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Antibodies reactive to self-antigens are an important component of posttransplant immune responses. The generation requirements and functions of autoantibodies, as well as the mechanisms of their influence on alloimmune responses, still remain to be determined. Our study investigated the contribution of autoimmunity during rejection of renal allografts. We have previously characterized a mouse model in which the acute rejection of a life-supporting kidney allograft is mediated by antibodies. At rejection, recipient sera screening against >4000 potential autoantigens revealed DNA topoisomerase I peptide 205-219 (TI-I205-219) as the most prominent epitope. Subsequent analysis showed TI-I205-219-reactive autoantibodies are induced in nonsensitized recipients of major histocompatibility complex–mismatched kidney allografts in a T cell–dependent manner. Immunization with TI-I205-219broke self-tolerance, elicited TI-I205-219immunoglobin G autoantibodies, and resulted in acute rejection of allogeneic but not syngeneic renal transplants. The graft loss was associated with increased priming of donor-reactive T cells but not with donor-specific alloantibodies elevation. Similarly, passive transfer of anti-TI-I205-219sera following transplantation increased donor-reactive T cell activation with minimal effects on donor-specific alloantibody levels. The results identify DNA topoisomerase I as a novel self-antigen in transplant settings and demonstrate that autoantibodies enhance activation of donor-reactive T cells following renal transplantation.

Diminishing homeostatic proliferation of memory T cells is essential for improving the efficacy of lymphoablation in transplant recipients. Our previous studies in a mouse heart transplantation model established that B lymphocytes secreting proinflammatory cytokines are critical for T cell recovery after lymphoablation. The goal of the current study was to identify mediators of B cell activation following lymphoablation in allograft recipients. Transcriptome analysis revealed that macrophage-inducible C-type lectin (Mincle, Clec4e) expression is up-regulated in B cells from heart allograft recipients treated with murine anti-thymocyte globulin (mATG). Recipient Mincle deficiency diminishes B cell production of pro-inflammatory cytokines and impairs T lymphocyte reconstitution. Mixed bone marrow chimeras lacking Mincle only in B lymphocytes have similar defects in T cell recovery. Conversely, treatment with a synthetic Mincle ligand enhances T cell reconstitution after lymphoablation in non-transplanted mice. Treatment with agonistic CD40 mAb facilitates T cell reconstitution in CD4 T cell-depleted, but not in Mincle-deficient, recipients indicating that CD40 signaling induces T cell proliferation via a Mincle-dependent pathway. These findings are the first to identify an important function of B cell Mincle as a sensor of damage-associated molecular patterns released by the graft and demonstrate its role in clinically relevant settings of organ transplantation.

Diminishing homeostatic proliferation of memory T cells is essential for improving the efficacy of lymphoablation in transplant recipients. Our previous studies in a mouse heart transplantation model established that B lymphocytes secreting proinflammatory cytokines are critical for T cell recovery after lymphoablation. The goal of the current study was to identify mediators of B cell activation following lymphoablation in allograft recipients. Transcriptome analysis revealed that macrophage‐inducible C‐type lectin (Mincle, Clec4e) expression is up‐regulated in B cells from heart allograft recipients treated with murine anti‐thymocyte globulin (mATG). Recipient Mincle deficiency diminishes B cell production of pro‐inflammatory cytokines and impairs T lymphocyte reconstitution. Mixed bone marrow chimeras lacking Mincle only in B lymphocytes have similar defects in T cell recovery. Conversely, treatment with a synthetic Mincle ligand enhances T cell reconstitution after lymphoablation in non‐transplanted mice. Treatment with agonistic CD40 mAb facilitates T cell reconstitution in CD4 T cell‐depleted, but not in Mincle‐deficient, recipients indicating that CD40 signaling induces T cell proliferation via a Mincle‐dependent pathway. These findings are the first to identify an important function of B cell Mincle as a sensor of damage‐associated molecular patterns released by the graft and demonstrate its role in clinically relevant settings of organ transplantation. In mouse heart allograft recipients, macrophage inducible C‐type lectin is an important pattern recognition receptor that drives proinflammatory cytokine production in B cells following lymphoablative treatment.

