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Peripheral Control of Alloreactive B cells
Anita ChongSection of Transplantation
Department of Surgery
Chong Lab
YiJin Li Dengping Yin
LianLi Ma Jamie Kim
JiKun Shen Anncy Varghese
Jingwen Ding
Tongmin Wang
Audrea Troutman
NextranGerry Byrne
Hui Xu
U ColoradoRoberta Pelanda
Alloreactive B cells andLong-term Allograft
Survival
• Alloantibodies mediate acute and chronic rejection
• Alloantibody-alloantigen complexes induce maturation of APC
• B cells can activate alloreactive T cells
Mechanisms of B cell tolerance
Calame, Nat Immunol 2001:1103
AntigenCD40L
Nature Immunology 2, 1126 - 1132 (2001)
B cells and professional APCs recruit regulatory T cells via CCL4Richard S. Bystry1, Varuna Aluvihare2, Katie A. Welch1, Marinos Kallikourdis1 & Alexander G. Betz1
Using gene expression profiling, we show here that activation of B cells and professional antigen-presenting cells (APCs) induces the expression of common chemokines. Among these, CCL4 was the most potent chemoattractant of a CD4+CD25+ T cell population, which is a characteristic phenotype of regulatory T cells. Depletion of either regulatory T cells or CCL4 resulted in a deregulated humoral response, which culminated in the production of autoantibodies. This suggested that the recruitment of regulatory T cells to B cells and APCs by CCL4 plays a central role in the normal initiation of T cell and humoral responses, and failure to do this leads to autoimmune activation.
Two-Phase model of
T cell-dependent allo-specific B cell activation
Modified from Baumgarth 2000 Immunol Rev.
SecretedIgM
Extrinsic control of alloreactive B cells
• Inhibiting the development of effector T helper cells
• Inhibiting effective T-B cell interaction
Tolerance Model
Day 0C57BL/6
Day 60+ C57BL/6
Anti-CD40L (1 mg MR1/mouse; D-7, 0, 7) + DST (B/6)
C57BL/6 (H-2b) into BALB/c (H-2d) mice
Anti-CD40L + DSTinduces allograft tolerance
Treatment Graft Survival
None 8 (x7), 9
+ Anti-CD40L > 160 (x11)
+ 2nd heart >60 (x5), >100 (x3)
•Tolerance can be dominantly transferred
•Tolerant mice reject third-party C3H hearts, exhibit linked tolerance to F1 (B/6 x C3H) hearts.
•A role of CD25+ T cells
•Allograft tolerance is associated with inhibited alloantibody production
Features of Allograft Tolerance
Peripheral B cell Tolerance
m
Donor-specific (B/6) heart
+ 3.83 BCR-Ki B cells
B/c recipients of B/6 heart
Day 60-80 Day 0: DST + MR1
Naïve
3.83 BCR-Ki B cells recognize Kb antigens
m
H2-K(b) H2-K(d)
Tetramer
CD19
m
Peripheral B cell Tolerance: 3.83 Cell Transfer + B/6 heart
Days Post-B Cell Transfer
0
0.25
0.5
0.75
1
1.25O
.D.
0 5 10 15 20
Anti-Id-IgM
Naïve - No TxpTolRej
Peripheral B cell Tolerance: Lack of T cell help
m
Third-party C3H heart + 3.83 BCR-Ki B cells
Day 60-80 Day 0: DST +
MR1
Naive
3.83 BCR-Ki B cells have dual
alloreactivity
m
H2-K(b) H2-K(d)H2-K(k)
Tetramer
CD19
Peripheral B cell Tolerance: Lack of T cell help
m
Third-party C3H heart + 3.83 BCR-Ki B cells
Day 60-80 Day 0: DST +
MR1
Naive
m
Days Post-B cell Transfer
Peripheral B cell Tolerance: Inhibition of T cell help
-0.25
0
0.25
0.5
0.75
1
1.25O
.D.
0 7 14 21
c) Anti-Id IgM
TolNaive
Peripheral B cell Tolerance: Inhibition of T cell help
m
B/6 heart + 3.83 BCR-Ki B cells +
Anti-CD40
Day 60-80 Day 0: DST +
MR1
Naive
m
Control of B cell Responses by limiting CD40-CD40L signals:
0
0.5
1
1.5
O.D
.
0 7 14 21 28
Days Post-B Cell Transfer
Anti-Id IgM
Tol + Anti-CD40
Tol
Rej
Peripheral B cell Tolerance: Regulation of T
cell help
m
F1 (B/6 x C3H) heart +3.83 BCR-Ki B cells
Day 60-80 Day 0 DST + MR1
Naive
m
Linked Regulation of T cell Helper Responses
Days Post-B Cell Transfer
-0.25
0
0.25
0.5
0.75
1O
.D.
