James S. Young

Spontaneous restoration of transplantation tolerance after acute rejection

Transplantation is a cure for end-stage organ failure but, in the absence of pharmacological immunosuppression, allogeneic organs are acutely rejected. Such rejection invariably results in allosensitization and accelerated rejection of secondary donor-matched grafts. Transplantation tolerance can be induced in animals and a subset of humans, and enables long-term acceptance of allografts without maintenance immunosuppression. However, graft rejection can occur long after a state of transplantation tolerance has been acquired. When such an allograft is rejected, it has been assumed that the same rules of allosensitization apply as to non-tolerant hosts and that immunological tolerance is permanently lost. Using a mouse model of cardiac transplantation, we show that when Listeria monocytogenes infection precipitates acute rejection, thus abrogating transplantation tolerance, the donor-specific tolerant state re-emerges, allowing spontaneous acceptance of a donor-matched second transplant. These data demonstrate a setting in which the memory of allograft tolerance dominates over the memory of transplant rejection.

Delayed Cytotoxic T Lymphocyte–Associated Protein 4–Immunoglobulin Treatment Reverses Ongoing Alloantibody Responses and Rescues Allografts From Acute Rejection

Antibody‐mediated rejection has emerged as the leading cause of late graft loss in kidney transplant recipients, and inhibition of donor‐specific antibody production should lead to improved transplant outcomes. The fusion protein cytotoxic T lymphocyte–associated protein 4–immunoglobulin (CTLA4‐Ig) blocks T cell activation and consequently inhibits T‐dependent B cell antibody production, and the current paradigm is that CTLA4‐Ig is effective with naïve T cells and less so with activated or memory T cells. In this study, we used a mouse model of allosensitization to investigate the efficacy of continuous CTLA4‐Ig treatment, initiated 7 or 14 days after sensitization, for inhibiting ongoing allospecific B cell responses. Delayed treatment with CTLA4‐Ig collapsed the allospecific germinal center B cell response and inhibited alloantibody production. Using adoptively transferred T cell receptor transgenic T cells and a novel approach to track endogenous graft‐specific T cells, we demonstrate that delayed CTLA4‐Ig minimally inhibited graft‐specific CD4+ and T follicular helper responses. Remarkably, delaying CTLA4‐Ig until day 6 after transplantation in a fully mismatched heart transplant model inhibited alloantibody production and prevented acute rejection, whereas transferred hyperimmune sera reversed the effects of delayed CTLA4‐Ig. Collectively, our studies revealed the unexpected efficacy of CTLA4‐Ig for inhibiting ongoing B cell responses even when the graft‐specific T cell response was robustly established.

Erosion of Transplantation Tolerance After Infection.

Recent clinical studies suggest that operational allograft tolerance can be persistent, but long‐term surviving allografts can be rejected in a subset of patients, sometimes after episodes of infection. In this study, we examined the impact of Listeria monocytogenes (Lm) infection on the quality of tolerance in a mouse model of heart allograft transplantation. Lm infection induced full rejection in 40% of tolerant recipients, with the remaining experiencing a rejection crisis or no palpable change in their allografts. In the surviving allografts on day 8 postinfection, graft‐infiltrating cell numbers increased and exhibited a loss in the tolerance gene signature. By day 30 postinfection, the tolerance signature was broadly restored, but with a discernible reduction in the expression of a subset of 234 genes that marked tolerance and was down‐regulated at day 8 post‐Lm infection. We further demonstrated that the tolerant state after Lm infection was functionally eroded, as rejection of the long‐term surviving graft was induced with anti‐PD‐L1 whereas the same treatment had no effect in noninfected tolerant mice. Collectively, these observations demonstrate that tolerance, even if initially robust, exists as a continuum that can be eroded following bystander immune responses that accompany certain infections.

Tracing donor-MHC class II reactive B cells in mouse cardiac transplantation: Delayed CTLA4-Ig treatment prevents memory alloreactive B-cell generation

Background The dual role of B cells as drivers and suppressors of the immune responses have underscored the need to trace the fate of B cells recognizing donor major histocompatibility complex class I and class II after allograft transplantation. Methods In this study, we used donor class II tetramers to trace the fate of I-Ed–specific B cells after immunization with BALB/c spleen cells or cardiac transplantation, in naive or sensitized C57BL/6 recipients. We combined this approach with genetic lineage tracing of memory B cells in activation-induced cytidine deaminase regulated Cre transgenic mice crossed to the ROSA26-enhanced yellow fluorescent protein reporter mice to track endogenous I-Ed–specific memory B cell generation. Results Immunization with BALB/c splenocytes or heart transplantation induced an expansion and differentiation of I-Ed–specific B cells into germinal center B cells, whereas BALB/c heart transplantation into sensitized recipients induced the preferential differentiation into antibody-secreting cells. A 10.8-fold increase in the frequency of I-Ed-specific memory B cells was observed by day 42 postimmunization. Treatment with CTLA4-Ig starting on day 0 or day 7 postimmunization abrogated I-Ed–specific memory B cell generation and sensitized humoral responses, but not if treatment commenced on day 14. Conclusions The majority of donor-specific memory B cells are generated between days 7 and 14 postimmunization, thus revealing a flexible timeframe whereby delayed CTLA4-Ig administration can inhibit sensitization and the generation of memory graft-reactive B cells.

