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Oral antigen exposure is a powerful, non-invasive route to induce immune tolerance to dietary antigens, and has been modestly successful at prolonging graft survival in rodent models of transplantation. To harness the mechanisms of oral tolerance for promoting long-term graft acceptance, we developed a mouse model where the antigen ovalbumin (OVA) was introduced orally prior to transplantation with skin grafts expressing OVA. Oral OVA treatment pre-transplantation promoted permanent graft acceptance and linked tolerance to skin grafts expressing OVA fused to the additional antigen 2W. Tolerance was donor-specific, as secondary donor-matched, but not third party allografts were spontaneously accepted. Oral OVA treatment promoted an anergic phenotype in OVA-reactive CD4(+) and CD8(+) conventional T cells (Tconvs) and expanded OVA-reactive Tregs pre-transplantation. However, skin graft acceptance following oral OVA resisted partial depletion of Tregs and blockade of PD-L1. Mechanistically, we revealed a role for the proximal gut draining lymph nodes (gdLNs) in mediating this effect, as an intestinal infection that drains to the proximal gdLNs prevented tolerance induction. Our study extends previous work applying oral antigen exposure to transplantation and serves as proof of concept that the systemic immune mechanisms supporting oral tolerance are sufficient to promote long-term graft acceptance.

Limiting CD4

Solid organ transplantation is the preferred treatment for end-stage organ failure. Although transplant recipients take life-long immunosuppressive drugs, a substantial percentage of them still reject their allografts. Strikingly, barrier organs colonized with microbiota have significantly shorter half-lives than non-barrier transplanted organs, even in immunosuppressed hosts. We previously demonstrated that skin allografts monocolonized with the common human commensal Staphylococcus epidermidis (S.epi) are rejected faster than germ-free (GF) allografts in mice because the presence of S.epi augments the effector alloimmune response locally in the graft. Here, we tested whether host immune responses against graft-resident commensal microbes, including S.epi, can damage colonized grafts independently from the alloresponse. Naive hosts mounted an anticommensal T cell response to colonized, but not GF, syngeneic skin grafts. Whereas naive antigraft commensal T cells modestly damaged colonized syngeneic skin grafts, hosts with prior anticommensal T cell memory mounted a post-transplant immune response against graft-resident commensals that significantly damaged colonized, syngeneic skin grafts. Importantly, allograft recipients harboring this host-versus-commensal immune response resisted immunosuppression. The dual effects of host-versus-commensal and host-versus-allograft responses may partially explain why colonized organs have poorer outcomes than sterile organs in the clinic.

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.

Background Solid organ transplant recipients show heterogeneity in the occurrence and timing of acute rejection episodes. Understanding the factors responsible for such variability in patient outcomes may lead to improved diagnostic and therapeutic approaches. Rejection kinetics of transplanted organs mainly depends on the extent of genetic disparities between donor and recipient, but a role for environmental factors is emerging. We have recently shown that major alterations of the microbiota following broad-spectrum antibiotics, or use of germ-free animals, promoted longer skin graft survival in mice. Here, we tested whether spontaneous differences in microbial colonization between genetically similar individuals can contribute to variability in graft rejection kinetics. Results We compared rejection kinetics of minor mismatched skin grafts in C57BL/6 mice from Jackson Laboratory (Jax) and Taconic Farms (Tac), genetically similar animals colonized by different commensal microbes. Female Tac mice rejected skin grafts from vendor-matched males more quickly than Jax mice. We observed prolonged graft survival in Tac mice when they were exposed to Jax mice microbiome through co-housing or fecal microbiota transplantation (FMT) by gastric gavage. In contrast, exposure to Tac mice did not change graft rejection kinetics in Jax mice, suggesting a dominant suppressive effect of Jax microbiota. High-throughput sequencing of 16S rRNA gene amplicons from Jax and Tac mice fecal samples confirmed a convergence of microbiota composition after cohousing or fecal transfer. Our analysis of amplicon data associated members of a single bacterial genus, Alistipes, with prolonged graft survival. Consistent with this finding, members of the genus Alistipes were absent in a separate Tac cohort, in which fecal transfer from Jax mice failed to prolong graft survival. Conclusions These results demonstrate that differences in resident microbiome in healthy individuals may translate into distinct kinetics of graft rejection, and contribute to interpersonal variability in graft outcomes. The association between Alistipes and prolonged skin graft survival in mice suggests that members of this genus might affect host physiology, including at sites distal to the gastrointestinal tract. Overall, these findings allude to a potential therapeutic role for specific gut microbes to promote graft survival through the administration of probiotics, or FMT. Electronic supplementary material The online version of this article (10.1186/s40168-018-0474-8) contains supplementary material, which is available to authorized users.