In 1963, Lepow and colleagues resolved C1, the first component of the classical pathway, into three components, which they named C1q, C1r, and C1s. All three of these components were demonstrated to be involved in causing hemolysis in vitro. For over 30 years after that seminal discovery, the primary function attributed to C1q was as part of the C1 complex that initiated the classical pathway of the complement cascade. Then, a series of papers reported that isolated C1q could bind to apoptotic cells and facilitate their clearance by macrophages. Since then, rheumatologists have recognized that C1q is an important pattern recognition receptor (PRR) that diverts autoantigen containing extracellular vesicles from immune recognition. This critical function of C1q as a regulator of immune recognition has been largely overlooked in transplantation. Now that extracellular vesicles released from transplants have been identified as a major agent of immune recognition, it is logical to consider the potential impact of C1q on modulating the delivery of allogeneic extracellular vesicles to antigen presenting cells. This concept has clinical implications in the possible use of C1q or a derivative as a biological therapeutic to down‐modulate immune responses to transplants. The authors review the evidence that the complement component C1q is a pattern recognition receptor and propose that C1q modulates the immune response to transplants by altering the delivery of allogeneic extracellular vesicles to antigen presenting cells.

In 1963, Lepow and colleagues resolved C1, the first component of the classical pathway, into three components, which they named C1q, C1r, and C1s. All three of these components were demonstrated to be involved in causing hemolysis in vitro. For over 30 years after that seminal discovery, the primary function attributed to C1q was as part of the C1 complex that initiated the classical pathway of the complement cascade. Then, a series of papers reported that isolated C1q could bind to apoptotic cells and facilitate their clearance by macrophages. Since then, rheumatologists have recognized that C1q is an important pattern recognition receptor (PRR) that diverts autoantigen containing extracellular vesicles from immune recognition. This critical function of C1q as a regulator of immune recognition has been largely overlooked in transplantation. Now that extracellular vesicles released from transplants have been identified as a major agent of immune recognition, it is logical to consider the potential impact of C1q on modulating the delivery of allogeneic extracellular vesicles to antigen presenting cells. This concept has clinical implications in the possible use of C1q or a derivative as a biological therapeutic to down-modulate immune responses to transplants.

Allogeneic transplants elicit dynamic T cell responses that are modulated by positive and negative co-stimulatory receptors. Understanding mechanisms that intrinsically modulate the immune responses to transplants is vital to develop rational treatment for rejection. Here, we have investigated the impact of programed cell death-1 (PD-1) protein, a negative co-stimulatory receptor, on the rejection of MHC incompatible kidney transplants in mice. T cells were found to rapidly infiltrate the kidneys of A/J mice transplanted to C57BL/6 mice, which peaked at six days and decline by day 14. The T cells primarily encircled tubules with limited infiltration of the tubular epithelium. Lipocalin 2 (LCN2), a marker of tubular injury, also peaked in the urine at day six and then declined. Notably, flow cytometry demonstrated that most of the T cells expressed PD-1 (over 90% of CD8 and about 75% of CD4 cells) at day six. Administration of blocking antibody to PD-L1, the ligand for PD-1, before day six increased T cell infiltrates and urinary LCN2, causing terminal acute rejection. In contrast, blocking PD-1/PD-L1 interactions after day six caused only a transient increase in urinary LCN2. Depleting CD4 and CD8 T cells virtually eliminated LCN2 in the urine in support of T cells injuring tubules. Thus, our data indicate that PD-1/PD-L1 interactions are not just related to chronic antigenic stimulation of T cells but are critical for the regulation of acute T cell responses to renal transplants.

Immunologic memory is the ability of adaptive immune system to quickly and specifically recognize previously encountered antigens and initiate an effector response. Alloreactive memory cells can mount rapid and robust responses to the transplanted organ resulting in allograft injury. Thus preexisting humoral or cellular memory alloresponses are typically associated with poor graft outcomes in experimental and clinical transplantation. While both B and T lymphocytes exhibit memory responses, this review discusses recent updates on the biology of memory T cells and their relevance to the field of transplantation. Three major areas of focus are the emergence and characterization of tissue resident memory T cells, manipulation of T cell metabolic pathways, and the latest promising approaches to targeting detrimental T cell memory in the settings of organ transplantation.

