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B-cell isotype switch and
the longevity of the IgE-
antibody response
Lone Hummelshøj
Allergy Clinic
Gentofte Hospital
Denmark
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
B cell differentiation in type I allergy
IL-4 & IL-13
TGF-b
IL-10
IFN-g
IL-10
2
IgM
IgD
Immunoglobulin genes
Signalling through IL-4R and CD40
Somatic hypermutation
Hypothetic function of germline transcripts
GLT (germline transcripts)
AID (Activation-Induced Deaminase)
UNG (Urasil DNA glycosylase)
EN (Endonuclease)
Double stranded break
Class switch recombination
dsDNA break dsDNA break
2
Class switch recombination
Class switch recombination
Class switch recombination
Transcription factors in B cell differentiation
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
Survival niches
Number of plasma cells in the bone marrow
Steiner and Pearson, J. Pediatrics, 1966
Plasma cell replacement
Radbruch A, Nature Rev Immunology, 2006
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!
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
Hiepe A, Nature Rev Rheumatol 2011
Strategies for targeting long-lived plasma cells
Method: Isolation and culture of IgD+ B cells
B cell culture, phenotype
Hummelshoj L, 2006
B cell culture, phenotype
Surface Ig Secreted Ig
Hummelshoj L, 2006
Plasma cell markers
Cord blood B cells
Hummelshoj L, 2007
Cord blood B cells
Hummelshoj L, 2007
Cord blood B cells vs. adult B cells
Hummelshoj L, 2007
Strategies for targeting long-lived plasma cells
IL-21
FcgRIIb
siRNA
+
The function of IL-21
1Mouse: IgG1 IgE
2Man: IgE
1Ozaki et al., Science, 2002. 2Wood et al., Cell Immunol, 2004.
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
IL-21 and transcription factors
Hummelshoj L, 2006
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)
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.
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
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
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…