cell development that
take place in the BM. In chapter 1, I examine the IL-7 response and, although I do not show a
specific role for CD22 in the loss of sensitivity to IL-7, my data suggest that cis interactions
involving sialic acids might modulate the IL-7 response. This section is followed by the analysis
of the effect of IL-21 on B cell progenitors in the BM. IL-21 is known to regulate the terminal
stages of B cell differentiation. In collaboration with Dr. Danijela Konforte, I present evidence
that B cell progenitors in the BM also express a functional IL-21 receptor and that stimulation of
this receptor with IL-21 accelerates the maturation pace of B cells. I further demonstrate that
proB cells stimulated with IL-21 and anti-CD40 can differentiate into immunoglobulin (Ig)-
secreting cells, and discuss the possibility that IL-21 plays a role during inflammation for the
development of B cell progenitors in peripheral lymphatic organs. Finally, in the last chapter, in
collaboration with the laboratory of Dr. Gommerman, I investigate how the microenvironment
can shape the development of B cells. It has been demonstrated by my collaborators that IgA+
PCs present in the gut produce iNOS and display traits commonly associated to the myeloid
lineage, and in this chapter, I describe a co-culture system with BM and gut stroma to study the
conditions that sustain the generation of IgA+iNOS+ cells. In particular, I show that the presence
of microbial products is one of the key factors required for their development.
ACKNOWLEDGEMENTS First, I would like to thank my thesis advisor, Dr. Christopher J. Paige, for his guidance
throughout this project, and for fostering such a unique and stimulating scientific environment at
the Paige lab.
I thank my coworkers at the Paige lab for their assistance, particularly Dr. Danijela Konforte, for
her valuable suggestion that contributed to the evolution of the IL-21 project; Dr. Ann Tran, for
her expertise in molecular biology; and Caren Furlonger, for her advice, experimental aid, and
friendship. I have had the privilege of working with and supervising an exceptional summer
student, Jessica Esufali, who not only excelled in her laboratory duties but, through her
enthusiastic disposition, made the laboratory a fun place to work.
In the latter half of my thesis work, I was fortunate to make the acquaintance of the Gommerman
lab, including Dr. Jennifer Gommerman, Dr. Jorg Fritz, Dr. Olga Rojas, and Dr. Douglas
McCarthy. Our discussions led to a fruitful scientific collaboration, and ultimately a publication
in a major scientific journal. I am also grateful to my thesis committee, consisting of Drs. Stuart
Berger, Robert Rottapel, and later Dr. Jennifer Gommerman, for their keen scientific insight and
constructive criticism.
ABSTRACT ................................................................................................................................... ii
ACKNOWLEDGEMENTS ........................................................................................................ iv
TABLE OF CONTENTS ..............................................................................................................v
LIST OF FIGURES ..................................................................................................................... ix
LIST OF APPENDICES ............................................................................................................. xi
LIST OF ABBREVIATIONS .................................................................................................... xii
1. INTRODUCTION......................................................................................................................1
1.1 IMMUNOGLOBULIN ........................................................................................................2
1.1.1 Structure of immunoglobulin .......................................................................................2
1.1.2 Generation of immunoglobulin diversity ....................................................................3
1.1.2.1 V(D)J recombination .............................................................................................3
1.1.2.2 Class switch recombination ...................................................................................8
1.1.2.3 Somatic hypermutation .........................................................................................9
1.2 B CELL DEVELOPMENT IN THE BONE MARROW ...............................................11
1.2.1 Lymphoid specification ...............................................................................................11
1.2.2 Upregulation of IL-7R is crucial for specification and commitment of B lineage .14
1.2.3 B cell specification and commitment: Pre-proB and proB cell stages ....................16
1.2.4 From preB cells to immature B cells ..........................................................................19
1.3 B CELL DEVELOPMENT IN THE PERIPHERY ........................................................22
1.3.1 Transitional B cells ......................................................................................................22
1.3.2 Mature B cells and beyond .........................................................................................24
1.3.3 The majority of plasma cells are located in the gut-associated lymphoid tissues and secrete IgA .....................................................................................................................28
1.3.4 Signals inducing IgA production ................................................................................30
1.4 IL-21 – AN IMPORTANT CYTOKINE FOR LATE STAGES OF B CELL DIFFERENTIATION ..............................................................................................................32
1.4.1 IL-21 expression and structure ..................................................................................32
1.4.2 IL-21R expression, structure, and signalling ............................................................33
1.4.3 IL-21 effects on B cell functions………………..…………………………………...34
1.4.3.1 Proliferation and survival ...................................................................................34
1.4.3.2 Plasma cell differentiation ...................................................................................36
1.4.3.3 Class-switch recombination and immunoglobulin production ........................37
1.4.3.4 IL-21 in hematopoiesis .........................................................................................39
1.5 THESIS OUTLINE…………………………...…………………………………………..39
2. MATERIALS AND METHODS ............................................................................................41
2.1 Cell lines ..............................................................................................................................42
2.2 Mice .....................................................................................................................................42
2.3 Isolation and culture of BM B cell progenitors ...............................................................42
2.4 Sorting of BM cells .............................................................................................................43
2.5 Isolation of intestinal lamina propria cells .......................................................................44
2.6 Co-culture of stromal cells and BM cells .........................................................................44
2.7 Thymidine-incorporation assay ........................................................................................45
2.8 Western blot analysis .........................................................................................................45
2.9 ELISA ..................................................................................................................................46
2.10 FACS analysis ...................................................................................................................46
2.11 Detection of IL-21 protein ...............................................................................................47
2.12 RNA isolation and analysis ..............................................................................................48
2.13 Class-switch recombination .............................................................................................49
2.14 Statistical analysis ............................................................................................................49
3. ANALYSIS OF THE ROLE OF CD22 IN THE REGULATION OF THE IL-7 RESPONSE ..................................................................................................................................50
3.1 Introduction ........................................................................................................................51
3.2 Results .................................................................................................................................53
3.2.1 BM B cell lines remain IL-7 responsive ........................................................................53
3.2.2 IL-7 dependent cell lines express variable levels of CD22 ...........................................55
3.2.3 Cross-linking of CD22 does not regulate the response to IL-7 .....................................55
3.2.4 Removal of sialic acid modulates the IL-7 response .....................................................58
3.3 Discussion ............................................................................................................................61
4. BONE MARROW B CELL PROGENITORS EXPRESS FUNCTIONAL IL-21 RECEPTOR .................................................................................................................................65
4.1 Introduction ........................................................................................................................66
4.2 Results .................................................................................................................................68
4.2.1 IL-21 is expressed and secreted by CD4+ T cells in BM ...............................................68
4.2.2 IL-21 receptor is expressed on BM B cell progenitors..................................................70
4.2.3 IL-21 induces tyrosine phosphorylation of STAT1, STAT3, and STAT5 in B cell progenitors .............................................................................................................................72
4.2.4 IL-21 accelerates the transition of proB cells towards the preB cell stage and the transition of preB cells toward the immature/mature B cell stage .........................................75
4.2.5 IL-21-mediated maturation of preB cells toward the immature/mature B cell stage does not correlate with alteration of sIgM signalling ............................................................76
4.2.6 IL-21 regulates the expression of Blimp1 and Aid, and induces the expression of germline transcrip progenitors .............................................81
4.2.7 Ig-secreting cells are generated from B cell progenitors stimulated with IL-21 and anti-CD40 ...............................................................................................................................83
4.3 Discussion ............................................................................................................................85
5. DEVELOPMENT OF AN IN VITRO SYSTEM FOR THE GENERATION OF B CELLS EXPRESSING IGA AND INOS ...................................................................................91
5.1 Introduction ........................................................................................................................92
5.2 Results .................................................................................................................................94
5.2.1 Small intestinal lamina propria contains a population of cells expressing markers characteristic of both B and myeloid lineages .......................................................................94
5.2.2 Generation of IgA+iNOS+ cells in vitro .........................................................................96
5.2.3 Characterization of the BM/gut stroma culture ............................................................96
5.2.4 iNOS expression is supported by lamina propria-derived stroma, but not by BM-derived stroma ........................................................................................................................99
5.2.5 Microbial exposure promotes the expression of iNOS in IgA+ plasma cells ..............102
5.3 Discussion ..........................................................................................................................105
6. GENERAL DISCUSSION ....................................................................................................110
6.1 The loss of IL-7 responsiveness .......................................................................................111
6.2 IL-21 promotes the maturation of B cell progenitors ...................................................114
6.3 Role of IL-21 in steady state B cell development ...........................................................115
6.4 Role of IL-21 in extramedullar hematopoiesis during inflammation ..........................117
6.5 Importance of the gut microbiota in the development of the humoral immune syst .120
CONCLUSION ..........................................................................................................................122
APPENDICES ............................................................................................................................123
REFERENCES ..........................................................................................................................125
LIST OF FIGURESCHAPTER 1
1.1 Basic structure of selected immunoglobulins .....................................................................4
1.2 V(D)J recombination ............................................................................................................5
1.3 Class switch recombination ...............................................................................................10
1.4 Phenotypic characteristics delineating stages of murine B cell development ...............12
CHAPTER 3
3.1 BM-derived B cell lines are responsive to IL-7 ................................................................54
3.2 CD22 expression on BM-derived B cell lines ...................................................................56
3.3 Effect of CD22 cross-linking on IL-7R-induced phospho-ERK .....................................57
3.4 Cross-linking CD22 on BM B cells does not regulate proliferation and survival induced by IL-7 .........................................................................................................................59
3.5 Removal of surface sialic acids on BM B cells leads to a decrease in IL-7-induced proliferation ..............................................................................................................................60
CHAPTER 4
4.1 IL-21 is produced by CD4+ T cells in BM ........................................................................69
4.2 IL-21R expression on BM B cell progenitors ...................................................................71
4.3 IL-21R stimulation increases tyrosine phosphorylation of STAT1, STAT3, and STAT5 in proB, preB, and immature/mature B cells ...........................................................73
4.4 IL-21 regulates maturation of different B cell progenitors ............................................77
4.5 IL-21R-/- mice have more proB and fewer mature B cells than WT mice in BM .........79
4.6 IgM signalling is similar in immature B cells grown with and without IL-21 ..............80
4.7 IL-21 regulates gene expression of Blimp1 and Aid in B cell progenitors .....................82
4.8 IL-21R stimulation induces the expression of germline transcripts .......................84
CHAPTER 5
5.1 Small intestinal lamina propria contains IgA+ cells that can produce iNOS ................95
5.2 IgA+iNOS+ cells are present in the lamina propria of WT and IL-7-/- mice .................97
5.3 Testing of different culture conditions to generate IgA+iNOS+ cells from B220+ BM progenitors ................................................................................................................................98
5.4 IgA+iNOS+ cells are detected in the B220- and B220+ fraction of BM cells ...............100
5.5 IgA+iNOS+ cells appears in culture around day 6 or 7 .................................................101
5.6 Gut stroma can support the development of IgA+iNOS+ cells ....................................103
5.7 The generation of iNOS-expressing IgA+ cells requires microbial exposure .............104
LIST OF APPENDICES
Appendix 1: IgA+iNOS+ cells are present in the lamina propria of IL-21R-/- mice .............124
LIST OF ABBREVIATIONS
AID
APRIL
BAFF
BCMA
BCR
Blimp1
BLNK
BM
BSAP
CD
CE
CLP
CDR
CSR
CT
DNA
DNA-PKCS
EBF
ELISA
ELP
ERK
FACS
FCS
Flt-3
FO
Foxo
GALT
GC
GF
Activation induced cytidine deaminase
A proliferation inducing ligand
B cell activation factor of the TNF family
B cell maturation antigen
B cell receptor
B lymphocyte-induced maturation protein-1
B cell linker protein
Bone marrow
B-cell-specific activator protein
Cluster of differentiation
Coding end
Common lymphoid progenitor
Complementarity-determining region
Class switch recombination
Circle transcript
Deoxyribonucleic acid
DNA-dependent protein kinase catalytic subunit
Early B cell factor
Enzyme-linked immunosorbent assay
Early lymphoid progenitor
Extracellular signal-regulated kinase
Fluoresence-activated cell sorting
Fetal calf serum
Fms-related tyrosine kinase-3
Follicular
Forkhead box
Gut-associated lymphoid tissue
Germinal centre
Germ-free
GLT
HSC
Ig
IL
IEL
ILF
INF
iNOS
IRF
ITAM
ITIM
J
JAK
kDa
LC
Lin
LMPP
LN
LP
LPS
LT
MACS
MALT
MAPK
MMP
MPP
MZ
NHEJ
NK
NO
PC
Germline transcript
Hematopoietic stem cell
Immunoglobulin
Interleukin
Intraepithelial lymphocytes
Isolated lymphois follicle
Interferon
Inducible nitric oxide synthase
Interferon regulatory factor
Immunotyrosine-based activation motif
Immunotyrosine-based inhibition motif
Joining
Janus associated kinase
kiloDalton
Light chain
Lineage
Lymphoid-primed multipotent progenitor
Lymph node
Lamina propria
Lipopolysaccharide
Lymphotoxin
Magnetic activated cell sorting
Mucosal-associated lymphoid tissue
Mitogen-activated kinase
Matrix metalloproteinase
Multipotent progenitor
Marginal zone
Non-homologous end-joining
Natural killer cell
Nitric oxide
Plasma cell
PCR
pIgR
PI3K
PKB
PP
PST
RAG
RNA
RSS
Sca
S
SCF
SCID
SE
SH2
SHM
SLC
SLE
SMAD
STAT
TACI
TGF
Tdt
TLR
TNF
TSLP
UPR
VLA-4
WT
XRCC4
XSCID
Polymerase chain reaction
Polymeric Ig receptor
Phosphatidylinositol 3-kinase
Protein kinase B
Peyer’s patches
Post-switch transcript
Recombination activating gene
Ribonucleic acid
Recombination signal sequence
Stem cell antigen
Switch
Stem cell factor
Severe combined immunodeficiency
Signal end
Src homology 2
Somatic hypermutation
Surrogate light chain
Systemic lupus erythematosus
Mothers against decapentaplegic homologue
Signal transducer and activator of transcription
Transmembrane activator and calcium-modulator and cyclophilin ligand interactor
Transforming growth factor
Terminal dideoxy-transferase
Toll-like receptor
Tumor necrosis factor
Thymic stromal lymphopoietin
Unfolded protein response
Very late antigen-4
Wild-type
X-ray repair cross-complementing protein 4
X-linked SCID
CHAPTER 1
INTRODUCTION
1
1.1 IMMUNOGLOBULIN
The production of immunoglobulin (Ig) is the fundamental characteristic that distinguishes B
cells from the others cells of the immune system. Igs play a central role in immune responses by
binding specifically to antigens and recruiting effector cells. The production of functional Igs is a
multistep process that takes place in the BM, beginning at the earliest stages of B cell
development in a reaction called V(D)J recombination. Originally, each B cell has the potential
to generate millions of different Igs, but ultimately, individual B cells will express Igs of a single
specificity. Igs are initially expressed in a membrane-bound form at the cell surface in
-cell receptor (BCR).
Given their short cytoplasmic tail, Igs do not transduce any signal. Cell signalling and activation
are propagate -based
activation motifs (ITAMs) in their cytoplamic tail. In terminally differentiated B cells, Igs are
synthesized and secreted in the blood as antibodies which can bind and neutralize antigens at a
distance.
1.1.1 Structure of immunoglobulin
The basic structure of all Igs consists of two identical polypeptide chains of approximately 55-77
kDa, termed heavy chains, and two identical light chains of approximately 25 kDa, linked
together by disulfide bounds (Fig. 1.1) (reviewed in (1)). Each heavy and light chain contains a
variable (V) domain at the N-terminus which is involved in the recognition of antigens. Both the
V domain of the heavy chain (VH) and the V domain of the light chain (VL) contain three short
sequences of amino acids mediating antigen recognition, referred to as complementarity-
determining regions (CDRs). These CDRs are highly variable between different Igs, reflecting
the ability of Igs to bind to a nearly infinite diversity of antigenic structures. Both chains also
contain relatively conserved C domains, whose amino acid sequence defines the Ig isotype. The
) isotype, whereas the
heavy chain contains three to four C domains depending on the isotype of the antibody. Based on
the heavy chain isotype, Igs are divided into five classes: IgM, IgG, IgA, IgD, and IgE. In mice,
minor variations in the sequence of IgG allow for further classification into IgG1, IgG2a, IgG2b,
2
and IgG3 subclasses. In humans, IgG is subdivided into IgG1, IgG2, IgG3, and IgG4 subclasses;
and IgA into IgA1 and IgA2. The constant region of the heavy chain confers the specific effector
functions to the Ig which differ amongst the different classes.
1.1.2 Generation of immunoglobulin diversity
Antibodies exhibit remarkable diversity at their antigen binding site. These differences arise
through the presence and rearrangement of different combinations of multiple gene segments
coding for the variable region of both the H and L chain. Diversity is further increased because
genes segments are assembled in an imprecise manner which leads to the insertion, deletion or
changes of amino acids at the junction sites. Once produced, H chains randomly pair with L
chains. After stimulation by antigen, Igs can furthermore undergo class switching and somatic
hypermutation, which leads to the production of antibodies with higher affinity and different
effector properties.
1.1.2.1 V(D)J recombination
The H chain and L chain proteins that form the Ig are each encoded at loci located on different
chromosomes. The variable region of the L chain is coded by a V (variable) and a J (joining)
segment of DNA, whereas the variable region of the H chain is made of a V, a D (diversity), and
a J segment. In the germline configuration, each locus contains several different V, D and J
segments, separated from one another by introns (Fig. 1.2A). Therefore, the genes coding for the
IgH and IgL chains cannot be transcribed directly from the genome. Indeed, the DNA sequences
coding for these proteins must first be generated in each B cell by productively assembling
together one copy of each type of segment in at least one heavy and one light chain locus. This
process is mediated by somatic recombination, and is tightly regulated at various checkpoints
during B cell differentiation in the BM (2).
Initiation of V(D)J recombination requires recognition of the Ig gene segments by lymphocyte-
specific enzymes named recombination-activating gene (RAG) 1 and RAG2. Both RAG1 and
3
VL
CL
VH
CH
1
CH
2
CH
3
CH
4
C C
NN
VL
CL
VH
CH
1
CH
2
CH
3
VL
CL
VH
CH
1
CH
2
CH
3
H4
4
V1 V2 V3 D1 D2 D3 J1 J2 J3
D
J
D J
D
J
D
J
NNN
N
D JNNN
NNN
5
6
RAG2 have been shown to be essential at the initial stages of V(D)J recombination as no Ig gene
rearrangement occurs in RAG1- or RAG2-deficient mice, and therefore no mature B cells are
produced (3-5). RAG1 and RAG2 form a complex that recognizes highly conserved
recombination signal sequences (RSSs) (6). These sequences are positioned 3’ of each V gene
segment, 5’ of each J segment, and on both sides of each D segment. Each RSS consists of a
stretch of 7 nucleotides (the heptamer) and a stretch of 9 nucleotides (the nonamer). These
highly conserved sequences are separated by a spacer of either 12 or 23 non-conserved
nucleotides, hence their names 12-RSS and 23-RSS (2). Efficient recombination occurs if a
segment containing a 12-nucleotide spacer joins a segment containing a 23-nucleotide spacer
located in the same locus (2). This constraint, called the 12/23 rule, ensures that genes recombine
appropriately, avoiding wasteful V-V and J-J rearrangements for either chain, or VH-JH (without
a D segment inserted in between) for the heavy chain.
In the first phase of V(D)J recombination, RAG proteins bind 12- and 23-RSSs and bring the two
targeted segments close to one another. Subsequently, RAGs introduce a nick in the DNA strand
immediately 5’ of the heptamer (Fig. 1.2B). The 3’hydroxyl group of the coding sequence reacts
with the corresponding phosphate on the opposite strand to create a hairpin structure called the
coding end (CE), leaving blunted ends at the extremities of both heptamers called signal ends
(SEs) (7).
In the second phase of V(D)J recombination, CEs and SEs are processed. This is mediated
principally by enzymes ubiquitously expressed in the organism and involved in the non-
homologous end-joining (NHEJ) DNA repair process (reviewed in (8)). To resolve the hairpin
junction at CEs, the DNA must first to be nicked by a complex containing the Ku70/80, DNA-
dependent protein kinase catalytic subunit (DNA-PKCS) and the endonuclease Artemis (9, 10).
Once open, the DNA is subject to modification via deletion and addition of nucleotides which
contributes to further increase the diversity of Igs. The insertion of nucleotides is done in two
different ways: palindromic (P) nucleotides are added by DNA polymerases and non-template
(N) nucleotides are added by the terminal deoxynucleotidyl transferase (TdT) to created stretches
7
called N-regions (reviewed in (11) and (12)). N-regions are usually found only in the heavy
chain expressed on B cells generated after birth. This is explained by the fact that TdT
expression is detected only after birth and before the rearrangement of the light chain. Finally, X-
ray repair cross-complementing protein 4 (XRCC4) and ligase IV rejoin the CEs to form a
coding joint (13). In contrast to the CEs, in which as described above modification is introduced
when resolved, the ligation of SEs is done with precision. This reaction is also mediated by an
XRCC4-DNA-ligase IV complex and results in the formation of a circular DNA called a “signal
joint” that will be eventually deleted when the cell divides. However, in some cases where the
two targeted signals are in the same orientation, the intervening DNA does not get deleted, but
instead becomes inverted and is therefore retained in the chromosome (Fig. 1.2C).
1.1.2.2 Class switch recombination
Following successful rearrangement and assortment of the heavy and light chains, B cells
produce Igs. Initially, alternative splicing of the mRNA transcript leads to the production of IgM
and IgD isotypes. However, different isotypes of the same antigen specificity as the original IgM
can be generated if an immune response is triggered and B cells undergo class switch
recombination (CSR). The selection of the isotype produced will depend on the cytokines to
which B cells are e
transcription which directs switching to IgA and IgG2b respectively. Alternatively, IL-4 favours
switching to IgG1 and IgE (14).
The genes coding for the different isotypes of Igs (CH genes) are located downstream of the VDJ
locus on the heavy chain chromosome. In mice, the CH locus is organized as follow: 5’-V(D)J-
C -C -C 3-C 1-C 2b-C 2a-C - -3’ (Fig. 1.3). Each CH gene, except for C , has a stretch of
DNA at its 5’ end called a switch (S) region. S regions differ between isotypes but have in
common a G-rich sequence in the non-template strand (15). Located upstream of each S region is
an exon called the I exon and a cytokine-inducible promoter that initiates the production of a
germline or sterile RNA transcript (GLT) of the associated CH gene (16, 17). The generation of a
GLT transcript is required for CSR to occur as demonstrated by the defect in the production of a
8
selective isotype in cells carrying a mutation in a given I exon or its promoter (18, 19). However,
the mere expression of GLTs is not sufficient. Indeed, studies have shown that the enzyme
activation-induced cytidine deaminase (AID) is clearly required for CSR (20, 21). GLTs
contribute to CSR by making the S region a substrate for AID, which deaminates cytosine
residues and thereby creates multiple DNA lesions (22). This action ultimately leads to double-
stranded DNA breaks in both the donor and the acceptor S regions that will be repaired by
NHEJ. The intervening DNA, which contains the sequence between S and the target S region, is
looped-out and excised from the genome as a circle. The Ix promoter present in the circle is
active and is regulated by the same signals that activate the promoter of the target S region (23).
Its activation triggers the transcription of a short half-life I-C mRNA, named circle transcript
(CT), whose detection is used as a hallmark of ongoing CSR (23). In addition to CTs, the
presence of post-switch transcripts (PST) is also used to detect CSR. PSTs are generated after
CSR has occurred and the I promoter becomes linked to a new CH gene. These PSTs are
mRNAs composed of the I exon spliced with the targeted CH gene (24). Figure 1.3 illustrates
the process of CSR with the example of a switch from the IgM to the IgA isotype.
1.1.2.3 Somatic hypermutation
The rearrangement of gene segments determines the primary repertoire of Igs. To further
increase the diversity of these proteins, V gene segments undergo somatic hypermutation (SHM)
(reviewed in (25)). As for CSR, SHM also occurs after encounter with an antigen. This process
requires the help of T cells via CD40:CD40-ligand interaction. It is initiated by AID which
deaminates cytosine to uracil at hot spot motifs (26). Mutations are then subsequently introduced
by an error-prone DNA polymerase during the replication or repair of the uracil residues (27).
Ultimately, B cells producing antibodies with higher specificity for a particular antigen will be
selected for survival in a competitive microenvironment called the germinal centres (GC).
