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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.

Hafnium-based ferroelectric films, remaining their ferroelectricity down to nanoscale thickness, present a promising application for low-power logic devices and nonvolatile memories. It has been appealing for researchers to reduce the required temperature to obtain the ferroelectric phase in hafnium-based ferroelectric films for applications such as flexible and wearable electronics. In this work, it demonstrates that a remanent polarization (P r) value of >5 μC/cm2 can be obtained in as-deposited Hf0.5Zr0.5O2 (HZO) films that are fabricated by thermal atomic layer deposition (TALD) under low temperature of 250 ℃. The ferroelectric orthorhombic phase (o-phase) in the as-deposited HZO films was detected by scanning transmission electron microscopy (STEM). This low fabrication temperature further extends the compatibility of ferroelectric HZO films to flexible electronics and avoids the cost imposed by following high-temperature annealing treatments.

Intestinal commensals can exert immunomodulatory effects on the host, with beneficial or detrimental consequences depending on underlying diseases. We have previously correlated longer survival of minor mismatched skin grafts in mice with the presence of an intestinal commensal bacterium, Alistipes onderdonkii. In this study, we investigated its sufficiency and mechanism of action. Oral administration of A. onderdonkii strain DSM19147, but not DSM108265, was sufficient to prolong minor mismatched skin graft survival through inhibition of TNF production. Metabolomics and metagenomics comparison between DSM19147 and DSM108265 identified candidate gene products associated with the anti-inflammatory effect of DSM19147. A. onderdonkii DSM19147 can lower inflammation both at steady state and after transplantation and may serve as an anti-inflammatory probiotic beneficial for transplant recipients.

Limiting CD4+T cell responses is important to prevent solid organ transplant rejection. In a mouse model of costimulation blockade-dependent cardiac allograft tolerance, we previously reported that alloreactive CD4+conventional T cells (Tconvs) develop dysfunction, losing proliferative capacity. In parallel, induction of transplantation tolerance is dependent on the presence of regulatory T cells (Tregs). Whether susceptibility of CD4+Tconvs to Treg suppression is modulated during tolerance induction is unknown. We found that alloreactive Tconvs from transplant tolerant mice had augmented sensitivity to Treg suppression when compared with memory T cells from rejector mice and expressed a transcriptional profile distinct from these memory T cells, including down-regulated expression of the transcription factor Special AT-rich sequence-binding protein 1 (Satb1). Mechanistically, Satb1 deficiency in CD4+T cells limited their expression of CD25 and IL-2, and addition of Tregs, which express higher levels of CD25 than Satb1-deficient Tconvs and successfully competed for IL-2, resulted in greater suppression of Satb1-deficient than wild-type Tconvs in vitro. In vivo, Satb1-deficient Tconvs were more susceptible to Treg suppression, resulting in significantly prolonged skin allograft survival. Overall, our study reveals that transplantation tolerance is associated with Tconvs’ susceptibility to Treg suppression, via modulated expression of Tconv-intrinsic Satb1. Targeting Satb1 in the context of Treg-sparing immunosuppressive therapies might be exploited to improve transplant outcomes.

Obstructive sleep apnea-hypopnea syndrome (OSAHS) is a sleep disorder causing cognitive impairments.We use the auditory verbal learning test (AVLT), clock drawing test (CDT), Wechsler intelligence scale for children (WISC) and Montreal cognitive assessment (MoCA) to evaluate the memory and spatial impairments of OSHAS in 6-12 years old children patients with different severity.A total of 137 children of snoring were enrolled following the inclusion criteria of this study. According to the apnea-hypopnea indices (AHI), they were divided into three groups. The AVLT, CDT, WISC and MoCA tests were executed by physicians. The self-rating depression scale (SDS) test was performed for depression screening.Compared with the children in the primary snoring group, the other two groups had higher body mass index (BMI), longer periods of snoring and older age. The AHI, oxygen desaturation index (ODI) and 90% oxygen saturation (TS90%) showed increasing trends whereas the lowest blood oxygen saturation (LSaOAVLT had clinical values for evaluation of impaired memory function in OSAHS children, suggesting a correlation between cognitive impairments and nocturnal hypoxia.

