Advancing mouse models for transplantation research.

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.

Autoantibodies against DNA topoisomerase I promote renal allograft rejection by increasing alloreactive T cell responses.

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-I205-219 broke self-tolerance, elicited TI-I205-219 immunoglobin 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-I205-219 sera 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.

Macrophage-inducible C-type lectin activates B cells to promote T cell reconstitution in heart allograft recipients.

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.

C1q as a potential tolerogenic therapeutic in transplantation.

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.

Early T cell infiltration is modulated by programed cell death-1 protein and its ligand (PD-1/PD-L1) interactions in murine kidney 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.

B cell-derived IL-1ß and IL-6 drive T cell reconstitution following lymphoablation.

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)-1ß and IL-6 is markedly upregulated after heart allotransplantation and lymphoablation. Neutralizing IL-1ß or IL-6 with mAb or the use of recipients lacking mature IL-1ß, 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-1ß on T lymphocytes. Furthermore, B cells incapable of IL-1ß 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-1ß and IL-6 during homeostatic T cell expansion in a clinically relevant model of lymphoablation.

Anti-donor MHC Class II Alloantibody Induces Glomerular Injury in Mouse Renal Allografts Subjected to Prolonged Cold Ischemia.

BACKGROUND: The mechanisms underlying the effects of prolonged cold-ischemia storage on kidney allografts are poorly understood. METHODS: To investigate effects of cold ischemia on donor-reactive immune responses and graft pathology, we used a mouse kidney transplantation model that subjected MHC-mismatched BALB/c kidney allografts to cold-ischemia storage for 0.5 or 6 hours before transplant into C57BL/6 mice. RESULTS: At day 14 post-transplant, recipients of allografts subjected to 6 versus 0.5 hours of cold-ischemia storage had increased levels of anti-MHC class II (but not class I) donor-specific antibodies, increased donor-reactive T cells, and a significantly higher proportion of transplant glomeruli infiltrated with macrophages. By day 60 post-transplant, allografts with a 6 hour cold-ischemia time developed extensive glomerular injury compared with moderate pathology in allografts with 0.5 hour of cold-ischemia time. Pathology was associated with increased serum levels of anti-class 2 but not anti-class 1 donor-specific antibodies. Recipient B cell depletion abrogated early macrophage recruitment, suggesting augmented donor-specific antibodies, rather than T cells, increase glomerular pathology after prolonged cold ischemia. Lymphocyte sequestration with sphingosine-1-phosphate receptor 1 antagonist FTY720 specifically inhibited anti-MHC class II antibody production and abrogated macrophage infiltration into glomeruli. Adoptive transfer of sera containing anti-donor MHC class II antibodies or mAbs against donor MHC class II restored early glomerular macrophage infiltration in FTY720-treated recipients. CONCLUSIONS: Post-transplant inflammation augments generation of donor-specific antibodies against MHC class II antigens. Resulting MHC class II-reactive donor-specific antibodies are essential mediators of kidney allograft glomerular injury caused by prolonged cold ischemia.

In the absence of natural killer cell activation donor-specific antibody mediates chronic, but not acute, kidney allograft rejection.

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 NK1.1low cells 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 NK1.1high cells 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.

The neonatal Fc receptor: Key to homeostasic control of IgG and IgG-related biopharmaceuticals.

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.

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.

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.

IL-1 Receptor Signaling on Graft Parenchymal Cells Regulates Memory and De Novo Donor-Reactive CD8 T Cell Responses to Cardiac Allografts.

Reperfusion of organ allografts induces a potent inflammatory response that directs rapid memory T cell, neutrophil, and macrophage graft infiltration and their activation to express functions mediating graft tissue injury. The role of cardiac allograft IL-1 receptor (IL-1R) signaling in this early inflammation and the downstream primary alloimmune response was investigated. When compared with complete MHC-mismatched wild-type cardiac allografts, IL-1R(-/-) allografts had marked decreases in endogenous memory CD8 T cell and neutrophil infiltration and expression of proinflammatory mediators at early times after transplant, whereas endogenous memory CD4 T cell and macrophage infiltration was not decreased. IL-1R(-/-) allograft recipients also had marked decreases in de novo donor-reactive CD8, but not CD4, T cell development to IFN-γ-producing cells. CD8 T cell-mediated rejection of IL-1R(-/-) cardiac allografts took 3 wk longer than wild-type allografts. Cardiac allografts from reciprocal bone marrow reconstituted IL-1R(-/-)/wild-type chimeric donors indicated that IL-1R signaling on graft nonhematopoietic-derived, but not bone marrow-derived, cells is required for the potent donor-reactive memory and primary CD8 T cell alloimmune responses observed in response to wild-type allografts. These studies implicate IL-1R-mediated signals by allograft parenchymal cells in generating the stimuli-provoking development and elicitation of optimal alloimmune responses to the grafts.

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.

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.

Natural killer cells play a critical role in mediating inflammation and graft failure during antibody-mediated rejection of kidney allografts.