Understanding the mechanisms of T cell homeostatic expansion is crucial for clinical applications of lymphoablative therapies. We previously established that T cell recovery in mouse heart allograft recipients treated with anti‐thymocyte globulin (mATG) critically depends on B cells and is mediated by B cell–derived soluble factors. B cell production of interleukin (IL)‐1β and IL‐6 is markedly upregulated after heart allotransplantation and lymphoablation. Neutralizing IL‐1β or IL‐6 with mAb or the use of recipients lacking mature IL‐1β, IL‐6, IL‐1R, MyD88, or IL‐6R impair CD4+and CD8+T cell recovery and significantly enhance the graft‐prolonging efficacy of lymphoablation. Adoptive co‐transfer experiments demonstrate a direct effect of IL‐6 but not IL‐1β on T lymphocytes. Furthermore, B cells incapable of IL‐1β or IL‐6 production have diminished capacity to mediate T cell reconstitution and initiate heart allograft rejection upon adoptive transfer into mATG treated B cell deficient recipients. These findings reveal the essential role of B cell–derived IL‐1β and IL‐6 during homeostatic T cell expansion in a clinically relevant model of lymphoablation. Using a mouse heart transplantation model of lymphoablative induction therapy, this study demonstrates that rapid homeostatic T cell reconstitution depends on proinflammatory cytokines IL‐1β and IL‐6 that are produced by recipient B cells.

Antibody-mediated rejection (AMR) is among the leading causes of graft failure in solid organ transplantation. However, AMR treatment options are limited by an incomplete understanding of the mechanisms underlying de novo donor-specific antibody (DSA) generation. The development of pathogenic isotype-switched DSA in response to transplanted allografts is typically attributed to follicular B cells undergoing germinal center reaction whereas the contribution of other B cell subsets has not been previously addressed. The current study investigated the role of recipient marginal zone B cells (MZ B cells) in DSA responses using mouse models of heart and renal allotransplantation. MZ B cells rapidly differentiate into antibody-secreting cells in response to allotransplantation. Despite the selective depletion of follicular B cells in heart allograft recipients, MZ B cells are sufficient for T-dependent IgM and early IgG DSA production. Furthermore, the presence of intact MZ B cell subset is required to support the generation of pathogenic isotype-switched DSA in renal allograft recipients containing donor-reactive memory helper T cells. These findings are the first demonstration of the role of MZ B cells in humoral alloimmune responses following solid organ transplantation and identify MZ B cells as a potential therapeutic target for minimizing de novo DSA production and AMR in transplant recipients.

Mouse models have been instrumental in understanding mechanisms of transplant rejection and tolerance, but cross-study reproducibility and translation of experimental findings into effective clinical therapies are issues of concern. The Mouse Models in Transplantation symposium gathered scientists and physician-scientists involved in basic and clinical research in transplantation to discuss the strengths and limitations of mouse transplant models and strategies to enhance their utility. Participants recognized that increased procedure standardization, including the use of prespecified, defined endpoints, and statistical power analyses, would benefit the field. They also discussed the generation of new models that incorporate environmental and genetic variables affecting clinical outcomes as potentially important. If implemented, these strategies are expected to improve the reproducibility of mouse studies and increase their translation to clinical trials and, ideally, new Food and Drug Administration–approved drugs.

IgG and albumin are the most abundant proteins in the circulation and have the longest half‐lives. These properties are due to a unique receptor, the neonatal Fc receptor (FcRn). Although FcRn is named for its function of transferring IgG across the placenta from maternal to fetal circulation, FcRn functions throughout life to maintain IgG and albumin concentrations. FcRn protects IgG and albumin from intracellular degradation and recycles them back into the circulation. Clinical trials have confirmed that pathogenic antibodies can be depleted by blocking this homeostatic function of FcRn. Moreover, understanding the molecular interactions between IgG and FcRn has resulted in the design of therapeutic monoclonal antibodies with more efficacious pharmacokinetics. As a result of genetic engineering these monoclonals can be delivered at lower doses and at longer intervals. More recent findings have demonstrated that FcRn enhances phagocytosis by neutrophils, immune complex clearance by podocytes and antigen presentation by dendritic cells, macrophages, and B cells. This minireview highlights the relevance of FcRn to transplantation. This minireview examines functions of the scavenger receptor for IgG that could be leveraged to manipulate the half‐life of therapeutic IgG biologics and pathogenic IgG antibodies in transplant recipients.

IgG and albumin are the most abundant proteins in the circulation and have the longest half-lives. These properties are due to a unique receptor, the neonatal Fc receptor (FcRn). Although FcRn is named for its function of transferring IgG across the placenta from maternal to fetal circulation, FcRn functions throughout life to maintain IgG and albumin concentrations. FcRn protects IgG and albumin from intracellular degradation and recycles them back into the circulation. Clinical trials have confirmed that pathogenic antibodies can be depleted by blocking this homeostatic function of FcRn. Moreover, understanding the molecular interactions between IgG and FcRn has resulted in the design of therapeutic monoclonal antibodies with more efficacious pharmacokinetics. As a result of genetic engineering these monoclonals can be delivered at lower doses and at longer intervals. More recent findings have demonstrated that FcRn enhances phagocytosis by neutrophils, immune complex clearance by podocytes and antigen presentation by dendritic cells, macrophages, and B cells. This minireview highlights the relevance of FcRn to transplantation.