9
V D J E S C C CS 3 SC 3 S 1 C 1 S 2b C 2b C 2aS 2a S C
C
C
S3
C3
S1
C 1S 2b C
2b
C2
a
S2
aS
C
I
I2a
I3
I1
I 2b
I
V D J E S CS
I I 3 I 1 I 2b I 2a I I
V D J E C
I
C
C
S3
C3
S1
C 1 S 2b C2b
C2
aS
2a
S
C
I
I2a
I3
I1
I 2b
I
10
1.2 B CELL DEVELOPMENT IN THE BONE MARROW
In mammals, the generation of B lymphocytes occurs primarily in the fetal liver prior to birth,
and in the BM during the postnatal period (28, 29). During this process, hematopoietic stem cells
(HSCs) gradually lose their properties while acquiring specialized functions associated with B
cells. This is not achieved in a rigid and irreversible manner, but occurs following a series of
stochastic decisions that allow HSCs to first differentiate into the lymphoid lineage, subsequently
commit to the B cell lineage, and finally become mature B cells (30, 31). Significant effort has
been put into characterizing and discriminating the various stages of B cell maturation and
determining their order of succession. This has led to the proposition of several nomenclatures
which dissect the different stages (pre-proB, proB, preB, immature and mature B cells) based on
the status of Ig gene rearrangement, the expression and silencing of diverse sets of genes,
including surface molecules, and growth factor requirements (Fig. 1.4) (28, 32, 33). The key
event that takes place during B cell development is the generation of a functional BCR which is
responsible for the diversity of antigen recognition and the selective survival of B cells with
appropriate specificities. Environmental factors, such as FMS-related tyrosine kinase-3 ligand
(Flt-3L) and IL-7, as well as various transcription factors, including Ikaros, PU.1, E2A, early B
cell factor (EBF), and Pax5, all contribute in the orchestration of B cell development, and their
role will be described in this section.
1.2.1 Lymphoid specification
All blood cells, including B cells, derive from a small number of HSCs present in the BM. It is
estimated that HSCs constitute about 1/30,000 of nucleated BM cells (34, 35). HSCs are the only
cell population that possesses the peculiar properties of unlimited self-renewal and the potential
for long-life replenishment of all types of blood cells (reviewed in (36) and (37)). These
properties grant these cells the ability to fully reconstitute the blood system upon transplantation
into a lethally irradiated host. The cell surface phenotype of HSCs is characterized by the
absence of lineage markers (Lin-), which comprise lymphoid (CD45R/B220, CD19, CD3, CD8),
myeloid (CD11b/Mac-1, Ly-6G/Gr-1), NK (DX-5 or NK1.1), and erythroid (TER119) markers.
However, HSCs do express stem cell antigen-1 (Sca-1) and high levels of c-Kit (CD117). Studies
11
B lineage
Specified B
lineage comm
itted
C
LP
Fr.A
P
re-proB
Fr.B
Early
ProB
Fr.C
Late
ProB
Fr.C
Large
PreB
Fr.D
Small
PreB
Fr.E
Imm
ature T
1 T
2 T
3 FO
II FO
I M
Z
c-kit +
+
low
/- low
/- -
- -
- -
- -
- -
IL-7R
+
+
+
+
+
+
+
-
- -
- -
- A
A4.1
+
+
+
+
+
+
+
+
+
+
low/-
- -
CD
43 +
+
+
+
+
/- -
- -
- -
- -
- T
dT
+
+
+
+
- -
- -
- -
- -
- R
ag 1/2 +
/- +
+
+
-
+
- -
- -
- -
- B
220 -
+
+
+
+
+
+
+
++
+
+
++
+
++
+
++
C
D19
- -
+
+
+
+
+
+
+
+
+
+
+
HSA
low
low
+
+
+
+
+
+
++
+
+
++
low
low
+
5/V
preB
- +
/- +
+
+
-
- -
- -
- -
- Ig
/
- -
+
+
+
+
+
+
+
+
+
+
+
BP
-1 -
- -
+
+
- -
- -
- -
- -
CD
22 -
- -
- +
+
+
+
+
++
+
+
++
+
++
+
++
+
CD
2 -
- -
- -
+
+
+
+
+
+
+
sIgM
-
- -
- -
- +
+
+
++
+
+
+
+
++
sIgD
-
- -
- -
- -
-/low
+
+
++
+
+
low
CD
21 -
- -
- -
- -
low
+
+
+
+
++
C
D23
- -
- -
- -
- -
+
+
++
+
+
- IgH
C
GL
(D
J) (D
J) D
J D
J (V
DJ)
(VD
J) V
DJ
VD
J V
DJ
VD
J V
DJ
VD
J V
DJ
VD
J V
DJ
VD
J
IgLC
G
L
GL
G
L
GL
G
L
VJ
VJ
VJ
VJ
VJ
VJ
VJ
VJ
Figure 1.4 P
henotypic characteristics delineating stages of murine B
cell development.
CL
P: Com
mon lym
phoid progenitor; T1-T
3: transitional 1-3 B cell; FO
II/I: Follicular type II/I B cell; M
Z: M
arginal zone B cell
12
of the murine phenotype resulting from mutations of either c-kit or its ligand stem cell factor
(SCF) have shown an important role for this receptor-ligand pair in the survival and self-renewal
of HSC (36, 38).
The initial event of B cell development occurs when some HSCs do not self-renew, but begin to
differentiate and progress down the B lineage pathway. This starts with the differentiation of
HSCs into multipotent progenitors (MPPs), a transition characterised by the gradual loss of self-
renewal potential and long-term repopulating activity. As they evolve toward a lymphoid fate,
MPPs express higher levels of Flt-3 (39, 40). The increased levels of Flt-3 appear to correlate
with a significant reduction in potential to generate megakaryocytes and erythrocytes. This
fraction of Flt-3hi MPP cells is sometimes referred to as lymphoid-primed multipotent
progenitors (LMPPs). LMPPs form a heterogeneous population, with some subsets able to
differentiate into T, B, or myeloid cells - while other subsets are restricted to B and/or T cells, or
NK cells (41).
It has been observed that a small subset of Flt-3hi LMPP cells is sensitive to estrogen, a negative
regulator of B and T lymphopoiesis (42). These cells are also characterized by the expression of
at least one gene associated exclusively with lymphoid cells, such as RAG1 or TdT, or in few
cases the presence of products derived from heavy chain D-JH rearrangement (42-45). Given
these properties, they have been designated early lymphoid progenitors (ELPs) (43). As they
differentiate and become gradually more restricted to a lymphoid fate, ELPs downregulate the
expression of c-kit and Sca-1, and upregulate the expression of IL-7R . This gives rise to
common lymphoid progenitors (CLPs), described as cells that mainly differentiate into B, T, or
NK cells - and which possess very weak myeloid cell potential.
The pattern of genes expressed in cells at a given time, and therefore the decision to progress
toward one lineage over another, depends on the concentration and combination of transcription
factors. Flt3, , rag1, and rag2 are key genes indicating the adoption of a lymphoid fate. It
13
has been observed that none of these genes are expressed in mice deficient for the zinc-finger
transcription factor Ikaros, implying a role for Ikaros in lymphoid specification (46, 47). In line
with these finding, Ikaros-/- mice do not generate CLP and display reduced potential to produce T
cells and an absence of B lineage cells (46, 48). Similar to Ikaros-/- hematopoietic cells, fetal liver
hematopoietic progenitors deficient for the transcription factor PU.1 exhibit reduced expression
of Flt-3 and IL-7R, and a severe reduction in their ability to generate lymphoid and myeloid
progenitors (49). These results can be explained by the observation that expression of IL-7R is
promoted by ligation of Flt-3 (50). Moreover, the promoter region of contains binding
motifs for Ikaros and PU.1 (51, 52).
1.2.2 Upregulation of IL-7R is crucial for specification and commitment of murine B
lineage
In mice, the appearance of functional IL-7R is a critical event in the progression through the B
lineage, as illustrated by the profound B cell development defects observed in mice deficient for
either IL-7 or IL-7R (53-55). A functional IL-7R is composed of two chains: the common ( c)
chain and the IL-7R chain (56). The c chain was originally identified as the IL-
has since been found as a component of the receptor for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21
(57). The IL-7R chain confers the specificity for binding IL-7, but it can also bind thymic
stromal lymphopoietin (TSLP) when paired with the TSLP receptor chain. This accounts for the
more pronounced lymphopenia seen in mice deficient for IL-7R chain compared to that
observed in IL-7-/- mice (58).
The ligand IL-7 is a 25kDa stromal cell-derived cytokine that was discovered by Namen et al. in
1988 as a molecule that promoted growth of cultured B cell progenitors (59, 60). Binding of IL-7
to the IL-7R rapidly triggers the activation of the Janus associated kinase (JAK)/signal
transducer and activator of transcription (STAT) pathway, more precisely of JAK1, JAK3,
STAT1, STAT3, and STAT5 (57, 61, 62). JAK3, associated with the c chain, and JAK1,
associated with the IL-7R chain, cross-activate each other and phosphorylate critical tyrosine
14
residues in the cytoplasmic portion of the IL-7R chain. Specific phosphotyrosine residues serve
as a docking site for the Src homology 2 (SH2) domains of STAT proteins. For example, STAT5
which is the predominant STAT protein activated by the IL-7/IL-7R system is recruited to Y449
of the IL-7R chain (63, 64). The STAT proteins are then phosphorylated on tyrosine residues
by the receptor-associated JAK kinases or Src kinases. This leads to the dimerization of the
phophorylated STATs, their dissociation from the IL-7R, and their migration to the nucleus. The
phosphorylated Y449 on IL-7R chain also serves as a docking site for the p85 subunit of
phosphatidylinositol 3-kinase (PI3K), which subsequently activates the catalytic subunit p110.
One substrate of PI3K is the serine/threonine kinase Akt, also called protein kinase B (PKB)
known to regulate pro- and anti-apoptotic factors and the transcription factor Forkhead box 1
(Foxo1) (65-67). Finally, stimulation of IL-7R also leads to the activation of the extracellular
signal-regulated kinase (ERK) mitogen-activated kinase (MAPK) pathway, which contributes to
the survival and proliferation of proB and preB cells (68).
It is now well established that IL-7 signalling promotes specification, commitment, proliferation,
survival, and differentiation of murine B cells in the BM (reviewed in (57)). However, it is
important to note that IL-7 signalling is only essential for the development of B cells in adult
mice. B cells that develop in murine fetal and perinatal life do not have the same requirement,
which accounts for the development of a small number of B cells in IL-7-/- mice (54). Indeed, IL-
7-/- mice lack conventional B2 B cells, but have B1 and marginal zone (MZ) B cells (69). The
presence of these residual B cells has been explained by the ability of TSLP to substitute for IL-7
in fetal liver and perinatal, but not adult, BM B cell development (58).
In humans, the requirement of IL-7 for the generation of B cells is still controversial. As in
murine fetal liver cells, it was originally thought to be dispensable. This hypothesis was
supported by the analysis of patients suffering from severe combined immunodeficiency (SCID)
attributed to a non-functional IL-7R. The most common form, X-linked SCID (XSCID) results
from mutations in the gene encoding the c chain (70). In these patients, the development of T
and NK cells is severely affected, whereas the number of B cells generated is normal. In another
15
form of SCID caused by mutations in the IL-
diminished number of T cells, but have normal numbers of NK and B cells (71, 72). In
agreement with the in vivo data, it was reported that B cells can be generated in vitro from
CD34+ human fetal BM cells without the addition of IL-7 (73). Nevertheless, IL-7R is expressed
on human B cells, and these cells respond to IL-7 by increased proliferation and survival (74-76).
Recently, using a co-culture model with human-only derived cells, Parrish et al. showed that IL-
7 enhances the production of B cells from adult BM and cord blood HSC (77). In addition,
reminiscent of murine B lymphopoiesis, a small population of B cells could develop from cord
blood HSC without the addition of IL-7, while the development of B cells from adult BM HSC
was dependent on IL-7 (77).
1.2.3 B cell specification and commitment: Pre-proB and proB cell stages
BM B cell precursors can be divided into subsets that correspond to different stage of maturation.
In 1991, Hardy et al. proposed to divide the earliest B cell progenitors that do not yet express
IgM into four subsets termed fraction A, B, C, and D based on the expression of surface markers
(32). Fraction A was originally resolved based on the expression of the surface markers B220 -
an isoform of CD45 whose expression is largely, but not only, restricted to the B cell lineage -
CD43, AA4.1 (CD93), low expression of heat stable antigen (HSA/CD24), and the absence of
CD19 (32). This fraction was suggested to represent the earliest identifiable stage restricted to B
cell development and hence was named the pre-proB cell fraction (32, 78). The B cell lineage
nature of this fraction was supported by the expression of the germline IgH transcript, mb1
B29 (which encodes Ig ) and , as well as transcription factors involved
in B cell development such as E2A (78). However, it is now apparent that although this fraction
likely includes the source of CD19+ proB cells, its cellular composition is rather heterogeneous
and can give rise to other cell lineages (40, 79, 80). Notably, it contains NK1.1+ natural killer
(NK) cell precursors, and a subset expressing Ly6C and/or CD4 that can generate plasmacytoid
dendritic cells (pDCs) (40, 80-83). Based on this new information, the pre-proB cell fraction has
been further defined as B220+CD19-HSAlowLy6C-AA4.1hiCD43medcKitmedIL- + (45). It has
been reported that cells included in this fraction are engaged (specified) in the B lineage
16
developmental process and differentiate predominantly into B cells. However, they are not yet
committed since they also have some potential to give rise to T cells in culture (45).
Cell specification occurs when genes associated with a particular cell fate program are turned on.
In B cells, this is characterized by the expression of genes coding for components of the preBCR
(mb-1, b29, 5, and VpreB) and the initiation of the rearrangement of the IgH locus. B cell
specification requires the action of the transcription factors E2A and EBF (reviewed in (84) and
(85)), as inactivation of either E2A or EBF results in a block of B cell development prior to the
pre-proB cell stage (86, 87). E2A comprises a homo- or- heterodimer of the basic helix-loop-
helix proteins E12 and E47 that bind to E-box DNA sequences. Ectopic expression of E2A in the
macrophage form of the cell line 70Z/3 induces Ebf expression, indicating that Ebf is a target
gene of E2A (88). In support of this, E box motifs have been identified and E2A interactions
detected in the distal promoter, one of the two promoters controlling the expression of EBF
(89, 90). The promoter is further up-regulated by STAT5 (89, 91), which itself is activated
by IL-7R signalling. IL- -/- and IL-7-/- mice have impaired EBF expression and BM B cells do
not mature beyond the pre-proB cell stage (91, 92). However B cell development can be partially
rescued upon enforced expression of EBF or STAT5 (91). Increasing the expression of EBF
through the activation of STAT5 represents one mechanism by which IL-7 controls the B lineage
development (91, 92). Evidence suggests that, once expressed, EBF can produce a positive
feedback loop by binding its own promoter (89). Another positive feedback loop for the
expression of EBF is created through the upregulation of the Pax5 gene which codes for the
transcription factor B cell-specific activator protein (BSAP). EBF induces the expression of
Pax5, which can in turn further increase the expression of EBF by binding to the proximal Ebf
promoter (85, 89). It has been proposed that this regulation mechanism of EBF evolved to
amplify B cell specific gene expression and ensure the B cell fate (85).
The induction of Pax5 expression is a critical step for the commitment of a progenitor to the B
lineage. In mice deficient for Pax5, B cell development is arrested at an early proB cell stage (93,
94). When cultured in vitro in the presence of IL-7 and stromal cells, Pax5-/- proB cells
17
proliferate, but are unable to mature further along the B lineage pathway (93). Moreover, Pax5-/-
proB cells maintain developmental plasticity, as evidenced by their ability to differentiate into
granulocytes, macrophages, natural killer cells, T cells, dendritic cells, and osteoclasts when
stimulated with the appropriate cytokines or after in vivo cell transfer (95-97). This pluripotency
is suppressed upon restoration of Pax5 expression (95). In contrast, conditional inactivation of
Pax5 in previously committed proB cells allow reacquisition of lineage plasticity (98).
Therefore, Pax5 not only plays an essential role for the commitment to the B lineage, it is also
required for the maintenance of B cell identity (98). In agreement with this, Pax5 expression is
induced at the proB cell stage (fraction B) and is maintained until mature B cells differentiate
into PCs (99). Pax5 consolidates the B cell fate by activating the transcription of B lineage-
associated genes such as Cd19, a key marker first expressed in cells of Hardy’s fraction B, mb-1
and Blnk; while suppressing other developmental options through the repression of B lineage-
inappropriate genes such as Notch and M-CSFR (93, 95, 100-104).
One important environmental cue that participates in the development of these early B cell
progenitors is the BM stromal cell-derived cytokine IL-7. As mentioned in the previous section,
mice deficient in IL-7 or IL-7R are severely deficient in B cells, a consequence of a block at the
pre-proB cell stage of development (53, 54). The mechanisms by which IL-7 supports the
development of B cells are well characterized. IL-7 signalling activates ERK and Akt which in
turn regulate the action of proteins involved in the cell cycle, such as c-myc and p27Kip, to
induce a robust proliferation of proB and large preB cells (57, 66, 105). Furthermore, IL-7
enhances proB cell survival by increasing the ratio of anti-apoptotic factors, including Bcl-2,
Bcl-xL, and Mcl-1, to pro-apoptotic molecules, such as Bax, Bad, and Bim (106-111). IL-7R
signalling is also essential for progression through the B lineage, as improving cell survival by
overexpressing Bcl-2 in IL- -/- -/- mice is insufficient to restore normal B cell
development (112, 113). Aside from promoting B cell maturation by inducing EBF expression,
IL-7 signals affect maturation by interfering with the process of VDJ recombination. Most cells
that have reached the proB stage have completed the rearrangement of D-JH gene segments and
are now beginning the recombination of VH segments to DJH-joined segments. IL-7R signalling
participate in this process by promoting the accessibility of the distal VH genes to the RAG
18
proteins through histone acetylation (114, 115). In addition, the signal delivered by IL-7R
interferes with the regulation of RAG protein expression (reviewed in (57, 116)). This is
achieved via the phosphorylation of Foxo1 by Akt. When phosphorylated, Foxo1 is retained in
the cytoplasm which prevents transcription of the rag genes. It has been proposed that during the
pre-proB cell stage, IL-7 signalling is controlled to reduce the activation of Akt and the
phosphorylation of Foxo1, allowing Foxo1 to migrate to the nucleus and stimulate the
transcription of the rag genes. As B cell mature toward the late proB cell stage, they become
more responsive to IL-7, and therefore less potent to activate Rag protein expression. IL-7
induced cell proliferation further compounds the suppression of Rag activity, due to the
decreased stability of Rag2 in cycling cells. Once a fully rearranged VDJH locus is formed, the
recombination machinery is suppressed and proB cells progress to the large preB stage. Cells
harbouring a successfully VDJH- rearranged allele and undergoing this transition are subject to a
few rounds of cell division prior to the recombination of the light chain locus. These cycling
cells can be distinguished by the presence of BP1, a B cell developmental marker known to be
induced by IL-7 (117).
1.2.4 From preB cells to immature B cells
The preB cell stage is a critical step in B cell development. The production of a chain
following the successful rearrangement of the IgH genes does not guarantee that it is a
structurally sound and functional protein; and therefore the chain must be tested before cells
progress past the preB cell stage (118). The fitness of a chain is assessed, in part, by its ability
to associate with the surrogate light chain (SLC). The SLC is a heterodimer made of the two non-
(119, 120). It
has been estimated that approximately half of the chains produced are unable to combine with
a SLC (121). Only those progenitors that express a chain able to pair with SLC and thus form a
signalling-competent preB cell receptor (preBCR) can be positively selected and progress down
the pathway of becoming mature B cells (122).
19
The preBCR is expressed transiently at the cell surface of large preB cells in complex with the
signal- (123). Several observations support the importance of a
functional preBCR for the normal development of B cells. For example, the absence of either
results in a complete developmental block at the proB cell stage (124). Deficiency in
the surface expression of the chain in mt-/- mice also leads to a profound block at the proB
cell stage, although a small number of B cells expressing other isotypes are still produced (125,
126). Finally, the proB to preB cell transition is impaired upon deletion of the genes encoding 5
and/or VpreB (127-129). The absence of these genes prevents the few rounds of cell division that
occur after a functional preBCR is expressed at the surface. However, this blockade is
incomplete as B cells are still found in the periphery. It has been suggested that prematurely
formed light chain might replace the SLC to select the chain repertoire and enable progression
to the preB cell stage. On the other hand, it is possible that the mere expression of signalling-
competent chains can occasionally support the development of preB cells, as chains have
been detected on the surface of preB cells in the absence of LC and SLC (118, 130).
Whether or not initiation of preBCR signalling depends on ligand-binding is still unclear. It is
thought that signalling is triggered upon aggregation of preBCR on the cell surface. However,
antibodies specific for the chain or the SLC do not affect the proliferation of large preB cells
(131). In addition, studies carried out on mice transgenic for a truncated form of the chain that
cannot associate with the SLC, but is expressed at the cell surface, have shown that the SLC is
dispensable for the development of B cells – supporting a ligand-independent activation of the
preBCR (132). On the other hand, others studies have shown that the non-Ig section of 5 is
important for the initiation of preBCR signalling, and this portion was recently shown to be also
polyreactive (133, 134). Heparin sulphate, galectin 1, insulin, DNA, and lipopolysaccharides
(LPS) have been identified as potential ligand candidates (133, 135, 136). Based on these results,
it has been suggested that the polyreactive potential of the non-Ig portion of 5 causes the
immediate aggregation of surface preBCR molecules, which in turn eases the initiation of
signalling (134, 137).
20
It is generally assumed that following preBCR crosslinking, ITAMs present in the ctoplasmic tail
are phosphorylated by protein tyrosine kinases of the Src family. However, single
loss mutants of the three Src kinases most highly expressed in B cells - Fyn, Lyn, and Blk - do
not show a significant effect on B cell development in the BM (138-141). Moreover, triple-
deficient Fyn-/- Lyn-/- Blk-/- mice exhibit a block at the proB to preB cell transition and, although
the activation of NF- B is impaired in anti-Ig -
and Ig is unaltered (142). While these results indicate that Fyn, Lyn, and Blk are involved in
ITAMs. Phosphorylated ITAMs serve as docking sites for the recruitment and activation of Syk,
which in turn stimulates numerous downstream signalling pathways leading to proliferation,
survival and differentiation of preB cells (143). One pathway activated by Syk is the PI3K
pathway. Phosphatidylinositol (3,4,5) triphosphate (PIP3), the product of PI3K activity, recruits
proteins containing pleckstrin-homology domains to the plasma membrane, including the
protein, which promotes cell survival and proliferation (144). Another target activated by Syk is
the B cell linker protein (BLNK, also named SLP65 or BASH), which upon activation forms a
2) which
regulates preB cell differentiation (145-147). BLNK also participates in the activation of the
Ras/MAPK pathway (148). We have previously shown that the ERK/MAPK pathway triggered
by the preBCR cooperates with IL-7-induced ERK activation for the selection of preBCR+ cells
(68). We have proposed a model in which the accessibility of IL-7 varies through the BM. Areas
with high IL-7 availability would support the expansion of proB cells, whereas adjacent areas
with limited IL-7 availability would favour the expansion of cells bearing a preBCR.
Cycling PreBCR+ cells are characterized by their large size and the initiation of CD22
expression, a molecule often associated with the inhibition of BCR signalling. They are thought
to undergo two to five rounds of cell division before entering the small preB cell stage (fraction
D), characterized by the loss of BP-1 and CD43; and the acquisition of CD2 (149). It has been
proposed that the number of cell divisions completed by a particular clone depends on the
stability of the chain-SLC complex (149). The preBCR-mediated downregulation of SLC
genes have been proposed to activate a negative feedback loop resulting in the cessation of
21
preBCR expression and signalling, and therefore account for the cell cycle exit observed during
the transition to the small preB cell stage (137, 150). However, the enforced expression of SLC
B cells has shown that inhibition of preB cell proliferation does not rely only on the silencing of
SLC genes (151). The transition of large preB cells toward the small preB cell stage is also
marked by the loss of the ability to proliferate in response to IL-7 (152, 153). The
recombinational machinery is reactivated in the small preB cells to allow the rearrangement of
the Ig and light chain loci. Successful rearrangement of either loci results in the generation of
a surface IgM+IgD- immature B cell.
1.3 B CELL DEVELOPMENT IN THE PERIPHERY
1.3.1 Transitional B cells
It is estimated that 10% of the cells that develop in the BM will exit to the periphery and, of
these, approximately 30% will enter the mature B cell pool and become marginal zone (MZ) B
cells or follicular (FO) B cells (154). Newly formed B cells that are released into the periphery
are termed transitional B lymphocytes (154-156). These cells can be distinguished from their
mature counterpart based on their higher susceptibility to apoptosis in response to BCR
engagement and the retention of expression of surface marker characteristic of BM immature
cells such as AA4 (CD93) and high levels of HSA (CD24) (156-158). Transitional B cells also
express lower amounts of CD22 and B220 than mature B cells and display low to intermediate
surface levels of CD21, which differentiates them from MZ B cell precursors (156, 157, 159).
Studies of splenic transitional B cell have led to the resolution of three different populations:
transitional 1 (T1) B cells IgMhighIgDlowCD23-, T2 IgMhighIgDhighCD23+ B and T3
IgMlowIgDhighCD23+ B cells (157).
It has been generally assumed that T1 B cells mature through the T2 stage before becoming
mature FO or MZ B cells and that these events takes place in the periphery. However recent
22
findings suggest that differentiation of BM immature B cells into mature B cells might not
necessarily follow this linear sequence but may also be achieved via alternative routes (160-163).