Solid organ transplantation can treat end-stage organ failure, but the half-life of transplanted organs colonized with commensals is much shorter than that of sterile organs. Whether organ colonization plays a role in this shorter half-life is not known. We have previously shown that an intact whole-body microbiota can accelerate the kinetics of solid organ allograft rejection in untreated colonized mice, when compared with germ-free (GF) or with antibiotic-pretreated colonized mice, by enhancing the capacity of antigen-presenting cells (APCs) to activate graft-reactive T cells. However, the contribution of intestinal versus skin microbiota to these effects was unknown. Here, we demonstrate that colonizing the skin of GF mice with a single commensal, Staphylococcus epidermidis, while preventing intestinal colonization with oral vancomycin, was sufficient to accelerate skin graft rejection. Notably, unlike the mechanism by which whole-body microbiota accelerates skin graft rejection, cutaneous S. epidermidis did not enhance the priming of alloreactive T cells in the skin-draining lymph nodes. Rather, cutaneous S. epidermidis augmented the ability of skin APCs to drive the differentiation of alloreactive T cells. This study reveals that the extraintestinal donor microbiota can affect transplant outcome and may contribute to the shorter half-life of colonized organs.

Background Obesity promotes a state of low-grade inflammation that exacerbates chronic inflammatory diseases, such as asthma and inflammatory bowel disease. In transplantation, the survival of organs transplanted into obese patients is reduced compared with allografts in lean recipients. However, whether this is due to increased alloimmunity remains to be addressed conclusively. Methods We used a mouse model of high-fat diet (HFD)-induced obesity and assessed immune responses to allogeneic stimulation in vitro, allogeneic splenocyte immunization in vivo, and allogeneic heart transplantation. Results Our results indicate that HFD altered the composition and phenotype of splenic antigen-presenting cells that led to their enhanced capacity to stimulate T cells. Immunization with allogeneic splenocytes in vivo resulted in increased alloreactivity, as determined by IFNγ production. Moreover, cardiac allograft rejection in HFD mice was modestly accelerated compared to aged-matched control animals fed a low-fat diet, correlating with enhanced alloreactive T cell function. Conclusions Our results highlight the increased alloresponse triggered by HFD-induced obesity and its negative impact on transplant outcome.

Regular exercise reduces risk of various chronic diseases and can prevent the development and recurrence of cancer, making it a promising non-pharmacological modulator of disease. Yet the effect of regular exercise on solid organ transplant outcome remains uncertain. Using a model of voluntary wheel running exercise and skin transplantation in mice, we hypothesized that exercise strengthens the alloimmune response, leading to an increased rate of rejection. Instead, we found that regular exercise in mice resulted in prolonged graft survival, with mean allograft survival time increasing by almost 50%. We observed this graft survival extension in exercised mice despite evidence of a slightly enhanced alloimmune response, comprised of increased proliferation of alloreactive CD4(+) T cells, as well as increased IFNγ production by these cells. Exercise was not associated with significant changes in numbers of conventional CD4(+) or CD8(+) T cells, NK cells, or Foxp3(+) regulatory T cells. In conclusion, our study suggests that exercise increases skin graft resistance to a similar or slightly higher level of alloimmunity and supports regular exercise as an important beneficial pursuit for transplant recipients.

