Gaoping Zhao

TGF-β-producing regulatory B cells induce regulatory T cells and promote transplantation tolerance

Regulatory B (Breg) cells have been shown to play a critical role in immune homeostasis and in autoimmunity models. We have recently demonstrated that combined anti‐T cell immunoglobulin domain and mucin domain‐1 and anti‐CD45RB antibody treatment results in tolerance to full MHC‐mismatched islet allografts in mice by generating Breg cells that are necessary for tolerance. Breg cells are antigen‐specific and are capable of transferring tolerance to untreated, transplanted animals. Here, we demonstrate that adoptively transferred Breg cells require the presence of regulatory T (Treg) cells to establish tolerance, and that adoptive transfer of Breg cells increases the number of Treg cells. Interaction with Breg cells in vivo induces significantly more Foxp3 expression in CD4+CD25− T cells than with naive B cells. We also show that Breg cells express the TGF‐β associated latency‐associated peptide and that Breg‐cell mediated graft prolongation post‐adoptive transfer is abrogated by neutralization of TGF‐β activity. Breg cells, like Treg cells, demonstrate preferential expression of both C‐C chemokine receptor 6 and CXCR3. Collectively, these findings suggest that in this model of antibody‐induced transplantation tolerance, Breg cells promote graft survival by promoting Treg‐cell development, possibly via TGF‐β production.

Anti‐CD45RB/Anti‐TIM‐1‐Induced Tolerance Requires Regulatory B Cells

The role of B cells in transplant tolerance remains unclear. Although B‐cell depletion often prolongs graft survival, sometimes it results in more rapid rejection, suggesting that B cells may have regulatory activity. We previously demonstrated that tolerance induction by anti‐CD45RB antibody requires recipient B cells. Here, we show that anti‐CD45RB in combination with anti‐TIM‐1 antibody has a synergistic effect, inducing tolerance in all recipients in a mouse islet allograft model. This effect depends on the presence of recipient B cells, requires B‐cell IL‐10 activity, and is antigen‐specific. These data suggest the existence of a regulatory B‐cell population that promotes tolerance via an IL‐10‐dependent pathway.

An Unexpected Counter-Regulatory Role of IL-10 in B-Lymphocyte-Mediated Transplantation Tolerance

Monoclonal antibody against the CD45RB protein induces stable transplantation tolerance to multiple types of allograft. We have previously established that this tolerance protocol relies on the regulatory function of B lymphocytes for its effect. B lymphocytes have also been reported to participate in immune regulation in several other settings. In most of these systems, the regulatory function of B lymphocytes depends on the production of IL‐10. Therefore, we investigated the role of IL‐10 in the anti‐CD45RB model of B‐cell‐mediated transplantation tolerance. Surprisingly, using antibody‐mediated neutralization of IL‐10, IL‐10‐deficient recipients and adoptive transfer of IL‐10‐deficient B lymphocytes, we found that IL‐10 actually counter‐regulates tolerance induced by anti‐CD45RB. Furthermore, neutralization of IL‐10 reduced the development of chronic allograft vasculopathy compared to anti‐CD45RB alone and reduced the production of graft reactive alloantibodies. These data suggest that the participation of regulatory B lymphocytes in transplantation tolerance may be distinct from how they operate in other systems. Identifying the specific B lymphocytes that mediate transplantation tolerance and defining their mechanism of action may yield new insights into the complex cellular network through which antigen‐specific tolerance is established and maintained.

Inhibition of Transplantation Tolerance by Immune Senescence Is Reversed by Endocrine Modulation