This notion was suggested by mathematical modeling which predicts that splenic transitional B
cells are derived from more than one source of BM B cells and that, under some circumstances,
the T1 stage may be bypassed (161). Recently, experimental data provided support to this model
by showing that immature B cells can mature in both compartments, the spleen and the BM, and
that the newly formed B cells released from the BM formed an heterogeneous pool of immature
and semimature B cells (160, 162, 163). It has been estimated that about two-thirds of newly
formed B cells exit the BM and pursue the maturation process in the spleen (160, 162, 163). In
the spleen transitional B cells give rise to AA4-/lowHSAlowIgMhighIgDhigh follicular type II B cells
(FO-II) which further differentiate either into AA4-HSAlowIgMlowIgDhigh follicular type I B cells
(FO-I) or MZ B cells (164, 165). The remaining immature B cells, about one third of BM
immature B cells, continue to mature in the BM to become FO-II and FO-I B cells (160, 162,
163). They do not differentiate into MZ B cells as MZ B cells are not produced at any other site
than spleen.
Different theories have been proposed regarding the nature of the T3 population (165). As
mentioned above, with the exception of CD93, this population expresses typical makers of
mature FO B cells. Based on BrdU labelling experiments, it has initially been proposed that T3 is
a developmental intermediate stage part of the sequence T1 T2 T3 mature (157). On the
other hand, it has recently been suggested that the T3 B cell population does not represent a
normal stage of B cell maturation as they do not give rise to mature FO B cells upon adoptive
transfer (166). Instead, this population could be formed mainly by cells rendered anergic due to
self-reactivity (167). Interestingly, it has been shown that this population is hyporesponsive to
BCR stimulation (166-168).
23
1.3.2 Mature B cells and beyond
Upon activation, mature B cells either die or increase in size and begin to proliferate. A fraction
of these proliferating cells also starts secreting detectable amounts of antibodies. Because these
cells have a mixed phenotype of blasts and Ig-secreting cells, they are called plasmablasts.
Plasmablasts ultimately exit from the cell cycle and differentiate into non-dividing Ig-secreting
PCs. PCs represent the final stage of B cell maturation and form a heterogeneous population of
cells which differ notably in their lifespan, localization, antigen affinity, and cell surface markers
(169). Any type of activated B cell can differentiate into PCs, however it is hypothesized that the
ability of B cells to develop into a specific type of PC is influenced by the nature of the antigen
engendering the response, the environment, and the subset of B cells from which they arise. For
instance, MZ B cells seem predisposed to differentiate rapidly after antigen exposure,
independently of T cell help, to yield short-lived PCs (170, 171). On the other hand, activated FO
B cells that have received T-cell help can either form plasmablasts which eventually develop into
short- or long-lived PCs with high affinity for an antigen; or they can become memory B cells
which contribute to the replenishment of the PC pool upon re-exposure to a specific antigen.
Although it has been commonly assumed that only B cells that differentiate in GCs in response
to T cell-dependent antigen could become long-lived PCs, recent data have shown that both T
cell-dependent and T cell-independent antigen responses can elicit the formation of long-lived
PCs in the absence of a GC reaction (172).
The discovery of B lymphocyte-induced maturation protein-1 (Blimp1) has been a critical event
for the understanding of the genetic program that governs PC differentiation (173). Blimp1 is a
zinc-finger transcription factor encoded by the prdm1 gene. Often described as the “master
regulator” of PC differentiation, its expression in B cell lines is sufficient to induce several traits
associated with PCs, including the central ability to secrete Ig (173, 174). In line with this,
deletion of Blimp1 in mice leads to a failure to generate a functional PC compartment (175, 176).
Blimp1 promotes PC differentiation by repressing genes involved in cell cycle, such as c-myc,
GC formation, such as bcl6, or commitment to and maintenance of the B cell identity, such as
pax5 (177-179). It is present in all Ig-secreting cells with long-lived PCs expressing higher levels
than cycling plasmablasts (180). Although important for the complete differentiation of B cells
24
into PCs, Blimp-1 is dispensable for the initial commitment to the PC fate (181). This is
suggested by the observation that Rag1-/- mice reconstituted with blimp1-/- B cells, and therefore
lacking a defined PC compartment, contained detectable quantities of Igs (181). In these mice, B
cells differentiate up to a preplasmablast stage which shows some, but not all, of the gene-
expression changes found in PCs, including up-regulation of xbp1, IgJ, splicing of IgH mRNA
into the secreted form, and downregulation of pax5. Based on these results, it was proposed that
the initiation step of PC differentiation was not the expression of Blimp1 but the inhibition of
Pax5 function.
Pax5 represses the PC program in mature B cells (182, 183). Therefore the inhibition of Pax5
function derepresses various Pax5-target genes involved in PC differentiation. One of them,
Xbp-1, is a transcription factor that drives the unfolded protein response (UPR) (reviewed in
(184, 185)). Xbp-1 was originally thought to be fundamental for the differentiation of B cells
into actively secreting PCs, as mouse lymphoid chimeras deficient in xbp1 displayed a
dramatically reduced number of PCs (186). It was proposed that its activation was triggered by
the accumulation of excess unfolded proteins and that Xbp-1 was acting by promoting the
synthesis of chaperones and other proteins believed to help the endoplamic reticulum (ER) to
handle the high demand of Ig synthesis (187-189). However, recent studies have demonstrated
that PCs can develop in the absence of Xbp1; though they fail to undergo the full program of
differentiation, as suggested by their severely compromised ability to secrete high levels of
antibodies (190). These results led to the new hypothesis that the activation of Xbp-1 is a
differentiation-dependent event that occurs prior the UPR (190). In this model, early activation
of the UPR could prepare the cells for the high demands of Ig synthesis in terminally
differentiated B cells (185).
Most PCs generated in secondary lymphoid organs during an immune response are lost within a
few days during a contraction phase (191). A limited number, however, can survive for several
weeks, particularly those residing in the BM. Nevertheless, it is estimated that PCs make up less
than 1% of the cells in lymphoid organs (192). Present data suggest that the lifespan of PCs
25
greatly depends on external signals present in the microenvironmental niches where they are
maintained (193-195). Numerous signals have been reported to promote survival of PCs in vitro
including tumor- -5, IL-6, CD44 ligands, and very late
antigen-4 (VLA-4) (194, 195). These factors are produced by BM stromal cells and could
therefore be components of the niches that support PCs in vivo. Interestingly, these molecules are
also present in inflamed tissues which could explained why PCs can persist at these sites (196-
198). It has been proposed that inflammation sites contain a number of niches where PCs can
survive and secrete relevant antibodies to fight the agent responsible for the inflammation (199).
Upon successful clearance of the pathogen and resolution of the inflammation, the survival
niches are destroyed which trigger the death of the PCs at the inflammation sites, the long-term
humoral protection being provided by the PCs that have migrated into the BM. In chronically
inflamed tissues, such as in autoimmune diseases, the stability of the survival niches would allow
PCs to persist and therefore maintain the production of autoantibodies (198, 199).
IL-6 has been reported to be one of the most efficient factors for enhancing the survival of PCs in
vitro (195). Indeed, addition of recombinant IL-6 to BM PCs cultured in media alone increases
their longevity (195, 200). In agreement with this finding, BM supernatant prepared from wild-
type (WT) mice can support PC survival, while BM supernatant prepared from IL-6-/- mice is
totally ineffective (195). IL-6 can be produced by BM stromal cells and BM DCs following
direct physical interaction with PCs (194, 200). It has been shown that, when deficient for IL-6,
both BM stromal cells and BM DCs are substantially impaired in their ability to promote IgG
production (194, 200). However, one study also reported that defective IgG production did not
correlate with decreased numbers of BM PCs (200). In vivo, although IL-6-/- mice require a
longer period to mount an antibody response, they ultimately reveal no significant differences in
either the serum levels of antigen-specific IgG or the number of BM antigen-specific PCs when
compared to WT mice (195). Together these observations suggest that although IL-6 can support
the survival of PCs and play an important role for the production of Ig in vitro, other factors
contribute to the maintenance of the PC pool and can compensate for the absence of IL-6 in vivo
(195).
26
CD44 forms a ubiquitously expressed family of molecules. The different isoforms are generated
by alternate splicing of multiple exons of a single gene which undergo additional modifications
at a post-translational level (reviewed in (201)). The principal ligand is hyaluronic acid, a
component of the extracellular matrix. CD44 is highly expressed on BM PCs (202, 203) and has
received further attention as expression of the CD44v9 isoform in patients with multiple
myeloma correlates with unfavourable prognosis (204). It was shown that engagement of some
CD44 isoforms on PCs, including CD44v9, can contribute to the induction of IL-6 secretion by
BM stromal cells and therefore represents one mechanism by which CD44 could promote PCs
survival (205). In addition, in vitro studies in which CD44 had been cross-linked with an anti-
CD44 antibody showed that engagement of CD44 itself can also provide a survival stimulus
(195).
CXCL12 is known to be important for the migration of newly formed PCs into the BM (206-
208). Both newly formed PCs and established BM PCs express CXCR4 which is the receptor for
CXCL12. However only newly generated PCs have the ability to migrate toward CXCL12 (208,
209). This could be a mechanism that allows some new PCs to dislodge previously established
BM PCs (169, 210). However, the maintenance of CXCR4 expression after the establishment of
PCs in the BM suggests that CXCR4/CXCL12 might have another function, which has been
proposed to be enhancement of survival based on in vitro data (195).
In vivo and in vitro experiments have also shown a role for a proliferation-inducing ligand
(APRIL) in the survival of PCs (211, 212). Of high relevance, APRIL-/- mice generate a normal
number of PCs but are impaired in their ability to establish a BM reservoir for PCs (211).
Furthermore, mice in which APRIL has been blocked by immunization with TACI-Ig (a fusion
of the APRIL receptor transmembrane activator and calcium-modulator and cyclophilin ligand
interactor (TACI) with human IgG) display a significant reduced number of BM PCs (213).
Since most BM PCs are associated with cells expressing CXCL12 and the CXCL12+ BM cells
do not co-express APRIL, the recruitment and survival of PCs in the BM seems to be governed
by at least two different types of cells (214, 215). In support of this, WT PCs adoptively
27
transferred into APRIL-/- mice, although unable to survive, are recruited into the proximity of
nursery cells in the BM (214). CXCL12-abundant reticular (CAR) cells have been proposed to
play a central role in the recruitment of PCs in the BM together with other hematopoietic cells
lineages that might contribute to the survival of PCs (214). In the BM, eosinophils have been
found to produce high levels of APRIL and be in close association with PCs (212). Experiments
carried out on eosinophil-depleted mice and reconstitution experiments suggested that these cells
may play a major role in PC survival (212). Other cells that have been reported to promote BM
PC survival, include reticular stromal cells, osteoblasts, osteoclasts, Gr1intCD11b+ monocytes,
megacaryocytes, and basophils (194, 206, 211, 216-218). In the periphery, F4/80+Gr1low
monocytes-macrophages have been described as the greatest source of APRIL in murine lymph
nodes (LNs), and in human mucosal-associated lymphoid tissue (MALT), APRIL is produced by
neutrophils, making these cells likely candidates involved in the creation of PCs niches (219,
220).
Finally, a recent study has also demonstrated a role for CD28 in the survival of PCs (200).
Interestingly, although similar levels of CD28 are expressed on splenic and BM PCs, its ability
to promote survival differed between these two populations. Notably, BM DCs significantly
increased the persistence in culture of WT BM PCs, but not CD28-/- BM PCs. In contrast, BM
DCs could not support the long-term survival of WT splenic PCs, but could improve their short-
term survival. Furthermore, when stimulated with anti-CD28, purified BM but not splenic PCs
were protected from serum starvation-induced death and showed induction of downstream
signaling, which may account for their differential responses (200).
1.3.3 The majority of plasma cells are located in the gut-associated lymphoid tissues and
secrete IgA
The major constituents of the gut-associated lymphoid tissues (GALT) are the Peyer’s patches
(PPs) and the isolated lymphoid follicles (ILFs). Other GALT components include the appendix,
mesenteric LNs, intraepithelial lymphocytes (IEL) and diffusely distributed cells located in the
lamina propria (LP), which is the layer of intestine between the epithelial cells and the superficial
28
smooth-muscle layer (221, 222). The importance of the GALT in mice and human is emphasized
by the fact that more than 80% of all PCs are located in the gut (221). The gastrointestinal tract
has to cope with an extremely dense and diverse community of microorganisms. It is estimated
that about 1014 microbes live in the intestines with densities that range up to 1012 microbes/mL
intestinal content in the distal gut (223, 224). In response to this microbial colonization, high
quantities of IgA are produced by the GALT. Indeed, it is estimated that 80-90% of the PCs
present in the gut produce IgA (221).
The IgA molecule is well adapted for the gut environment. It is secreted mainly in the lamina
propria as a dimer consisting of two basic Ig units linked by a joining (J) chain (225). Once
released by PCs, the IgA J chain binds to the polymeric Ig receptor (pIgR) expressed on the
basolateral surface of epithelial cells (reviewed in (226)). This binding triggers the endocytosis
of IgA from clathrin-coated pits and the subsequent translocation through the cellular
compartment to the apical surface of epithelial cells. At the apical surface, pIgR is subjected to
proteolytic cleavage which releases IgA into extracellular fluid, in addition to ensuring a
unidirectional transport (226). After cleavage, a portion of the pIgR, called the secretory
component (SC), remains attached to the IgA molecule and protects it against the activity of
proteases present in the gut (227). In the intestinal lumen, IgA neutralizes toxins and binds to
bacteria to control their growth and limit their access beneath the gut epithelium. Since IgA is
unable to fix and activate the complement cascade, its function is performed without inducing
inflammation (226). A link between the production of IgA and the presence of intestinal
microbes was established by the finding that germ-free (GF) mice have extremely low, but not
absent, numbers of IgA+ PCs in their intestinal lamina propria, but that the content of IgA
increases after colonization with commensals (228, 229). Similarly, IgA-secreting B cells are not
present in neonatal mice that have not yet been exposed to bacteria (228, 230). The composition
of the intestinal flora is also likely to influence the magnitude of the IgA response since some
bacteria, like segmented filamentous bacteria, are more potent inducers of mucosal IgA (231,
232).
29
It is generally accepted that PPs play a major role in the differentiation of B2 cells into IgA+ B
cells. However, it is now becoming clear that IgA+ B cells can also be generated in other sites in
the GALT from either B1 or B2 cells (224). However, the importance of each site in the
production of IgA have been hard to address since their relative contribution can be affected by
factors such as gene deficiencies or the environment (225). For example, mice devoid of PPs
have larger ILFs than WT mice, but wild-caught feral mice have enlarged PPs and a fairly
insignificant number of ILFs (225). Furthermore, mice deficient for CD40 or T cells have PPs
and ILF structure and produce significant but lower levels of IgA (229, 233, 234). This implies
that intestinal IgAs are generated via both T-dependent and T-independent pathways. It has been
hypothesized that the nature of the microbial populations present in the gut might affect the
relative contribution of each pathway (229). Based on the set of data available to date, one model
proposed is that the largest proportion of IgA+ cells are generated from IgM+ B cells in PPs
through a T-dependent pathway before migrating to the LP, yet T-independent CSR can also
occur in PPs (225). IgA+ cells can also be generated in other sites of the GALT independently of
T cell help. Although under normal conditions, the development of IgA+ cells is most prominent
in PPs, if insufficient numbers are produced, then the effectiveness of IgA+ cell generation can
be increased at other sites of the GALT to compensate (225).
1.3.4 Signals inducing IgA production
As mentioned above PPs host the majority of IgA CSR events. PPs are organized lymphoid
structures that are mainly found in the terminal section of the small intestine, the ileum (235).
Most lymphocytes present in PPs are activated and reside in GCs. In contrast to other lymphoid
tissues, GCs are found continuously in PPs due to constant stimulation by microbial products
(236, 237). Recent studies suggested that formation of GCs in PPs also likely depends on the
presence of CD4+ follicular B helper T (TFH) cells (238, 239). PP GCs have the particularity to
harbour conditions that cause B cells to undergo preferential CSR to IgA.
30
It is well established that TGF is one of the most important factors that regulates IgA
production (240-245). In vivo, mice in which the receptors for TGF have been depleted have
undetectable levels of serum IgA and extremely low number of IgA+ cells in their PPs (242,
243). It was demonstrated by Coffman et al. and Sonada et al. that TGF stimulates IgA
production from IgA- B cells, but not IgA+ B cells, indicating that TGF acts as an IgA-switch
factor rather than a stimulator for IgA post-switch cells (241, 246). In support of this hypothesis,
it was later shown using in vitro systems that TGF signalling activates the transcription factors
mothers against decapentaplegic homologue 2 (SMAD2), SMAD3, SMAD4, and runt-related
(230, 240, 241, 244, 247).
Subsequently, the induction candidates have
been proposed as potential source of TGF in the gut including B cells, TFH cells, antigen
presenting cells, and stromal cells (245, 248-251). However, since TGF is secreted as part of a
latent complex from which it must be released, its function also depends on the presence of other
molecules such as matrix metalloproteinase (MMP)9, MMP13, integrins, or thrombospondin-1
(252). TGF is known to direct CSR to IgA, but also IgG2b (241, 253). As an explanation for the
bias toward IgA switching observed in PP, it has been proposed that IL-21, which is secreted by
TFH cells, synergizes with TGF to enhance the proliferation and differentiation of IgA PC
precursors (254). In mice, it has been shown that IL-21 inhibits the expression of TGF -induced
expression of GLTy2b and consequently the production of the IgG2b isotype (255). Other
cytokines, such as IL-5, IL-6, and IL-10 have been reported to augment the production of IgA by
acting on B cells that have already undergone class switching (246, 248, 256-259).
Two additional factors that that can act as switch factors for IgA due to their capacity to induce
-cell activation factor of the tumor necrosis
factor family (BAFF) (also known as B lymphocyte stimulator (BLyS)) (260). Both APRIL and
BAFF molecules bind to the receptors B cell maturation antigen (BCMA) and TACI. In addition,
BAFF binds to a third receptor called BAFF receptor (BAFF-R) (Reviewed in (261)). All these
receptors have been detected on the surface of B cells, however their degree of participation to
IgA production varies (262). BCMA does not seem to affect the induction of CSR, as suggested
by the normal serum levels and antibody response, but might be involved in post-switch events
31
given its ability to enhance PC survival (213, 263). BAFF-R is also well known for delivering a
survival signal to peripheral B cells upon binding to BAFF (264). In contrast to BCMA,
signalling through BAFF-R contributes to some extent to CSR (260, 264). However, in vitro
experiments showed that the switch to IgA appears largely controlled by TACI (260). In
agreement with this, TACI-/- and APRIL-/- mice have lower serum levels of IgA (225, 262, 265).
Both APRIL and BAFF are produced by DC and epithelial cells that have received stimulation
through Toll-like receptors (TLRs) (229, 230, 266, 267). They act directly on B cells to promote
IgA switching without the requirement for CD40-T cell help, which has led to the proposition
that it could represent an alternative pathway used to generate IgA+ B cells in the non-organized
gut tissue (260, 266, 268).
1.4 IL-21 – AN IMPORTANT CYTOKINE FOR LATE STAGES OF B CELL
DIFFERENTIATION
IL-21 receptor (IL-21R), originally named novel interleukin receptor (NILR), was discovered in
2000 independently by two groups through the identification of an open reading frame on a
human genomic sequence (269) and by expressed-sequence tag (EST)-based sequencing (270).
The activity of a ligand was detected in the culture supernatants of activated CD3+ T cells which
led to the cloning and identification of IL-21 (270). Il-21 is a small protein of 15 kDa (270). It is
the last member added to the chain ( c) -dependent cytokine family (271). The effects of IL-21
are diverse and occur on various hematopoietic and non-hematopoietic cells (reviewed in (272)).
This section focuses on the action of IL-21 in B cell biology.
1.4.1 IL-21 expression and structure
The gene coding for IL-21 is located only few kilobases away of Il-2 on chromosome 3 in mice
and chromosome 4 in humans (270, 273). The similarity between the Il-21 and Il-2 genes and
their proximity to each other on the chromosome have led to the hypothesis that they may have
arisen by gene duplication (274). IL-21 is expressed by activated CD4+ T cells as well as NKT
32
cells (275-278). The mRNA contains many motifs that create instability which might explain
why the message is absent in resting cells but is detected after cell treatments such as ionomycin,
or a combination of anti-CD3, and anti-CD28 (274). The polypeptide encoded by Il-21 cDNA
contains 131 and 122 amino acid residues in human and mice respectively and shares 57%
identity between the two species (270). The protein has a structure related to the four-helix
bundle type I cytokine and a peptide sequence particularly well conserved in the regions
-helices A and D (270). These regions have been shown to play a role in
receptor interactions for other cytokines (279, 280). Likewise, the importance of helix D in IL-21
function is demonstrated by the incapacity of mutant IL-21 protein to activate IL-21R following
their binding (274).
1.4.2 IL-21R expression, structure and signalling
IL-21R is expressed by B, T, NK, and dendritic cells and some non-immune cells, including
fibroblasts, keratinocytes and intestinal epithelial cells (269, 270, 281-285). In murine BM, the
levels of IL-21R expressed at the surface of B cells gradually increase as the cells progress
toward more mature stages (281). In the periphery, T1 B cells, which correspond to the earliest
developing B cells to exit the BM, express lower levels of IL-21R than their more mature
counterparts T2 and follicular B cells, but similar levels to that of MZ B cells (286). After
activation of naïve B cells through TLRs or CD40, the expression of IL-21R is further up-
regulated and reaches its highest expression (281). In human, IL-21R has also been detected on
naïve and GC B cells, but not on memory or terminally differentiated PCs from the BM (287,
288). Activation of human naïve B cells or memory cells through CD40 increases the surface
levels of IL-21R (287). The developmental regulation and higher density of IL-21R found on the
surface mature B cells suggest that the ability of IL-21 to induce a response varies according to
the stage of differentiation and has important functions late in B cell development. Moreover, a
role during immune response is suggested by the up-regulation of the receptor expression
following B cell activation.
33
The receptor for IL-21 is a heterodimer that consists of IL-
(289). The amino acid sequence of the murine IL-21R is 62% identical to
the sequence of human IL-21R (274). Like other c-dependent cytokines, IL-21 activates
members of the Janus family tyrosine kinases. More precisely, IL- c binds
JAK3. Once phosphorylated, these kinases activate signal transducer and activator of
transcription 3 (STAT3), STAT1, and to a lesser extend STAT5. In agreement with this, our lab
has previously shown that in EBV-infected human B cells, stimulation with IL-21 activates
STAT3 as early as 5 min after treatment and this activation persists up to 6 days. In these cells,
activation of STAT1 is weaker, while activation of STAT5 is transient ( 60 min) (290). The
dominant activation of STAT3 and STAT1 distinguishes IL-21 from the other c-dependent
cytokines. Effectively, IL-2, IL-7, IL-9, and IL-15 predominantly activate STAT5, whereas IL-4
principally activates STAT6 (272). The cytoplasmic domain of IL-21R contains six tyrosine
residues. Five of them, Y281, Y361, Y369, Y397, and Y510 are conserved between mouse and
human (291). Biochemical studies carried on B and T cells transfected with a series of IL-21R in
which tyrosine of the cytoplasmic tail have been mutated to phenylalanine established that Y510
mediates activation of STAT1 and STAT3 and plays a critical role in IL-21-induced cell
proliferation (291).
1.4.3 IL-21 effects on B cell functions
1.4.3.1 Proliferation and survival
Many cytokines play an important role in the regulation of cell proliferation and survival. In vitro
experiments have shown that IL-21 can both positively and negatively regulate B cell outcome
depending on the signal context and the developmental stage. For instance, in the presence of
signalling through both BCR and CD40, IL-21 promotes growth and differentiation of murine
splenic B cells into antibody-secreting cells (281, 292). However, growth arrest and apoptosis
occurs when lipopolysaccharides (LPS)-, CpG-, or IgM-treated B cells are stimulated with IL-21
(281, 286). Similarly, IL-21 inhibits proliferation of cells stimulated with anti-IgM and IL-4 in
34
the absence of anti-CD40. However addition of anti-CD40 under these condition reverses the
effect and leads to increased proliferation (292). The ability of IL-21 to regulate B cell
proliferation has also been demonstrated in human in vitro systems. In fact, IL-21 has been
proven to be more potent at increasing proliferation of anti-CD40-stimulated B cells than IL-2,
IL-4, IL-10, and IL-13, cytokines also known to induce B cell proliferation (287, 293, 294).
Analogous to murine B cells, BCR stimulated human B cells are protected from IL-21-mediated
apoptosis by the addition of anti-CD40 (293).
Analysis of mice overexpressing IL-21 indicates that IL-21 also regulates the proliferation and
survival of B cells in vivo (292). Overexpression of IL-21 was achieved using either transgenic
methods to overexpress human IL-21 or by injection of plasmid DNA encoding mouse IL-21
into WT mice. Both systems created similar effects on B cell populations - mice showed
increased numbers of immature B cells, post-switch B cells, and PCs, but not mature B cells.
Consistent with the higher numbers of PCs, there was an increase production of total serum IgM
and IgG. In spleen, the percentage of follicular B cells was notably lower than that observed in
WT mice, whereas the percentage of marginal zone B cells was normal though cells were
relocated outside the MZ area (292, 294).
