Dirk Mielenz

University of Erlangen-Nuremberg Division of Molecular Immunology

B cell activation and long-lived plasma cells

ca. 1000 Children were given dried pustules of convalescent Pock patients (Variolation, „Variola“ from lat. varius = blurry, checkered)

ca. 1500 variolation in Harems

1717  Mary Wortley Montagu introduces from Turkey variolation in Europe

1760 Variolation of the famillies of Maria Theresia and George III. make variolation popular

1776 Washington variolates its continental army

Lady Mary Montagu, wife of the British ambassodour to Turkey

Generation of antibodies is the basis for the vast majority of succesful vaccination strategies

Nutt et al., Nat. Rev. Immunol., 2015 H.M. Jäck, Division of Molecular Immunology, Erlangen

How succesful vaccination reduced incidence of infectious diseases

Duration of humoral immunity to common viral and vaccine antigens

Latent infection, recurrent re-exposure, repetitive infections, memory cell numbers or bystander memory is not necessarily an indicator for antibody titer longevity; it is likely the nature of the antigen

Viral antigens Very long halflife Protein antigens Shorter half-life

Amanna et al., NEJM, 2007

Relationship between long-term immunity and long term protection

M.K. Slifka, Vaccine, 2014

Are there different kinds of antibodies? What are the cells that secrete antibodies? Is there only one type of B cells? Is a B cell completely autonomous? How does a B cell develop into a plasma cell? How does a B cell sense an antigen – and where? Does a normal B cell make „good“ antibodies?

Questions – principles of antibody formation

Antibodies

Antibodies are bifunctional molecules secreted by differentiated B cells

Ig-Classes IgM (penta/hexameric) - blood

IgG blood

[IgD] IgA (dimer) - gut

IgE - mucosa

H-chain L-chain

VL

VH

CL

CH

CH

CH

CH

Effector function - constant “Bullet Part”

Pathogen/antigen recognition - variable

(„magic Part“)

IgM

H.M. Jäck

IgM - Antigen receptor on immature and naive/IgM memory B cells - First line defense during immune reaction - Very good agglutination - anti-inflammatory (artherosclerosis)

IgD - Antigen receptor on mature B cells

IgG - Main antibody in blood → Internal defense - Passes placenta

IgA - Main Ab on mucosal and gut surfaces (lung, gut, urogenital tract) → External defense - In tears, sputum, mother milk (passive immunity important for newbornes)

IgE - activates effector cells (mast cells, eosinophils) during worm infection - allergy

Antibodies

•  Agglutination of bacteria (IgM)

•  Neutralisation (IgG, IgA)

•  Phagocytosis and Opsonization (IgM und IgG)

•  Killing (antibody dependent cytotoxicity, ADCC; antibody dependent respiratory burst, ADRB)

•  Mast cell activation (IgE) – inflammation

Antibody effector functions

•  Immune regulation via activating and inhibitory Fc receptors

Hematopoietic stem cell

B cell Monocyte Erythocyte

Bone marrow, fetal liver (mouse, human)

CD4+ T cell

Thymus

CD8+ T cell

Lymphocyte Maturation and Migration

Bursa fabricii (chicken)

Primary lymphatic

organs

H.M. Jäck

Antibodies

Antibodies are bifunctional molecules secreted by differentiated B cells

Ig-Classes IgM (penta/hexameric) - blood

IgG blood

[IgD] IgA (dimer) - gut

IgE - mucosa

H-chain L-chain

VL

VH

CL

CH

CH

CH

CH

Effector function - constant “Bullet Part”

Pathogen/antigen recognition - variable

(„magic Part“)

IgM

H.M. Jäck

µH chain N C

B cell

B cell

VH Cµ

Transcription Translation

VH Exon

V(D)J recombination generates antibody diversity

Recombination

stem cell

J segments Cµ

V segments D segments

ca. 2.5 Mb (mouse) HC locus

κL chain N C

VL Exon

J V Cκ

LC locus

Generation of primary antibody diversity (repertoire)

S. Tonegawa Nobel Price 1987

Basel Institute of Immunology

H.M. Jäck

antigen

B cell repertoire (~ 1011 specificies)

B cell receptor

Clonal selection & expansion by proliferation B cell clone

Short-lived plasma cell

Differentiation

Memory B cell

Long-lived plasma cell

+TH - TH

Burnet Jerne Talmage

CLONAL Selection Theories (1956-58)