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.

Novel CD8 T cell alloreactivities in CCR5-deficient recipients of class II MHC disparate kidney grafts.

Recipient CD4 T regulatory cells inhibit the acute T cell-mediated rejection of renal allografts in wild-type mice. The survival of single class II MHC-disparate H-2(bm12) renal allografts was tested in B6.CCR5(-/-) recipients, which have defects in T regulatory cell activities that constrain alloimmune responses. In contrast to wild-type C57BL/6 recipients, B6.CCR5(-/-) recipients rejected the bm12 renal allografts. However, donor-reactive CD8 T cells rather than CD4 T cells were the primary effector T cells mediating rejection. The CD8 T cells induced to bm12 allografts in CCR5-deficient recipients were reactive to peptides spanning the 3 aa difference in the I-A(bm12) versus I-A(b) ß-chains presented by K(b) and D(b) class I MHC molecules. Allograft-primed CD8 T cells from CCR5-deficient allograft recipients were activated during culture either with proinflammatory cytokine-stimulated wild-type endothelial cells pulsed with the I-A(bm12) peptides or with proinflammatory cytokine-simulated bm12 endothelial cells, indicating their presentation of the I-A(bm12) ß-chain peptide/class I MHC complexes. In addition to induction by bm12 renal allografts, the I-A(bm12) ß-chain-reactive CD8 T cells were induced in CCR5-deficient, but not wild-type C57BL/6, mice by immunization with the peptides. These results reveal novel alloreactive CD8 T cell specificities in CCR5-deficient recipients of single class II MHC renal allografts that mediate rejection of the allografts.

B cells in cardiac transplants: from clinical questions to experimental models.

After many years of debate, there is now general agreement that B cells can participate in the immune response to cardiac transplants. Acute antibody-mediated rejection (AMR) is the best defined manifestation of B cell responses, but diagnostic and mechanistic questions still surround AMR. Many complement dependent mechanisms of antibody-mediated injury have been elucidated. C5 has become a therapeutic target that may not just truncate complement activation, but also may tip the balance away from inflammation by altering macrophage function. Additional complement independent effects have been identified. These may escape diagnosis and progress to chronic graft injury. The function of B cell infiltrates in cardiac transplants is even more enigmatic. Nodular endocardial infiltrates that contain B cells and plasma cells have been described in protocol biopsies of cardiac transplants for decades, but an understanding of their significance is still evolving based on more critical morphological and molecular evaluation of these infiltrates. A range of infiltrates containing B cells has also been described in the epicardial fat in transplants with advanced chronic rejection. B cells have been observed in endocardial and epicardial tertiary lymphoid nodules, but their impact on antigen presentation or antibody production remains to be determined. Experimental models in small and large animals suggest that B cells could be essential for the formation of lymphoid nodules through cytokine production. Similarly, the role of proinflammatory adipokines in the formation or function of epicardial lymphoid nodules has not been studied. These clinical observations provide critical questions to be addressed in experimental models.

Mechanisms of antibody-mediated acute and chronic rejection of kidney allografts.

PURPOSE OF REVIEW: Antibody-mediated rejection is responsible for up to half of acute rejection episodes in kidney transplant patients and more than half of late graft failures. Antibodies cause acute graft abnormalities that are distinct from T cell-mediated rejection and at later times posttransplant, a distinct pathologic lesion is associated with capillary basement membrane multilayering and glomerulopathy. Despite the importance of donor-reactive antibodies as the leading cause of kidney graft failure, mechanisms underlying antibody-mediated acute and chronic kidney graft injury are poorly understood. Here, we review recent insights provided from clinical studies as well as from animal models that may help to identify new targets for therapy. RECENT FINDINGS: Studies of biopsies from kidney grafts in patients with donor-specific antibody versus those without have utilized analysis of pathologic lesions and gene expression to identify the distinct characteristics of antibody-mediated rejection. These analyses have indicated the presence of natural killer cells and their activation during antibody-mediated rejection. The impact of studies of antibody-mediated allograft injury in animal models have lagged behind these clinical studies, but have been useful in testing the activation of innate immune components within allografts in the presence of donor-specific antibodies. SUMMARY: Most insights into processes of antibody-mediated rejection of kidney grafts have come from carefully designed clinical studies. However, several new mouse models of antibody-mediated kidney allograft rejection may replicate the abnormalities observed in clinical kidney grafts and may be useful in directly testing mechanisms that underlie acute and chronic antibody-mediated graft injury.

B cells regulate antidonor T-cell reactivity in transplantation.

The analyses of indirect T-cell responses in patients with antibody-mediated renal transplant injury by Shiu et al. emphasize the complex contribution of B cells in alloimmunity. The data suggest at least three distinct but potentially overlapping consequences of T cell-B cell interactions: antigen presentation by B cells, alloantibody production, and immune regulation. These multifaceted functions of B cells should be taken into consideration in developing diagnostic tools and therapeutic strategies.