Antibody mediated rejection (ABMR) is a major barrier to long-term kidney graft survival. Dysregulated donor-specific antibody (DSA) responses are induced in CCR5-deficient mice transplanted with complete major histocompatibility complex (MHC)-mismatched kidney allografts, and natural killer (NK) cells play a critical role in graft injury and rejection. We investigated the consequence of high DSA titers on kidney graft outcomes in the presence or absence of NK cell activation within the graft. Equivalent serum DSA titers were induced in CCR5-deficient B6 recipients of complete MHC mismatched A/J allografts and semi-allogeneic (A/J x B6) F1 kidney grafts, peaking by day 14 post-transplant. A/J allografts were rejected between days 16-28, whereas B6 isografts and semi-allogeneic grafts survived past day 65. On day 7 post-transplant, NK cell infiltration into A/J allografts was composed of distinct populations expressing high and low levels of the surface antigen NK1.1, with NK1.1lowcells reflecting the highest level of activation. These NK cell populations increased with time post-transplant. In contrast, NK cell infiltration into semi-allogeneic grafts on day 7 was composed entirely of NK1.1highcells that decreased thereafter. On day 65 post-transplant the semi-allogeneic grafts had severe interstitial fibrosis, glomerulopathy, and arteriopathy, accompanied by expression of pro-fibrogenic genes. These results suggest that NK cells synergize with DSA to cause acute kidney allograft rejection, whereas high DSA titers in the absence of NK cell activation cannot provoke acute ABMR but instead induce the indolent development of interstitial fibrosis and glomerular injury that leads to late graft failure.

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.

Prolonged cold ischemia storage (CIS) is a leading risk factor for poor transplant outcome. Existing strategies strive to minimize ischemia–reperfusion injuryin 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 CISheart 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. Using a robust mouse model of heart allograft rejection, this study provides the first evidence that aquaporin water channels regulate adaptive alloimmunity and can be targeted to improve transplant outcome.

Lymphoablation is routinely used in transplantation, and its success is defined by the balance of pathogenic versus protective T cells within reconstituted repertoire. While homeostatic proliferation and thymopoiesis may both cause T cell recovery during lymphopenia, the relative contributions of these mechanisms remain unclear. The goal of this study was to investigate the role of the thymus during T cell reconstitution in adult allograft recipients subjected to lymphoablative induction therapy. Compared with euthymic mice, thymectomized heart allograft recipients demonstrated severely impaired CD4 and CD8 T cell recovery and prolonged heart allograft survival after lymphoablation with murine anti‐thymocyte globulin (mATG). The injection with agonistic anti‐CD40 mAb or thymus transplantation only partially restored T cell reconstitution in mATG‐treated thymectomized mice. After mATGdepletion, residual CD4 T cells migrated into the thymus and enhanced thymopoiesis. Conversely, depletion of CD4 T cells before lymphoablation inhibited thymopoiesis at the stage of CD4−CD8−CD44hiCD25+immature thymocytes. This is the first demonstration that the thymus and peripheral CD4 T cells cooperate to ensure optimal T cell reconstitution after lymphoablation. Targeting thymopoiesis through manipulating functions of depletion‐resistant helper T cells may thus improve therapeutic benefits and minimize the risks of lymphoablation in clinical settings. This study demonstrates that depletion‐resistant memory CD4 T cells migrate into the thymus and facilitate thymopoiesis following antibody‐mediated lymphoablation. Snyder and Farber's editorial is on page 1970.

