Ran Fan

Interleukin-17 Promotes Early Allograft Inflammation

Acute cellular rejection of organ transplants is executed by donor-reactive T cells, which are dominated by interferon-gamma-producing cells. As interferon-gamma is dispensable for graft destruction, we evaluated the contribution of interleukin-17A (IL-17) to intragraft inflammation in major histocompatibility complex-mismatched heart transplants. A/J (H-2(a)) cardiac allografts placed into wild-type BALB/c (H-2(d)) mice induced intragraft IL-17 production on day 2 after transplant. Allografts placed into BALB/c IL-17(-/-) recipients demonstrated diminished production of the chemokines CXCL1 and CXCL2 and delayed neutrophil and T cell recruitment. However, by day 7 after transplant, allografts from IL-17(-/-) and wild-type recipients had comparable levels of cellular infiltration. The priming of donor-specific T cells was not affected by the absence of IL-17, and the kinetics of cardiac allograft rejection were similar in wild-type and IL-17(-/-) recipients. In contrast, IL-17(-/-) mice depleted of CD8 T cells rejected A/J allografts in a delayed fashion compared with CD8-depleted wild-type recipients. Although donor-reactive CD4 T cells were efficiently activated in both groups, the infiltration of effector T cells into allografts was impaired in IL-17(-/-) recipients. Our data indicate that locally produced IL-17 amplifies intragraft inflammation early after transplantation and promotes tissue injury by facilitating T cell recruitment into the graft. Targeting the IL-17 signaling network in conjunction with other graft-prolonging therapies may decrease this injury and improve the survival of transplanted organs.

The Spleen Is the Major Source of Antidonor Antibody-Secreting Cells in Murine Heart Allograft Recipients

Antibody‐mediated allograft rejection is an increasingly recognized problem in clinical transplantation. However, the primary location of donor‐specific alloantibody (DSA)‐producing cells after transplantation have not been identified. The purpose of this study was to test the contribution of allospecific antibody‐secreting cells (ASCs) from different anatomical compartments in a mouse transplantation model. Fully MHC‐mismatched heart allografts were transplanted into three groups of recipients: nonsensitized wild type, alloantigen‐sensitized wild‐type and CCR5−/− mice that have exaggerated alloantibody responses. We found that previous sensitization to donor alloantigens resulted in the development of antidonor alloantibody (alloAb) with accelerated kinetics. Nevertheless, the numbers of alloantibody‐secreting cells and the serum titers of antidonor IgG alloantibody were equivalent in sensitized and nonsensitized recipients 6 weeks after transplantation. Regardless of recipient sensitization status, the spleen contained higher numbers of donor‐reactive ASCs than bone marrow at days 7–21 after transplantation. Furthermore, individual spleen ASCs produced more antidonor IgG alloantibody than bone marrow ASCs. Taken together, our results indicate that the spleen rather than bone marrow is the major source of donor‐reactive alloAb early after transplantation in both sensitized and nonsensitized recipients.

Pretransplant antithymocyte globulin has increased efficacy in controlling donor-reactive memory T cells in mice

Antibody‐mediated lymphocyte depletion is frequently used as induction therapy in sensitized transplant patients. Although T cells with an effector/memory phenotype remain detectable after lymphoablative therapies in human transplant recipients, the role of preexisting donor‐reactive memory in reconstitution of the T cell repertoire and induction of alloimmune responses following lymphoablation is poorly understood. We show in a mouse cardiac transplantation model that antidonor immune responses following treatment with rabbit antimouse thymocyte globulin (mATG) were dominated by T cells derived from the preexisting memory compartment. Administration of mATG 1 week prior to transplantation (pre‐TP) was more efficient in targeting preexisting donor‐reactive memory T cells, inhibiting overall antidonor T cell responses, and prolonging heart allograft survival than the commonly used treatment at the time of transplantation (peri‐TP). The failure of peri‐TP mATG to control antidonor memory responses was due to faster recovery of preexisting memory T cells rather than their inefficient depletion. This rapid recovery did not depend on T cell specificity for donor alloantigens suggesting an important role for posttransplant inflammation in this process. Our findings provide insights into the components of the alloimmune response remaining after lymphoablation and may help guide the future use of ATG in sensitized transplant recipients.

