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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.

T cell receptor transgenic (TCR-Tg) T cells are often used as tracer populations of antigen-specific responses to extrapolate findings to endogenous T cells. The extent to which TCR-Tg T cells behave purely as tracer cells or modify the endogenous immune response is not clear. To test the impact of TCR-Tg T cell transfer on endogenous alloimmunity, recipient mice were seeded with CD4+ or CD8+ TCR-Tg or polyclonal T cells at the time of cardiac allograft transplantation. Only CD4+ TCR-Tg T cells accelerated rejection and, unexpectedly, led to a dose-dependent decrease in both transferred and endogenous T cells infiltrating the graft. In contrast, recipients of CD4+ TCR-Tg T cells exhibited enhanced endogenous donor-specific CD8+ T cell activation in the spleen and accelerated alloantibody production. Introduction of CD4+ TCR-Tg T cells also perturbed the intragraft accumulation of innate cell populations. Transferred CD4+ TCR-Tg T cells alter many aspects of endogenous alloimmunity, suggesting that caution should be used when interpreting experiments using these adoptively transferred cells because the overall nature of allograft rejection may be altered. These results also may have implications for adoptive CD4+ T cell immunotherapy in tumor and infectious clinical settings because cell infusion may have additional effects on natural immune responses.

Although many microbial infections elicit an adaptive immune response that can protect against reinfection, it is generally thought that Staphylococcus aureus infections fail to generate protective immunity despite detectable T and B cell responses. No vaccine is yet proven to prevent S. aureus infections in humans, and efforts to develop one have been hampered by a lack of animal models in which protective immunity occurs. Our results describe a novel mouse model of protective immunity against recurrent infection, in which S. aureus skin and soft tissue infection (SSTI) strongly protected against secondary SSTI in BALB/c mice but much less so in C57BL/6 mice. This protection was dependent on antibody, because adoptive transfer of immune BALB/c serum or purified antibody into either BALB/c or C57BL/6 mice resulted in smaller skin lesions. We also identified an antibody-independent mechanism, because B cell-deficient mice were partially protected against secondary S. aureus SSTI and adoptive transfer of T cells from immune BALB/c mice resulted in smaller lesions upon primary infection. Furthermore, neutralization of interleukin-17A (IL-17A) abolished T cell-mediated protection in BALB/c mice, whereas neutralization of gamma interferon (IFN-γ) enhanced protection in C57BL/6 mice. Therefore, protective immunity against recurrent S. aureus SSTI was advanced by antibody and the Th17/IL-17A pathway and prevented by the Th1/IFN-γ pathway, suggesting that targeting both cell-mediated and humoral immunity might optimally protect against secondary S. aureus SSTI. These findings also highlight the importance of the mouse genetic background in the development of protective immunity against S. aureus SSTI.

Self-assembling peptides and peptide derivatives have received significant interest for several biomedical applications, including tissue engineering, wound healing, cell delivery, drug delivery, and vaccines. This class of materials has exhibited significant variability in immunogenicity, with many peptides eliciting no detectable antibody responses but others eliciting very strong responses without any supplemental adjuvants. Presently, strategies for either avoiding strong antibody responses or specifically inducing them are not well developed, even though they are critical for the use of these materials both within tissue engineering and within immunotherapies. Here, we investigated the molecular determinants and immunological mechanisms leading to the significant immunogenicity of the self-assembling peptide OVA-Q11, which has been shown previously to elicit strong antibody responses in mice. We show that these responses can last for at least a year. Using adoptive transfer experiments and T cell knockout models, we found that these strong antibody responses were T cell-dependent, suggesting a route for avoiding or ensuring immunogenicity. Indeed, by deleting amino acid regions in the peptide recognized by T cells, immunogenicity could be significantly diminished. Immunogenicity could also be attenuated by mutating key residues in the self-assembling domain, thus preventing fibrillization. A second self-assembling peptide, KFE8, was also non-immunogenic, but nanofibers of OVA-KFE8 elicited strong antibody responses similar to OVA-Q11, indicating that the adjuvant action was not dependent on the specific self-assembling peptide sequence. These findings will facilitate the design of self-assembled peptide biomaterials, both for applications where immunogenicity is undesirable and where it is advantageous.

