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Following antigen stimulation, naïve T cells differentiate into memory cells that mediate antigen clearance more efficiently upon repeat encounter. Donor-specific tolerance can be achieved in a subset of transplant recipients, but some of these grafts are rejected after years of stability, often following infections. Whether T cell memory can develop from a tolerant state and whether these formerly tolerant patients develop antidonor memory is not known. Using a mouse model of cardiac transplantation in which donor-specific tolerance is induced with costimulation blockade (CoB) plus donor-specific transfusion (DST), we have previously shown that systemic infection with Listeria monocytogenes (Lm) months after transplantation can erode or transiently abrogate established tolerance. In this study, we tracked donor-reactive T cells to investigate whether memory can be induced when alloreactive T cells are activated in the setting of tolerance. We show alloreactive T cells persist after induction of cardiac transplantation tolerance, but fail to acquire a memory phenotype despite becoming antigen experienced. Instead, donor-reactive T cells develop T cell-intrinsic dysfunction evidenced when removed from the tolerant environment. Notably, Lm infection after tolerance did not rescue alloreactive T cell memory differentiation or functionality. CoB and antigen persistence were sufficient together but not separately to achieve alloreactive T cell dysfunction, and conventional immunosuppression could substitute for CoB. Antigen persistence was required, as early but not late surgical allograft removal precluded the acquisition of T cell dysfunction. Our results demonstrate transplant tolerance-associated T cell-intrinsic dysfunction that is resistant to memory development even after Lm-mediated disruption of tolerance.

Solid organ transplantation tolerance can be achieved following select transient immunosuppressive regimens that result in long-lasting restraint of alloimmunity without affecting responses to other antigens. Transplantation tolerance has been observed in animal models following costimulation or coreceptor blockade therapies, and in a subset of patients through induction protocols that include donor bone marrow transplantation, or following withdrawal of immunosuppression. Previous data from our lab and others have shown that proinflammatory interventions that successfully prevent the induction of transplantation tolerance in mice often fail to break tolerance once it has been stably established. This suggests that established tolerance acquires resilience to proinflammatory insults, and prompted us to investigate the mechanisms that maintain a stable state of robust tolerance. Our results demonstrate that only a triple intervention of depleting CD25+ regulatory T cells (Tregs), blocking programmed death ligand-1 (PD-L1) signals, and transferring low numbers of alloreactive T cells was sufficient to break established tolerance. We infer from these observations that Tregs and PD-1/PD-L1 signals cooperate to preserve a low alloreactive T cell frequency to maintain tolerance. Thus, therapeutic protocols designed to induce multiple parallel mechanisms of peripheral tolerance may be necessary to achieve robust transplantation tolerance capable of maintaining one allograft for life in the clinic.

Clinical correlations between bacterial infections and rejection suggest a hypothesis that innate immune stimulation by bacterial infections results in the production of inflammatory cytokine that facilitate bystander T cell activation, increased alloreactivity and inhibition of tolerance induction. Previous studies demonstrated that IFNs produced during an infection with a model bacterium, Listeria monocytogenes, prevented the induction of transplantation tolerance in mice with anti-CD154 and donor-specific transfusion (DST) (1). We investigated the impact of two clinically relevant bacterial infections at the time of transplantation on the ability of anti-CD154 and DST to induce skin allograft acceptance in mice. Staphylococcus aureus (SA) infection prevented skin allograft acceptance whereas maximally tolerated doses of Pseudomonas aeruginosa infection had no effect. SA induced an acute production of IL-6, which was necessary and sufficient for the prevention of skin allograft acceptance. Furthermore, a single pulse of methylprednisolone modulated IL-6 production during SA infection and facilitated skin allograft acceptance in SA-infected recipients. Taken together, our results suggest that bacterial infections elicit specific pro-inflammatory cytokines signatures that can serve as barriers to tolerance induction, and that inhibiting the production of or neutralizing these inflammatory cytokines can synergize with co-stimulatory blockade-based therapies to facilitate the development of transplantation tolerance.

Transplantation of solid organs across histocompatibility barriers in the absence of immunosuppression is invariably followed by acute allograft rejection. Although several immunosuppressive regimens have been developed to prevent allograft rejection, these global immunosuppressive agents effectively inhibit all T cells, leaving the host vulnerable to infections. Thus, a major goal in transplantation immunology is to induce donor-specific tolerance that results in the extended suppression of allograft-specific immune responses, while leaving the remainder of the immune system competent to fight infections and malignancies. Initial successes in identifying approaches that successfully induce transplantation tolerance in experimental models have led to a newer research focus of identifying potential barriers to the induction of such tolerance as well as events that may reverse established allograft tolerance. Both clinical and experimental studies have identified bacterial infections as a possible trigger of allograft rejection. Recently, experimental models of transplantation tolerance have identified that bacterial signals can promote acute allograft rejection either by preventing the induction of transplantation tolerance or by reversing tolerance after it has been stably established. This review summarizes experimental and clinical literature supporting the hypothesis that bacterial infections and innate immunity can qualitatively and quantitatively alter adaptive alloreactivity through effects on innate immune responses.

There is an error in the author contributions. KMB should be listed in "wrote the paper" rather than DME. The correct author contributions are: Conceived and designed the experiments: KMB CLE DME. Performed the experiments: KMB JMT MDD. Analyzed the data: KMB. Contributed reagents/materials/analysis tools: KMB. Wrote the paper: KMB. Edited the manuscript: CLE DME.

The angiotensin converting enzyme inhibitor captopril prevents myosin-induced experimental autoimmune myocarditis. Captopril inhibits production of angiotensin II and increases bradykinin signaling, among other actions. To test whether captopril inhibits disease through blockade of angiotensin signaling, we tested the ability of losartan, an angiotensin II receptor blocker, to prevent myosin-induced myocarditis. A/J mice immunized with the heavy chain of cardiac myosin in complete Freund's adjuvant develop acute myocarditis by day 21 post immunization, consisting of severe focal inflammation, necrosis and fibrosis. Administration of losartan (250 mg/L in the drinking water) or captopril (75 mg/L in the drinking water) significantly reduced inflammation, necrosis, and fibrosis in myosin-immunized mice. The heart weights and the heart weight-to-body weight ratios were also significantly reduced in both treatment groups. However, whereas captopril reduced myosin-specific delayed-type hypersensitivity, losartan did not. Both captopril treated mice and losartan treated mice showed a decrease in myosin-specific autoantibody production. Because losartan treatment significantly reduced myocarditis, fibrosis and autoantibody production in EAM, it is likely that prevention of angiotensin II receptor stimulation is a major mechanism underlying the inhibition of myosin-induced myocarditis by captopril.