To date, data obtained in mice suggest that IL-21-mediated apoptosis involves the mitochondrial
death pathway. In support to this, IL-21-induced cell death was blocked in mice overexpressing
Bcl-2, but was unaffected in mice deficient for FAS, FAS ligand, TNFRI or TNFRII (281). In
addition, in LPS or CpG activated murine B cells, IL-21 induces Bim-dependent apoptosis (281).
The ability of IL-21 to induce apoptosis appears to be influenced to some extend by the genetic
background of mice. It was shown that B cells from C57BL/6 were approximately twice more
susceptible to IL-21-mediated apoptosis after treatment with LPS, CpG, or anti-CD40 than B
cells from BALB/c mice (281). In humans, Il-21 has been shown to induce cell death in B cells
stimulated with anti-IgM, although Bim did not seem to be involved in this process (295).
35
-dependent cytokine family activate prosurvival and proliferation
signals. In this matter, IL-21 differs from the other members of this cytokine family by its ability
to induce apoptosis, although complete protection from IL-21-mediated cell death can be
achieved when B cells are treated with anti-CD40 prior to culture with IL-21 (296). Collectively,
the data currently available suggest that IL-21 can control the fate of activated B cells by
promoting proliferation and survival of B cells that have received stimulation through the BCR
and cognate T cell help; while promoting apoptosis in incompletely activated B cells that have
received BCR or TLR stimulation in the absence of T cell help. In a clinical setting, these effects
on B cells suggest that dysregulation of the IL-21-IL-21R system could impair the elimination of
autoreactive B cells and, therefore contributes to the development of antibody-mediated
autoimmune disease like systemic lupus erythematosus (SLE). In this regard, BXSB.B6-Yaa+/J
mice suffering from SLE showed elevated levels of serum IL-21 (292).
1.4.3.2 Plasma cell differentiation
Besides its role in B cell proliferation and survival, IL-21 also regulates the terminal
differentiation of B cells into Ig-secreting cells called PCs. Support for a role for IL-21 in the
generation of Ig-secreting cells was provided by data obtained from IL-21R-/- mice, which lack
IL-21R extracellular and transmembrane domains (297). In steady-state, these mice have normal
serum levels of IgM, IgG2a, and IgG3, but lower amount of IgG1 and IgG2b than WT mice
(297). After immunization with the T cell-dependent antigens ovalbumin or keyhole limpet
hemocyanin, total serum levels of IgM, IgG2a, IgG2b, and IgA were normal, whereas the
production of antigen-specific IgG2b and IgG3 was significantly impaired. The IgG1 isotype
was the most severely affected with both total serum and antigen-specific IgG1 present in
reduced quantities (297). Independent of immunization, the amount of serum IgE detected in IL-
21R-/- mice was noticeably higher than that in WT mice (297). Moreover, increased production
of IgE was observed in IL-21R-/- mice infected with Toxoplasma gondii (297). These
observations were unexpected given that the production of IgE is usually associated with IL-4,
and that infection of mice with the parasite Toxoplasma gondii does not induce an IgE response
(298).
36
Using EBV-infected human B cell lines, our lab has shown that IL-21-induced differentiation of
B cells into late plasmablast/PCs relied on the activation of the JAK/STAT pathway (290). More
recently, a study performed on B cells isolated from humans carrying inactivating mutations in
Stat1 or Stat3 has shown that STAT3, but not STAT1, played a major role in the IL-21-driven
differentiation of naïve B cells into PCs (299). The involvement of STAT3 in this process can be
explained by its ability to cooperate with IRF4 to induce the expression of Blimp1, which is
encoded by the Prdm1 gene (300). STAT3 binds to an IL-21 response element located 3’ of
Prdm1 (300). To be active, this element required the binding of both STAT3 and IRF4 (300).
The binding of STAT3 to this element is greatly diminished in the absence of IRF4 (300). Since
IRF4 has been frequently found constitutively bound to STAT3 binding sites, it has been
suggested that prebound IRF4 may guide STAT3 or enhanced STAT3 binding to these sites
(300). In addition to Blimp1, IL-21 promotes differentiation of murine and human B cells into
PCs by repressing transcription factors such as Pax5 (292, 300-303). Interestingly, IL-21 also
induces the expression of Bcl6, a transcription factor that directs the GC program and represses
the differentiation of B cell into PCs (292, 304-306). Although it is not yet clear whether both
Blimp1 and Bcl6 are induced in the same cell, it is possible that the duration of the signals
derived from the IL-21R determine the Blimp1/Bcl6 ratio (307).
1.4.3.3 Class-switch recombination and immunoglobulin production
As described in an earlier section, CSR and SHM are mechanisms used by B cells to increase the
diversity of the antibody produced. These reactions required the activity of AID, whose
expression was shown to be induced by IL-21 in mouse and human naïve B cells stimulated with
anti-CD40 and anti-IgM antibodies (293, 308). Interestingly, although AID catalyzes both CSR
and SHM, only evidence for a role of IL-21 in CSR has been reported (293). Based on this
observation, it has been hypothesized that IL-21 induces activity of AID only at the C-terminus,
which contains a domain important for CSR but not involved in SHM (293, 309, 310).
37
Cytokines also affect the CSR process by directing which isotype will be generated. The
outcome of CSR following IL-21 stimulation varies according to the developmental stage of the
cells. For example, IL-21 favours CSR of CD40-stimulated human naïve splenic IgM+ B cells to
IgG1 and IgG3, while anti-CD40 stimulated IgM memory B cells switch predominantly to IgG1,
and CD40-stimulated cord blood B cells to IgA (311, 312). In addition, IL-21 treatment of
CD40-stimulated PBMC isolated from patients with selective IgA deficiency triggered the
production of IgA (313). The selection of the isotype is also influenced by the presence of other
cytokines. In this regard, the interplay between IL-21 and IL-4 on the production of IgE has been
one of the most studied. The interest for this combination was originally stimulated by the
observation that Il-21r-/- mice produce larger quantities of serum and antigen-specific IgE than
WT mice (297). The production of IgE is usually associated with the activity of IL-4 (314). The
generation of Il-4-/-Il21r-/- mice has revealed that IL-4 was responsible for the overproduction of
IgE in Il21r-/- mice. Indeed, similar to Il4-/- mice, these mice did not produce IgE (297).
Consistent with these findings, it was subsequently shown that IL-21 suppressed the expression
of C in B cells active with IL-4 (315) and induces apoptosis of IgE+ cells (277). In addition, a
smaller production of antigen-specific IgE was detected in mice following their injection with
IL-21 (315). Surprisingly, overexpression of IL-21 in mice contrasted with these results as it did
not result in a reduced concentration of serum IgE (292). In human B cells, depending on the
culture conditions, addition of IL-21 resulted in both negative and positive effects on IL-4-driven
production of IgE (316-318). Differences in cell culture density as been suggested as a potential
explanation for this discrepancy (318). The interplay between IL-21 and IL-4 has also been
analysed for its effect on IgA production in some human cell culture systems. Notably, the
addition of IL-4 prevented IL-21-mediated class switching to IgA in cord blood B cell cultures
(312), but increased IgA production in PBMC (313).
More recently, the interaction between IL-21 and TGF has attracted attention as it could explain
to some degree the prevalence of IgA+ cells in peyer’s patches. In contrast to the results obtained
in human systems, Seo et al. did not induce switching to IgA when IL-21 was added to cultures
of murine splenic B cells (255). However they showed that treatment of these cells with TGF
directed switching to IgA and IgG2b, and that switching to IgG2b, but not IgA, was decreased
38
when IL-21 was present in the culture. Since elevated levels of IL-21 have been found in CD4+ T
cells isolated from peyer’s patches, it is thought to play an important role in the selective IgA+ B
cell commitment occuring in this lymphoid tissue (255).
1.4.3.4 IL-21 in hematopoiesis
As mentioned above, IL-21R has been detected on BM B cells progenitors. The comparable BM
B cell phenotype observed between IL-21R-/- and WT mice supports a non-critical role for IL-21
in B cell development (297). On the other hand, other observations suggests that IL-21 can
influence the development of certain cell populations in the BM, including murine hematopoietic
progenitor c-kit+sca+lin-/low (KSL) cells and BM CD11b- lymphoid cells (319). More importantly,
it was also reported that mice overexpressing IL-21 exhibited increased numbers of peripheral
immature B cells (292), which could potentially be derived from an alteration in the development
of B cell progenitors in the BM. An analysis of the effects of IL-21 on BM B cell progenitors
will be presented in chapter IV.
1.5 THESIS OUTLINE
B lymphocytes develop from HSCs and mature to plasma cells in reaction to signals from the
supportive microenvironment. To sense these signals and differentiate successfully, maturing B
cells must acquire and use receptors in a carefully prescribed sequence. The work presented in
this thesis is centered on factors regulating the differentiation of murine B cells from progenitors
to Ig-secreting plasma cells.
In the first two data chapers, I focus on the early stages of B cell development in the BM, more
specifically on the involment of CD22 and IL-21R, whose expression has been reported to begin
at the preB cell stage. CD22 is a sialic-acid binding lectin known to recruit negative effectors of
cell activation. Previous work in our lab has established that the appearance of CD22 is inversely
39
correlated with the ability of B cells to respond to IL-7, which led me to investigate whether the
two events were interrelated. In chapter 3, I show that the ligation of CD22 with an antibody
does not modulate the B cell response to IL-7. However, the reduced proliferation observed
following the disruption of the interactions involving sialic acids suggests a role for these
interactions in the regulation of the IL-7 response. In chapter 4, I describe the effect of IL-21R
stimulation on B cell progenitors. IL-21R was initially thought to be expressed at low levels on
preB cells, with levels increasing as the cells mature. In this chapter, I demonstrate that a
functional IL-21R is expressed as early as the proB cell stage. When present in the environment,
IL-21 accelerates the development of B cell progenitors, and can furthermore drive their
differentiation to class switched Ig-secreting cells when CD40 is simultaneously stimulated.
These data suggest that IL-21 may shape B lymphopoiesis in health and, under some
circumstances, in disease. For example, the conditions present in the environment during
inflammation could potentially allow the differentiation of the B cell precursors that have
recently migrated to the periphery into Ig-secreting plasma cells, thereby evading important
checkpoints where autoreactive cells are eliminated.
In the last data chapter, I describe an example of how the environmental conditions found in the
periphery at steady-state can also affect the development of B cells. It is known that the unique
conditions present in the GALT favor class switching to IgA. Lately, it has been reported that
some of these IgA+ cells also express iNOS. In chapter 5, I describe a BM/gut stroma co-culture
system that allows the generation of IgA+iNOS+ cells. Importantly, I show that microbial
products present in the gut are required for the induction of iNOS expression in IgA+ cells.
40
CHAPTER 2
MATERIALS AND METHODS
41
2.1 Cell lines
H33, B62, B62c, B62.1, R5b, mu23 and H24 are IL-7-dependent cell lines generated by limiting
dilution of B220+ cells as described in (68). They were cultured in IMDM with 5% heat
inactivated FCS, 100 g/mL kanamycin or penicillin-sptreptomycin, and 50 M -
mercaptoethanol. Supernatant from the stably transfected J558 cell line was used as a source of
IL-7 (supplied by Dr. A. Cumano, Institut Pasteur, Paris). In some experiments, anti-CD22
(dialyzed Cy34.1; Pharmingen) or neuraminidase (Sigma) were added to the culture at the
indicated concentration. The adherent cell line S17 was cultured in OptiMEM supplemented with
5% FCS, 2.4 g/L NaHCO3, 100 penicillin-sptreptomycin, and 50 M -mercaptoethanol. Cells
were cultured in a humidified atmosphere at 37 C and 5% CO2.
2.2 Mice
CD45.2 C57Bl/6, RAG-/-, and TCR -/- mice were obtained from Jackson Laboratory (Bar
Harbor, ME, USA). CD45.1 C57Bl/6 mice and LT R-/- mice were provided by Dr. JL.
Gommerman (University of Toronto, Toronto). Germ-free (GF) mice were obtained through KD.
McCoy (McMaster University, Hamilton). AID-YFP mice were provided by Dr. R. Casellas
(National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of
Health, Bethesda) and were bred at the University of Toronto animal facility. IL-21R-/- mice
were provided by Dr. WJ. Leonard and were bred at the Ontario Cancer Institute (OCI) animal
facility (Toronto). Mice were sacrificed at between 6 to 8 weeks of age (except mice used to
isolate cells from the intestinal lamina propria that were sacrificed between 10 to 12 weeks of
age) according to a protocol approved by the animal care committee of the Ontario Cancer
Institute.
2.3 Isolation and culture of BM B cell progenitors
B cell progenitors were isolated from BM. B220+ or CD19+ cells were selected by MACS
(Miltenyi). Briefly, single cells suspension from BM was prepared by crushing and gently
grinding both femurs and tibiae using a mortar and pestle. The cells were then flushed with
42
MACS buffer (PBS –Ca2+, -Mg2+, 1 mM EDTA, 0.25% BSA). B cell progenitors were isolated
directly using anti-B220 (clone RA3-6B2, Miltenyi) coupled to beads, or indirectly using anti-
CD19 (supernatant from clone ID3) together with goat anti-rat bead-coupled antibody (Miltenyi).
A VarioMACS magnet with an LS adaptor was used to positively select the cells. Cells were
washed three times with MACS buffer and eluted in OptiMEM (Life Technologies). Typically,
8-14 x 106 B220+ cells are recovered from BM (two femurs and two tibiae) of a mouse.
Recovered B cells were cultured in freshly reconstituted OptiMEM supplemented with 10% non-
heat inactivated FCS, 50 M -mercaptoethanol, 2.4 g/L NaHCO3, 100 g/mL penicillin-
streptomycin, and grown at 37 C in a 5% CO2 atmosphere. Supernatant from the stably
transfected J558 line was used as a source of IL-7. 30 ng/mL IL-21 and/or 2 μg anti-CD40 was
added to the culture where indicated.
2.4 Sorting of BM cells
B cell populations - B220+ cells isolated by MACS were cultured with IL-7 for 4 days (day 4IL-
7) as described above. On day 4, cells were labeled with appropriate antibodies and sorted using a
FASC Aria (BD Bioscience) to a greater than 98% purity. In order to stain proB (CD2- / -),
preB (CD2+ / -), and immature/mature (CD2+ / -) BM B cells rat anti-mouse CD2 (RM2-5;
eBioscience), rat anti-mouse (Southern Biotech) and rat anti-mouse (southern Biotech) IgM
(33.60; made in-house) were used. For experiments presented in Fig. 4.6, B220+ cells were
grown 4 days in IL-7 IL-21 (30 ng/mL). Rat anti-IgM (33.60) and rat anti-IgD (SBA.1;
Southern) antibodies were used to select for the immature IgM+IgD- B cell fraction.
Other populations—For experiments presented in Fig.1, freshly isolated BM or BM B220
cells were stained with rat anti-mouse-B220-APC (RA3-6B2), -CD3-FITC (145-2C11), -CD4-
biotin (GK1.5), -CD8-PE (53–6.7), -CD44-APC (IM7), -CD69-PE, and - NK1.1-APC (PK136)
(BD Bioscience). Cells were then sorted into CD4 T cells (CD3+CD4+NK1.1 ), CD8 T cells
43
(CD3+CD8+NK1.1 ), NK cells (NK1.1+) and B cells (B220+) (Fig.1B). To sort naive CD4 T
cells (CD3+CD4+CD44loCD69 ) and memory CD4 T cells (CD3+CD4+CD44hiCD69 ) (Fig.
1C), BM from 10 mice was first enriched for CD4+ cells by negative selection using EasySep
(StemCell Technologies) according to the manufacturer instructions.
2.5 Isolation of intestinal lamina propria cells
Small and large intestines were cut out and mesentery and fat removed, flushed, Peyer’s patches
were removed and the intestine was cut open longitudinally and into pieces of ~5 mm. The
caecum was opened, the content removed and the tissue cut in pieces. Tissue pieces were washed
twice by gentle vortexing for a few seconds in ice-cold buffer (HBSS (Gibco) supplemented with
2% FBS (PAA) and 15 mM HEPES pH 7.4). The epithelial cells and intestinal epithelial
lymphocytes were then removed by transferring the gut pieces to an EDTA-containing buffer
(HBSS (Gibco) supplemented with 10% FBS (PAA), 5 mM EDTA, 15 mM HEPES, buffered
with NaOH at pH 7.4) and shaken vigorously at room temperature for 10 minutes, vortexed
gently for a few seconds, before decanting the supernatant. This wash step was repeated three
times. Gut pieces were then washed three times in cold HBBS buffer (Gibco) to remove residual
EDTA before transfer into RPMI 1640 supplemented with 10% FBS (PAA), 15 mM HEPES
pH7.4, Collagenase type IV (0.25 mg/ml, Sigma) and DNase I (0.05 mg/ml, Roche) for digestion
of tissue for approximately 1-2 hours at 37°C with occasional vortexing. The resulting
suspension was filtered through a 70 mm nylon cell strainer to obtain a single cell suspension.
Finally cells were washed and resuspended in ice-cold FACS buffer containing PBS
supplemented with 2% FBS (PAA) and analyzed by flow cytometry or used for cytospin
preparations.
2.6 Co-Culture of stromal cells and bone marrow cells
For co-culture experiments, either 200 BM-derived S17 cells, or 80,000 (live) BM-derived
stromal cells, or 80,000 (live) lamina propria-derived stromal cells were plated together with
300,000 BM-derived B220+ cells. Cultures were performed in 24 well plates with OptiMEM
44
(Life Technologies) supplemented with 10% non-heat inactivated FBS (Gibco), -
mercaptoethanol (50 M), NaHCO3 (2.4 g/l), penicillin-streptomycin (100 g/ml), IL-21 (30
ng/ml, R&D Systems) TGF- (2.5ng/ml, R&D Systems), IL-7 (0.5 ng/ml), and 2 μg of rat IgG2a
anti-mouse CD40 (clone : 3/23; BD Biosciences) or rat IgG2a (clone : HB9419) as isotype
control. In some experiments, LPS (2 Cells were
initially plated in a total volume of 1 ml/well, and were fed on day 4 by adding 0.5 ml of culture
media/well. On day 7, cells were harvested and analyzed by flow cytometry.
2.7 Thymidine-incorporation assay
Cells were plated in triplicate at 5000 cells/well in 96-well flat-bottom plates with various
concentrations of IL-7. Proliferation was measured on day 4 by addition of 0.5 Ci 3H-thymidine
(perkin Elmer, Wellesley, MA) 6 hours prior to cell lysis onto microplate filters. Scintillation
fluid was added to each well and radioactivity was measured in a scintillation counter (Topcount
Systems, Canberra Packard, Meriden, CT).
2.8 Western blot analysis
In chapter 3, cell lines were stimulated with 25 ng/mL IL-7 for the indicated amount of time with
or without prior cross-linking of CD22 molecules for 10 min using 10 g/mL of CY34.1
antibody (Pharmingen). In chapter 4, sorted BM B cell progenitors were stimulated with 50
ng/mL IL-21 (Fig. 4.3) or 2.5μg/mL anti-IgM F(ab)2 (Fig.4.6) for 15 min. Cells were then lysed
in 1% NP40, 150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 10 mM NaF, 1 mM sodium
orthovanadate, 5 mM sodium pyrophosphate, 1 mM PMSF, 5 g/mL aprotinin and leupeptin
(Roche) on ice for 30 min. Equal amount of cell lysates were separated onto a 4-12% gradient
NuPAGE gel, and then transferred to a PVDF membrane. Membranes were blocked with 5%
milk in PBS/0.05% Tween/5% BSA (TBST) for 1 hour at room temperature and then probed
overnight at 4 C for pSTAT3, pSTAT1, pSTAT5, pERK, ERK (Cell signaling) or actin
(NeoMarkers). After several washes in TBST, membranes were subsequently probed with a
horseradish peroxidase-coupled goat anti-rabbit IgG diluted 1:10,000 in TBST containing 5%
45
milk for 45 min. Detection was performed using the enhanced chemiluminescence (ECL)
substrate (Amersham Pharmacia Biotech) as described by the manufacturer.
2.9 ELISA
Enzyme immunoabsorbant (EIA) plates (Costar; no. 3590) were coated with 5 μg/mL goat anti-
mouse IgM (Jackson ImmunoResearch Laboratories), IgG1, IgG2a, IgG2b, IgG3 or IgA (Sigma)
overnight at 4 C. Plates were washed with distilled water several times and blocked for 40 min at
room temperature with 3%FCS/PBS. After washing, 50 μL of culture supernatant was added and
plates were incubated at room temperature for 40 min. A standard curve was established using
purified mouse IgM (Pharmingen), IgG1 (Sigma), IgG2a , IgG2b , IgG3 and IgA
isotype standards (Pharmingen). Plates were washed several times with distilled water. Plate-
bound antibodies were detected after a 40 min incubation with anti-mouse IgM, anti-mouse IgG,
or anti-mouse IgA conjugated to peroxydase (Sigma). After washing, BD OptEIA substrate (BD
biosciences) was added to the plates and absorbance was read according to the manufacturer
instructions.
2.10 FACS analysis
For experiments presented in chapter 3 and 4, cells were washed with ice-cold PBS containing
3% (v/v) FCS and then incubated for 30 min on ice with predetermined concentration of FACS
antibodies in a total volume of 100 μL. The following antibodies (clone) were used: IgM-biotin
(33.60), B220-FITC, B220-APC (RA3-6B2; eBioscience), CD2-PE (RM2-5; eBiosciene),
CD43-PE (S7-5; BD Bioscience), IgD-FITC (clone SBA.1; Southern), IL-21R-biotin (eBio4A9;
eBioscience), CD22-PE (Cy34.1; Pharmingen), kappa-FITC (Southern Biotech), lambda-FITC
(Southern Biotech), and rat IgG-biotin (R35-95; Pharmingen). For indirect staining, cells were
washed twice after binding of the primary antibody and incubated with streptavidin-PECy5 or
PerCP (BD Bioscience) for 15 min on ice. Samples were kept at 4 C in the dark and analyzed
using a FACS calibur or FACScan (BD Bioscience). 10,000 cellular events were analyzed for
46
each sample. Data were analysed using CellQuest software version 3.1 or FlowJo (Tree Star
Inc.).
For experiments presented in chapter 5, cells were washed with ice-cold PBS and a live/dead
stain was performed using the fixable aqua dead cell stain kit (In Vitrogen). Cells were then
wash with PBS containing 3% FCS and incubated with 1 μg/ml of purified rat anti-mouse
CD16/CD32 antibody (“Fc Block”, clone: 2.4G2, BD Pharmingen) for 5 minutes at 4 C.
Predetermined concentrations of fluorochrome labeled antibodies were added directly to cells in
the presence of Fc Block and incubated for 15 minutes at 4 C. The following antibodies were
used: murine anti-mouse CD45.1-Pacific Blue (A20, ebioscience), murine anti mouse CD45.2-
Pacific blue (104, Biolegend), rat anti-mouse Ly6C-PerCP-Cy5.5 (HK1.4, eBioscience), hamster
anti-mouse CD11c-APC (N418, eBioscience), rat anti-mouse CD19-FITC (1D3, eBioscience),
rat anti-mouse Ly6G-Alexa Fluor 700 (1A8, BD Pharmingen), and rat anti-mouse CD138-APC
(281-2, BD Pharmingen). After washing with FACS buffer, cells were fixed and permabilized
using a cytofix/cytoperm kit from BD Biosciences according to the manufacturer’s protocol.
Intracellular staining was then performed for 30 min at 4 C using the following antibodies: rat
anti-mouse IgA-FITC (11-44-2, Southern Biotech), rat anti-mouse IgA-biotin (11-44-2) followed
by Streptavidin-APC-Cy7 or Streptavidin PerCp-Cy5.5 (Biolegend), murine anti-mouse iNOS-
FITC (6, BD Pharmingen), rabbit anti-mouse iNOS-PE (N-20, Santa Cruz Biotechnology). Cells
were then washed twice with Perm/Wash buffer, resuspended in FACS buffer, and filtered prior
analysis by flow cytometry using either a Canto II or a LSR-II (BD Biosciences). Acquired data
were analyzed and processed using FlowJo.
2.11 Detection of IL-21 protein
BM cells from 10 mice were incubated in red blood cell lysis buffer (150 mM NH4Cl, 100 mM
NaHCO3, 1 mM EDTA pH 8.0) for 1 minute on ice and washed in PBS/FCS. To isolate the
CD4+ population, BM cells were enriched for the CD4+ T cell population by negative selection
using EasySep (StemCell Technologies) according to the manufacturer instructions and then
47
labeled with an anti-CD4-PE Ab for sorting on a FACS Aria (BD Bioscience). Sorted BM CD4+
T cells were culture for 3 days with anti-CD3 (10 g/mL), anti-CD28 (2 g/mL) ± human IL-6
(100 ng/mL). On day 3, the supernatants were collected and the presence of IL-21 was detected
by cytometric bead array (CBA) according to the manufacturer protocol (BD Bioscience), with
the following modification: 75 L of supernatant were added to 25 L of beads.