CD40-Independent Help by Memory CD4 T Cells Induces Pathogenic Alloantibody But Does Not Lead to Long-Lasting Humoral Immunity

CD40/CD154 interactions are essential for productive antibody responses to T‐dependent antigens. Memory CD4 T cells express accelerated helper functions and are less dependent on costimulation when compared with naïve T cells. Here, we report that donor‐reactive memory CD4 T cells can deliver help to CD40‐deficient B cells and induce high titers of IgG alloantibodies that contribute to heart allograft rejection in CD40−/− heart recipients. While cognate interactions between memory helper T and B cells are crucial for CD40‐independent help, this process is not accompanied by germinal center formation and occurs despite inducible costimulatory blockade. Consistent with the extrafollicular nature of T/B cell interactions, CD40‐independent help fails to maintain stable levels of serum alloantibody and induce differentiation of long‐lived plasma cells and memory B cells. In summary, our data suggest that while CD40‐independent help by memory CD4 T cells is sufficient to induce high levels of pathogenic alloantibody, it does not sustain long‐lasting anti‐donor humoral immunity and B cell memory responses. This information may guide the future use of CD40/CD154 targeting therapies in transplant recipients containing donor‐reactive memory T cells.

Platelets in Early Antibody-Mediated Rejection of Renal Transplants

Antibody-mediated rejection is a major complication in renal transplantation. The pathologic manifestations of acute antibody-mediated rejection that has progressed to functional impairment of a renal transplant have been defined in clinical biopsy specimens. However, the initial stages of the process are difficult to resolve with the unavoidable variables of clinical studies. We devised a model of renal transplantation to elucidate the initial stages of humoral rejection. Kidneys were orthotopically allografted to immunodeficient mice. After perioperative inflammation subsided, donor-specific alloantibodies were passively transferred to the recipient. Within 1 hour after a single transfer of antibodies, C4d was deposited diffusely on capillaries, and von Willebrand factor released from endothelial cells coated intravascular platelet aggregates. Platelet-transported inflammatory mediators platelet factor 4 and serotonin accumulated in the graft at 100- to 1000-fold higher concentrations compared with other platelet-transported chemokines. Activated platelets that expressed P-selectin attached to vascular endothelium and macrophages. These intragraft inflammatory changes were accompanied by evidence of acute endothelial injury. Repeated transfers of alloantibodies over 1 week sustained high levels of platelet factor 4 and serotonin. Platelet depletion decreased platelet mediators and altered the accumulation of macrophages. These data indicate that platelets augment early inflammation in response to donor-specific antibodies and that platelet-derived mediators may be markers of evolving alloantibody responses.

IFN-γ Production by Memory Helper T Cells Is Required for CD40-Independent Alloantibody Responses

Cognate T–B cell interactions and CD40–CD154 costimulation are essential for productive humoral immunity against T-dependent Ags. We reported that memory CD4 T cells can deliver help to B cells and induce pathogenic IgG alloantibodies in the absence of CD40–CD154 interactions. To determine cytokine requirements for CD40-independent help, we used CD40−/− mice containing differentiated subsets of donor-reactive memory Th cells as heart allograft recipients. Th1 and Th17, but not Th2, memory CD4 T cells elicited high titers of anti-donor Ab. Abs induced by Th17 memory CD4 T cells had decreased reactivity against donor MHC class I molecules and inferior ability to cause complement deposition in heart allografts compared with Abs induced by Th1 cells, suggesting a requirement for IFN-γ during CD40-independent help. IFN-γ neutralization inhibited helper functions of memory CD4 T cells in both CD40−/− recipients and wild type recipients treated with anti-CD154 mAb. Our results suggest that IFN-γ secreted by pre-existing memory helper cells determines both isotype and specificity of donor-reactive alloantibodies and can thus affect allograft pathology. This information may be valuable for identifying transplant patients at risk for de novo development of pathogenic alloantibodies and for preventing alloantibody production in T cell–sensitized recipients.

B cell activating factor (BAFF) and a proliferation inducing ligand (APRIL) mediate CD40-independent help by memory CD4 T cells

Donor‐reactive memory T cells undermine organ transplant survival and are poorly controlled by immunosuppression or costimulatory blockade. Memory CD4 T cells provide CD40‐independent help for the generation of donor‐reactive effector CD8 T cells and alloantibodies (alloAbs) that rapidly mediate allograft rejection. The goal of this study was to investigate the role of B cell activating factor (BAFF) and a proliferation‐inducing ligand (APRIL) in alloresponses driven by memory CD4 T cells. The short‐term neutralization of BAFF alone or BAFF plus APRIL synergized with anti‐CD154 mAb to prolong heart allograft survival in recipients containing donor‐reactive memory CD4 T cells. The prolongation was associated with reduction in antidonor alloAb responses and with inhibited reactivation and helper functions of memory CD4 T cells. Additional depletion of CD8 T cells did not enhance the prolonged allograft survival suggesting that donor‐reactive alloAbs mediate late graft rejection in these recipients. This is the first report that targeting the BAFF cytokine network inhibits both humoral and cellular immune responses induced by memory CD4 T cells. Our results suggest that reagents neutralizing BAFF and APRIL may be used to enhance the efficacy of CD40/CD154 costimulatory blockade and improve allograft survival in T cell–sensitized recipients.