T cells are essential for immune defenses against pathogens, such that viability of naïve T cells before antigen encounter is critical to preserve a polyclonal repertoire and prevent immunodeficiencies. The viability of naïve T cells before antigen recognition is ensured by IL-7, which drives expression of the prosurvival factor Bcl-2. Quiescent naïve T cells have low basal activity of the transcription factor NF-κB, which was assumed to have no functional consequences. In contrast to this postulate, our data show that basal nuclear NF-κB activity plays an important role in the transcription of IL-7 receptor α-subunit (CD127), enabling responsiveness of naïve T cells to the prosurvival effects of IL-7 and allowing T-cell persistence in vivo. Moreover, we show that this property of basal NF-κB activity is shared by mouse and human naïve T cells. Thus, NF-κB drives a distinct transcriptional program in T cells before antigen encounter by controlling susceptibility to IL-7. Our results reveal an evolutionarily conserved role of NF-κB in T cells before antigenic stimulation and identify a novel molecular pathway that controls T-cell homeostasis.

Acute allograft rejection has often been correlated with Th1 differentiation, whereas transplantation tolerance is frequently associated with induction of regulation. The discovery of the Th17 phenotype has prompted its scrutiny in transplant rejection. Although IL-17 has recently been observed in settings of acute allograft rejection and drives rejection in T-bet-deficient mice that have impaired type 1 T cell responses, there is little evidence of its requirement during acute rejection in wild-type animals. We and others have previously shown that TLR9 signaling by exogenous CpG at the time of transplantation is sufficient to abrogate anti-CD154-mediated acceptance of fully mismatched cardiac allografts. In this study, we investigated the mechanism by which acute rejection occurs in this inflammatory context. Our results indicate that CpG targets recipient hemopoietic cells and that its pro-rejection effects correlate both with prevention of anti-CD154-mediated conversion of conventional CD4+ T cells into induced regulatory T cells and with the expression of IFN-γ and IL-17 by intragraft CD4+ T cells. Moreover, the combined elimination of IL-6 and IL-17 signaling abrogated the ability of CpG to promote acute cardiac allograft rejection. Thus, proinflammatory signals at the time of transplantation can change the quality of the effector immune response and reveal a pathogenic function for IL-6 and IL-17 in wild-type recipients.

Transplantation is the only cure for end-stage organ failure, but without immunosuppression, T cells rapidly reject allografts. While genetic disparities between donor and recipient are major determinants of the kinetics of transplant rejection, little is known about the contribution of environmental factors. Because colonized organs have worse transplant outcome than sterile organs, we tested the influence of host and donor microbiota on skin transplant rejection. Compared with untreated conventional mice, pretreatment of donors and recipients with broad-spectrum antibiotics (Abx) or use of germ-free (GF) donors and recipients resulted in prolonged survival of minor antigen-mismatched skin grafts. Increased graft survival correlated with reduced type I IFN signaling in antigen-presenting cells (APCs) and decreased priming of alloreactive T cells. Colonization of GF mice with fecal material from untreated conventional mice, but not from Abx-pretreated mice, enhanced the ability of APCs to prime alloreactive T cells and accelerated graft rejection, suggesting that alloimmunity is modulated by the composition of microbiota rather than the quantity of bacteria. Abx pretreatment of conventional mice also delayed rejection of major antigen-mismatched skin and MHC class II-mismatched cardiac allografts. This study demonstrates that Abx pretreatment prolongs graft survival, suggesting that targeting microbial constituents is a potential therapeutic strategy for enhancing graft acceptance.