2.12 RNA isolation and analysis
Total RNA was isolated from total BM of C57Bl/6, Rag2-/- and TCR -/- mice or from BM B cell
progenitors from C57Bl/6 mice using the Trizol reagent (GIBCO BRL) or RNeasy kit (Quiagen)
according to the manufacturer’s instructions. First-strand cDNA was prepared from 0.5 to 3.0 g
of total RNA in 20 L reaction volume using the Superscript II (Gibco Life Technology). After
reverse transcription, Il21, Blimp1 and Aid were amplified by real-time PCR according to
manufacturer instruction (Applied Biosystems). Amplification of actin was used for sample
normalization. PCR primers used: Il21 5’-CGCCTCCTGATTAGACTTCG-3’ (sense) and 5’-
TGGGTGTCCTTTTCTCATACG-3’ (anti-sense), Blimp1 5’-TAGACTTCACCGATGAGGGG-
3’ (sense) and 5’-GTATGCTGCCAACAACAGCA-3’ (anti-sense), Aid 5’-
GCGGACATTTTTGAAATGGTA-3’ (sense) and 5’-TTGGCCTAAGACTTTGAGGG-3’ (anti-
sense), and -Actin 5’-GCCAACCGTGAAAAGATGACCCAG-3’ (sense) and 5’-
ACGACCAGAGGCATACAGGGACAG-3’ (anti-sense).
Semi-quantititative RT-PCR for Il21r and Actin was performed on three serial dilutions of cDNA
isolated from sorted into proB (CD2-LC-), preB (CD2+LC-), and immature/mature (CD2+LC+) B
cell populations. PCR products were amplified using the following conditions: for Il21r: Ta =
67°C, 34 cycles, and for -Actin: Ta = 58°C, 22 cycles. Amplification of -Actin was used as a
cDNA loading control. The -Actin specific primers were 5’-TCCCTGGAGAAGAGCTACGA-
3’ (sense) and 5’-ATCTGCTGGAAGGTGGACAG-3’ (anti-sense). Primers for Il21r were 5’-
ATGCCCCGGGGCCCAGTGGCTG-3’ (sense) and 5’-CACAGCATAGGGGTCTCTGAGGT
TC-3’ (anti-sense).
48
2.13 Class-switch recombination
BM from C57Bl/6 was cultured for 4 days in IL-7 and then sorted into proB (B220+CD2 ),
preB (B220+CD2+ ), and immature/mature (B220+CD2+ +) B cells. Cells were then
cultured without supplements (ctr), with IL-21 (30ng/mL), with anti-CD40 (2ug/ml), or anti-
CD40/IL21 for 24 hours prior to RNA extraction. After cDNA synthesis samples were analysed
for class-switch recombination. Primers for GLT 2b were previously described (18).
Amplification of GLT 2b was done using the following conditions: for: Ta = 62°C, 40 cycles.
2.14 Statistical analysis
Statistical significance was assessed by a 2-tailed Student t test and the level of significance was
established at p 0.05.
49
CHAPTER 3
ANALYSIS OF THE ROLE OF CD22 IN THE REGULATION
OF THE IL-7 RESPONSE
______________________________________________________________________________
I performed all of the experiments presented in this chapter.
50
3.1 Introduction
The successful differentiation of progenitors into functional, mature B cells requires the presence
of critical stromal cell-derived cytokines. One of them, IL-7, plays an important role for growth,
survival and maturation, especially at the earliest stages of development (55, 110, 320, 321). B-
cell progenitors are initially sensitive to IL-7, but during the transition from the proB to the preB
cell stage, this sensitivity is lost (153, 322-324). However, IL-7R is still expressed on all
developing B cells in the BM (325), and thus the inability to respond to IL-7 is not a
consequence of the downregulation of IL-7R expression.
We have previously shown that the regulatory lectin CD22 is present on BM B cell progenitors.
Stoddart et al. have validated that CD22 is a maturation marker for B-cell development by
showing that CD22 expression, which first occurs in the late proB/early preB stage, increases as
the cells mature(322). Interestingly, they have also demonstrated that the appearance of CD22
correlates with the loss of IL-7 responsiveness. This was done by experiments in which freshly
isolated BM cells were fractionated into three populations all expressing B220: -CD22-, -
CD22lo and +CD22hi. Each population was plated by limiting dilution to determine the
frequency of cells that were responsive to IL-7, and the frequency that were non-responsive to
IL-7 but still able to respond to mitogen in the presence of stromal cells. It was concluded that
higher CD22 expression levels correlated with an increased mitogen response and a decreased
IL-7 response. However, it was not determined whether CD22 played a causal role in the loss of
IL-7 responsiveness, or if it was merely a marker of the event.
CD22 is a 140 kDa sialic acid binding Ig-like lectin (siglec) member of the Ig superfamily (326)
that is expressed only on B cells and specifically binds to 2-6-linked sialic acid-containing N-
glycans (327, 328). Numerous ligands have been identified including CD22 itself, IgM, and
B220 (329-333). CD22 binding can occur in cis (with ligands expressed on the same cell
surface) or in trans (with ligands expressed on other cells) (334-338) and appears to be at least
51
partly modulated by enzymes that regulate 9-O-acetylation. Indeed, it has been shown that the
presence of a 9-O-acetyl group on sialic acid prevents the binding of CD22 (339). More
recently, mice in which the esterase activity of 9-O-acetyl sialic acid esterase has been abrogated
have been generated and displayed increased 9-O-acetylation of 2-6-linked sialic acids in B
cells (340). Our lab has previously reported evidence that 9-O-acetylation is temporally
regulated in primary cells, by demonstrating that expression of the gene encoding 9-O-
acetylesterase was upregulated during differentiation of fetal liver bipotential precursors into B
cells and macrophages (341). In line with this finding, we also found that the 9-O-acetylesterase
gene was differentially expressed in proB and preB cell lines, which raises the possibility that 9-
O-acetylesterase plays a role in early B cell development (341, 342).
CD22 is primarily known as a negative regulator of BCR signalling, where upon stimulation of
BCR the immunoreceptor tyrosine-based inhibition motif (ITIM) of CD22 is phosphorylated by
the src kinase, Lyn (343, 344). This enables the recruitment of the tyrosine phosphatase Src
homology 2 domain-containing phosphatase 1 (SHP-1), an important negative regulator of BCR
signalling (345-348) that notably inhibits BCR-induced Ca2+ (349-353). Although results vary
between different experimental systems, CD22 also appears to have the potential to regulate the
activity of the extracellular signal-regulated kinase (ERK). Poe et al. found normal activation
kinetics for ERK1/2 in CD22-/- B cells stimulated with anti IgM (354). However, Tuscano et al.
reported cross-linking of CD22 alone resulted in a modest increase in ERK activity, but co-
ligation of CD22 and BCR resulted in a slight increase and significant prolongation of ERK
activation (355). In contrast, Wakabayashi et al. and Zhu et al. showed that transfection of cell
lines with CD22 resulted in impaired IgM-induced ERK phosphorylation (348, 356). CD22
inhibition of BCR signalling was specific to IgM and IgD as it did not occur following surface
IgG stimulation (356).
Based on the fact that IL-7R activates signalling pathways susceptible to modulation by CD22,
we investigated whether the loss of IL-7 responsiveness observed in B cell progenitors was a
consequence of negative regulation by CD22. We show that BM-derived immature B cell lines
52
respond to IL-7R stimulation by inducing ERK, regardless of the presence of CD22 at the cell
surface. CD22 binding in trans also does not appear to influence the outcome of IL-7R
signalling. However, IL-7-induced proliferation was reduced following sialidase treatment,
suggesting that cis interactions involving sialic acids could modulate IL-7R signalling.
3.2 Results
3.2.1 BM B cell lines remain IL-7 responsive.
IL-7 is a key player for the successful differentiation of early B cell progenitors. Although B cell
progenitors express IL-7R on their surface, they lose the ability to respond to IL-7 as they
progress towards more mature stages (153, 322). To study B cell development, our lab has
previously generated cell lines representing different stages of B cell differentiation. These cell
lines were obtained by growing B220+ BM cells for several days in the presence of IL-7. In order
to establish clones, the resulting culture was plated by limiting dilution and cultured in the
presence of IL-7 for 6 to 8 weeks.
To determine whether these cell lines could be used as a tool to study the loss of IL-7
responsiveness, we first tested them to determine if they were still responsive to IL-7 by
measuring their proliferation and ability to activate ERK in the presence of IL-7. The ability to
proliferate in response to IL-7 was measured by thymidine incorporation. Figure 3.1A shows
representative results obtained with three different cell lines, and illustrates that increase IL-7
dilution leads to decreased proliferation. In addition, western blot analysis confirmed that
stimulation of these cell lines with IL-7 activates the ERK pathway within ~15 min, although the
peak of ERK phosphorylation varied between cell lines (Fig. 3.1B). Together, these results
indicate that these cell lines are not only responsive to IL-7, they also depend on IL-7 for growth.
53
54
3.2.2 IL-7 dependent cell lines express variable levels of CD22.
Since it was demonstrated that the expression of CD22 correlates with the loss of IL-7
responsiveness (322), we screened the IL-7 dependent cell lines for the presence of CD22 using
single color flow cytometry. In many cases, CD22 molecules were either absent, or expressed at
very low levels (Fig 3.2). However, we also found three cases where substantial levels of CD22
were expressed. The fact that we found cell lines that are still IL-7 dependent while expressing
CD22 indicates that the mere expression of CD22 is not sufficient to block IL-7R response. It
may also be that this molecule is not involved in the loss of IL-7 responsiveness at all. However,
data obtained from mice carrying mutant CD22 molecules that lacked ligand-binding activity
revealed a complex activity of CD22, and demonstrated that CD22 can perform both ligand-
independent functions and ligand-dependent functions (357). Moreover, it is known that the
interaction of CD22 with its ligand can occur in trans, but also in cis with other B cell surface
sialoglycoproteins, resulting in the “masking” of CD22 (334-338). Therefore, the fact that we
observed expression of CD22 on IL-7-dependent cell lines does not rule out a potential role for
CD22 in the regulation of IL-7 response. It is possible that the IL-7 response is regulated only
following ligand-binding to CD22. Thus we conducted more experiments to test this hypothesis.
3.2.3 Cross-linking of CD22 does not regulate the response to IL-7
We first tested the effect of CD22 binding in trans on the IL-7R signalling by cross-linking
CD22 with an external ligand. In a first set of experiments, IL-7-dependent cell lines were
washed and starved for 1 hour. CD22 was bound with an anti-CD22 biotinylated antibody and
cross-linked with avidin-coated beads 10 minutes prior to stimulation with IL-7. IL-7 response
was assessed by western blotting of phospho-STAT5 as well as phospho-ERK, two major
pathways activated downstream of IL-7R. As expected, stimulation of BM-derived B cell lines
with IL-7 only induced the phosphorylation of STAT5 and ERK within 30 minutes (Fig. 3.3).
However, we did not detect any modulation of the STAT5 response among the cells that were
co-stimulated with anti-CD22. Variable effects in the phosphorylation status of ERK were
observed. In some cases, we noted a reduction of phospho-ERK while in other cases the peak of
phospho-ERK was reached at an earlier time point (Figure 3.3).
55
56
57
IL-7 is known to increase cell proliferation of early B cell progenitors via activation of PI3K, as
was demonstrated by experiments carried on IL-7R -/- BM progenitors transduced with different
mutant IL-7R chains (321). In addition, IL-7 has been reported to increase the probability of
cell survival by inducing an increase in the ratio Bcl-2/Bax (110). We therefore tested
proliferation and survival to clarify whether binding of CD22 in trans influences the IL-7
response. First, we coated wells with anti-CD22 and cultured B62.1 cell line for four days, and
measured proliferation and survival by counting cells and applying the trypan blue exclusion
method. We found that cross-linking CD22 on B62.1 had no effect on IL-7-induced proliferation
or survival (Fig. 3.4A). Given that cell lines can compensate by modifying a signalling pathway
and use different pathways that lead to proliferation, we also tested the effect of anti-CD22 on
primary BM cells. We selected for B220+ BM cells and grew them for 7 days in the presence of
IL-7 in wells coated with anti-CD22 or isotype control. On day 7, cells were counted and
survival assessed using trypan blue. Consistent with the data obtained on progenitor cell lines,
we found that cross-linking of CD22 on primary BM progenitors did not affect the proliferation
induced by IL-7. The same conclusion was reached for the survival rate; it was also not affected
by the presence or absence of anti-CD22 (Fig. 3.4B). Altogether, these results suggest that CD22
binding in trans is not involved in the loss of IL-7 responsiveness observed in maturing BM B
cells.
3.2.4 Removal of sialic acid modulates the IL-7 response
Although the cross-linking of CD22 did not modulate the IL-7 response, it is possible that the
absence of an effect was due to the fact that CD22 was already bound to other ligands. In
particular, CD22 has been shown to bind to other ligands on the cell surface in cis. This
“masking” of CD22 by cell surface ligands can be reversed by costimulating the cells with anti-
CD40, or by treating the cells with a sialidase (337). To gain insight into whether cis ligand
binding of CD22 had an impact on the IL-7 response, we treated cells with the sialidase
neuraminidase. We first tested the effect of eliminating sialic acids on the H33 and B62 cell
lines. As shown above, these cells lines express respectively high and low levels of CD22 (Fig.
3.2). We found that treating these cell lines with sialidase decreases the ability of both cell lines
to proliferate in response to IL-7 (Fig. 3.5A-right panel). This effect was independent of survival
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since sialidase treatment did not interfere with the survival rate of the cell lines (Fig. 3.5A-left
panel). To determine if a similar effect occurs in primary cells, we selected BM B220+ cells and
grew them in the presence of IL-7 and different concentrations of neuraminidase. Similar to what
was observed in the cell lines, we found that greater concentrations of neuraminidase were
associated with decreased proliferation (Fig.3.5B-left panel). Compared to the cell lines, the
survival rate was more variable in primary BM B cells. At higher concentration of
neuraminidase, survival diminished by up to 22% (Fig.3.5B-right panel). This sialidase
modulation of IL-7 response suggests that a significant fraction of CD22 might be bound in cis to
cell surface ligands, and regulation of the IL-7 response by CD22 cannot be ruled out.
3.3 Discussion
Numerous studies have documented the essential role that IL-7 plays in the development of
murine B cells in the BM. However less is known about the mechanism that causes B cells to
lose their ability to respond to IL-7 as they differentiate. Evidence suggests that it involves
molecules acting as negative regulators of the signalling pathways activated by IL-7R, since the
failure to respond to IL-7 is not concomitant with the down-regulation of IL-7R. Former students
in our lab have demonstrated that the loss of IL-7 responsiveness occurs when cells transit from
the proB cell stage to the small preB cell stage (153, 322). Stoddart et al. have shown that it
coincides with the expression of CD22 at the cell surface (153), a molecule often depicted as a
negative regulator of BCR signalling (326, 358-360). To study the IL-7 response in detail, we
used BM-derived B cell lines that we have previously generated in our lab. These cell lines
display phenotypes corresponding to various stages of early B cell development that occur
between the proB and the immature B cell stage. Our results show that, similar to primary BM B
cell progenitors, the BM-derived B cell lines generated in vitro are dependent on IL-7 for
growth. Furthermore, stimulation of these cell lines with IL-7 activated MAPK and JAK/STAT
pathways. These cells are ideal for studying the response to IL-7, as stimulation of these cell
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lines with IL-7 reproduces the activation pattern observed in IL-7 stimulated primary BM B
cells.
We have found that the expression of CD22 on IL-7-dependent B cell lines was variable. Cross-
linking of CD22 on these cell lines did not have an impact on the proliferation or survival
response that is associated with IL-7R stimulation; neither did cross-linking of CD22 on primary
BM B cells. These results suggest that binding of CD22 in trans is not involved in the loss of IL-
7 responsiveness. However, we cannot exclude the possibility that the absence of effect on the
IL-7 response following cross-linking of CD22 was because CD22 was already bound by a
ligand expressed on the same cell surface, a phenomenon often referred as “masking”. Further
experiments in which cis binding ligands are removed prior to CD22 cross-linking could address
this possibility.
In mice, only one isoform of CD22 is expressed (361). The molecule contains seven Ig domains
(361). Together, the membrane-distal domains 1 and 2 form the binding site for sialic acid (332).
These domains have also been identified as the domains bound by the CY34.1 antibody used in
our study (332). However, mutations within these domains that disrupt binding of Cy34.1 do not
affect binding of CD22 to its CD45 ligand, and neither does the binding of Cy34.1 antibody to
CD22 (362). More recently, it has been shown that mutation of CD22 arginine residue required
for sialic-acid binding did not affect CD22 association with CD45 or CD22, suggesting that these
molecules may interact via a different interface (363). Together, these data indicate that Cy34.1,
sialic acid, and CD22/CD45 bind to different sites on CD22. Thus, in spite of the evidence that
cross-linking of CD22 was successful in our system, we cannot rule out the possibility that it
only partially reproduced the effect of a natural ligand, or that it triggered a signal that is
normally not induced by a physiological ligand. Differences between the responses induced by
antibodies versus natural ligands have been observed in other systems. For example, it has been
reported that in B cells stimulated with anti-Ig antibody, ERK is strongly activated and CD22
weakly phosphorylated, contrary to what is observed in B cells stimulated with antigens (364). In
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addition, in contrast to antigen-stimulated B cells, the activation of B cells with anti-Ig by-passes
the requirement for CD40 stimulation to induce proliferation (364).
Our results obtained on neuraminidase treated cells suggest that a good proportion of CD22
might be involved in cis interactions with sialic acids present on the surface of the same cell,
which is consistent with previous predictions (358). This concept is supported by the observation
that the affinity of CD22 for sialic acid is very low regardless of the protein backbone to which it
is attached to, suggesting that binding is determined only by the density of sialic acid. Since the
concentration of the ligand (L) sialic acid on the surface of B cells is estimated to be
approximately 100-fold higher than the dissociation constant (Kd) of CD22 (P), CD22 is
expected to interact mainly in cis to form ligand-protein complex (C) (333, 335, 358, 365)
according to the Kd definition:
therefore
Other studies report evidence that enzymes regulating 9-O-acetylation could modulate the cis
interaction of CD22, since the presence of a 9-O-acetyl group prevents ligand binding to CD22
(339, 340). Our lab has also previously observed that B-cell precursors do not express significant
levels of 9-O-acetylated sialic acids (341, 342), supporting the hypothesis that a sizable
proportion of the CD22 expressed on BM-derived cell lines or primary BM B cells could already
be bound in cis to endogenous ligands. Finally, it has been reported that on most B cells, CD22 is
unable to bind exogenous sialoside probes unless the cells were first pretreated with sialidase
(337, 338, 366). Exceptions included activated B cells, transitional, MZ, and a subset of IgD+
BM B cells (337, 366, 367). To circumvent this problem, some studies have found that binding
of CD22 in trans could be achieved without sialidase treatment if high-affinity multivalent
probes were used (336).
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We found that disrupting cis-bound CD22 decreased the IL-7 response. Removal of sialic acids
on H33 and B62.1 cell lines resulted in a dose-dependent decrease of cell proliferation. The
effect was even more pronounced on neuraminidase treated primary BM B cells, and consistent
with previous reports (342). Although a non-specific neuraminidase induced toxic effect could
not be ruled out in earlier studies, our data suggests that neuraminidase does not affect survival at
doses up to 10mU. However, our results do not rule out the possibility that removal of the sialic
acid influences other functions of B cells. Indeed, expression of another member of the siglec
family, siglec-G, is under the control of the B-cell specific transcription factor Pax-5 (183).
Siglec-G was found to be expressed from the proB to the mature B cell stage (183). Therefore, to
confirm that the inhibition of proliferation observed after removal of sialic acid is specific to
CD22, further experiments using BM B cells derived from transgenic or knockout mice would be
required. Of interest, a study directly addressing the physiological relevance of CD22 binding
has been released. In that report, two lines of mice expressing mutant CD22 molecules were
generated: a truncated form lacking the 1 and 2 domains required for sialic acid ligand binding,
and a form containing point mutations in certain amino acids required for the binding of sialic
acid (357). Using these mice, the authors demonstrated that CD22 interactions with sialic acid
are important for normal B cell physiology, but that not all functions of CD22 are sialic acid-
dependent. IL-7 stimulation of BM cells derived from these mutant mice would help to clarify
whether CD22 binding to sialic acid in cis has an impact on IL-7-induced proliferation.
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CHAPTER 4
BONE MARROW B CELL PROGENITORS EXPRESS
FUNCTIONAL IL-21 RECEPTOR
_____________________________________________________________________________
Some contents of this chapter appear in:
Nathalie Simard, Danijela Konforte, Anne H. Tran, Jessica Esufali, Warren J. Leonard,
Christopher J (2011) Paige. Journal of Immunology May 1; 186(9) :5244-5253.
Available at http://www.jimmunol.org/content/186/9/5244.long
N.S. performed all of the experiments presented with the exception of figure 4.8A which was
generated in collaboration with A.H.T who did the RT-PCR analysis.
65
4.1 Introduction
The generation of mature, antigen-responsive B cells from pluripotent stem cells is directed by
signals provided by the supportive microenvironment in the BM. Some of these signals are
mediated by the action of soluble cytokines. At each developmental stage, B cells express a
distinct profile of cytokine receptors on their cell surface to sense these signals. In mice, one
critical phase of B cell development in the BM is regulated by the cytokine IL-7. It has been
established that IL-7 promotes proliferation, survival, and development of proB cells toward the
preB cell stage (55, 110, 320, 321). Furthermore, our lab has previously shown that once the
preBCR is expressed at the cell surface, integrated signals from both the IL-7R and the preBCR
allow cells to proliferate in reduced concentrations of IL-7 (68).
IL-7 is a member of the common -chain-dependent cytokine family, which includes IL-
2, IL-4, IL-9, IL-15, and IL-21 (272). Like IL-7, IL-21 has also been shown to play a key role in
B cell development. However, in contrast to IL-7, current data show that IL-21 exerts its effect at
later stages when B cells differentiate into PCs (272, 294, 368). The first evidence for the
importance of IL-21 in B cell differentiation was provided by studies carried out on IL-21R-
deficient (IL-21R-/-) mice (297). IL-21R-/- mice exhibited a severe defect in IgG1 production,
while the secretion of IgE was augmented, both at steady-state and following immunization with
T cell-dependent antigen. Using human EBV-infected B cell lines, we have demonstrated that
IL-21-mediated signalling through the JAK/STAT pathway was required for the differentiation
of B cells into late plasmablasts/early PCs (290). In mature B cells, binding of IL-21 to the IL-
21R induces activation of STAT1, STAT3, and to a lesser extent STAT5 (290, 369).
Multiple studies have demonstrated that the effects of IL-21 stimulation of B cells depend
on the cell signalling context (281, 286, 292). For instance, in the presence of signalling through
both the BCR and CD40, IL-21 promotes growth and differentiation of murine splenic B cells
into Ig-secreting cells (281, 292). Conversely, growth arrest and apoptosis occur following
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addition of IL-21 to B cells stimulated with lipopolysaccharides (LPS), CpG, or anti-IgM in the
absence of T cell help (281, 286). These and other results have led to a consensus that the IL-21-
IL-21R system is context-dependent and plays an important role in maintaining B cell
homeostasis. One consequence of a breakdown in this system could be impaired elimination of
autoreactive B cells and the subsequent development of antibody-mediated autoimmune diseases
like systemic lupus erythematosus (SLE). In this regard, lupus prone BXSB.B6-Yaa+/J mice
showed elevated levels of serum IL-21 (292). In addition, a recent study has shown that BXSB-
Yaa+/J mice deficient for IL-21R failed to develop the disease (370).
To date, the majority of IL-21 studies have focused on its role in the final stage of B cell
differentiation. However, IL-21R has also been reported to be expressed on early B cells
progenitors in the BM (281), although it is not known whether it is functional and involved in the
regulation of B cell development in the BM. One study reported that IL-21R-/- mice have normal
numbers and phenotypes of B cells in the BM (297), although several specific populations of B
cell progenitors, of interest to us, were not examined. While this study shows that IL-21 does not
have an essential, non-redundant role in B cell development, more recent data show that IL-21
can contribute to early hematopoiesis. It has been found that murine hematopoietic progenitor c-
kit+sca+lin-/low (KSL) cells express low levels of IL-21R (371). When grown in vitro with a
cocktail of c-kit ligand, Flt-3L and IL-7, proliferation of KSL cells is enhanced in the presence of
IL-21 (371). In addition, overexpression of IL-21 in vivo increased the number of KSL cells in
the spleen (371). Another study showed that IL-21 did not have any mitogenic effect on total
murine BM cells. However, apoptosis of BM CD11b- lymphoid cells that expressed IL-21R was
delayed when the cells were cultured in the presence of IL-21 (319). Based on this information
and the fact that very few B220- cells express IL-21R (281), the authors hypothesized that IL-21
acts mainly on lymphoid B220+ B cell subsets in the BM. Finally, it has been reported that IL-21
transgenic mice have increased number of immature B cells in the spleen (292). One explanation
for this phenotype could be increased maturation of BM B cell precursors. Collectively, these
studies indicate that further investigation is required to determine the exact role of IL-21 in
development of B cell progenitors in the BM.