Immune senescence causes resistance to transplantation tolerance that can be functionally restored by targeting sex steroid production. A Fountain of Youth for the Immune System Raging hormones are often blamed for the sometimes erratic behavior of teenagers; yet, hormonal fluctuations aren’t saddled with the stigma they deserve for altering biological behaviors in older adults. An example of such a phenomenon is the senescent immune system, which responds poorly to new stimuli. This failure may stem in part from the shrinking of the thymus that begins by puberty (thymic involution). However, in contrast to what one might intuit, this age-related muting of the immune response does not improve tolerance induction and reduce rejection after cell or organ transplantation. Instead, an active immune system is required to promote tolerance to novel antigens. Zhao et al. now show that modifying hormone concentrations can restore the induction of transplant tolerance in aged mice. The authors found that after the age of 12 months, mice became resistant to tolerance induction for cardiac transplants. However, surgical castration led to long-term graft acceptance and restoration of thymic cellularity. They then confirmed their findings with a procedure more likely to elicit patient assent—Lupron Depot injections, which temporarily disrupt gonadal function and are used clinically in prostate cancer care. This hormone-dependent transplant tolerance required the thymic production of regulatory T cells. Although it’s hard to extrapolate a mouse’s age to a human’s, these results suggest that hormone modification may improve transplant acceptance in older patients by restoring youthful exuberance to the immune system. The senescent immune system responds poorly to new stimuli; thymic involution, accumulation of memory cells against other specificities, and general refractoriness to antigen signaling all may contribute to poor resistance to infection. These same changes may pose a significant clinical barrier to organ transplantation, as transplantation tolerance requires thymic participation and integrated, tolerance-promoting responses to novel antigens. We found that after the age of 12 months, mice became resistant to the tolerance-inducing capacity of the monoclonal antibody therapy anti-CD45RB. This resistance to tolerance to cardiac allografts could be overcome by surgical castration of male mice, a procedure that led to thymic regeneration and long-term graft acceptance. The potential for clinical translation of this endocrine-immune interplay was confirmed by the ability of Lupron Depot injections, which temporarily disrupt gonadal function, to restore tolerance in aged mice. Furthermore, we demonstrated that the restoration of tolerance after surgical or chemical castration depended on thymic production of regulatory T cells (Tregs); thymectomy or Treg depletion abrogated tolerance restoration. The aging of the immune system (“immune senescence”) is a significant barrier to immune tolerance, but this barrier can be overcome by targeting sex steroid production with commonly used clinical therapeutics.

Generation of adaptive regulatory T cells by alloantigen is required for some but not all transplant tolerance protocols.

Background. Because CD4+CD25+Foxp3+ regulatory T cells (Tregs) are essential for the maintenance of self-tolerance, significant interest surrounds the developmental cues for thymic-derived natural Tregs (nTregs) and periphery-generated adaptive Tregs (aTregs). In the transplant setting, the allograft may play a role in the generation of alloantigen-specific Tregs, but this role remains undefined. We examined whether the immune response to a transplant allograft results in the peripheral generation of aTregs. Methods. To identify generation of aTregs, purified graft-reactive CD4+CD25− T cells were adoptively transferred to mice-bearing skin allograft. To demonstrate that aTregs are necessary for tolerance, DBA/2 skin was transplanted onto C57BL/6-RAG-1-deficient recipients adoptively transferred with purified sorted CD4+CD25− T cells; half of the recipients undergo tolerance induction treatment. Results. By tracking adoptively transferred cells, we show that purified graft-reactive CD4+CD25− T lymphocytes up-regulate Foxp3 in mice receiving skin allografts in the absence of any treatment. Interestingly, cotransfer of antigen-specific nTregs suppresses the up-regulation of Foxp3 by inhibiting the proliferation of allograft-responsive T cells. In vitro data are consistent with our in vivo data—Foxp3+ cells are generated on antigen activation, and this generation is suppressed on coculture with antigen-specific nTregs. Finally, blocking aTreg generation in grafted, rapamycin-treated mice disrupts alloantigen-specific tolerance induction. In contrast, blocking aTreg generation in grafted mice treated with nondepleting anti-CD4 plus anti-CD40L antibodies does not disrupt graft tolerance. Conclusions. We conclude that graft alloantigen stimulates the de novo generation of aTregs, and this generation may represent a necessary step in some but not all protocols of tolerance induction.

B cell depletion improves islet allograft survival with anti-CD45RB

A short course of anti-CD45RB leads to long-term islet allograft survival and donor-specific tolerance in approximately half of immunocompetent mice. We have previously demonstrated that anti-CD45RB antibody-mediated tolerance requires B-cells for cardiac allograft survival. We therefore asked whether B-cells were also required for anti-CD45RB antibody-mediated survival of islets. Unexpectedly, we found that nearly 100% of islet allografts survive long term in B-cell-deficient mice. Similarly, B-cell depletion by anti-CD22/cal augmented anti-CD45RB-mediated tolerance when administered pretransplant, although it had no effect on tolerance induction when administered posttransplant. Our results demonstrate that the role of B-cells in promoting tolerance with anti-CD45RB is graft specific, promoting tolerance in cardiac grafts but resisting tolerance in islet transplantation. These findings may help elucidate the varied action of B-cells in promoting tolerance versus rejection.