Both type-2 CD4+ Th cells (CD4+Th2) and type-2 innate effector cells play critical roles in generating type-2 immunity that can either be protective against parasitic infection or cause tissue damage in allergy and asthma. How innate effector cells acquire the capacity to produce Th2 cytokines is not entirely known. We previously showed that IL-4 induced differentiation of Th2 cytokine-producing eosinophils. To determine whether other Th2 cytokines can also induce Th2 cytokine-producing capacity in innate effector cells, we cultured bone marrow progenitor cells in the presence of various Th2 cytokines. IL-5, but not IL-13 or IL-25, primed bone marrow progenitor cells to differentiate into robust IL-4-producing cells. The majority of IL-4-producing cells induced by IL-5 were eosinophils. Importantly, IL-5 completely depended on STAT5 to promote IL-4-producing capacity in eosinophils. Thus, our study demonstrates that IL-5 functions as a potent factor that drives bone marrow progenitor cells into IL-4-producing eosinophils.

Infections and TLR signals at the time of transplantation have been shown to prevent the induction of tolerance, but their effect on allografts after tolerance has been established is unclear. We here report that infection with Listeria monocytogenes precipitated the loss of tolerance and the MyD88- and T cell-dependent rejection of accepted cardiac allografts in mice. This loss of tolerance was associated with increases in the numbers of graft-infiltrating macrophages and dendritic cells, as well as CD4(+)FoxP3(-) and CD8(+) T cells. Rejection was also associated with increased numbers of graft-infiltrating alloreactive as well as Listeria-reactive IFNgamma-producing T cells. Rejection of the established grafts required both IL-6 and IFNss, cytokines produced during acute Listeria infection. However, IL-6 and IFNss alone, even when present at higher concentrations than during Listeria infection, were insufficient to break tolerance, while the combination of IL-6 and IFNss was sufficient to break tolerance. These and in vitro observations that IL-6 but not IFNss enhanced T cell proliferation while IFNss but not IL-6 enhanced IFNgamma production support a hypothesis that these cytokines play nonredundant roles. In conclusion, these studies demonstrate that the proinflammatory effects of infections can induce the loss of tolerance and acute rejection of accepted allografts.

Exposure to certain viruses and parasites has been shown to prevent the induction of transplantation tolerance in mice via the generation of cross-reactive memory T cell responses or the induction of bystander activation. Bacterial infections are common in the perioperative period of solid organ allograft recipients in the clinic, and correlations between bacterial infections and acute allograft rejection have been reported. However, whether bacterial infections at the time of transplantation have any effect on the generation of transplantation tolerance remains to be established. We used the Gram-positive intracellular bacterium Listeria monocytogenes (LM) as a model pathogen because its effects on immune responses are well described. Perioperative LM infection prevented cardiac and skin allograft acceptance induced by anti-CD154 and donor-specific transfusion in mice. LM-mediated rejection was not due to the generation of cross-reactive T cells and was largely independent of signaling via MyD88, an adaptor for most TLRs, IL-1, and IL-18. Instead, transplant rejection following LM infection was dependent on the expression of the phagosome-lysing pore former listeriolysin O and on type I IFN receptor signaling. Our results indicate that bacterial exposure at the time of transplantation can antagonize tolerogenic regimens by enhancing alloantigen-specific immune responses independently of the generation of cross-reactive memory T cells.

In many experimental models, heart, pancreas and kidney allografts are accepted long-term following costimulation-targeting therapies, whereas skin, lung and intestine resist the induction of tolerance under the same regimens. We noted that a common feature of the resistant organs is their constant exposure to commensal microbes and hypothesized that these microorganisms may stimulate Toll-like receptors (TLRs), promote alloresponses and prevent tolerance induction. This hypothesis prompts the predictions that TLR engagement at the time of transplantation should avert tolerance to heart allografts in animals treated with costimulation-targeting therapies, whereas inhibition of TLR signaling should promote tolerance to skin allografts under the same conditions. Indeed, engagement of a single TLR was sufficient to prevent anti-CD154-mediated long-term cardiac allograft acceptance and correlated with abolished intragraft recruitment of CD4+/FoxP3+ regulatory T cells and the development of linked-suppression. Conversely, a lack of donor and recipient MyD88-dependent signaling led to successful skin allograft acceptance in anti-CD154-treated animals. Thus, the status of TLR signaling contributes to the resistance versus susceptibility of organs to transplantation tolerance.