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In this study, we show that IL-21 message is constitutively expressed in murine BM
CD4+ T cells. IL-21R is expressed and is functional on all subsets of B cell progenitors,
including proB, preB and immature/mature B cells. In vitro culture of B cell progenitors with IL-
21 is sufficient to induce expression of Aid and Blimp1, and germline transcripts (GLTs) in these
cells, and to accelerate their development into mature B cells. A role for IL-21 in promoting B
cell maturation is further supported by data that show increase in numbers of proB cells and
decrease in number of mature B cells in BM of IL-21R-/- mice. Finally, stimulation of the IL-21R
and CD40 on B cell progenitors results in the formation of Ig secreting cells.
4.2 Results
4.2.1 IL-21 is expressed and secreted by CD4+ T cells in BM
To determine whether B cell progenitors could encounter IL-21 during development in the BM,
we performed RT-PCR on BM cells isolated from WT mice kept in pathogen-free conditions.
Our results show that Il21 mRNA was expressed in total BM of WT mice (Fig. 4.1A). To further
define the source of Il21 message, we analysed BM of RAG-/- mice, which lack mature B and T
cells but contain normal numbers of stromal cells and other hematopoietic cells, as well as BM
of TCR -/- mice, which lack mature T cells. We failed to detect the expression of Il21 mRNA
in either RAG-/- and TCR -/- BM (Fig. 4.1A). These results suggest that Il21 message is
expressed by BM lymphocyte subsets, most likely T cells. To establish whether BM T cells
produce Il21 we sorted different populations of BM cells including CD4+ and CD8+ T cells. We
found expression of Il21 mRNA only in the CD4+ T cell population (Fig. 4.1B). To further
characterize which subpopulation of T cells is producing Il21, we performed real-time PCR on
sorted naive and memory BM T cells (Fig. 4.1C). We found that Il21 mRNA was produced
mainly by memory T cells.
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It has been reported that IL-21 protein is secreted by splenic CD4+ T cells activated with anti-
CD3, -CD28, and IL-6 (372). Therefore to determine whether IL-21 protein was secreted by BM
CD4+ T cells, we cultured BM CD4+ T cells with anti-CD3, anti-CD28 with or without IL-6.
Data collected by CBA assay show that CD3- and CD28-mediated signals were sufficient to
induce IL-21 secretion by BM CD4+ T cells (Fig. 4.1D). The greater number of cells observed in
wells containing IL-6 likely explains the higher level of IL-21 detected in these wells.
4.2.2 IL-21 receptor is expressed on BM B cell progenitors
The developmental stages of B lymphopoiesis can be characterized by different profiles of cell
surface proteins. We have previously shown that CD2 and μ heavy chain, or CD2 and + light
chains, constitute reliable developmental markers to follow the progression of progenitors from
proB to preB to immature and mature B cell stages in in vitro culture (152). In order to analyse
the expression of IL-21R on different BM B cell subsets, B220+ BM cells were isolated and
grown in IL-7 for 4 days (day 4IL-7). We used a combination of antibodies that recognize CD2,
and light chains (anti-LC) to select different B cell populations, including CD2-LC- (proB),
CD2+LC- (preB), and CD2-LC+ (immature/mature B cells). Semi-quantitative RT-PCR results
show that Il21r mRNA was present at low levels in proB cells, and at higher levels in preB and
immature/mature B cells (Fig. 2A). As expected, Il21r mRNA was not expressed in S17 stromal
cells but was expressed in total spleen cells (Fig. 4.2A). Next, FACS analysis was performed on
day 4IL-7 B220+ BM cells isolated from WT and IL-21R-/- mice to determine the expression of
IL-21R on the cell surface of B cell progenitors. We found that IL-21R expression progressively
increased with the developmental stage of B cell progenitors. IL-21R was below detection on
CD2-LC- proB cells, but it was expressed at low levels on CD2+LC- preB cells and at high levels
on CD2+LC+ immature/mature B cells (Fig. 4.2B – upper panel). This pattern of expression
closely matched the expression pattern on freshly isolated BM B cells stained with the same
markers (Fig. 4.2B – lower panel).
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4.2.3 IL-21 induces tyrosine phosphorylation of STAT1, STAT3 and STAT5 in B cell progenitors
To determine whether IL-21R was functional, we carried out signalling experiments on different
subsets of B cell progenitors. Day 4IL-7 B220+ cells were sorted as described above. CD2-LC-
proB cells, CD2+LC- preB cells, and CD2+LC+ immature/mature B cells were stimulated with
IL-21 (50 ng/mL for 15 min). Western blot analysis shows that the tyrosine phosphorylation of
STAT3, STAT1, and STAT5 was induced after stimulation in all B cell populations (Fig. 4.3A).
Moreover, the intensity of the signal correlated with the expression levels of IL-21R in the
different B cell populations. It was the weakest in proB cells and the strongest in
immature/mature B cells (Fig. 4.2B). To rule out the possibility that contaminating preB cells
could account for the phosphorylation of STAT3 observed in the proB cell fraction, we spiked an
IL-21 unresponsive B progenitor cell line with different numbers of sorted preB cells. We then
measured the phosphorylation of STAT3 following IL-21 stimulation. Fig. 4.3B shows that
contamination with preB cells greater than 10% was necessary to achieve the level of STAT3
phosphorylation observed in IL-21-stimulated proB cells. Since the purity of the sorted proB cell
fraction was 99.98%, this result indicated that the source of the phosphorylated STAT3 was an
IL-21 stimulated proB cell and not a contaminating preB cell. To further test this finding, we
isolated B220+ BM cells from RAG-/- mice, which have a B cell developmental block at the proB
cell stage. We found that IL-21 treatment of day4IL-7 RAG-/- B220+ BM cells activated
phosphorylation of STAT3 (Fig. 4.3C). To exclude the possibility that a receptor other than the
IL-21R was involved in the activation of STAT3, we stimulated proB, preB and
immature/mature B cells from WT and IL-21R-/- mice with IL-21. Our results show that
phosphorylation of STAT3 occurred only in WT cells (Fig. 4.3D). Finally, we showed that
phosphorylation of STAT3 could be induced by IL-7, but not IL-21 in IL-21R-/- cells indicating
that STAT3 signalling is functional in IL-21R-/- cells (Fig. 4.3E). Together, these results clearly
indicate that the IL-21R expressed on the surface of proB, preB and immature/mature B cells is
functional, and that its stimulation can result in the phosphorylation of downstream proteins.
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Figure 4.3. IL-21R stimulation increases tyrosine phosphorylation of STAT1, STAT3 and STAT5 in proB, preB and immature/mature B cells. Day 4IL-7 B220+ BM cells from WT mice were sorted for proB (CD2-LC-), preB (CD2+LC-) and immature/mature B (CD2+LC+) cells. A, Tyrosine-phosphorylated forms of STAT1 (pY701), STAT3 (pY705), and STAT5 (pY694) were detected by Western blotting. Actin represents a loading control. B, The IL-21 non-responsive B62c proB cell line was spiked with different numbers of sorted preB cells, stimulated for 15 min. with IL-21 (50 ng/ml), and analyzed for STAT3 phosphorylation by Western blotting. C, B220+ BM cells were isolated from RAG-/- mice and grown in IL-7. On day 4, cells were harvested and stimulated for 15 min with IL-21 (50 ng/ml). Phosphorylation of STAT3 was detected by Western blotting. D, Day 4IL-7 BM B220+ cells from WT and IL-21R-/- mice were sorted into proB (CD2-LC-), preB (CD2+LC-) and immature/mature B (CD2+LC+) populations. Phosphorylation of STAT3 was detected by Western blotting. E, Day 4IL-7 BM B220+ cells from WT and IL-21R-/- mice were simulated with IL-7 or IL-21. Phospho-STAT3 was detected by Western blotting. Data are representative of two independent experiments.
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4.2.4 IL-21 accelerates the transition of proB cells toward the preB cell stage and the transition
of preB cells toward the immature/mature B cell stage
To examine whether IL-21-mediated signals affect development of B cell progenitors, day 4IL-7
BM B220+ cells isolated from WT and IL-21R-/- mice were sorted into proB, preB and
immature/mature B cell populations as previously described, and cultured with or without IL-21
for 48 hours. Absolute numbers of viable cells for each population were determined using trypan
blue exclusion. No significant difference in total cell numbers was observed between the control
and IL-21-containing cultures (data not shown). However, FACS analysis showed that IL-21
treatment affected the percentages of maturing cells that arose from WT proB and WT preB
cultures. In the cultures initiated from CD2-LC- proB cells, there were more preB cells and fewer
proB cells in the presence of IL-21 after 48 hours (Fig. 4.4A and 4.4B). Similarly, we observed a
trend towards increased percentages of immature/mature B cells and fewer preB cells in cultures
initiated from CD2+LC- preB cells in the presence of IL-21 (Fig. 4.4C; top panel and 4.4D; top
panel). Further analysis showed that the percentage of immature/mature B cells expressing high
levels of light chain tended to be higher in the IL-21-containing wells than in the control wells
(Fig. 4.4C; top panel; CD2+LChi population and 4.4D; lower panel). We hypothesized that this
population (CD2+LChi) likely represents cells that express both IgM and IgD on the surface. In
agreement with this, FACS analysis showed that the percentage of IgM+IgD+ mature B cells was
higher in cultures grown with IL-21 than the percentage observed in the control cultures (Fig.
4.4C; lower panel). In support of the finding that the increase in percentages of maturing B cells
is IL-21 mediated, we did not observe an increase in B cell development with IL-21 treatment of
cultures initiated with IL-21R-/- cells (Fig. 4.4A to 4.4D). Together, these results show that IL-21
accelerates the transition of proB toward the preB cell stage and of preB cells toward the mature
B cell stage.
To determine whether IL-21 affects B cell development in a similar way in vivo, we performed
FACS analysis on BM B cell progenitors freshly isolated from WT and IL-21R-/- mice. In
agreement with the in vitro analysis, we found that IL-21R-/- mice have increased proportion of
B220+CD2-IgM- proB cells (p=0.0376) and smaller proportion of B220+CD2+IgM+
immature/mature B cells (p=0.0131) than WT mice (Fig. 4.5A-top panel). We confirmed this
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result using a different set of B cell development markers and found that both the percentage and
absolute number of B220loCD43hiIgM- proB were increased (p<0.0001 and p=0.0021
respectively) and the percentage and absolute number of B220hiCD43-IgM+ mature B cells were
decreased (p=0.0232 and p=0.0756 respectively) in IL-21R-/- (Fig. 4.5A and 4.5B-middle panel).
The decrease in IgM+ cells was due to a decrease in the IgM+IgDhi mature B cell population,
more specifically in the IgMhiIgDhi cell population (percentage: p=0.0355; absolute number:
p=0.0053) (Fig. 4.5A and 4.5B-bottom panel). These results confirm that IL-21 has an impact on
B cell development in the BM and support the hypothesis that IL-21 accelerates the maturation
of B cell progenitors.
4.2.5 IL-21-mediated maturation of preB cells toward the immature/mature B cell stage does not
correlate with alteration of sIgM signaling
B cell progenitors pass critical checkpoints as they mature. One such checkpoint is the assembly
of a heavy and light chain to form the IgM receptor at the surface of immature B cells. The
productive rearrangement and assembly of Ig heavy and light chains is a positive signal that
allows B cells to continue their development (20). However, the quality of IgM-mediated signals
can be affected by other signals transmitted through co-expressed surface molecules. For
instance, CD22 is a transmembrane glycoprotein member of the Ig superfamily that is known to
regulate signals generated by IgM BCR expressed on mature B cells through the recruitment of
SHP-1 and SHIP (21). Moreover it has been shown that CD22 uses ligand-dependent and ligand-
independent mechanisms to regulate B cell functions (22). We performed FACS analysis on BM
CD19+ cells and found that CD22 expression was clearly higher in IgM+IgD- immature B cells
cultured with IL-21 for 24hr (Fig. 4.6A). FACS analysis also showed that IL-21 had no effect on
the surface expression of IgM on IgM+IgD- immature B cells (Fig. 4.6B). To determine whether
the higher expression of CD22 correlated with changes in the quality of IgM signalling, we
compared IgM signalling by immature IgM+IgD- cells grown with and without IL-21 for 24
hours. IgM signal quality was analysed by stimulating IgM BCR with anti-μ F(ab)2 and
measuring the resulting phosphorylation of ERK by western blot. ERK1/2 is a kinase activated
downstream of the BCR. A previous study showed that CD22 expression alone, even in the
absence of an anti-CD22 antibody-mediated crosslinking, reduces ERK activation downstream
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Figure 4.4. IL-21 regulates maturation of different B cell progenitors. Day 4IL-7 B220+ BM cells from WT and IL-21R-/- mice were sorted into proB (CD2-LC-), preB (CD2+LC-) and immature/mature B CD2+LC+) cell populations. Each population was plated with or without IL-21 (30 ng/ml) for 48 hours. Control and IL-21-containing cell cultures initiated with proB (A and B) and preB (C and D) cells from IL-21R-/- and WT mice were harvested on day 2 of culture and analyzed by FACS using a CD2-LC and IgM-IgD Ab combination. A,C) Percentages of cells in each population are shown on the plots of a representative experiment. B,D) Scatter plot of cell population percentages from separate experiments. Lines connect pairs of experiments performed. Statistical significance of the observed change in the number of preB and immature/mature B cells between control and IL-21-treated WT cell cultures was assessed by a 2-tailed paired Student t-test. The analysis shows that there was a significant increase in the percentage of preB cells in IL-21-containing culture started with proB cells, as compared to control (p < 0.0001*) (B; lower panel), and a trend toward an increased percentage of immature/mature B cells in IL-21-containing culture initiated with preB cells (percentage of CD2+LC+, p=0.09 (D; lower left panel); percentage of CD2+LChi, p=0.08 (D; lower right panel)).
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of IgM-BCR stimulation (23). However, we detected no significant difference in ERK
phosphorylation between the control samples and the samples stimulated with IL-21 (Fig. 4.6C).
4.2.6 IL-21 regulates the expression of Blimp1 and Aid, and induces the expression of germline
transcript gamma 2b (GLT 2b) in B cell progenitors
In mature B cells, stimulation of the IL-21R has been shown to induce the expression of
BLIMP1 (292, 301). BLIMP1 is a DNA binding zinc finger protein that can associate with
certain methyl transferases and is important in the regulation of PC differentiation (373). IL-21
also induces the expression of activation-induced cytidine deaminase (AID), an enzyme essential
for class switch recombination (CSR) and somatic hypermutation, when costimulated with anti-
CD40 or anti-CD40 and anti-IgM (293, 308, 311). Although no evidence of somatic
hypermutation has been reported, CSR to IgG and IgA has been observed in human cord blood
and mature B cells in response to IL-21 and anti-CD40 (293, 311, 312). Since BLIMP1 and AID
are typically expressed by the peripheral B cells, it was of interest to determine whether IL-21
can activate a similar pattern of genes in B cell progenitors from the BM. For this purpose, day
4IL-7 B220+ BM cells were stimulated with IL-21 for 24 hours prior to being sorted into proB,
preB and immature/mature B cells. Real-time PCR results showed that IL-21 increased
expression of Blimp1 in preB cells (Fig. 4.7A). In contrast, we did not observe any changes in
Blimp1 expression in proB or immature/mature B cells. In addition, IL-21 clearly induced Aid
expression in preB cells and immature/mature cells, but no significant effect was observed in the
cultures initiated with proB cells (Fig. 4.7B).
In mature B cells, IL-21-induced expression of Aid has been associated with the initiation of
class switch recombination (CSR) (25). CSR begins with the initiation of transcription of the
germline transcripts (GLTs) from the promoter of a specific isotype. Therefore we measured this
early hallmark of CSR by RT-PCR. We searched for GLTs in unstimulated B cells, B cells
stimulated with anti-CD40, IL-21, or anti-CD40 and IL-21. For this purpose, day 4IL-7 B220+ BM
cells were sorted into proB, preB, and immature/mature B cells and then stimulated for 24 hours.
Interestingly, IL-21 treatment of sorted preB and sorted immature/mature B cells resulted in
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increased levels of GLT 2b compared to the untreated cells (Fig 4.8A). Given that B cell
progenitors continuously mature when cultured in vitro, we wanted to determine whether IL-21
exposure resulted in increased transcription of GLTs in the preB cells themselves or in
immature/mature B cells that develop from preB cells. We treated bulk BM B220+ day 3IL-7
cultures with IL-21 for 24 hours and then sorted the proB, preB and immature/mature B
population. Although there was a trend for increased GLT2 b in IL21 treated cells, we did not
observe a significant difference between stimulated versus unstimulated cells in progenitor
populations that were treated with IL-21 prior to sorting (data not shown). Under these
conditions we also examined later hallmarks of CSR and did not observe IL-21-induced
differences in postswitch transcripts (PSTs) and circular transcripts (CTs) (data not shown).
Collectively, our data show that IL-21 induces aid in preB cells but suggest that IL-21-initiated
CSR occurs at later stages of B cell development.
4.2.7 Ig-secreting cells are generated from B cell progenitors stimulated with IL-21 and anti-
CD40
It is known that IL-21 induces PC differentiation and Ig secretion of human cord blood and
CD19+ peripheral blood and splenic B cells when used in combination with anti-CD40 (293,
311). IL-21-mediated induction of Blimp1, Aid, and GLTs described above is consistent with the
hypothesis that IL-21 can drive the differentiation of BM B cell progenitors into Ig-secreting
cells. To test this, we plated day 4IL-7 B220+ BM cells with IL-7, and combination of IL-21 and
anti-CD40. In this experiment, IL-7 was included in the culture to allow for continued expansion
of proB subset which kept maturing and replenishing preB and immature/mature subsets. This
extended the survival of the overall cell culture, thereby giving it sufficient time to respond to
IL-21 and anti-CD40. ELISAs were performed on supernatants collected on day 7 of culture.
Fig. 4.8B (left panel) shows that both IL-21 and anti-CD40 were required to induce secretion by
B220+ cells. In these cultures, we detected mostly IgM and IgG3, but also IgG1, IgG2b, and
some IgG2a. However, we did not detect IgA (data not shown).
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To determine whether early B cell progenitors can differentiate into Ig-producing cells, we sorted
day 4IL-7 B220+ BM cells into proB cells. ProB cells were then cultured with IL-7 and anti-CD40
with or without IL-21. ELISAs were performed on supernatants harvested on day 9 of culture.
We show that IL-21 and CD40 signals induced differentiation of proB cells into cells that
secreted mostly IgG3, but also IgM, IgG1, IgG2a, and IgG2b in the presence of IL-21 and anti-
CD40. IgA was below detection level of our assay (Fig. 4.8B - right panel).
4.3 Discussion
It is well established that IL-21 strongly influences the differentiation of murine and human B
cells. To date, this has been demonstrated at the end stages of B cell development where IL-21
induces the transition from fully mature B cells to Ig-secreting PCs. In this report we
significantly extend the role of IL-21 by showing, for the first time, that IL-21 accelerates the
maturation of murine B cell progenitors. Such cells are induced to express Blimp1 and Aid,
genes normally expressed in peripheral mature B cells. In addition, we show that IL-21 induces
the first step of CSR in B cell progenitors by inducing GLTs and, together with anti-CD40,
enables cells to differentiate into Ig-secreting cells.
Careful analysis of expression of IL-21R on different BM B cell progenitors revealed a gradual
increase of cell surface IL-21R from proB cells to immature/mature B cells, consistent with the
previous observations reported by Jin et al (281). We extend these findings by showing that IL-
21R is functional on these populations of B cell progenitors. The intensity of IL-21-induced
STAT phosphorylation signals correlated with the level of IL-21R expression. The signals were
the weakest in proB cells and the strongest in the immature/mature B cells. Even though we and
others (281) failed to detect IL-21R protein at the surface of proB cells by FACS, the results
from both RT-PCR and functional signalling experiments clearly show that IL-21R is also
expressed on these cells. Given the unexpected nature of this finding, it was important to ensure
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that the observation was not based on contaminating preB cells. To examine this question we
used an IL-21 unresponsive proB cell line to which we added different numbers of FACS sorted
IL-21 responsive preB cells. We found that a contamination level of at least 10% preB was
required to attain the level of STAT3 phosphorylation observed in sorted proB cells stimulated
with IL-21. This threshold is much higher than the 0.5-1 % contamination levels we routinely
achieve when we enrich for proB cells. Further, we found that IL-21 stimulation of BM B cells
isolated from RAG-/- mice, where B cell development is blocked at the proB cell stage, also
induces phosphorylation of STAT3. Finally, IL-21 stimulation failed to induce phoshorylation of
STAT3 in IL-21R-deficient proB cells confirming that the phosphorylation of STAT3 in IL-21-
stimulated proB cells occurs through the IL-21R.
We also found that Il21 message is constitutively expressed in murine BM, providing evidence
that B cell progenitors may well encounter IL-21 during development. Activated Th17, T
follicular helper (Tfh), and NKT cells are thought to be main sources of IL-21 in the periphery
(275-278). Consistent with a T cell origin of the Il21 transcripts detected in the BM, we detected
Il21 message specifically in CD4+ T cells. Furthermore, we showed that BM CD4+ T cells
require fewer stimuli to secrete IL-21 protein than splenic CD4+ T cells. In contrast to splenic
naïve and memory CD4+ T cells, which require stimulation through CD3, CD28 and IL-6R
(372), IL-6R stimulation was not required to induce secretion of IL-21 protein in BM CD4+ T
cells.
One of the main findings of our study is that IL-21 promotes the development of B cells
progenitors. This is supported by in vivo data showing that IL-21R-/- mice have more proB cells
which is likely a consequence of slower maturation. These mice also have fewer mature B cells
in their BM than WT mice, although based on current models (160, 162, 163) we cannot
distinguish whether this difference in IgMhiIgDhi cells comes from the newly arising B cells or
from the recirculating B cells.
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In further support of our in vivo observation, in vitro stimulation of sorted proB and preB
progenitors by IL-21 accelerates their development. The increased percentage of B cells at a
more advanced developmental stage is unlikely to be a consequence of enhanced proliferation or
survival in response to IL-21. This is supported by the finding that the total number of cells
recovered after 48 hours of culture is similar between IL-21-treated and control wells. Moreover,
after 48 hours treatment with IL-21, annexin-V and 7-AAD staining showed variable cell
survival with no observable trend in cultures initiated with proB or preB cells (data not shown).
It should be noted that while the experiments reported here show an effect of IL-21 on purified B
cell progenitors in tissue culture, it may be the case that other factors are involved in vivo. For
example, it has been shown that IL-21 synergizes with Flt3L and IL-15 to increase proliferation
and promote differentiation of NK cells from BM progenitors (270).
Several lines of evidence show that IL-21 can induce Blimp1 and, when used in combination
with anti-CD40 or anti-IgM, IL-21 can induce Aid in different populations of mature B cells
(292, 293, 308, 311). We show that IL-21 increases expression of Blimp1 in developing B cells,
and in particular, in preB cells. It is possible that the increase in Blimp1 expression contributes to
the acceleration of maturation of preB cell progenitors observed in presence of IL-21 by inducing
genes normally found in mature B cells and repressing genes associated with early B cell
progenitors. We also show that IL-21 alone induces Aid as early as the preB cell stage. This
result contrasts with data obtained on other type of B cells where anti-CD40 or anti-CD40 and
anti-IgM are required. While this is not the first report of early B lineage cells expressing Aid and
Blimp1 (374-376), it is the first study to suggest a method of induction in early B cell
progenitors.
Importantly, our study shows that IL-21 alone was sufficient to initiate early steps of CSR in BM
B cell progenitors. This is in contrast to what has been reported for human cord blood B cells, in
which both IL-21 and anti-CD40 are required for GLTs to occur (311, 312). However, similar
effects on GLTs have been observed in bulk human CD19+ spleen cells even though Aid
expression was absent (311). While expression and function of Aid is generally associated with
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the GC reaction in the secondary lymphoid organs, a recent study has shown that aid message is
expressed in early proB/preB cell progenitors in vivo, and is responsible for CSR observed in
these cells (374). Moreover, other studies have shown that CSR can occur in some of the
Abelson-transformed preB cell lines (377-379).
We believe it is highly unlikely that IL-21R, Aid, Blimp1, GLT 2b messages come from the
contaminating BM plasma and BM memory B cells. First, BM B cells used in our study are
selected using an anti-B220 antibody, a molecule absent from BM PCs (180). Second, we used
anti- and anti- antibodies instead of anti- to negatively select proB and preB cells, and
thereby avoid possible contamination of these populations with memory cells expressing IgM or
any other heavy chain isotype.
Our results show that proB cells differentiate into PCs secreting IgM and IgG1, IgG2a, IgG2b
and IgG3 Igs when costimulated with IL-21 and anti-CD40. Seagal et al. have proposed that
isotype-switched B cell precursors are deleted under normal physiological conditions by a
mechanism involving Fas/FasL interaction, presumably to prevent autoimmunity (380).
However, presence of IgA and IgG in MT mice (125, 126) suggests that at least some isotype-
switched cells can bypass this mechanism. Two hypotheses have been proposed to explain the
presence of CSR in BM B cell progenitors. One hypothesis is these cells could be the product of
an alternative B cell development pathway (125). Alternatively, signals through BCR and TLRs
can induce aid expression, which is involved in CSR observed in some B cell progenitors. Class-
switched B cell progenitors could responsible for enhanced innate immunity by generating IgG-
or IgA- producing cells (374).