H.M. Jäck

The IgM BCR is a signaling machine and an endocytotic receptor

Activation Apoptosis

plasma membrane

Cytosol

L

lyn Syk

BLNK Btk PLCγ2

PKCβ

NF-AT NF-κB

IP3

Ca++

cdc42 rac rho

WASP

G/F Actin

Igα/β µ

antigen Costimulation (CD40, IL-4R, TLR)

µ

Antigen presentation

MHCII

Nucleus

,,lipid raft“

MHCII loading compartment

Integration of adaptive and innate signals in B cells – relevance for antiviral immunity and autoimmunity

Hobeika et al., J. Mol. Med., 2015

IgM BCR signaling

The BCR provides a „tonic“ survival signal The BCR provides signals for B cell activation by ultimatively triggering transcription factors The BCR organizes B cell signaling and its machinery integrates signals from CD40 and TLRs The BCR is an endocytotic receptor which delivers antigen super-efficiently into the MHCII loading compartment

B cell receptor signaling, CTMI, 2016, Kurosaki, Wienands (eds.)

Naive B cells

Plasmablast

1 µm

1 µm

anti

CD

40 /

IL4

1 µm LP

S

Grötsch B et. al, JEM 2014

antibody factory

naive B cell

?

Morphological and functional diversification of B cells

Morphological and functional diversification of B cells

Mature B cells are resting and have a low metabolic activity, consuming mainly fatty acids Activated B cells proliferate and have a high metabolic activity Plasma cells are quiescent but have an enormous energy turnover and a high, very specific metabolic activity Plasma cells need a good redox balance

Caro-Maldonado et al., J. Immunol., 2014 Anelli et al., Free Rad. Biol. Med., 2015 Lam et al., Immunity, 2016

103 IgM molecules / s x 102 disulfide bonds/IgM = 105 disulfide bonds / s / cell!! 105 sugar molecules / s / cell!

•  Rapid T independent response against polysaccharides (encapsulated bacteria) (TLR)

•  Natural IgM

•  Very rapid differentiation into plasma cells

MZ

•  Shuttle between MZ and follicle; transport antigen

•  Prone to interact with T cells

•  Give rise to class switched (IgG, IgA, IgE) antibodies + memory

Naive B cell subsets: B1 and B2 (marginal zone, MZ, and follicular, FO)

Cyster et al., Nat. Immunol., 2000

B1 (pleura) and MZ (spleen)

FO

IgG

IgG Log

Cab

in S

erum

primary secondary

3 2 1

0 4 8

IgM 10Ag 20Ag

IgM

0 4 8 t(d)

Primary and secondary antibody responses

•  Normally not T cell help

•  Bacterial components (PAMPs) (i.e., LPS=Lipopolysaccharide)

TLR3/7/9 (mouse)

•  Highly repetitive antigens

•  B1- and MZ B cells

→ No memory

Thymus-independent (TI) and dependent (TD) activation – The nature of the antigen matters!

•  T cell help

•  Antigen needs to contain protein in a digestable and presentable form

•  Mainly FO B cells

•  High affine IgG

• → Memory

(Thymus- Dependent)

(Thymus- Independent)

Klaus D. Elgert: „Immunology“, 2nd ed., WILEY-Blackwell, Hoboken, NJ, 2009

B cells at the crossroads of innate and adaptive immunity

Primary follicle Secondary

follicle +

Germinal center

CXCR5

IgM

Antigen

+/-TH

Expansion IgM

Naive B cell B cell

focus

Short-lived Plasma

cell

Long-lived plasma

cells

Memory B cell

IgG IgA IgE

IgG IgA IgE

Anatomy of the B Cell Response

IgD - B cells PNA - GC B cells CD3 - T cells

Spleen section - 7 days SRBC

B

T HEV

GC

T C

ell Z

one

B C

ell Z

one

+TH

White pulp

Fates of activated B cells

Muramatsu et al., Cell, 2016 Victora & Nussenzweig, Ann. Rev. Immunology, 2012

Weisel et al., Immunity, 2016

high affine antibodies class switched late

lower affinity class switched early

competition for T cells

AID

AID: activation induced deaminase

Molecular Changes at the Ig locus (pre GC/dark zone)