Ferroelectric resistive switching (RS) devices with functional oxide electrodes allow controlled emergent phenomena at an interface. Here, we demonstrate RS polarity reversal due to ferroelectrically induced phase transition at a doped charge transfer insulator interface. For BiFeO3/Ca0.96Ce0.04MnO3 bilayers grown on a NdAlO3 substrate, by applying voltages to a Ca0.96Ce0.04MnO3 bottom electrode, the resistance changes from a high resistance state (HRS) to a low resistance state (LRS) during a positive voltage cycle (0 → 3 → 0 V), and from a LRS to a HRS during a negative voltage cycle (0 → −3 → 0 V). The RS polarity is completely opposite the expected RS behavior in ferroelectric heterostructures induced by polarization reversal. It is proposed that the unique resistance switching polarity is attributed to the band-filling controlled metal-insulator transition in a Ca0.96Ce0.04MnO3 film, triggered by ferroelectric based electrostatic doping. The results address the importance of ferroelectric field effect on the electronic properties of the interfacial system in ferroelectric/complex oxide-based resistive memory devices.

Molecular ferroelectrics (MFs) have been proven to demonstrate excellent properties even comparable to those of inorganic counterparts usually with heavy metals. However, the validation of their device applications is still at the infant stage. The polycrystalline feature of conventionally obtained MF films, the patterning challenges for microelectronics and the brittleness of crystalline films significantly hinder their development for organic integrated circuits, as well as emerging flexible electronics. Here, a large-area flexible memory array is demonstrated of oriented molecular ferroelectric single crystals (MFSCs) with nearly saturated polarization. Highly-uniform MFSC arrays are prepared on large-scale substrates including Si wafers and flexible substrates using an asymmetric-wetting and microgroove-assisted coating (AWMAC) strategy. Resultant flexible memory arrays exhibit excellent nonvolatile memory properties with a low-operating voltage of5 V, i.e., nearly saturated ferroelectric polarization (6.5 µC cm

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.

Patients weaned from immunosuppressive drugs can reject their graft after years of stability, often after an infection. We have modeled this in mice: infection with Listeria monocytogenes (Lm) at the maintenance phase of tolerance can precipitate rejection of a cardiac allograft. Here, we investigated which alloreactive Tconvs may mediate rejection after Lm infection. To track the function of allospecific Tconvs, we seeded tracer TCR75 cells, which recognize I-Ab-restricted donor Kd peptide, into B6 mice prior to Balb/c cardiac transplantation with or without tolerance induction by anti-CD154 + Balb/c splenocytes. ≥35 days post-transplantation, TCR75 cells persisting in tolerant mice developed a PD-1hi CD127lo exhausted-like phenotype and expressed the anergy markers FR4 and CD73. TCR75 cells were dysfunctional, exhibiting 10-fold less recall expansion compared to memory cells and lacked IFNγ and TNF production upon re-stimulation. Intriguingly, TCR75 cells in tolerant hosts remained dysfunctional after Lm infection, suggesting that they may not participate in infection-triggered rejection. Having found that programming T cell dysfunction in tolerance required persistence of the allograft for ~3 weeks, we tested whether T cells specific for alloantigens expressed transiently after transplantation, such as donor MHC Class II, remain functional during tolerance and may mediate rejection after Lm infection. We used tracer TEa cells specific for a donor Class II Eα peptide presented on I-Ab. During tolerance, TEa cells remained PD-1lo and CD73lo, and retained recall expansion and TNF production. We conclude that functional allospecific Tconvs present during tolerance may pose a previously unappreciated risk to the graft.