Lymphoablation is routinely used in transplantation, and its success is defined by the balance of pathogenic versus protective T cells within reconstituted repertoire. While homeostatic proliferation and thymopoiesis may both cause T cell recovery during lymphopenia, the relative contributions of these mechanisms remain unclear. The goal of this study was to investigate the role of the thymus during T cell reconstitution in adult allograft recipients subjected to lymphoablative induction therapy. Compared with euthymic mice, thymectomized heart allograft recipients demonstrated severely impaired CD4 and CD8 T cell recovery and prolonged heart allograft survival after lymphoablation with murine anti-thymocyte globulin (mATG). The injection with agonistic anti-CD40 mAb or thymus transplantation only partially restored T cell reconstitution in mATG-treated thymectomized mice. After mATG depletion, residual CD4 T cells migrated into the thymus and enhanced thymopoiesis. Conversely, depletion of CD4 T cells before lymphoablation inhibited thymopoiesis at the stage of CD4−CD8−CD44hiCD25+immature thymocytes. This is the first demonstration that the thymus and peripheral CD4 T cells cooperate to ensure optimal T cell reconstitution after lymphoablation. Targeting thymopoiesis through manipulating functions of depletion-resistant helper T cells may thus improve therapeutic benefits and minimize the risks of lymphoablation in clinical settings.

Despite recent advances in immunosuppression, donor‐reactive memory T cells remain a serious threat to successful organ transplantation. To alleviate damaging effects of preexisting immunologic memory, lymphoablative induction therapies are used as part of standard care in sensitized recipients. However, accumulating evidence suggests that memory T cells have advantages over their naive counterparts in surviving depletion and expanding under lymphopenic conditions. This may at least partially explain the inability of existing lymphoablative strategies to improve long‐term allograft outcome in sensitized recipients, despite the well‐documented decrease in the frequency of early acute rejection episodes. This minireview summarizes the insights gained from both experimental and clinical transplantation as to the effects of existing lymphoablative strategies on memory T cells and discusses the latest research developments aimed at improving the efficacy and safety of lymphoablation. The authors summarize the known effects of lymphoablation on alloreactive memory T cells and discuss new approaches for improving the efficacy of induction therapies in sensitized transplant recipients.

Despite recent advances in immunosuppression, donor-reactive memory T cells remain a serious threat to successful organ transplantation. To alleviate damaging effects of preexisting immunologic memory, lymphoablative induction therapies are used as part of standard care in sensitized recipients. However, accumulating evidence suggests that memory T cells have advantages over their naive counterparts in surviving depletion and expanding under lymphopenic conditions. This may at least partially explain the inability of existing lymphoablative strategies to improve long-term allograft outcome in sensitized recipients, despite the well-documented decrease in the frequency of early acute rejection episodes. This minireview summarizes the insights gained from both experimental and clinical transplantation as to the effects of existing lymphoablative strategies on memory T cells and discusses the latest research developments aimed at improving the efficacy and safety of lymphoablation.

While the incidence of antibody-mediated kidney graft rejection has increased, the key cellular and molecular participants underlying this graft injury remain unclear. Rejection of kidney allografts in mice lacking the chemokine receptor CCR5 is dependent on production of donor-specific antibody. Here we determine if cells expressing cytotoxic function contributed to antibody-mediated kidney allograft rejection in these recipients. Wild-type C57BL/6, B6.CCR5-/-, and B6.CD8-/-/CCR5-/-mice were transplanted with complete MHC-mismatched A/J kidney grafts, and intragraft inflammatory components were followed to rejection. B6.CCR5-/-and B6.CD8-/-/CCR5-/-recipients rejected kidney allografts by day 35, whereas 65% of allografts in wild-type recipients survived past day 80 post-transplant. Rejected allografts in wild-type C57BL/6, B6.CCR5-/-, and B6.CD8-/-/CCR5-/-recipients expressed high levels of VCAM-1 and MMP7 mRNA that was associated with high serum titers of donor-specific antibody. High levels of perforin and granzyme B mRNA expression peaked on day 6 post-transplant in allografts in all recipients, but were absent in isografts. Depletion of natural killer cells in B6.CD8-/-/CCR5-/-recipients reduced this expression to background levels and promoted the long-term survival of 40% of the kidney allografts. Thus, natural killer cells have a role in increased inflammation during antibody-mediated kidney allograft injury and in rejection of the grafts.

We have reported that B6.CCR5−/−mice reject renal allografts with high serum donor-specific antibody (DSA) titers and marked C4d deposition in grafts, features consistent with antibody-mediated rejection (AMR). B6.huCD20/CCR5−/−mice, where human CD20 expression is restricted to B cells, rejected A/J renal allografts by day 26 posttransplant with DSA first detected in serum on day 5 posttransplant and increased thereafter. Recipient treatment with anti-huCD20 mAb prior to the transplant and weekly up to 7 weeks posttransplant promoted long-term allograft survival (>100 days) with low DSA titers. To investigate the effect of B cell depletion at the time serum DSA was first detected, recipients were treated with anti-huCD20 mAb on days 5, 8, and 12 posttransplant. This regimen significantly reduced DSA titers and graft inflammation on day 15 posttransplant and prolonged allograft survival >60 days. However, DSA returned to the titers observed in control treated recipients by day 30 posttransplant and histological analyses on day 60 posttransplant indicated severe interstitial fibrosis. These results indicate that anti-huCD20 mAb had the greatest effect as a prophylactic treatment and that the distinct kinetics of DSA responses accounts for acute renal allograft failure versus the development of fibrosis.

