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