Elevated Levels of Interferon-γ Production by Memory T Cells Do Not Promote Transplant Tolerance Resistance in Aged Recipients

Immunosenescence predisposes the elderly to infectious and autoimmune diseases and impairs the response to vaccination. We recently demonstrated that ageing also impedes development of transplantation tolerance. Unlike their young counterparts (8-12 weeks of age) aged male recipients (greater than 12 months of age) transplanted with a full MHC-mismatched heart are resistant to tolerance mediated by anti-CD45RB antibody. Surprisingly, either chemical or surgical castration restored tolerance induction to levels observed using young recipients. Based on the strong impact of endocrine modulation on transplant tolerance, we explored the impact of ageing and castration on the immune system. Here we report a significant increase in the percentage of T cells that produce interferon-γ (IFN-γ) in aged male versus young male animals and that the overall increase in IFN-γ production was due to an expansion of IFN-γ-producing memory T cells in aged animals. In contrast to IFN-γ production, we did not observe differences in IL-10 expression in young versus old male mice. We hypothesized that endocrine modulation would diminish the elevated levels of IFN-γ production in aged recipients, however, we observed no significant reduction in the percentage of IFN-γ+ T cells upon castration. Furthermore, we neutralized interferon-γ by antibody and did not observe an effect on graft survival. We conclude that while elevated levels of interferon-γ serves as a marker of tolerance resistance in aged mice, other as yet to be identified factors are responsible for its cause. Defining these factors may be relevant to design of tolerogenic strategies for aged recipients.

An immunosufficient murine model for the study of human islets.

For the sake of therapy of diabetes, it is critical to understand human beta cell function in detail in health and disease. Current studies of human beta cell physiology in vivo are mostly limited to immunodeficient mouse models, which possess significant technical limitations. This study aimed to create a new model for the study of human islets through induction of transplant tolerance in immunosufficient mice. B6 diabetic mice were transplanted with human islets and treated with anti‐CD45RB. To assess whether anti‐CD45RB‐induced transplant tolerance requires B cells, B6 recipients received additional anti‐CD20 or B6μMT−/− mice were used. For some anti‐CD45RB‐treated B6μMT−/− mice, additional anti‐CD25 mAb was applied at the early or late stage post‐transplant. Immunohistology was performed to show the Foxp3 cells in grafted anti‐CD45RB/anti‐CD20‐treated Foxp3‐GFP B6 mice. The results showed that anti‐CD45RB alone allowed indefinite graft survival in 26.6% of B6 mice, however 100% of xenografts were accepted in mice treated simultaneously with anti‐CD20, and 88.9% of xenografts accepted in anti‐CD45RB‐treated μMT−/− mice. These μMT−/− mice accepted the islets from another human donor but rejected the islets from baboon. Additional administration of anti‐CD25 mAb at the time of transplantation resulted in 100% rejection, whereas 40% of grafts were rejected while the antibody was administrated at days 60 post‐transplant. Immunohistologic examination showed Foxp3+ cells accumulated around grafts. We conclude that induction of tolerance to human islets in an immunosufficient mouse model could be generated by targeting murine CD45RB and CD20. This new system will facilitate study of human islets and accelerate the dissection of the critical mechanisms underlying islet health in human disease.

TGF-b-Producing Regulatory B Cells Induce Regulatory T Cells and Promote Transplantation Tolerance.: Abstract# 658