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.

Using microdissection methods on cardiac transplants, the investigators establish that the balance between inflammatory and wound‐healing macrophages is different in the interstitial and arterial compartments, and that this balance shifts with time.

Using a murine model of heart transplantation, the authors demonstrate that neutralizing B cell activating factor and a proliferation‐inducing ligand inhibits CD40‐independent helper functions of donor‐reactive memory CD4 T cells and prolongs allograft survival in T cell‐sensitized recipients treated with anti‐CD154 monoclonal antibody.

Acute and chronic rejection impact distinct compartments of cardiac allografts. Intramyocardial mononuclear cell infiltrates define acute rejection, whereas chronic rejection affects large arteries. Hearts transplanted from male to female C57BL/6 mice undergo acute rejection with interstitial infiltrates at 2 weeks that resolve by 6 weeks when large arteries develop arteriopathy. These processes are dependent on T cells because no infiltrates developed in T cell–deficient mice and transfer of CD4 T cells restored T cell as well as macrophage infiltrates and ultimately neointima formation. Markers of inflammatory macrophages were up-regulated in the interstitium acutely and decreased as markers of wound healing macrophages increased chronically. Programmed cell death protein, a negative costimulator, and its ligand PDL1 were up-regulated in the interstitium during resolution of acute rejection. Blocking PDL1:PD1 interactions in the acute phase increased interstitial T cell infiltrates. Toll-like receptor (TLR) 4 and its endogenous ligand hyaluronan were increased in arteries with neointimal expansion. Injection of hyaluronan fragments increased intragraft production of chemokines. Our data indicate that negative costimulatory pathways are critical for the resolution of acute interstitial infiltrates. In the arterial compartment recognition of endogenous ligands including hyaluronan by the innate TLRs may support the progression of arteriopathy.

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.

These results in a murine model of heart transplantation show that donor‐reactive memory CD4 T cells induce pathogenic IgG alloantibody in the absence of CD40/CD154 interactions but fail to promote differentiation of longlived plasma cells and memory B cells under these conditions.

Induction of lymphopenia at day 60 posttransplant in recipients carrying long‐term surviving cardiac allografts stimulates the generation of donor‐reactive effector‐memory T cells that break the tolerance and reject the allograft.

Lymphopenia is induced by lymphoablative therapies and chronic viral infections. We assessed the impact of lymphopenia on cardiac allograft survival in recipients conditioned with peritransplant costimulatory blockade (CB) to promote long-term graft acceptance. After vascularized MHC-mismatched heterotopic heart grafts were stably accepted through CB, lymphopenia was induced on day 60 posttransplant by 6.5 Gy irradiation or by administration of anti-CD4 plus anti-CD8 mAb. Long-term surviving allografts were gradually rejected after lymphodepletion (MST = 74 ± 5 days postirradiation). Histological analyses indicated signs of severe rejection in allografts following lymphodepletion, including mononuclear cell infiltration and obliterative vasculopathy. Lymphodepletion of CB conditioned recipients induced increases in CD44higheffector/memory T cells in lymphatic organs and strong recovery of donor-reactive T cell responses, indicating lymphopenia-induced proliferation (LIP) and donor alloimmune responses occurring in the host. T regulatory (CD4+Foxp3+) cell and B cell numbers as well as donor-specific antibody titers also increased during allograft rejection in CB conditioned recipients given lymphodepletion. These observations suggest that allograft rejection following partial lymphocyte depletion is mediated by LIP of donor-reactive memory T cells. As lymphopenia may cause unexpected rejection of stable allografts, adequate strategies must be developed to control T cell proliferation and differentiation during lymphopenia.

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.

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.

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.

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.

Memory T cells have emerged as a major threat to transplant survival; they are well equipped and well positioned to respond to antigens in an accelerated fashion. They participate in transplant rejection and resist interventions that usually contain naïve T cells. Thus, the means to prevent memory T cells from attacking allotransplants are an important issue in transplantation.