Having demonstrated that developing B cell progenitors express IL21R and show accelerated
maturation in response to IL-21, it is of critical importance to determine what role the IL-21/IL-
21R pathway plays in the life of a B cell. One possibility is that T cell-derived IL-21 contributes
to the “normal” development of B cells in the BM. CD8+ and CD4+ mature T cells constitute
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approximately 3-8% of total BM nucleated cells (381). Several studies reported that, at steady
state, most CD4+ T cells in murine BM have an activated phenotype (382, 383). Maintenance of
the activation state of BM T cells does not require antigen stimulation, but occurs in response to
factors produced by the local microenvironment, such as IL-7, IL-15, and 4-1BBL (381, 383-
386). There is evidence for interaction between BM T cells and BM B cells through
CD40/CD40L that would be required for maintaining bone homeostasis (387). In addition, it has
been reported that hematopoiesis is severely impaired in T cell-deficient nude mice, and that
restoration of the BM CD4+ T cells rescued normal development of the myeloid compartment
(383). Our discovery that IL-21 in the murine BM is produced by T cells reinforces the potential
importance of a T cell-dependent B cell developmental pathway option.
An alternative, but not mutually exclusive, interpretation is that IL-21 influences B cell
development in the context of an inflammatory response. It is known that leukocyte production is
affected during infection and inflammation. Experimental inflammation caused by the injection
of adjuvants leads to a remarkable decline in BM B cell development and a corresponding onset
of extramedulary development in the spleen (388). This phenomenon is mediated by a rapid
reduction of CXCL12 which is thought to be involved in the sequestration of developing B cell
progenitors in the BM (388). Under these conditions, B cell progenitors might be found in areas
with active CD4+ T cells in the spleen. Interaction of B cell progenitors with T cells through
CD40-CD40 ligand interaction in the presence of IL-21 could allow B cell progenitors that have
been mobilized to the periphery to continue their development outside the BM and participate in
the immune response. It has been noted that such extramedulary development might bypass
checkpoints which normally eliminate autoreactive cells in the BM (389). Indeed, there is a
growing body of evidence linking IL-21 with the development of some autoimmune diseases
with strong humoral component. For example, BXSB.B6-Yaa+/J murine model of SLE shows
elevated levels of serum IL-21 (292). In contrast, BXSB-Yaa+ mice deficient for IL-21R do not
develop SLE (370).
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We do not yet know if our observations have uncovered a “normal” process that may contribute
to the immune response or even a “dangerous” anomaly that may contribute to autoimmunity by
allowing immature cells to bypass regulatory mechanisms that normally eliminate autoreactive
cells. However our results clearly suggest that IL-21 regulates maturation of B cell progenitors
and, in combination with anti-CD40, can lead to the formation of Ig-secreting cells.
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CHAPTER 5
DEVELOPMENT OF AN IN VITRO SYSTEM FOR THE
GENERATION OF B CELLS EXPRESSING IGA AND INOS
___________________________________________________________________________
Some contents of this chapter appear in:
Jörg H. Fritz, Olga Lucias Rojas, Nathalie Simard, Douglas D. McCarthy, Siegfried
Hapfelmeier, Stephen Rubino, Susan J. Robertson, Mani Larijani, Jean Gosselin, Ivaylo I.
Ivanov, Alberto Martin, Rafael Casellas, Dana J. Philpott, Stephen E. Girardin, Kathy D.
McCoy, Andrew J. Macpherson, Christopher J. Paige, Jennifer L. Gommerman (2012) Nature
January 12; 481: 199-203.
N.S. performed all of the experiments presented in this chapter with the exception of figure 5.1
with was generated in collaboration with J.H.F., O.LR., and D.D.M.
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5.1 Introduction
Every day the body is confronted by a variety of potentially harmful environmental agents,
including microorganisms such as viruses, bacteria and parasites; and internal threats like tumor
cells. To cope with the wide diversity of possible situations, various mechanisms of defense have
evolved to form collectively the immune system. The components of the immune system are
often classified as belonging to the innate immune system or the adaptive immune system.
The protection provided by the innate immune system exists from birth in all multicellular
organisms, including primitive organisms. It is present before infection and constitutes the first
line of defense against microbes. Innate mechanisms of defense are relatively nonspecific and
respond the same way to repeated infections. They include physical and chemical barriers, such
as skin and acidic pH; blood proteins, such as the complement system; and effector cells (390).
Phagocytic cells (neutrophils, macrophages) and natural killer (NK) cells are examples of cells
conventionally associated with the innate immune system. These cells express germline-encoded
receptors that recognize conserved molecular structures shared by microbes, and rapidly react to
their presence by producing antimicrobial substances and stimulating the adaptive immune
response (390).
To supplement the protection provided by the innate immune system, vertebrates also produces
highly specialized effector cells that develop a protective response only upon encounter with a
foreign agent. This response, called adaptive immune response, is triggered by the activation of
BCR or T cell receptors (TCR) on the surface of lymphocytes, and although it is relatively slow
to develop when the cells are first exposed to an antigen, it becomes much more rapid and robust
upon repeated exposure to the same antigen (390). The BCR and TCR recognize a vast array of
antigens, but each individual one is highly specific. They are not encoded directly in the
germline DNA, but are the products of V(D)J gene segments that have undergone somatic
recombination. Only one allele is expressed, which confers unique specificity to each cell (390).
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Although these two categories, innate and adaptive immunity, are sufficient to describe most
common immune cells, a small but growing number of cell populations are not so readily
categorized, including T cells, B-1 B cells, and
marginal zone B cells (102). Although these cells express either a BCR or a TCR, they
nonetheless only recognize a limited number of antigens. Another distinctive feature is that,
similar to conventional innate immune cells, they rapidly execute their effector function (102).
Recent research has identified a new population of IgA+ PCs in the small intestinal lamina
propria that display characteristics of both adaptive and innate immune cells (391). These cells
express IgA, which can protect the host by neutralizing toxins and preventing bacterial adhesion
to the mucosal surface (1), but they also exhibit traits conventionally associated with
monocyte/granulocyte cell types (391). In particular, these cells produce the anti-microbial
mediators inducible nitric oxide synthase (iNOS) and , and therefore could be actively
involved in the homeostasis between the intestinal microflora and the host immune system.
Indeed, iNOS is the enzyme that synthesizes nitric oxide (NO) from L-arginine (392). NO is a
cytotoxic component that can kill microbes, but has also other functions in the immune system
such as the regulation of AID expression (392, 393). In various cell types, iNOS expression is
stimulated by the presence of microbial products and/or TNF . It has been postulated that the
particular conditions present in the gut could promote the development of the “monocytic”
potential observed in some intestinal IgA+ PCs during the process of differentiation (391). In this
chapter, I show that IgA+iNOS+ cells can be generated in vitro from BM B220+ cells. The
generation of these cells is dependent on the presence of gut stroma. More specifically,
experiments carried on germ-free (GF) mice suggest that it relies on the presence of microbial
agents.
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5.2 Results
5.2.1 Small intestinal lamina propria contains a population of cells expressing markers
characteristic of both B and myeloid lineages
A group led by Dr. Jennifer Gommerman at the University of Toronto has identified a small
population of IgA+ cells in the lamina propria that also exhibit characteristics of
monocyte/granulocytes (391). In collaboration with the Gommerman lab, we have confirmed the
presence of this population by performing FACS staining of lamina propria isolated from
reporter mice expressing the AID-YFP reporter (Fig. 5.1) (394). As reported (391), we found that
these cells also express low levels of CD19 and CD138, which are usually associated with the
PC phenotype. Molecules conventionally considered markers of monocytes or granulocytes were
also detected, including low levels of CD11c, the expression of Ly6C and Ly6G, as well as the
anti-microbial mediator iNOS. This pattern was specific to the IgA+YFP+ population, and was
not observed in the IgA-YFP- or IgA-YFP+ populations. These results confirm the presence of
IgA+ cells in the lamina propria, and characterize the phenotype of a new cell population subset,
as described by Fritz et al.(391).
The Gommerman group has also demonstrated that B lineage cells contribute to the expression
of iNOS in the gut (391). Indeed, JH-/- mice and Rag2-/- mice, in which B cell development is
arrested at the proB cell stage (5, 395), do not express iNOS in their gut. Similarly MT B6
mice, which have a profound B cell deficiency but can still produce some IgA+ cells in some
colonies (125), also present a strongly reduced iNOS expression in their gut (391). During
haematopoiesis two types of B-lineage cells are generated. They are referred to as B1 and B2 B
cells. It is generally accepted that B1 cells are predominantly generated in the fetal liver whereas
B2 cells develop principally in the BM (396-398). In contrast to B cell development in the BM,
B cell development in the fetal liver does not depend on IL-7 (69) and therefore mice deficient
for IL-7 are still able to produce B1 cells, but lack B2 cells (69). Since gut IgA+ cells can be of
either phenotype (224), we sought to determine whether IgA+iNOS+ cells were derived from B1
and/or B2 B cells by staining lamina propria from WT and IL-7-/- mice. Although we found
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IgA+iNOS+ cells in the lamina propria of IL-7-/- mice, the percentage of IgA+iNOS+ cells was
much lower in IL-7-/- compared to WT mice (Fig. 5.2). These results indicate that IgA+iNOS+
cells are not uniquely of B1 or B2 origin.
5.2.2 Generation of IgA+iNOS+ cells in vitro
Having confirmed that the lamina propria harbours a population of PCs possessing monocytic
traits, we sought to determine whether these cells could be produced in vitro. To recapitulate the
development of IgA+iNOS+ cells, we cultured B220+ BM cells isolated from CD45.1 mice for
seven days on gut-derived stroma extracted from CD45.2 mice. Cells were plated with different
mixtures of factors promoting B-cell development, as well as cytokines promoting CSR to IgA
(Fig. 5.3). We also added IFN- to some wells, since it has been reported to induce expression of
iNOS in macrophages and dendritic cells (399, 400); and lipopolysaccharides (LPS) to simulate
one of the bacterial components naturally found in the gut. We found that the minimum
requirements to generate IgA+iNOS+ cells in vitro on WT gut stroma (see materials and methods)
were achieved with a cocktail composed of IL-7, IL-21, and anti-CD40 (Figure 5.3). Addition of
TGF and IFN- to this cocktail further increased the percentage of IgA+iNOS+ cells generated.
We then compared results obtained with WT gut stroma to results obtained with BM cells grown
on Rag2-/- gut stroma, which is devoid of B and T lymphocytes. Similar to WT gut stroma, the
Rag2-/- gut stroma was efficient at generating IgA+ cells (Fig. 5.3), but the generation of
IgA+iNOS+ cells was rarely observed, suggesting that intestinal lymphocytes might be important
to condition the stroma. Thus for the remainder of our study, we decided to use the combination
of IL-7, IL-21, anti-CD40 and TGF , which together gave the highest proportion of IgA
switched cells, and also constituted the minimum requirement to generate IgA+iNOS+ cells
efficiently.
5.2.3 Characterization of the BM/gut stroma culture
The IgA+iNOS+ cell population observed in in vitro culture could be the result of newly
generated IgA+iNOS+ cells, or proliferation of pre-existing IgA+iNOS+ in the starting BM
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Fig. 5.3. T
esting of different culture conditions to generate IgA+iN
OS
+ cells from B
220+ B
M progenitors
F
actors added to the culture W
T gut strom
a + B220
+ BM
cells R
AG
-/- gut stroma + B
220+ B
M cells
T
GF
IL-21
CD
40 IN
FL
PS
iNO
S+
IgA+
IgA+iN
OS
+ iN
OS
+ IgA
+ IgA
+iNO
S+
x
+
/- -
- +
/- -
-
x
+/-
+/-
- -
+/-
-
x
+/-
+
+/-
+/-
+
-
x
+
+/-
- N
A
NA
N
A
x x
+
/- +
-
+
++
+
/- x
x
+/-
+/-
+/-
- -
- x
x
x
+
+/-
+/-
- -
-
- -
- -
- -
x
+
/- +
/- -
+/-
+/-
-
x x
+
++
+
/- +
+
-
x x
x
+
/- +
++
+
/- +
/- +
++
-
x x
x
x +
/- +
++
+
/- +
+
++
-
x x
x x
+
+
++
+
+
+
-
x x
x x
x +
+
++
+
+
+
+
+
B220
+ BM
cells from C
D45.1
+ mice w
ere co-cultured with gut strom
a from C
D45.2
+ mice w
ith the indicated factors. On day 7, cells w
ere harvested and FA
CS analysis w
as performed. B
M-derived cells w
ere selected by gating the CD
45.2- population and analysed for their
expression of IgA and iN
OS. U
pper section = poor cell grow
th. Low
er section = good cell grow
th
98
population. To determine whether IgA+iNOS+ cells were present in our starting population, we
stained whole BM with B220, IgA, and iNOS antibodies. We then gated on B220- and B220+
cells to determine the distribution of IgA+iNOS+ cells between these fractions. We established
that IgA+iNOS+ cells were present in both fractions (Fig. 5.4A), and consequently also present in
our starting population. To further confirm that IgA+iNOS+ cells were present in the starting
population, and to get a clearer idea of the proportion of this population, we selected BM B220+
using MACS purification and stained them with IgA and iNOS antibodies. The percentage of
B220+ cells in the purified sample was greater than 98%, and IgA+iNOS+ cells accounted for
0.01% of the cells (Fig. 5.4B), suggesting that some IgA+iNOS+ cells grown in culture might
derived from pre-existing IgA+iNOS+ BM cells.
To further characterise our in vitro system, we performed time course experiments. BM B220+
cells were plated with WT gut stroma and cultured for 8 days. Cells were harvested and stained
for IgA and iNOS every day starting at day 5 (Fig. 5.5). Typically, by day 5 a relatively high
proportion of B cells had switched to IgA; however, they did not yet express iNOS. IgA+iNOS+
cells appeared on day 6 or day 7 of culture. The number of days IgA+iNOS+ cells persisted in
culture showed a high degree of variation, ranging from from a single day to at least three days.
The rapidity at which the peak is reach and the duration of the population suggest that although
some IgA+iNOS+ cells are initially seeded in culture, it is likely that that some or all cells
recovered at day 6-7 are due to de novo generation of IgA+iNOS+ cells.
5.2.4 iNOS expression is supported by lamina propria-derived stroma, but not by BM-derived
stroma
From the data presented in Fig.5.5, it is clear that gut stroma is able to support the expression of
iNOS in IgA+ cells. However, it is not clear whether this property is specific to gut stroma or
whether other types of stroma share the same potential. Since IgA+iNOS+ cells are also naturally
found in the BM, we were particularly interested to know whether BM stroma can trigger iNOS
expression. For this purpose, we first used the BM stromal cell line S17 as a support to grow BM
B220+ cells and compared S17 monolayer to gut stroma for its capacity to support iNOS
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101
expression in IgA+ cells. The BM B220+ cells were isolated from the fate map reporter AID-YFP
mice to determine whether, similar to the in vivo situation, prior expression of AID correlated
with the production of iNOS. We found that although both gut stroma and S17 were efficient at
generating IgA+YFP+ cells, only gut stroma could promote the development of IgA+iNOS+ cells
(Fig. 5.6A). Since S17 is a cell line (401), and therefore does not have a complexity comparable
to gut stroma, we also grew B220+ cells on ex vivo BM-derived stroma. Results obtained with ex
vivo BM stroma were comparable to data shown for S17 and did show development of
IgA+iNOS+ cells (Fig. 5.6B). Taken together, these results suggest that gut stroma is unique in its
ability to promote the development of IgA+iNOS+ cells.
Since the Gommerman group demonstrated that lymphotoxin receptor -/- (LT R-/-) mice had a
reduced number of IgA+iNOS+ cells in the lamina propria (391), we tested the contribution of the
LT pathway in the generation of IgA+iNOS+ cells in vitro. We grew BM CD45.2 B220+ cells on
gut stroma isolated from CD45.1 LT R-/- mice or WT mice. We found that LT R-/- gut stroma
was as efficient as WT gut stroma at supporting the development of IgA+iNOS+ cells (Fig. 5.6B).
This result demonstrates that, in contrast to the in vivo situation, LT R is not critical for the
development of IgA+iNOS+ cells in vitro, and that the presence of specific exogenous factors can
overcome the lack of a functional LT pathway.
5.2.5 Microbial exposure promotes the expression of iNOS in IgA+ plasma cells
One of the particularities of the gut environment is the high prevalence of normally harmless
microbes. Our results showed that gut stroma, but not BM stroma, was efficient at generating
IgA+iNOS+ cells, thus it is plausible that this difference is due to the presence of residual
microbial agents present in the gut stroma preparation. In support of this, it has been shown that
IgA+ PCs present in the lamina propria of GF mice do not express iNOS, and that reconstitution
of the flora, even with a limited diversity of microbes, restored the expression of iNOS (391). To
test whether microbes were contributing to the expression of iNOS in vitro, we cultured BM
CD45.1 B220+ cells on gut stroma isolated from specific pathogen free (SPF) or GF CD45.2
mice. We found that gut stroma from SPF but not GF mice sustained the development of
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IgA+iNOS+ cells (Fig. 5.7). Moreover, addition of fecal matter derived from SPF mice to culture
with GF gut stroma restored the development of IgA+iNOS+ cells. These results suggest that
microbes present in the gut environment contribute to the development IgA+ PCs expressing
iNOS.
5.3 Discussion
In this chapter, we characterized the in vitro generation of a newly identified population of IgA+
PCs that share characteristics with the monocyte/granulocyte lineages. In vivo, IgA+iNOS+ PCs
have been detected in the gut (391). Our results show that the generation of a B cell population
expressing iNOS and IgA can be achieved by growing BM B220+ cells on lamina propria-
derived stroma with factors promoting the growth of B cell progenitors as well as class-switching
to IgA.
We found that a number of BM B220+ cells were able to differentiate into IgA+iNOS+ cells when
grown on gut stroma in the presence of IL-7, which ensured the survival and proliferation of BM
B cells progenitors, as well as TGF , IL-21, and anti-CD40, which favoured CSR to IgA. The
pro-IgA CSR factors used in this study are naturally present in the gut environment and could
therefore be participating in the generation of the IgA+iNOS+ cells found in the lamina propria.
Related to this, TGF has been shown to cooperate with CD40L to generate IgA+ cells in peyer’s
patches (PP) (402). Furthermore, TGF -/- mice present a marked defect in IgA-committed cells
in the gut, which is particularly pronounced in the lamina propria (402). In contrast, the role of
CD40 in the production of intestinal IgA is less clear. CD40-/- mice exhibit near normal levels of
gut IgA, arguing against a critical role of CD40 for intestinal IgA production (233). However,
propria were not likely to be major sites of ongoing IgA CSR (233). Finally, we have conducted
FACS analysis on the lamina propria of IL-21R-/- mice to assess the contribution of IL-21
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signalling in the generation of IgA+iNOS+ cells in vivo. IgA+iNOS+ cells were present in the
lamina propria of IL-21R-/- mice (Appendix 1), indicating that although addition of IL-21 aided
the generation of IgA+ cells in vitro, this factor is not critical for the production of IgA+iNOS+
cells in vivo. Aside from the pro-IgA CSR factors that we added to the culture, it is also possible
that the induction of iNOS in some cells further favors the production of IgA+ B cells, as
evidenced by previous observations that iNOS-/- mice express lower levels of AID and are
impaired in their ability to produce IgA (393, 403). Since treatment of iNOS-/- B cells with
SNAP, a NO donor, was shown to restore the expression of AID, it has been suggested that NO
synthesized by iNOS can regulate the expression of AID, and therefore promotes CSR (393).
When properly stimulated, B cells proliferate and differentiate into Ig-secreting plasmablasts,
and ultimately into non-dividing PCs. We were able to generate a substantial number of IgA+
cells in vitro, but only a small proportion of these cells expressed iNOS. We attempted to
promote the expression of iNOS by adding
iNOS production in myeloid cells (399, 400), but the addition of these factors did not
significantly increase the proportion of IgA+iNOS+ cells. Interestingly, it has been shown that L-
arginine, which is used to synthesize NO, also regulates the expression of iNOS at the protein
level in astrocytes and macrophages (404, 405). Therefore it is possible that an insufficient
concentration of L-arginine in the culture media had impaired the generation of higher numbers
of iNOS-expressing IgA+ cells.
Besides the suboptimal conditions used to stimulate iNOS expression, the low percentage of
IgA+iNOS+ cells obtained in our culture could also be the results of plasmablasts not dividing,
the intrinsic short lifetime of IgA+iNOS+, or because IgA+iNOS+ cells require other factors to
survive in addition to the stromal cells and cytokines provided. There is mounting evidence that
the survival of PCs is determined by micro-environmental rather than intrinsic factors (193-195).
Indeed, when cultured without feeder cells or exogenous factors, PCs die rapidly (194, 195). It is
also well established that the BM microenvironment provides a niche that supports the extended
survival of PCs. The ability of BM-derived stromal cells to maintain plasma cell longevity in
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culture has also been demonstrated and appears to rely on cell contact-dependent signals and
cytokines such as IL-5 and IL-6 (169, 194). We sought to increase the numbers of IgA+iNOS+
cells by adding IL-4, IL-5 and IL-6 to our cultures. IL-4 and IL-5 have been shown to increase
the probability of commitment to the plasmacytic fate (406-408). IL-5 and IL-6 are known to
increase the survival rate of PCs in vitro, with IL-6 being the most efficient factors (194, 195).
However the percentages of IgA+iNOS+ cells obtained in the cultures containing IL-4, IL-5, and
IL-6 were similar to that observed in control cultures, implying that these cytokines did not
increase the proliferation or survival of IgA+iNOS+ cells. Recently, a study has shown that
eosinophils play a crucial role for the survival of PCs by secreting a proliferation-inducing ligand
(APRIL) and IL-6 (212). In addition, APRIL-/- mice, but not IL-6-/- mice, show a significant
defect in PC survival in their BM compartment (195, 211). Hence, it would be interesting to test
whether APRIL can enhance the survival of IgA+iNOS+ cells in our in vitro system.
An alternative explanation for our findings is that once in culture, the gut stroma gradually loses
its ability to support the development of IgA+iNOS+ cells. For example, this could happen if
microorganisms present in the gut are necessary to condition the lamina propria stromal cells to
enable them to support the growth of IgA+iNOS+ cells. In this regard, a role for the microbiota in
the generation of IgA+iNOS+ cells in the lamina propria was suggested by our in vitro
experiment performed with GF stroma. Interestingly, gut stroma derived from RAG-/- mice
housed in a cleaner room than WT mice were also less efficient at supporting the development of
IgA+iNOS+ cells. Furthermore, in vivo data obtained on GF mice demonstrated that the
appearance of IgA+iNOS+ cells in the lamina propria was dependent on the presence of intestinal
bacteria (391). However, it could also be that the microbiota acts directly on IgA+ B cells to
induce the expression of iNOS. Further experiments using mutant gut stroma and/or BM
progenitors will help identifying the mechanisms responsible for the recognition of the
microbiota and how it leads to iNOS production.
Our in vitro data show that primary gut stroma, but not BM stroma, could support the
development of BM B220+ cells into IgA+iNOS+ cells. Since we found small numbers of
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IgA+iNOS+ cells in the BM in vivo, our results suggest that IgA+iNOS+ PCs are first generated in
the gut and then migrate to a different localization. Furthermore, it implies that conditions
present in the microenvironment, such as the accessory cells, the factors secreted, and the nature
of antigens encountered, influence the transcriptional program activated in B cells and the type
of PCs generated. Therefore PCs that arise in the gut may differ from those developing in other
compartment (169). In line with this, it is has been postulated that the gut environment imparts a
restricted monocytic potential to IgA+ cells, and that these cells contribute either directly or
indirectly to maintain the composition of the intestinal microflora (391).
Another factor that might influence the type of PCs generated is the subset of B cells from which
it derives. The presence of IgA+iNOS+ cells in IL-7-/- mice indicates that their origin is not
specific to B1 or B2 cell precursors. This is in line with findings reported by other groups who
showed that, in mice, intestinal IgA+ PCs can derive from both B1 and B2 cells (409, 410). One
possibility is that these cells could derive from a rarer subset B cell progenitors. Indeed, although
it is thought that most B cells derive from CLP, evidence for an alternative route of B cell
development has been previously reported – specifically, it has been shown that the fetal liver
contains B lineage cells with limited myeloid developmental potential and that these cells
represent normal intermediates in fetal hematopoiesis (411). Bipotential B-macrophage
progenitors have also been identified in adult murine BM (412). Supporting the notion that B
cells might share a common progenitor with myeloid cells is the findings that some leukemic
cells display traits of both B and myeloid cells (413). IgA+iNOS+ cells differ in that they
represent an example of mature B cells with monocytic properties that occurs normally in the
gut.
