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B-cell isotype switch and
the longevity of the IgE-
antibody response
Lone Hummelshøj
Allergy Clinic
Gentofte Hospital
Denmark
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Antibodies (Immunoglobulins)
Variable region, rearranged VDJ
antibody specificity.
Constant region
antibody biological function.
Allergen (e.g. pollen)
The biological function can be replaced and still keeping the
specificity: Isotype switch/class switch recombination
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B cell differentiation in type I allergy
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IL-4 & IL-13
TGF-b
IL-10
IFN-g
IL-10
2
IgM
IgD
Immunoglobulin genes
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Signalling through IL-4R and CD40
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Somatic hypermutation
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Hypothetic function of germline transcripts
GLT (germline transcripts)
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AID (Activation-Induced Deaminase)
UNG (Urasil DNA glycosylase)
EN (Endonuclease)
Double stranded break
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Class switch recombination
dsDNA break dsDNA break
2
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Class switch recombination
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Class switch recombination
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Class switch recombination
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Transcription factors in B cell differentiation
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CXCR3 (CXCL9, CXCL10, CXCL11): Inflamed tissue.
CXCR4 (CXCL12): Bone marrow.
CCR9/CCR10 (CCL25/CCL28): Mucosal sites (IgA)
1-2 weeks after their generation, plasmablasts lose their ability to
migrate towards ligands.
Migration of plasmablasts
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Survival niches
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Number of plasma cells in the bone marrow
Steiner and Pearson, J. Pediatrics, 1966
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Plasma cell replacement
Radbruch A, Nature Rev Immunology, 2006
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Radbruch 2006, Nature reviews immunology:
Bone marrow: 0,1 – 1 % plasma cells Total of 109 plasma cells
Immunization studies: 1 x 106 specific plasma cells in the bone marrow.
That gives space for 1000 different main specificities.
Plasma cells for an old antigen would wane at a frequency of 0.1% for
each generation of cells with a new specificity.
50% reduction in plasma cell number: ~700 challenges
30 estimated challenges per year 23 years to reach 50% of the
original steady-state concentration.
Plasma cell repertoire – the rough calculations!
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Factors that may influence the composition of the plasma cell
repertoire in the bone marrow:
Different antigens might induce different number of plasma cells –
some specificities might wash out sooner than others
Plasma cells of different antibody specificity might have different
migratory capacity (IgG >> IgE)
Plasma cells of different antibody specificity might have a different
capability in staying settled in the survival niches
Competition for survival niche space
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Hiepe A, Nature Rev Rheumatol 2011
Strategies for targeting long-lived plasma cells
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Method: Isolation and culture of IgD+ B cells
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B cell culture, phenotype
Hummelshoj L, 2006
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B cell culture, phenotype
Surface Ig Secreted Ig
Hummelshoj L, 2006
Plasma cell markers
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Cord blood B cells
Hummelshoj L, 2007
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Cord blood B cells
Hummelshoj L, 2007
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Cord blood B cells vs. adult B cells
Hummelshoj L, 2007
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Strategies for targeting long-lived plasma cells
IL-21
FcgRIIb
siRNA
+
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The function of IL-21
1Mouse: IgG1 IgE
2Man: IgE
1Ozaki et al., Science, 2002. 2Wood et al., Cell Immunol, 2004.
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IL-21 and germline transcripts
anti-CD40
IL-21 + anti-CD40
IL-4 + anti-CD40
IL-21 + IL-4 + anti-CD40
Germeline transcripts (GLTs)
Hummelshoj L, 2006
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IL-21 and transcription factors
Hummelshoj L, 2006
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IL-21 and immunoglobulin production
10000
1000
100
10
1
0.1
0 10 100
Imm
un
glo
bu
lin
(n
g/m
l)
IL-21 (ng/ml)
Anti-CD40
IgA
IgGtotal
IgG4
IgE
Hummelshoj L, 2006
10000
1000
100
10
1
0.1
0 10 100
IL-21 (ng/ml)
Anti-CD40 + IL-4
IgA
IgGtotal
IgG4
IgE
imm
un
glo
bu
lin
(n
g/m
l)
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IL-21 conclusion
IL-21 alone increase the production of all immunoglobulins
• No GLTs
• No significant change in AID
• No significant change in XBP-1
Does IL-21 effect pre-shifted B cells by cell division?
IL-21 increase the IL-4 induced production of IgE and IgG4.
• Increased XBP-1
IL-21 increases the general plasma cell differentiation.
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Plasma cells and FcgRIIb
ng/ml FcgRIIb blocking antibody
% p
osit
ive c
ells
00,
5 5 50
0
5
10
15
20
25
% CD19 positive cells
% CD138 positive cells
Hansen T, Hummelshoj L, 2011
Nimmerjahn, Nat Rev Immonol, 2008
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Plasma cells and IgE siRNA
SiRNA concentration
To
tal Ig
E (
pg
/ml)
0 nM
20 n
M
50 n
M
Contr
ol
0
5000
10000
15000
*
*
Hansen T, Hummelshoj L, 2011
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Once settled in their survival niches, long-lived plasma cells are
beyond the reach of available therapies.
Curative therapy might require elimination of long-lived IgE producing
plasma cells.
Take home message…