Induction therapy is used in kidney transplantation to inhibit the activation of donor‐reactive T cells which are detrimental to transplant outcomes. The choice of induction therapy is decided based on perceived immunological risk rather than by direct measurement of donor T‐cell reactivity. We hypothesized that immune cellular alloreactivity pretransplantation can be quantified and that blocking versus depleting therapies have differential effects on the level of donor and third‐party cellular alloreactivity. We studied 31 kidney transplant recipients treated with either antithymocyte globulin (ATG) or an IL‐2 receptor blocker. We tested pre‐ and posttransplant peripheral blood cells by flow cytometry to characterize T‐cell populations and by IFN‐γ ELISPOT assays to assess the level of cellular alloreactivity. CD8+T cells were more resistant to depletion by ATG than CD4+T cells. Posttransplantation, frequencies of donor‐reactive T cells were markedly decreased in the ATG‐treated group but not in the IL‐2 receptor blocker group, whereas the frequencies of third‐party alloreactivity remained nearly equivalent. In conclusion, when ATG is used, marked and prolonged donor hyporesponsiveness with minimal effects on nondonor responses is observed. In contrast, induction with the IL‐2 receptor blocker is less effective at diminishing donor T‐cell reactivity.

Induction therapy is used in kidney transplantation to inhibit the activation of donor-reactive T cells which are detrimental to transplant outcomes. The choice of induction therapy is decided based on perceived immunological risk rather than by direct measurement of donor T-cell reactivity. We hypothesized that immune cellular alloreactivity pretransplantation can be quantified and that blocking versus depleting therapies have differential effects on the level of donor and third-party cellular alloreactivity. We studied 31 kidney transplant recipients treated with either antithymocyte globulin (ATG) or an IL-2 receptor blocker. We tested pre- and posttransplant peripheral blood cells by flow cytometry to characterize T-cell populations and by IFN-γ ELISPOT assays to assess the level of cellular alloreactivity. CD8+T cells were more resistant to depletion by ATG than CD4+T cells. Posttransplantation, frequencies of donor-reactive T cells were markedly decreased in the ATG-treated group but not in the IL-2 receptor blocker group, whereas the frequencies of third-party alloreactivity remained nearly equivalent. In conclusion, when ATG is used, marked and prolonged donor hyporesponsiveness with minimal effects on nondonor responses is observed. In contrast, induction with the IL-2 receptor blocker is less effective at diminishing donor T-cell reactivity.

Acute cellular rejection of organ transplants is executed by donor-reactive T cells, which are dominated by interferon-γ-producing cells. As interferon-γ 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-2a) cardiac allografts placed into wild-type BALB/c (H-2d) 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.

Studies in the past decade advanced our understanding of the development, execution and regulation of T-cell-mediated allograft rejection. This review outlines recent progress and focuses on three major areas of investigation that are likely to guide the development of graft-prolonging therapies in the future. The discussed topics include the contribution of recently discovered molecules to the activation and functions of alloreactive T cells, the emerging problem of alloreactive memory T cells and recently gained insights into the old question of transplantation tolerance.

Decades of experiments in small animals had tipped the balance of opinion away from antibodies as a cause of transplant rejection. However, clinical experience, especially with sensitized patients, has convinced basic immunologists of the need to develop models to investigate mechanisms underlying antibody-mediated rejection (AMR). This resurgent interest has resulted in several new rodent models to investigate antibody-mediated mechanisms of heart and renal allograft injury, but satisfactory models of chronic AMR remain more elusive. Nevertheless, these new studies have begun to reveal many insights into the molecular and pathological sequelae of antibody binding to the allograft endothelium. In addition, complement-independent and complement-dependent effects of antibodies on endothelial cells have been identified in vitro. As small animal models become better defined, it is anticipated that they will be more widely used to answer further questions concerning mechanisms of antibody-mediated tissue injury as well as to design therapeutic interventions.

Studies in the past decade advanced our understanding of the development, execution and regulation of T‐cell‐mediated allograft rejection. This review outlines recent progress and focuses on three major areas of investigation that are likely to guide the development of graft‐prolonging therapies in the future. The discussed topics include the contribution of recently discovered molecules to the activation and functions of alloreactive T cells, the emerging problem of alloreactive memory T cells and recently gained insights into the old question of transplantation tolerance.