657 B Cell Depletion of Naïve Recipients Enhances Graft Reactive T Cell Responses. T. Singh,1 K. Jiang,1 R. Ippolito,1 B. Ramaswami,1 F. Lund,2 G. Chalasani.1 1Medicine (Renal), Surgery, Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA; 2Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL. B cells play both regulatory and infl ammatory roles in alloimmunity. B cell depletion as induction therapy in unsensitized kidney transplant patients is associated with increased rejection. It is not known if this is due to enhanced T cell responses. Here, we examined the impact of B cell depletion on graft reactive T cell responses in naïve mice. Methods: B6 mice were adoptively transferred with 1 x 106 OT-I and OT-II transgenic T cells followed by either anti-CD20 treatment (0.25mgX1) or nothing. Mice were transplanted with H2b/d-ActmOVA skin grafts to study both endogenous and transferred OVA-reactive T cell responses. Results: Anti-CD20 treated mice showed effective B cell depletion; >99% in peripheral blood and >90% in secondary lymphoid tissues. First H2b/d-mOVA skin graft rejection was not different between groups (MST = 17 days). However, at day 17 post transplantation, graft reactive T cells were signifi cantly increased in B cell depleted mice when compared to control (OT-I 30-fold and OT-II 7-fold; p<0.005), suggesting enhanced effector differentiation. At >8 weeks after graft rejection, B cell depleted mice showed increased endogenous (p<.05), and OT-I and OT-II T cell (p<.005) memory populations. Endogenous memory T cells were 1.5-fold (p<0.05) and OT-I and OT-II T cells were 7-fold more enriched in effector memory (CD44hiCD62Llow TEM) cells in B cell depleted mice. No signifi cant differences in T cell IFNγ and TNFα were observed, whereas IL-2 and IL-17 in CD4+ T cells on exvivo restimulation with donor splenocytes was signifi cantly increased in B cell depleted mice as compared to controls (p<0.05 and p<0.0005). No differences were seen in T regulatory cells between the two groups. Upon rechallenge with H2b/d or H2bd-ActmOVA skin grafts under costimulation blockade (CTLA4Ig+MR1), rejection was signifi cantly faster in B cell depleted mice compared to controls (MST 14 vs. 20, p<0.005), consistent with increased endogenous and OT memory T cell frequencies, respectively. Conclusions: B cell depletion at the time of transplantation in naïve recipients leads to enhanced differentiation of graft reactive effector and memory T cells enriched in TEM. This implies an important role for B cell regulatory functions early in the alloimmune response. Abstract# 658 TGF-b-Producing Regulatory B Cells Induce Regulatory T Cells and Promote Transplantation Tolerance. J. Kim,1 K. Lee,1 R. Stott,1 G. Zhao,1,2 J. SooHoo,1 W. Xiong,1,2 M. Lian,1 L. Fitzgerald,1 S. Shi,1 S. Deng,1,2 H. Yeh,1 J. Markmann.1 1Surgery, Massachusetts General Hospital/Harvard Medical School, Boston, MA; 2Surgery, Sichuan Provincial People’s Hospital, Chengdu, Sichuan, China. Regulatory B cells (Bregs) have been shown to play a critical role in immune homeostasis and in autoimmunity models. We have recently demonstrated that combined anti-TIM-1 and anti-CD45RB antibody treatment results in tolerance to 658 TGF-b-Producing Regulatory B Cells Induce Regulatory T Cells and Promote Transplantation Tolerance. J. Kim,1 K. Lee,1 R. Stott,1 G. Zhao,1,2 J. SooHoo,1 W. Xiong,1,2 M. Lian,1 L. Fitzgerald,1 S. Shi,1 S. Deng,1,2 H. Yeh,1 J. Markmann.1 1Surgery, Massachusetts General Hospital/Harvard Medical School, Boston, MA; 2Surgery, Sichuan Provincial People’s Hospital, Chengdu, Sichuan, China. Regulatory B cells (Bregs) have been shown to play a critical role in immune homeostasis and in autoimmunity models. We have recently demonstrated that combined anti-TIM-1 and anti-CD45RB antibody treatment results in tolerance to full MHC-mismatched islet allografts in mice by generating Bregs that are necessary for tolerance. Bregs are antigen-specifi c, IL-10-dependent, and are capable of transferring tolerance to untreated, transplanted animals. Our aim was to identify mechanism(s) by which regulatory B cells prolong graft survival. METHODS After 100 days of islet allograft survival by dual antibody treatment, B cells were enriched by CD90.2 magnetic bead depletion. For Breg-Treg Foxp3 induction experiment, naive CD4+Foxp3-GFPT cells are sorted by FACSAria, and each B6.RAG receives 4.5 x 106 T cells on day 0. To generate Bregs, on day -14, C57BL/6 animals are injected i.p. withirradiated BALB/c splenocytes and receive standard anti-CD45RB plus anti-TIM-1 antibody treatment. On day 0, B cells are magnetically sorted from splenocyte-injected mice or from naive mice. Grafted B6.RAG recipients receive GFPT cells alone or plus either 12 x 106 Bregs or naive B cells. Graft survival between experimental groups was compared using Kaplan-Meier survival curves and Wilcoxon statistics. Foxp3-GFP