1.  Somatic hypermutation

2.  IgH class switch

IgM IgG, IgA, IgE

VH

CH

VL

CL

AID

Anti HIV broadly neutralizing Ab (bnAb) are highly mutated and have long CDR3

Escolano et al., Cell, 2016 Tian et al., Cell, 2016

Fates of activated B cells

Victora & Nussenzweig, Ann. Rev. Immunology, 2012 Weisel et al., Immunity, 2016

high affine antibodies class switched late

lower affinity class switched early

competition for T cells

M.K. Slifka, Vaccine, 2014

Characteristics of a vaccine antigen determine subsequent levels and duration of immunity – or: the

antigen has to go THROUGH the B cell in a BCR specific manner

A principle of plasma cell differentiation is proliferation, increasing the probability for plasma cell differentiation

Nutt et al., Sem. Immunology, 2011

The gene network controlling terminal plasma cell differentiation

Nutt et al.,Sem. Immunology, 2011 Ochiai et al., Immunity, 2013

IgG1 IgG3 IgG2a IgG2b IgA IgM0

200

400

600

800

1000

controlFra1tg

*** *** *

***

Ig (µ

g/m

l)

0 10 21 310.0

0.5

1.0

1.5

2.0controlFra1tg

*** *** ***

day post-immunization

NP-

spec

ific

IgG

11/

1600

0 (A

490)

0 100.0

0.1

0.2

0.3

controlFra1tg

*

day post-immunizationNP-

spe

cific

IgM

1/4

000

(A49

0)

0 10 21 310.0

0.2

0.4

0.6

0.8controlFra1tg

*** *** ***

day post-immunizationNP

-spe

cific

IgG

1/6

000

(A49

0)

Fra1(FosL1) tg mice do not respond well to immunization

Grötsch et al., JEM, 2014 H2-Fra1-LTRtg

Fra1 inhibits the formation of RER containing plasmablasts in vitro

1 µm 5 µm 5 µm

10 µm 2 µm 2 µm 5 µm

Fra1

tgcontrol

Uns'mulatedLPSCD40/BCR/IL-4

020406080

100

LPSunst. CD40/BCR/IL-4

Fra1

tg c

ell n

umbe

r

020406080

100

cells with prominent RERcells without prominent RER

unst. LPS CD40/BCR/IL-4

control c

ell n

umbe

r

Grötsch et al.,JEM, 2014

Ficoll purified cells

0 2 50.000

0.005

0.010

0.015

control

*Fra1ΔB cell

day post-stimulation

Blimp1

(rel

. exp

ress

ion)

0 2 50.000

0.002

0.004

0.006

0.008

0.010

controlFra1tg

*

day post-stimulation

Blimp1

(rel

. exp

ress

ion)

Blimp1IRF4

µmµs

wttgwttgwttgFra1

d0d2d3LPS

9745

kDa

66

+/+-/-+/+-/-+/+-/-Fra197

45

66

kDa

Blimp1

IRF4

µmµs

d0d2d3LPS

Fra1 controls Blimp1 and µs expression in vitro

Grötsch et al., JEM, 2014

Nutt et al., Nat. Rev. Immunol., 2015

Different kinds of plasma cells

Nutt et al., Nat. Rev. Immunol., 2015

Plasma cells need a niche

Nutt et al., Nat. Rev. Immunol., 2015

Generation of long lived plasma cells - overview

IgG

IgM

Naive Cell

Long-lived plasma cells

(IgG, IgA)

Long-lived plasma cells

(IgG, IgA)

Memory B cell (IgG, IgA) GC reaction

IgG Log

Cab

in

Ser

um

primary secondary

3 2 1

0 4 8

IgM 10Ag 20Ag

IgM

0 4 8 t(d)

Long-lived plasma cells

(IgG, IgA)

Memory B cell (IgG, IgA)

GC reaction

Short-lived plasma cell

(IgM) Ag+TH

IgM Short-lived plasma cell

(IgM) Naive B-Cell

Ag+TH

Summary

Duration of humoral immunity to common viral and vaccine antigens

Latent infection, recurrent re-exposure, repetitive infections, memory cell numbers or bystander memory is not necessarily an indicator for antibody titer longevity; it is likely the nature of the antigen

Viral antigens Very long halflife Protein antigens Shorter half-life

Amanna et al., NEJM, 2007