Decades of experiments in small animalshad tipped the balance of opinion away from antibodies as a cause of transplant rejection. However, clinical experience, especially with sensitized patients, has convinced basic immunologists of the need to develop models to investigate mechanisms underlying antibody‐mediated rejection (AMR). This resurgent interest has resulted in several new rodent models to investigate antibody‐mediated mechanisms of heart and renal allograft injury, but satisfactory models of chronic AMR remain more elusive. Nevertheless, these new studies have begun to reveal many insights into the molecular and pathological sequelae of antibody binding to the allograft endothelium. In addition, complement‐independent and complement‐dependent effects of antibodies on endothelial cells have been identified in vitro. As small animal models become better defined, it is anticipated that they will be more widely used to answer further questions concerning mechanisms of antibody‐mediated tissue injury as well as to design therapeutic interventions.

Alloreactive T-cell memory is present in every transplant recipient and endangers graft survival. Even in the absence of known sensitizing exposures, heterologous immunity and homeostatic T-cell proliferation generate 'endogenous' memory T cells with donor-reactivity. We have recently shown that endogenous donor-reactive CD8 memory T cells infiltrate murine cardiac allografts within hours of reperfusion and amplify early posttransplant inflammation by producing IFN-?. Here, we have tested the role of ICOS co-stimulation in eliciting effector function from these memory T cells. ICOS is not expressed on the cell surface of circulating CD8 memory T cells but is rapidly upregulated during cell division within the allograft parenchyma. Donor-reactive CD8 memory T-cell infiltration, proliferation and ICOS expression are regulated by donor class I MHC molecule expression. ICOS blockade significantly reduced IFN-? production and other proinflammatory functions of the activated CD8 memory T cells. Our data demonstrate that this induction of ICOS expression within peripheral tissues is an important feature of CD8 memory T-cell activation and identify ICOS as a specific target for neutralizing proinflammatory functions of endogenous CD8 memory T cells.

Alloreactive T-cell memory is present in every transplant recipient and endangers graft survival. Even in the absence of known sensitizing exposures, heterologous immunity and homeostatic T-cell proliferation generate ’endogenous’ memory T cells with donorreactivity. We have recently shown that endogenous donor-reactive CD8 memory T cells infiltrate murine cardiac allografts within hours of reperfusion and amplify early posttransplant inflammation by producing IFN-γ . Here, we have tested the role of ICOS costimulation in eliciting effector function from these memory T cells. ICOS is not expressed on the cell surface of circulating CD8 memory T cells but is rapidly upregulated during cell division within the allograft parenchyma. Donor-reactive CD8 memory T-cell infiltration, proliferation and ICOS expression are regulated by donor class I MHC molecule expression. ICOS blockade significantly reduced IFN-γ production and other proinflammatory functions of the activated CD8 memory T cells. Our data demonstrate that this induction of ICOS expression within peripheral tissues is an important feature of CD8 memory T-cell activation and identify ICOS as a specific target for neutralizing proinflammatory functions of endogenous CD8 memory T cells.

Normal immune responses stimulated by pathogenic and environmental antigens generate memory T cells that react with donor antigens and no currently used immunosuppressive drug completely inhibits memory T-cell function. While donor-reactive memory T cells clearly compromise graft outcomes, mechanisms utilized by memory T cells to promote rejection are largely unknown. In this study, we investigated how early endogenous memory cells infiltrate and express effector function in cardiac allografts. Endogenous CD8 memory T cells in nonsensitized recipients distinguish syngeneic versus allogeneic cardiac allografts within 24 h of reperfusion. CD8-dependent production of IFN-? and CXCL9Mig was observed 24 to 72 h posttransplant in allografts but not isografts. CXCL9 was produced by donor cells in response to IFN-? made by recipient CD8 T cells reactive to donor class I major histocompatibility complex (MHC) molecules. Activated CD8 T cells were detected in allografts at least 3 days before donor-specific effector T cells producing IFN-? were detected in the recipient spleen. Early inflammation mediated by donor-reactive CD8 memory T cells greatly enhanced primed effector T-cell infiltration into allografts. These results suggest that strategies for optimal inhibition of alloimmunity should include neutralization of infiltrating CD8 memory T cells within a very narrow window after transplantation.

The need to control donor-reactive T cell memory for successful organ transplantation is widely acknowledged. Alloreactive memory T cells are present in many human transplant recipients prior to transplantation and are less susceptible to the effects of currently used immunosuppression than naïve T cells. This review brings together recent advances in various fields of immunology that are potentially applicable for targeting memory T cells in sensitized transplant patients.