Evolving Approaches in the Identification of Allograft-reactive T and B Cells in Mice and Humans

Abstract Whether a transplanted allograft is stably accepted, rejected, or achieves immunological tolerance is dependent on the frequency and function of alloreactive lymphocytes, making the identification and analysis of alloreactive T and B cells in transplant recipients critical for understanding mechanisms, and the prediction of allograft outcome. In animal models, tracking the fate of graft-reactive T and B cells allows investigators to uncover their biology and develop new therapeutic strategies to protect the graft. In the clinic, identification and quantification of graft-reactive T and B cells allows for the early diagnosis of immune reactivity and therapeutic intervention to prevent graft loss. In addition to rejection, probing of T and B cell fate in vivo provides insights into the underlying mechanisms of alloimmunity or tolerance that may lead to biomarkers predicting graft fate. In this review, we discuss existing and developing approaches to track and analyze alloreactive T and B cells in mice and humans and provide examples of discoveries made utilizing these techniques. These approaches include mixed lymphocyte reactions, trans-vivo delayed-type hypersensitivity, enzyme-linked immunospot assays, the use of antigen receptor transgenic lymphocytes, and utilization of peptide-major histocompatibility multimers, along with imaging techniques for static multiparameter analysis or dynamic in vivo tracking. Such approaches have already refined our understanding of the alloimmune response and are pointing to new ways to improve allograft outcomes in the clinic.

Equal Expansion of Endogenous Transplant-Specific Regulatory T Cell and Recruitment Into the Allograft During Rejection and Tolerance

Despite numerous advances in the definition of a role for regulatory T cells (Tregs) in facilitating experimental transplantation tolerance, and ongoing clinical trials for Treg-based therapies, critical issues related to the optimum dosage, antigen-specificity, and Treg-friendly adjunct immunosuppressants remain incompletely resolved. In this study, we used a tractable approach of MHC tetramers and flow cytometry to define the fate of conventional (Tconvs) and Tregs CD4+ T cells that recognize donor 2W antigens presented by I-Ab on donor and recipient antigen-presenting cells (APCs) in a mouse cardiac allograft transplant model. Our study shows that these endogenous, donor-reactive Tregs comparably accumulate in the spleens of recipients undergoing acute rejection or exhibiting costimulation blockade-induced tolerance. Importantly, this expansion was not detected when analyzing bulk splenic Tregs. Systemically, the distinguishing feature between tolerance and rejection was the inhibition of donor-reactive conventional T cell (Tconv) expansion in tolerance, translating into increased percentages of splenic FoxP3+ Tregs within the 2W:I-Ab CD4+ T cell subset compared to rejection (~35 vs. <5% in tolerance vs. rejection). We further observed that continuous administration of rapamycin, cyclosporine A, or CTLA4-Ig did not facilitate donor-specific Treg expansion, while all three drugs inhibited Tconv expansion. Finally, donor-specific Tregs accumulated comparably in rejecting tolerant allografts, whereas tolerant grafts harbored <10% of the donor-specific Tconv numbers observed in rejecting allografts. Thus, ~80% of 2W:I-Ab CD4+ T cells in tolerant allografts expressed FoxP3+ compared to ≤10% in rejecting allografts. A similar, albeit lesser, enrichment was observed with bulk graft-infiltrating CD4+ cells, where ~30% were FoxP3+ in tolerant allografts, compared to ≤10% in rejecting allografts. Finally, we assessed that the phenotype of 2W:I-Ab Tregs and observed that the percentages of cells expressing neuropilin-1 and CD73 were significantly higher in tolerance compared to rejection, suggesting that these Tregs may be functionally distinct. Collectively, the analysis of donor-reactive, but not of bulk, Tconvs and Tregs reveal a systemic signature of tolerance that is stable and congruent with the signature within tolerant allografts. Our data also underscore the importance of limiting Tconv expansion for high donor-specific Tregs:Tconv ratios to be successfully attained in transplantation tolerance.