Memory CD4 T cells induce antibody-mediated rejection of renal allografts

Despite advances in immunosuppression, antibody-mediated rejection is a serious threat to allograft survival. Alloreactive memory helper T cells can induce potent alloantibody responses and often associate with poor graft outcome. Nevertheless, the ability of memory T cells to elicit well characterized manifestations of antibody-mediated rejection has not been tested. We investigated helper functions of memory CD4 T cells in a mouse model of renal transplantation. Whereas the majority of unsensitized C57Bl/6 recipients spontaneously accepted fully MHC-mismatched A/J renal allografts, recipients containing donor-reactive memory CD4 T cells rapidly lost allograft function. Increased serum creatinine levels, high serum titers of donor-specific alloantibody, minimal T cell infiltration, and intense C4d deposition in the grafts of sensitized recipients fulfilled all diagnostic criteria for acute renal antibody-mediated rejection in humans. IFNγ neutralization did not prevent the renal allograft rejection induced by memory helper T cells, and CD8 T cell depletion at the time of transplantation or depletion of both CD4 and CD8 T cells also did not prevent the renal allograft rejection induced by memory helper T cells starting at day 4 after transplantation. However, B cell depletion inhibited alloantibody generation and significantly extended allograft survival, indicating that donor-specific alloantibodies (not T cells) were the critical effector mechanism of renal allograft rejection induced by memory CD4 T cells. Our studies provide direct evidence that recipient T cell sensitization may result in antibody-mediated rejection of renal allografts and introduce a physiologically relevant animal model with which to investigate mechanisms of antibody-mediated rejection and novel therapeutic approaches for its prevention and treatment.

CD4 T Cell Help via B Cells Is Required for Lymphopenia-Induced CD8 T Cell Proliferation

Ab-mediated lymphoablation is commonly used in solid organ and hematopoietic cell transplantation. However, these strategies fail to control pathogenic memory T cells efficiently and to improve long-term transplant outcomes significantly. Understanding the mechanisms of T cell reconstitution is critical for enhancing the efficacy of Ab-mediated depletion in sensitized recipients. Using a murine analog of anti-thymocyte globulin (mATG) in a mouse model of cardiac transplantation, we previously showed that peritransplant lymphocyte depletion induces rapid memory T cell proliferation and only modestly prolongs allograft survival. We now report that T cell repertoire following depletion is dominated by memory CD4 T cells. Additional depletion of these residual CD4 T cells severely impairs the recovery of memory CD8 T cells after mATG treatment. The CD4 T cell help during CD8 T cell recovery depends on the presence of B cells expressing CD40 and intact CD40/CD154 interactions. The requirement for CD4 T cell help is not limited to the use of mATG in heart allograft recipients, and it is observed in nontransplanted mice and after CD8 T cell depletion with mAb instead of mATG. Most importantly, limiting helper signals increases the efficacy of mATG in controlling memory T cell expansion and significantly extends heart allograft survival in sensitized recipients. Our findings uncover the novel role for helper memory CD4 T cells during homeostatic CD8 T cell proliferation and open new avenues for optimizing lymphoablative therapies in allosensitized patients.

Aquaporin 4 blockade improves survival of murine heart allografts subjected to prolonged cold ischemia

Prolonged cold ischemia storage (CIS) is a leading risk factor for poor transplant outcome. Existing strategies strive to minimize ischemia–reperfusion injury in transplanted organs, yet there is a need for novel approaches to improve outcomes of marginal allografts and expand the pool of donor organs suitable for transplantation. Aquaporins (AQPs) are a family of water channels that facilitate homeostasis, tissue injury, and inflammation. We tested whether inhibition of AQP4 improves the survival of fully MHC‐mismatched murine cardiac allografts subjected to 8 hours of CIS. Administration of a small molecule AQP4 inhibitor during donor heart collection and storage and for a short‐time posttransplantation improves the viability of donor graft cells, diminishes donor‐reactive T cell responses, and extends allograft survival in the absence of other immunosuppression. Furthermore, AQP4 inhibition is synergistic with cytotoxic T lymphocyte–associated antigen 4–Ig in prolonging survival of 8‐hour CIS heart allografts. AQP4 blockade markedly reduced T cell proliferation and cytokine production in vitro, suggesting that the improved graft survival is at least in part mediated through direct effects on donor‐reactive T cells. These results identify AQPs as a promising target for diminishing donor‐specific alloreactivity and improving the survival of high‐risk organ transplants.