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.

The specificity and function of T helper (Th) immune responses underlying the induction, progression, and resolution of experimental autoimmune myocarditis (EAM) in A/J mice are unclear. Published data suggest involvement of both Th1 and Th2 responses in EAM; however, the previous inability to assess antigen-specific in vivo and in vitro T-cell responses in cardiac myosin-immunized animals has confounded our understanding of this important model of autoimmune myocarditis. The goal of our study was to develop an alternative model of EAM based on a recombinant fragment of cardiac myosin, in hopes that the recombinant protein will permit measurement of functional T-cell responses that is not possible with purified native protein. A/J mice immunized with a recombinant fragment of cardiac myosin spanning amino acids 1074-1646, termed Myo4, developed severe myocarditis characterized by cardiac hypertrophy, massive mononuclear cell infiltration and fibrosis, three weeks post-immunization. The mice also developed an IgG1 dominant humoral immune response specific for both Myo4 and purified cardiac myosin. The in vitro stimulation of splenocytes harvested from Myo4-immunized animals with Myo4 resulted in cellular proliferation with preferential production of the Th1- and Th17-associated cytokines, IFN-gamma, IL-17, and IL-6, respectively. Production of IL-4 was negligible by comparison. This study describes a new model of EAM, inducible by immunization with a specific fragment of cardiac myosin, from which antigen-specific analyses reveal an importance for both Th1 and Th17 immunity.

Chagas’ heart disease (CHD), caused by the parasiteTrypanosoma cruzi, is the most common form of myocarditis in Central America and South America. Some humans and experimental animals develop both humoral and cell-mediated cardiac-specific autoimmunity during infection. Benznidazole, a trypanocidal drug, is effective at reducing parasite load and decreasing the severity of myocarditis in acutely infected patients. We hypothesized that the magnitude of autoimmunity that develops followingT. cruziinfection is directly proportional to the amount of damage caused by the parasite. To test this hypothesis, we used benznidazole to reduce the number of parasites in an experimental model of CHD and determined whether this treatment altered the autoimmune response. Infection of A/J mice with the Brazil strain ofT. cruzileads to the development of severe inflammation, fibrosis, necrosis, and parasitosis in the heart accompanied by vigorous cardiac myosin-specific delayed-type hypersensitivity (DTH) and antibody production at 21 days postinfection. Mice succumbed to infection within a month if left untreated. Treatment of infected mice with benznidazole eliminated mortality and decreased disease severity. Treatment also reduced cardiac myosin-specific DTH and antibody production. Reinfection of treated mice with a heart-derived, virulent strain ofT. cruzior immunization with myosin led to the redevelopment of myosin-specific autoimmune responses and inflammation. These results provide a direct link between the levels ofT. cruziand the presence of autoimmunity and suggest that elimination of the parasite may result in the reduction or elimination of autoimmunity in the chronic phase of infection.

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

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.

African trypanosomes express a family of dually acylated, EF-hand calcium-binding proteins called the calflagins. These proteins associate with lipid raft microdomains in the flagellar membrane, where they putatively function as calcium signaling proteins. Here we show that these proteins bind calcium with high affinity and that their expression is regulated during the life cycle stage of the parasite, with protein levels approximately 10-fold higher in the mammalian bloodstream form than in the insect vector procyclic stage. We also demonstrate a role for the calflagins in mammalian infection, as inhibition of the entire calflagin family by RNA interference dramatically increased host survival and attenuated parasitemia in a mouse model of sleeping sickness. In contrast to infection with parental wild-type parasites, which demonstrated an unremitting parasitemia and death within 6 to 10 days, infection with calflagin-depleted parasites demonstrated prolonged survival associated with a sudden decrease in parasitemia at approximately 8 days postinfection. Subsequent relapsing and remitting waves of parasitemia thereafter were associated with alternate expression of the variant surface glycoprotein, suggesting that initial clearance was antigen specific. Interestingly, despite the notable in vivo phenotype and flagellar localization of the calflagins, in vitro analysis of the calflagin-deficient parasites demonstrated normal proliferation, flagellar motility, and morphology. Further analysis of the kinetics of surface antibody clearance also did not demonstrate a deficit in the calflagin-deficient parasites; thus, the molecular basis for the altered course of infection is independent of an effect on parasite cell cycle progression, motility, or degradation of surface-bound antibodies.

Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a major public health problem in Central and South America. The pathogenesis of Chagas disease is complex and the natural course of infection is not completely understood. The recent development of bioluminescence imaging technology has facilitated studies of a number of infectious and non-infectious diseases. We developed luminescent T. cruzi to facilitate similar studies of Chagas disease pathogenesis. Luminescent T. cruzi trypomastigotes and amastigotes were imaged in infections of rat myoblast cultures, which demonstrated a clear correlation of photon emission signal strength to the number of parasites used. This was also observed in mice infected with different numbers of luminescent parasites, where a stringent correlation of photon emission to parasite number was observed early at the site of inoculation, followed by dissemination of parasites to different sites over the course of a 25-day infection. Whole animal imaging from ventral, dorsal and lateral perspectives provided clear evidence of parasite dissemination. The tissue distribution of T. cruzi was further determined by imaging heart, spleen, skeletal muscle, lungs, kidneys, liver and intestines ex vivo. These results illustrate the natural dissemination of T. cruzi during infection and unveil a new tool for studying a number of aspects of Chagas disease, including rapid in vitro screening of potential therapeutical agents, roles of parasite and host factors in the outcome of infection, and analysis of differential tissue tropism in various parasite-host strain combinations.

The renin-angiotensin system is primarily responsible for regulating vascular tone. Drugs that inhibit this pathway, angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists, are widely used to treat hypertension and a variety of cardiomyopathies. Recent studies have shown that, in addition to reducing blood pressure, these drugs also modulate inflammation, adhesion molecule expression, and fibrosis. To assess the therapeutic potential of these inhibitory agents for the treatment of inflammatory heart disease, the drugs have been tested in experimental models of infectious and autoimmune myocarditis. This review summarizes the results of studies examining the efficacy of angiotensin converting enzyme inhibitors and angiotensin receptor antagonists for the treatment of mouse models of virus-induced and parasite-induced myocarditis, as well as autoimmune cardiomyopathy. The collective results strongly support the use of renin-angiotensin modulation for the treatment of myocarditis. Importantly, this therapeutic approach seems to downregulate autoimmunity without causing immune suppression which may enhance the survival of the disease-initiating infectious agent.

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.

Chagas heart disease, caused by the protozoan parasite Trypanosoma cruzi, is a potentially fatal cardiomyopathy often associated with cardiac autoimmunity. T. cruzi infection induces the development of autoimmunity to a number of antigens via molecular mimicry and other mechanisms, but the genesis and pathogenic potential of this autoimmune response has not been fully elucidated. To determine whether exposure to T. cruzi antigens alone in the absence of active infection is sufficient to induce autoimmunity, we immunized A/J mice with heat-killed T. cruzi (HKTC) emulsified in complete Freund's adjuvant, and compared the resulting immune response to that induced by infection with live T. cruzi. We found that HKTC immunization is capable of inducing acute cardiac damage, as evidenced by elevated serum cardiac troponin I, and that this damage is associated with the generation of polyantigenic humoral and cell-mediated autoimmunity with similar antigen specificity to that induced by infection with T. cruzi. However, while significant and preferential production of Th1 and Th17-associated cytokines, accompanied by myocarditis, develops in T. cruzi-infected mice, HKTC-immunized mice produce lower levels of these cytokines, do not develop Th1-skewed immunity, and lack tissue inflammation. These results demonstrate that exposure to parasite antigen alone is sufficient to induce autoimmunity and cardiac damage, yet additional immune factors, including a dominant Th1/Th17 immune response, are likely required to induce cardiac inflammation.