experiment was analyzed using ANOVA. P values less than 0.05 were considered statistically signifi cant. RESULTS We demonstrate that adoptively transferred Bregs require the presence of Tregs to establish tolerance, and that adoptive transfer of Bregs increases the number of Tregs. Interaction with Bregs in vivo induces signifi cantly more Foxp3 expression in CD4+CD25T cells than with naive B cells. We also show that Bregs express the TGF-β associated latency-associated peptide (LAP) and that Breg-mediated graft prolongation post-adoptive transfer is abrogated by neutralization of TGF-β activity. Regulatory B cells, like regulatory T cells, demonstrate preferential expression of both CCR6 and CXCR3. CONCLUSIONS Collectively, these findings suggest that in this model of antibody-induced transplantation tolerance, Bregs promote graft survival by promoting Treg development, possibly via TGF-β production. Abstract# 659 Novel NKT Cell-Mediated Help for Alloantibody Production. J. Zimmerer,1 P. Swamy,2 P. Sanghavi,2 C. Wright,1 S. Elzein,1 R. Brutkiewicz,3 G. Bumgardner.1 1Department of Surgery, The Ohio State University Wexner Medical Center, Columbus, OH; 2Medical Student Research Program and the College of Medicine, The Ohio State University, Columbus, OH; 3Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN. IntroductionWhile it is well known that CD4+ T cells and B cells collaborate for antibody production, work by our group suggests that this process is more complex and involves cells of the innate and adaptive immune system. In the current studies, we evaluated the role of NKT cells on the magnitude of alloantibody production posttransplant based on some preliminary observations that mice defi cient in NKT cells (CD1 KO mice) produced less alloantibody than other transplant recipients. MethodsCD8-defi cient wild-type (C57BL/6) recipients, those which lacked all NKT cells (CD1 KO mice), or type 1 NKT cells (Ja18 KO mice; all H-2b) were transplanted with allogeneic hepatocytes (H-2q) and assessed for alloantibody production on day 14 posttransplant. Some experimental groups received adoptive transfer of type I NKT cells on the day of transplant. B cells pooled from wild-type and Ja18 KO recipients were analyzed by ELISPOT for IgG1 as an indicator of in vivo priming. ResultsWhile CD8-deficient wild-type recipients produced high levels of alloantibody posttransplant (76.9±5.0%; n=7, p<0.0002), CD8-defi cient recipients lacking all NKT cells (CD1 KO; 34.8±8.8%, n=5, p<0.0001) or specifi cally lacking type I NKT cells (Ja18 KO mice; 2.0±0.4%, n=5, p<0.0001) produced signifi cantly reduced levels of posttransplant alloantibody compared to wild-type. The adoptive transfer of wild-type type I NKT cells reconstituted maximal alloantibody production in CD8-depleted Ja18 KO recipients (33.3±2.2%; n=7, p<0.0001). Unexpectedly, type I NKT cell contribution to enhanced alloantibody levels was IFN-γ-dependent and IL-4-independent. In addition, more alloprimed B cells were producing IgG1 in wild-type versus type I NKT cell-defi cient recipients on days 8 (288±42 vs. 169±28 IgG1 spot forming cells per million splenocytes) and day 11 (266±53 vs. 94±30) posttransplant (n=4-5, p<0.05 for both comparisons), suggesting type I NKT cells may enhance B cell activation and isotype switching. ConclusionTo our knowledge, this is the fi rst report to substantiate a critical role for type I NKT cells in enhancing in vivo (allo)antibody production in response to endogenous antigenic stimuli. 659 Novel NKT Cell-Mediated Help for Alloantibody Production. J. Zimmerer,1 P. Swamy,2 P. Sanghavi,2 C. Wright,1 S. Elzein,1 R. Brutkiewicz,3 G. Bumgardner.1 1Department of Surgery, The Ohio State University Wexner Medical Center, Columbus, OH; 2Medical Student Research Program and the College of Medicine, The Ohio State University, Columbus, OH; 3Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN. IntroductionWhile it is well known that CD4+ T cells and B cells collaborate for antibody production, work by our group suggests that this process is more complex and involves cells of the innate and adaptive immune system. In the current studies, we evaluated the role of NKT cells on the magnitude of alloantibody production posttransplant based on some preliminary observations that mice defi cient in NKT cells (CD1 KO mice) produced less alloantibody than other transplant recipients. MethodsCD8-defi cient wild-type (C57BL/6) recipients, those which lacked all NKT cells (CD1 KO mice), or type 1 NKT cells (Ja18 KO mice; all H-2b) were transplanted with allogeneic hepatocytes (H-2q) and assessed for alloantibody production on day 14 posttransplant. Some experimental groups received adoptive transfer of type I NKT cells on the day of transplant. B cells pooled from wild-type and Ja18 KO recipients were analyzed by ELISPOT for IgG1 as an indicator of in vivo priming. ResultsWhile CD8-de