0 5 10 15 20
Anti-Id IgM
TolNaive
Cells Maintaining Peripheral B cell Tolerance
m
Spleen Cells from:i. Naïveii. Tolerant (d60-120)iii. CD25-Depl Tolerant
Scid recipients of B/6 heart
m
Peripheral B cell Tolerance: Role of CD25 Cells
0
50
100
150
All
o-I
gG
0 10 20 30
Tolerant + Htx
Naive + Htx
Naive-No Txp
0
25
50
75
100
All
o I
gG
0 10 20 30
CD25 Depl Tolerant
Tolerant
Peripheral suppression of alloantibody production:
1.Inhibiting the generation of T cell help
2.Dependent on CD25+ cells
m
Regulation of T cell help
C3H-TH cell
Recpt. APC
F1 antigensB/6xC3H
3.83 B cell
B/6-TTol/Reg
C3H-TH cell
No T cell Help
T cell Zone
B cell ZoneB/6-TTol/Reg
Phase 1
Phase 2
Anti-carbohydrate Antibodies in Allograft
transplantation • Naturally occurring and elicited
anti-A or anti-B antibodies
• Donor-specific B-cell tolerance after ABO-incompatible infant heart transplantation (Fan et al. 2004)
• Can peripheral mechanisms control anti-carbohydrate antibody responses?
Structure of Gal1,3Gal and Blood Group A and B antigens
CH2OH
ROH
O O O
OO
CH2OH CH2OH
NHCOCH3
OH
OH
OH
OH O
H
CH2OH
ROH
O O O
OO
CH2OH CH2OH
NHCOCH3
OH
OH
OH O
H
O
OH
OH
OH
C3H
O
CH2OH
ROH
O O O
OO
CH2OH CH2OH
NHCOCH3
OH
NHCOCH3
OH O
H
O
OH
OH
OH
C3H
O
Gal1,3Gal-ß1,4GlcNAc
Blood Group BGal1,3(Fuc1,2)Gal
Blood Group AGalNAc1,3(Fuc1,2)Gal
Tolerance Model
Day 0Gal+BALB/c Heart +Intact Active Bone
Day 60Gal+BALB/c Heart
Anti-CD40L (1 mg MR1/mouse; D 0, 7, 14)
C57BL/6-Gal-/- recipients tolerant to BALB/c-Gal+/+ grafts
IAB and anti-CD40L therapy induces allograft tolerance
Treatment Mean
None 7 d
Anti-CD40L + IAB - 1st BALB/c heart > 130 dAnti-CD40L + IAB - 2nd BALB/c heart > 60 d
Features of IAB/Anti-CD40L-induced tolerance
•Tolerance can be dominantly transferred
•Tolerant mice reject third-party C3H hearts, exhibit linked tolerance to F1 (B/c x C3H) hearts.
•A role of CD25+ T cells
•Allograft tolerance is associated with suppressed alloantibody production
Anti-Gal IgM production in IAB/Anti-CD40L-induced tolerant
mice
0
0.25
0.5
0.75
1
1.25
O.D
.
0 30 60 90
Days Post-Transplant
Anti-Gal IgMb
None
MR1 + IAB
Peripheral B cell Tolerance
m
Donor-specific (B/c) heartOr Third-Party C3H heart
+ Anti-Gal BCR-Tg B cells
B/6 GT-/- recipient of B/c heart
Day 60-80 Day 0: IAB + MR1
Naïve
IgMa+ CD43+ CD21+
Madcam-1IgMa
Anti-Gal IgMa production following challenge with BALB/c
hearts
00.5
1
1.52
0 10 20 30
f) Anti-Gal IgMa
Days Post-B cell Transfer
Naive
Tolerant
Anti-Gal IgMa production following challenge with C3H
hearts
0
0.25
0.5
0.75
1O
.D.
0 7 14
b) Anti-Gal IgMa
Days Post-B cell Transfer
Naive
Tolerant
Conclusion
•Anti-Gal cell responses are inhibited even in the presence of effector T helper cells
m
Regulation of B cell responses
C3H-TH cell
Recpt. APC
Balb/c-Galantigens
T cell Help
B/c-TTol/Reg C3H-Gal
antigens
Recpt. APC
Gal-B cell
B/c-TTol/Reg
C3H-TH cell
T cell Zone
B cell Zone
Phase 1
Phase 2
Extrinsic control of alloreactive B cells
• Inhibiting the generation of effector T helper cells
• Inhibiting the development of effector B cells
Histology of Spleen
0.0
20.0
40.0
60.0
80.0
Native B/6 C3H F1
2nd Graft
% G
erm
inal
Cen
ter
B/6
C3H
F1 FDC-M1B220
Peripheral B cell Tolerance:
Effect of TI-B cell stimulation
m
Donor-specific (B/c) heart+ Anti-Gal BCR-Tg B cells
+ CpG
B/6 GT-/- recipient of B/c heart
Day 60-80 Day 0: IAB + MR1
Naïve
Inhibition of Anti-Gal IgMa production is reversed by CpG
0
0.5
1
1.5
O.D
.
0 7
Days Post-B cell Transfer
Anti-Gal IgMa
Tol + CpG
Tol
Naive-No Txp