Our gut stroma culture system measures the potential of different BM progenitors to give rise to
IgA+iNOS+ cells, and potentially identifies the stimuli necessary for their development via the
addition of factors to the culture, the blocking of cytokines produced by gut stromal cells, the
testing of cell-cell contact requirements, or the use of gut stroma or BM progenitors derived from
mutant mice. Mutant mice may be particularly useful to determine how microbiota interacts
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within the system to induce expression of iNOS in IgA+ cells. Optimizing the number of
IgA+iNOS+ cells generated would facilitate their study and potentially their purification. So far, it
has not been possible for us to sort this population due to the necessity to perform intracellular
stains to label the cells properly. It would be useful to push further the phenotype
characterisation of IgA+iNOS+ cells from both in vivo and in vitro origin to identify a set of
surface markers that are specific for this population. This would allow us to compare the in vitro
generated cells with the in vivo cells, and enable further RNA and effector function studies.
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CHAPTER 6
GENERAL DISCUSSION
110
The course of B cell development in the BM and periphery is largely influenced by signals
received from the environment. The developmental pathway described in the introduction applies
to B cells maturing in young, healthy mice. However, this pattern can be altered under some
circumstances, for example, following infection. This thesis has investigated factors regulating B
cell development in steady state conditions, and discussed hypothetical roles for some of these
factors in B lymphopoiesis in infected hosts.
6.1 The loss of IL-7 responsiveness
IL-7R is expressed on all developing BM B cell progenitors, and its activation is known to play
an important role in the early stages of murine B cell development (325). As B cells mature,
there is a marked drop in IL-7 responsiveness. Many studies have been carried out on this
subject, and the mechanisms underlying this sensitivity loss are beginning to be revealed. It was
observed that a + - preB-like cells isolated from normal murine BM proliferated in the presence
of IL-7, but that this ability to respond to IL-7 was lost in the small fraction of cells that
spontaneously differentiated to the + + stage (414). The failure to proliferate in response to IL-
7 was not due to the downregulation of IL-7R; instead, it was shown to be dependent on the
Tyr410 present in the cytoplasmic domain of IL-7R. Since the transfection of these cells with a
rearranged light chain gene led to the inhibition of IL-7-induced proliferation, it was suggested
that the assembly of a functional BCR triggers the recruitment of an inhibitory molecule to
Tyr410. However, the existence of some IL-7-dependent cell lines that express a BCR argues
against this view (415). In addition, our lab has shown that B cells lose their ability to proliferate
in response to IL-7 when they reach the small preB cell stage, further supporting the hypothesis
that signalling derives from another molecule than the BCR (152).
The magnitude of the response to IL-7 stimulation is likely influenced by signals originating
from other receptors, whose combination and levels of expression varies with the stage of B cell
development. Our lab has previously shown that the loss of sensitivity to IL-7 observed in
developing B cells is coincident with the appearance of the surface marker CD22 (322). CD22 is
a well conserved ITIM-containing molecule expressed in mammals that recognizes sialic acids.
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It is mainly known for its ability to associate with the BCR and modulate BCR signalling. CD22
has been shown to recruit both negative effectors of cell activation, such as SHP-1 and SHIP, as
well as positive regulators, such as Syk, PLC 2, and PI3-K (354, 416). SHP-1 is believed to be
the main effector mediating CD22 inhibition (349). Interestingly, evidence supports a role for
SHP1 in the regulation of ERK and JAK/STAT proteins, including those involved in IL-7R
signalling, notably JAK1, JAK3, and STAT5. Indeed, transfection of cell lines with CD22
reduced the IgM-induced phosphorylation of ERK (348, 355, 356). Furthermore, co-
immunoprecipitation experiments have shown that SHP-1 can physically associate with JAK3
(417, 418), and SHP1-deficient macrophages show increased JAK1 phosphorylation (419).
Finally, introduction of WT SHIP1 in Ba/F3 cell lines reduces IL-3-induced STAT5
phosphorylation (420).
The finding that CD22 expression is inversely correlated with the magnitude of the IL-7 response
prompted us to determine whether signals delivered through CD22 contributed to the inhibition
of IL-7 response. Since IL-7 stimulation of bone-marrow derived B cell lines with IL-7 activates
similar pathways to those in primary BM B cells, a large part of our analysis was performed on
cell lines. These cell lines exhibit phenotypes associated to diverse stages of B cell development
arising between the proB and the immature B cell stage. The lack of correlation between the
expression of CD22 on these cell lines and the stage of B cell development suggests that CD22 is
not involved in the inhibition of the IL-7 response. Experiments in which CD22 molecules have
been cross-linked support this conclusion, although we could not rule out the possibility that the
antibody induced cross-linking mimicked completely the response triggered by the binding of a
natural ligand. In addition, it is possible that the absence of modulation of the IL-7 response upon
CD22 cross-linking was due to the fact that CD22 was already bound in cis. The presence of cis
interactions with sialic acids is supported by the observation that cells treated with
neuraminidase proliferate less in the presence of IL-7 than non-treated cells. However our data
did not demonstrate that this effect was specific to CD22. One candidate that should be
considered when interpreting the results obtained on cells treated with neuraminidase is Siglec G
(reviewed in (421, 422)). Murine Siglec-G is found predominantly on B cells, including BM B
cell progenitors (423, 424). Similar to CD22, Siglec- -6 Sia residues of N-linked
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oligosaccharides. Its cytoplasmic tail also contains an ITIM that is believed to serve as a docking
site for SHP-1, as it has been demonstrated for Siglec-10, the human ortholog of Siglec-G (425).
The intensity of the IL-7R response is carefully regulated throughout B cell development. In fact,
it has been shown that B cell development is arrested at an early stage in either the absence or
excess of IL-7 signalling (53, 54, 426). One regulator of IL-7 signalling is CD45 (B220), which
has been shown to dephosphorylate and thereby inactivate all JAK proteins in murine cells (427).
In agreement with this, the IL-7 mediated activation of the JAK/STAT pathway is sustained over
a longer period of time in proB cell lines derived from the BM of CD45-/- mice compared to WT
mice (428). This prolonged JAK/STAT activation translated into a higher number of early proB
cells generated both in vivo and in vitro as a result of improved survival. The increased number
of proB cells in CD45-/- mice indicates that CD45 participates in the inhibition of IL-7R
signalling; however, the observation that complete abrogation of the IL-7 response is achieved in
later stage of development implies that the inhibition by other molecules is required.
In preB cells, it has been established that the PreBCR can cooperate with the IL-7R to activate
the ERK/MAP kinase pathway, which initially allows for the selective expansion and survival of
preBCR+ cells in low amount of IL-7 (68). However, a newly published report suggests that
preBCR signalling ultimately inhibits preBCR and IL-7-induced proliferation by suppressing the
expression of c-myc (429). The suppression of c-myc subsequently leads to the downregulation
of cyclin D3 and the upregulation of the negative cell cycle regulator p27Kip.
Most recently, our lab has proposed an additional mechanism that could contribute to the
modulation of the IL-7 response (430). Corfe et al. have shown that suppressor of cytokine
signalling-1 (SOCS-1) can inhibit signals transmitted by the IL-7R in proB and preB cells by
hampering the phosphorylation of JAK/STAT. The expression of SOCS1 can be induced by the
IL-7R itself thereby creating a negative auto-regulatory loop. IL-21 and INF were also able to
regulate the expression of SOCS-1 and as a result the sensitivity of B cells to IL-7.
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6.2 IL-21 promotes the maturation of B cell progenitors
Signals present in the microenvironment influence the decision of B cells to progress further
down the B lineage pathway. In chapter 4, we provide evidence that IL-21 affects the
development of B cells. This hypothesis was based on flow cytometry data presented in a report
published by Jin et al. that showed the expression of IL-21R on BM B cell progenitors, starting
at the B220lowCD43intIgM- preB cell stage (281). We have confirmed the expression of IL-21R in
freshly isolated BM B cells by FACS using B220, CD43, and IgM markers. However, CD43
does not constitute a suitable marker to distinguish the different cell stages in cultured BM B
cells as, for unknown reasons, its expression is not downregulated when late proB/large preB
cells mature to the small preB stage in vitro (415). We have therefore also analysed the
expression of IL-21R on the different populations identified based on the expression of CD2 and
IgM. The expression pattern of IL-21R detected on cultured BM B cell progenitors closely
matched the pattern observed on ex vivo progenitors. In conjunction with signalling experiments,
our results clearly show that IL-21R is expressed and is functional on all B cell progenitors,
including proB cells. We have also found that ex vivo BM CD4+ T cells express Il21 mRNA and
have the ability to secrete the protein when properly stimulated. This observation provides
evidence for a ready source of IL-21 at the site of B cell development.
Our FACS data on ex vivo cells from IL-21R-/- and WT mice as well as with in vitro sorted BM
B cell progenitors support the idea that IL-21 can accelerate the pace of B cell development. The
mechanism through which IL-21 mediates this effect is still to be determined. It is possible that
the induction of Blimp1 expression observed upon treatment of the progenitors contributes to this
effect, as it is known to regulate a large pool of genes involved in diverse functions (431).
Preliminary experimental data obtained on the cultured B cell progenitors suggests that levels of
Irf4 might also be elevated in IL-21-treated cells, particularly in proB and preB cells. The action
of IRF4, in collaboration with STAT3, could account for the upregulation of Blimp1 (300).
Interestingly, IRF4 has also been shown to play a role at several stages of B cell development,
including the transition from large preB to small preB cells (432, 433). IRF4 function overlaps
greatly with IRF8 as reexpression of one of these genes is sufficient to restore the development
of preB cells in IRF4-/-IRF8-/- mice (434). However, they differ in their expression pattern, IRF8
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expression being relatively stable throughout B cell development, whereas IRF4 expression is
relatively low in proB cells but significantly elevated in preB cells (433). Both genes play a
critical role for the transition from large preB to small preB by limiting preB cells expansion and
promoting light chain genes rearrangement (432, 434-436). IRF4, whose expression is also
induced by signalling through the preBCR, induces the expression of Ikaros and Aiolos (432,
433). Ikaros and Aiolos limit preB cell expansion by downregulating preBCR, through the
suppression of surrogate light chain expression, and impeding cell cycle progression at the G1-S
transition (437). Furthermore, IRF4 and IRF8 support the development of B cells by promoting
the rearrangement and transcription of the Ig light chain (434, 435). This effect is achieved either
directly by activating light chain enhancers or indirectly by attenuation of IL-7R signalling,
which is thought to be a consequence of CXCR4 upregulation leading to the subsequent
migration of preB cells away from IL-7-secreting stromal cells (435). The modulation of IRF4
during B cell development has been proposed to be important for the orchestration of the
sequential rearrangement of the Ig heavy chain and light chain (433, 435). In this model, IL-7
signalling promotes the rearrangement of the heavy chain in proB cells. The rearrangement of
the light chain is disfavored by IL-7 signalling and the low levels of IRF4. In preB cells, the
rearrangement of the light chain is promoted by the increased levels of IRF4 which activates
light chain enhancers in addition to its contribution to cell cycle exit and IL-7 signalling
reduction. Based on this, increased levels of IRF4 following IL-21 stimulation to levels sufficient
to activate light chain enhancers and thereby promote light chain rearrangement is one potential
mechanism that could explain the accelerated preB to immature B transition in our in vitro
culture.
6.3 Role of IL-21 in steady state B cell development
Data presented in this thesis show that IL-21 signalling is dispensable for the development of B
cells, but when present, can nevertheless affect their development. Furthermore, CD4+ BM T
cells express IL-21 transcripts, suggesting the possibility that IL-21 gets secreted and affects the
maturation of B cells in normal conditions. We showed that to secrete IL-21, CD4+ T cells need
to be activated. A number of reports indicate that an increased proportion of CD4+ T cells
present in mouse BM display an activated phenotype (382, 383). It is thought that these cells get
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primed by cognate antigen stimulation, as opposed to endogenous weak ligands (383, 438).
Priming can take place directly in the BM if blood-borne antigens get captured by BM dendritic
cells, or if antigen-loaded circulating dendritic cells migrate to the BM (438-441). Alternatively,
T cells can be primed in peripheral lymphoid organs and then migrate to the BM, which could be
the case for antigens that enter the body through oral, mucosal or subcutaneous routes (438, 440).
The maintenance of the activation state in the BM does not necessarily require further antigenic
stimulation as it can be provided by factors, such as cytokines, present in the microenvironment
(381, 438).
Despite their relatively low number (3-8% of total BM nucleated cells), it is recognized that T
cells play an important role to maintain bone and BM homeostasis. An example of this is
described by Monteiro et al. who shows that in the absence of T cells, mice exhibit a low number
of granulocytes in the periphery and accumulate immature myeloid cells in the BM, reflecting a
severely impaired granulopoiesis (383). This defect is not due to an intrinsic incapacity of the
progenitors to differentiate, but results from the lack of CD4+ T cell-derived stimuli. A similar
phenotype was observed in non-immunized DO11.10 RAG-/- mice carrying a transgenic TCR
specific for OVAp presented by the I-Ad major histocompatibility complex (383). Restoration of
granulopoiesis in these mice was obtained following intravenous immunization with OVA. This
was accompanied by a five-fold increase in the number of activated BM CD4+ T cells, without
changes in the total absolute number of BM T cells, pointing at the importance of the activation
state of T cells to support hematopoiesis. In the case of granulopoiesis, it has been proposed that
IL-17 secreted by activated T cells induces the production of G-CSF by BM stromal cells, which
in turn stimulates the production of granulocytes (383, 442). Our data, as well as studies
published by others, suggest that production of IL-21 is an additional mechanism used by CD4+
T cells to regulate hematopoiesis (275, 371, 443, 444). It has been shown to provide an
alternative to the IL-6 requirement for the differentiation and maintenance of the TH17 cell pool,
a sub-population of CD4+ T cells that produces large amounts of IL-21 compared to the TH1 and
TH2 subsets (275, 444). Furthermore, based on the observation that IL-21R-/- mice have lower
frequencies of memory TH17 cells, it was suggested that IL-21 might be important for their
generation and maintenance in the absence of inflammation, when levels of IL-6 are lower (444,
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445). Aside from its effect on TH17 cells, IL-21 has been reported to regulate lin-Sca-1+c-Kit+
hematopoietic progenitor cells (371, 443). Mice transgenic for IL-21 showed an expansion of
these early progenitors (371). Shortly after the publication of our results on IL-21, it was shown
that injection of WT mice with anti-IL-21 results in a decrease number and cycling of
hematopoietic progenitors in the BM (443). Our data now show that the development of B cell
progenitors is also affected by the presence of IL-21, and since activated T cells are present in
BM, it might support the steady-state production of B cells.
6.4 Role of IL-21 in extramedullar hematopoiesis during inflammation
B cell development in the BM is generally described as a process that is unaffected by exogenous
antigen. However, several studies report that this is not the case (reviewed in (389)). Infection
and inflammation alter BM hematopoiesis favouring the development of granulocytes over
lymphocytes. One mechanism that is thought to contribute to this effect is the mobilization of B
cell progenitors to the periphery. It has been shown that the action of adjuvants or some
inflammatory cytokines can contribute to the reduction in the number of B cell progenitors in the
BM. For example, decreased numbers of B cells are observed in the BM after intraperitonale
diminution of CXCL12 protein, a chemokine important for the homing of hematopoietic cell
progenitors in the BM, including cells of the B lineage (388, 446, 447).
The reduction of B cell populations in the BM coincides with the appearance of developing B
cells in the spleen, raising the question of whether or not B cell progenitors can pursue their
development in the periphery and ultimately take part in the immune response to pathogens
(388). Support for a role of B cell progenitors in the immune response was provided by a study
published by Ueda et al. that analysed the secretion of antibodies by immature/T1 B cells (375).
It was shown that inflammation triggers the mobilization of immature/T1 B cells in the spleen.
The immature/T1 population is known to be susceptible to apoptosis following IgM stimulation
alone (448, 449). However, proliferation and secretion of antibodies was elicited upon in vitro
stimulation with LPS or CpG (375, 450, 451). Furthermore, using GFP+ immature/T1 cells
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transferred to BL/6 mice, it was shown that this cell population can differentiate into CD138+
plasmacytes following administration of bacterial vaccine. Although this study showed that these
effects can occur independently of T cell help, our results suggest that signals provided by CD4+
T cells could also support the development in the periphery of newly mobilized B cell
progenitors. Indeed, addition of anti-CD40 and IL-21 to proB cell cultures led to their
differentiation into Ig-secreting cells. Therefore, is possible that in addition to its contribution to
B cell development in the BM, IL-21 plays a role in the development of B cell progenitors in the
periphery when BM B cell progenitors get mobilized in the spleen during an immune response.
Although continued development of the mobilized B cell progenitors in the periphery might be
beneficial to a humoral immune response, it might also trigger autoimmunity. The spleen
microenvironment, unlike the BM, might allow the survival of self-reactive cells that would have
otherwise died. In the BM, the generation of auto-reactive clones is limited by three prominent
mechanisms: 1) receptor editing that allows the replacement of a self-reactive BCR by a non-self
reactive one, 2) deletion, and 3) anergy. The strength of BCR signalling appears to play a major
role in the selection, with strong signals directing the deletion of the B cell clones, whereas low
affinity interactions render the clone anergic (452). In spite of this, it is clear that a number of
autoreactive B cells escape these control checkpoints and migrate to the periphery. Therefore,
tolerance mechanisms must also be in place in the periphery to prevent the development of
autoimmune diseases. A number of these mechanisms operating at different stages of B cell
development have been described, including an important contribution played by the maturation
and survival factor BAFF at the transitional stage (453-458). The role for BAFF in the selection
of the B cell repertoire is supported by the observation that BAFF transgenic mice develop anti-
DNA autoantibodies and other autoimmune-like anomalies (456, 459). Importantly, the
effectiveness of BAFF to delete self-reactive clones varies with the degree of competition from
non-self reactive clones. It has been proposed that when autoreactive B cells are in competition
with non-autoreactive B cells for BAFF, the survival of non-autoreactive cells is favored due to a
lower dependence on BAFF signalling. In this model, autoreactive B cell clones require greater
BAFF stimulation to avoid deletion because chronic BCR stimulation increases the amount of
pro-apoptotic molecules. Alternatively, since expression of BAFF-R is regulated by BCR
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signalling (460, 461), partly anergized auto-reactive cells would express lower levels of BAFF-R
(456).
Interestingly, BAFF expression is upregulated by pro-inflammatory responses, which might
increase the quantity of BAFF available (450, 451, 462). This temporary excess of BAFF could
allow the survival of mobilized BM progenitor cells in the spleen that have reached a given stage
of maturation. In support to this, it was shown that the presence of BAFF enhances cell numbers
and secretion of Igs by LPS-stimulated immature/T1 B cells (375). In our system, the
differentiation of B cell progenitors into Ig-secreting cells depends only on CD40 and IL-21R
stimulation. Similar to the LPS system, the requirement of antigenic stimulation of the BCR is
bypassed, which suggest that cells of any specificity could survive and differentiate into Ig-
secreting cells. The development of B cells in such conditions clearly poses a potential threat,
and a pathogenic role of IL-21 has actually been suggested for the development of autoimmune
diseases in which excessive production of autoantibodies play a central role (292, 370, 463). On
the other hand, it is possible that the development of B cell progenitors in the periphery is part of
the normal immune response. It has been proposed that the production of self-reactive antibodies
could aid in the elimination of microbes that evade the immune system by mimicking host
antigens, and in the efficient clearance of cellular debris from infection sites (375). In this model,
the production of autoantibodies is not thought to be deleterious to the host, as a return to normal
levels of BAFF following resolution of inflammation would disfavor survival of the substantially
more BAFF-dependent immature/T1 B cells (375). Relevant to this context is the observation
that immature B cells expressing a transgenic non-autoreactive BCR express higher levels of
BAFF-R compared to immature B cells expressing a transgenic auto-reactive BCR (464). It is
not known yet to what extent cells generated from anti-CD40/IL-21-stimulated B cells
precursors, self-reactive or not, would be susceptible to deletion through this mechanism, as the
levels of BAFF-R expression present on the different population of B cells produced under these
conditions has not yet been ascertained. The levels of the inhibitory receptor Fc RIIB expressed
by Ig-secreting cells generated in our system is another factor that could influence the fate of
these cells. Fc RIIB is normally up-regulated on antigen-exposed B cells, and it has been shown
to suppress T-cell dependent, but not T-cell independent, B cell responses in the late IgG, but not
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the early IgM response. Furthermore, failure to up-regulate Fc RIIB has been suggested to
contribute to autoantibody production (453, 454).
6.5 Importance of the gut microbiota in the development of the humoral immune system
B cell development is typically described as a process that starts in the BM and continues in the
spleen until cells become fully mature. The mature follicular B cells then circulate in the spleen,
LNs, and BM, until they either die or encounter an antigen which triggers the final differentiation
program that leads to the formation of PCs. While the initial steps are often studied at steady-
state conditions, the final steps of maturation are often analysed in the context of a response to
infectious pathogens. Yet the body constantly has to deal with vast numbers of harmless
microorganisms that co-exist in the gastrointestinal tract. Research shows that these
microorganisms act not only locally to influence the development of the intestinal immune
system (465, 466), but also affect the host systemic immune system, as evidenced by the ability
of a microbiota-derived peptidoglycan to modulate the function of BM neutrophils (467).
IgA is the main antibody isotype produced in the gut where it contributes to the maintenance of
homeostasis between the host and the microbiota. Its production is largely driven by the
commensal flora, as reflected by the low numbers of IgA+ cells generated in GF mice (468).
Recently, it has been reported that IgA-secreting PCs residing in the lamina propria are endowed
with characteristics commonly associated with myeloid cells (391). It is well established that the
development of B cells is subject to factors present in their microenvironment, and hence, with
the billions of microorganisms living in the gut, it might not be surprising that PCs residing in
this area have developed distinctive characteristics to maintain homeostasis. Notably, IgA-
secreting PCs were found to be the dominant expressers of intestinal iNOS (391). In addition, it
was shown that GF mice do not express iNOS unless the mice are colonized with commensal
bacteria, indicating that the production of iNOS by the IgA+ PCs present in the lamina propria is
dependent on microbial exposure. In agreement with this, we showed in chapter 5 that it is
possible to induce the expression of iNOS in IgA+ cells in vitro when BM progenitors are co-
cultured with lamina propria-derived stroma. The induction of iNOS in IgA+ cells was triggered
120
only in the presence of microbes, a stimulus known to induce iNOS expression in other cells,
notably inflammatory cells (392). Interestingly, NO, a product generated by iNOS, has been
shown to regulate IgA CSR in the gut (403). Therefore, aside from the pro-IgA CSR factors
added in the culture, the upregulation of iNOS and subsequent NO production might have also
contributed to the generation of IgA+ cells by favoring CSR to IgA.
The production of the cytotoxic molecule NO by local residents of the gut can be advantageous
by providing an additional mechanism, aside from the secretion of IgA, to control the commensal
microflora as well as mucosal infection by pathogens. We do not know whether or not in vitro
generated IgA+iNOS+ cells have the ability to kill microorganisms, and if deletion of iNOS
would prevent such an effect. However, in vivo, the importance of -producing IgA
PCs for gut homeostasis and immunity against infections was demonstrated by the low
production of IgA, the altered composition of the microflora, and the inefficiency at resolving
Citrobacter rodentium intestinal infection in mice carrying a B cell-specific deletion of iNOS
(391). It could also be interesting to determine whether other myeloid traits expressed
by these cells contribute to their function, and, using our in vitro system, what signals induce
their expression. For example, it has recently been shown that Ly6G modulates the recruitment
of neutrophils to inflamed tissues (469). Therefore, it is possible that Ly6G has a similar function
in IgA+iNOS+ PCs in the case of mucosal infection.
The population of IgA+iNOS+ PCs is present mainly in the small intestine lamina propria, but is
also found in small numbers in the BM (391). However, we found that BM stroma was
inefficient at generating IgA+iNOS+, suggesting that in vivo, at steady-state conditions, only B
cells that have migrated to the gut are exposed to the proper stimuli that will endow them with
that particular phenotype. Once formed, some of these cells might return in the circulation and
seed other organs. The precise nature of the microbial stimuli that leads to the development of
the IgA+iNOS+ cells, and whether the microbial stimuli act directly on B cells or indirectly via
factors released from stimulated lamina propria stromal cells, are still to be determined. In
addition, the data accumulated so far indicate that microbial stimuli are necessary, but it does not
121
rule out the possibility that other stroma-derived tissue is efficient at generating these cells.
Therefore it remains unclear whether IgA+iNOS+ PCs can be generated in other tissues during
infection by some types of microorganisms outside the mucosal system.
CONCLUSION
The data presented in this thesis has expanded our knowledge of the environmental factors that
influence the development of B cells in the BM and in the periphery. In the BM, IL-7R
signalling promotes survival, proliferation, and differentiation of early B cell progenitors. The
intensity of the signal is kept under control during B cell development, and we provide evidence
that interactions involving sialic acid contribute to its regulation. In addition, we showed that IL-
21 is expressed by BM CD4+ T cells and its receptor is present at the surface of proB, preB, and
immature/mature B cells. Stimulation of IL-21R activates the JAK/STAT signalling pathway and
accelerates the maturation of B cells. Finally, we describe a BM B cells/gut stroma co-culture
system that allows the generation of IgA+iNOS+ cells in the presence of microbial products,
thereby showing how unique conditions present in a given environment can shape B
lymphopoiesis.
122
APPENDICES
123
124
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