Innate effector cells that produce Th2-type cytokines are critical in Th2 cell-mediated immune responses. However, it is not known how these cells acquire the ability to produce Th2 cytokines. IL-4 is a potent inducer that directs differentiation of naive CD4+ T cells into CD4+ Th2 effector cells. To determine whether IL-4 can induce differentiation and expansion of Th2 cytokine-producing innate cells, we used mice whose il-4 gene was replaced by a knock-in green fluorescence protein (gfp) gene. We found that, directly ex vivo, IL-4 increased the number of GFP+ cells in the airway and the lung tissue in an Ag-specific manner. The majority of GFP+ cells were eosinophils, suggesting that IL-4 plays a pivotal role in expanding IL-4-producing eosinophils in vivo. IL-4-producing eosinophils showed some unique features compared with IL-4-producing CD4+ T cells. They exhibited biallelic expression of the il-4 gene when stimulated and were more dominant IL-4- and IL-5-producing cells. Furthermore, we show that IL-4 drove bone marrow progenitor cells to differentiate into Th2 cytokine-producing eosinophils in vitro. These results strongly suggest IL-4 is a potent factor in directing bone marrow progenitor cells to differentiate into Th2 cytokine-producing eosinophils.

We analyzed the responses of several T cell fractions reactive with superantigenic toxins (SAGTs), staphylococcal enterotoxin A (SEA), or Yersinia pseudotuberculosis-derived mitogen (YPM) in mice implanted with mini-osmotic pumps filled with SEA or YPM. In mice implanted with the SEA pump, SEA-reactive Vβ3+CD4+ T cells exhibited a high-level protracted expansion for 30 days, and SEA-reactive Vβ11+CD4+ T cells exhibited a low-level protracted expansion. SEA-reactive CD8+ counterparts exhibited only a transient expansion. A similar difference in T cell expansion was also observed in YPM-reactive T cell fractions in mice implanted with the YPM pump. Vβ3+CD4+ and Vβ11+CD4+ T cells from mice implanted with the SEA pump exhibited cell divisions upon in vitro restimulation with SEA and expressed surface phenotypes as memory T cells. CD4+ T cells from mice implanted with the SEA pump exhibited high IL-4 production upon in vitro restimulation with SEA, which was due to the enhanced capacity of the SEA-reactive CD4+ T cells to produce IL-4. The findings in the present study indicate that, in mice implanted with a specific SAGT, the level of expansion of the SAGT-reactive CD4+ T cell fractions varies widely depending on the TCR Vβ elements expressed and that the reactive CD4+ T cells acquire a capacity to raise a memory response. CD8+ T cells are low responders to SAGTs.

Program death-1 (PD-1), an inhibitory receptor upregulated on T cells upon TCR stimulation, has been shown to attenuate a number of immune responses in vivo, including acute allograft rejection. We tested whether constitutive expression of PD-1 would further inhibit allograft rejection. To this end, we generated transgenic mice expressing T-cell-restricted PD-1 under the control of the Lck proximal promoter and CD2 locus control. PD-1 transgenic (PD-1-Tg) mice did not develop gross abnormalities of thymic development and displayed normal numbers of thymocyte subsets and peripheral T cells. In vitro, PD-1-Tg T cells had reduced proliferative and cytokine secretion capacity upon TCR stimulation and cross-linking of PD-1 resulted in diminished phosphorylation of protein kinase C-theta and Akt, as well as increased activation of the phosphate and tensin homolog. However, only T-cell responses to minor but not major mismatches were reduced in vitro. Similarly, PD-1-Tg mice exhibited prolonged survival of cardiac allografts only in mice transplanted with heart allografts expressing multiple minor mismatches and treated with suboptimal doses of cyclosporine A. We conclude that genetic engineering of T cells to express PD-1 constitutively has only a mild impact on allograft survival.