CTLA4-Ig in combination with FTY720 promotes allograft survival in sensitized recipients

Despite recent evidence of improved graft outcomes and safety, the high incidence of early acute cellular rejection with belatacept, a high-affinity CTLA4-Ig, has limited its use in clinical transplantation. Here we define how the incomplete control of endogenous donor-reactive memory T cells results in belatacept-resistant rejection in an experimental model of BALB/c.2W-OVA donor heart transplantation into C57BL/6 recipients presensitized to donor splenocytes. These sensitized mice harbored modestly elevated numbers of endogenous donor-specific memory T cells and alloantibodies compared with naive recipients. Continuous CTLA4-Ig treatment was unexpectedly efficacious at inhibiting endogenous graft-reactive T cell expansion but was unable to inhibit late CD4+ and CD8+ T cell infiltration into the allografts, and rejection was observed in 50% of recipients by day 35 after transplantation. When CTLA4-Ig was combined with the sphingosine 1-phosphate receptor-1 (S1PR1) functional antagonist FTY720, alloantibody production was inhibited and donor-specific IFN-γ-producing T cells were reduced to levels approaching nonsensitized tolerant recipients. Late T cell recruitment into the graft was also restrained, and graft survival improved with this combination therapy. These observations suggest that a rational strategy consisting of inhibiting memory T cell expansion and trafficking into the allograft with CTLA4-Ig and FTY720 can promote allograft survival in allosensitized recipients.

Successful Treatment of T Cell-Mediated Acute Rejection with Delayed CTLA4-Ig in Mice

Clinical observations that kidney transplant recipients receiving belatacept who experienced T cell-mediated acute rejection can be successfully treated and subsequently maintained on belatacept-based immunosuppression suggest that belatacept is able to control memory T cells. We recently reported that treatment with CTLA4-Ig from day 6 posttransplantation successfully rescues allografts from acute rejection in a BALB/c to C57BL/6 heart transplant model, in part, by abolishing B cell germinal centers and reducing alloantibody titers. Here, we show that CTLA4-Ig is additionally able to inhibit established T cell responses independently of B cells. CTLA4-Ig inhibited the in vivo cytolytic activity of donor-specific CD8+ T cells, and the production of IFNγ by graft-infiltrating T cells. Delayed CTLA4-Ig treatment did not reduce the numbers of graft-infiltrating T cells nor prevented the accumulation of antigen-experienced donor-specific memory T cells in the spleen. Nevertheless, delayed CTLA4-Ig treatment successfully maintained long-term graft acceptance in the majority of recipients that had experienced a rejection crisis, and enabled the acceptance of secondary BALB/c heart grafts transplanted 30 days after the first transplantation. In summary, we conclude that delayed CTLA4-Ig treatment is able to partially halt ongoing T cell-mediated acute rejection. These findings extend the functional efficacy of CTLA4-Ig therapy to effector T cells and provide an explanation for why CTLA4-Ig-based immunosuppression in the clinic successfully maintains long-term graft survival after T cell-mediated rejection.

Acquired Resistance to Transplantation Tolerance as a Result of Prior Pregnancy Requires B Cells

Introduction Clinical data suggest allogeneic pregnancy is a sensitizing event; yet, recent data from mouse models indicates it induces fetal-specific regulatory T cell (Treg) expansion that mediates systemic, fetal-specific immune regulation post-partum (PP). In this study, we aimed to define the mechanism for acquired resistance to allograft tolerance after allogeneic pregnancy. Methods Virgin female wild-type (WT) B/6, &mgr;KO, or anti-HEL BCR-tg (MD4) mice were mated with male B/c-2W-OVA transgenic mice. Fetus-specific IgG were monitored by flow cytometry, while donor-specific cellular responses were tracked using Kd+IEd+Ld tetramers for B cells, and 2W:I-Ab and OVA:Kb tetramers for CD4+ and CD8+ T cells, respectively. At PP day 30–45, F1 (B/6 x B/c)-2W-OVA heart transplants (HTx) were performed and anti-CD154 (POD 0, 7, 14) and donor splenocytes (DST, POD 0) were used to induce tolerance. Results Fetus-specific IgG increased during and post-pregnancy to a 9-fold peak by PP day 21. Prior donor-matched pregnancy, but not syngeneic pregnancy, prevented the induction of tolerance in 60% of PP-WT B/6 recipients, despite a significant increase in percentage of fetal-specific FOXP3+ cells and a modest reduction in number of fetal-specific conventional CD4+ T cells (Tconvs) examined at POD 21–189, compared to PP-WT B/6 untransplanted controls. In contrast, ongoing donor-specific germinal center B cell responses were detected suggesting a B cell-mediated resistance to transplant tolerance. Strikingly, anti-CD154/DST successfully achieved long-term graft acceptance in PP-&mgr;KO B/6 and PP-MD4 (normal B cell numbers but ⩽5% donor-specific B cells compared to WT) recipients. Conclusion Despite the sustained expansion of maternal FoxP3+ Tregs with fetal/allograft-specificity, allogeneic pregnancy results in subsequent resistance to transplant tolerance induced with anti-CD154/DST. These studies provide new insights into the mechanism of pregnancy-induced sensitization by demonstrating a necessity for B cells in preventing subsequent induction of fetal/allograft-specific transplantation tolerance. American Society of Transplant Surgeons.