Background: Although the relevance of humoral immunity for protection against S. aureus skin and soft tissue infections (SSTIs) has been suggested by several animal and human studies, the question of which human antibodies may be protective has so far impeded the development of a safe and effective vaccine. Because most adults have developed certain anti-S. aureus antibodies due to S. aureus colonization or infection, we hypothesized that the titers of antibodies to S. aureus in uninfected controls would differ from those in infected patients and would also differ in infected patients from the time of acute infection to a 40-day convalescent serum. Methods: To test these hypotheses, we measured human antibody levels against a panel of 134 unique antigens comprising the S. aureus surfome and secretome in subjects with active culture-confirmed S. aureus SSTIs (cases) and in controls with no infection, using a novel S. aureus protein microarray. Results: Most S. aureus SSTI patients (n = 60) and controls (n = 142) had antibodies to many of the tested S. aureus antigens. Univariate analysis showed statistically weak differences in the IgG levels to some antigens in the SSTI patient (case) sera compared with controls. Antibody levels to most tested antigens did not increase comparing acute with 40-day serum. Multiple logistic regression identified a rich subset of antigens that, by their antibody levels, together correctly differentiated all cases from all controls. Conclusions: Antibodies directed against S. aureus antigens were present both in patients with S. aureus SSTIs and in uninfected control patients. We found that SSTI patients and controls could be distinguished only based on differences in antibody levels to many staphylococcal surface and secreted antigens. Our results demonstrate that in the studied population, the levels of anti-S. aureus antibodies appear largely fixed, suggesting that there may be some level of unresponsiveness to natural infection.

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.

Alpha diversity of the fecal microbiota is not associated with prolonged skin transplant survival in Jax mice. Figure showing Shannon entropy and inverse Simpson index of fecal microbiota from female Jax and Tac skin transplant recipients A. before and after cohousing, and B. before and after FMT from male Jax or Tac mice. (PDF 788Â kb)

Relative abundance of ASV_5969 in skin transplant donors is associated with prolonged skin transplant survival. Figure showing percent relative abundance of transplant survival-associated ASVs in male Jax and Tac skin transplant donors A. before and after cohousing, B. before and after FMT from male Jax or Tac mice, and C. after FMT from male Tac or Fast Jax mice. (PDF 764Â kb)

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.

Staphylococcus aureus is both a commensal and a pathogen, and USA300, a strain that is usually methicillin-resistant but can sometimes be methicillin-susceptible, has been causing skin and soft tissue infections (SSTIs) in epidemic proportions among otherwise healthy individuals. Although many people are colonized with S. aureus strains, including some with USA300, few of these colonized individuals develop SSTIs. This prompts the hypothesis that infections may develop in individuals with somewhat reduced innate and/or adaptive immune responses to S. aureus, either because prior S. aureus colonization has dampened such responses selectively, or because of more globally reduced immune reactivity. In this study, we analyzed the S. aureus colonization status and PBMC responses to innate and adaptive stimuli in 72 patients with SSTIs and 143 uninfected demographically matched controls. Contrary to the hypothesis formulated, PBMCs from infected patients obtained at the time of infection displayed enhanced innate cytokine production upon restimulation compared with PBMCs from controls, a difference that disappeared after infection resolution. Notably, PBMCs from patients infected with a documented USA300 SSTI displayed greater innate cytokine production than did those from patients infected with documented non-USA300 genotypes. Moreover, colonization with USA300 in infected patients, regardless of their infecting strain, correlated with increased production of IL-10, IL-17A, and IL-22 compared with patients colonized with non-USA300 subtypes. Thus, our results demonstrate that infected patients associated with USA300 either as an infecting strain, or as a colonizing strain, have systemic immune responses of greater magnitude than do those associated with other S. aureus subtypes.

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.

Background There is considerable interest in investigating the role of the microbiota in various diseases, including transplant rejection. Germ-free (GF) and gnotobiotic mice are powerful models for this line of investigation, but performing surgery within the confines of a sterile housing isolator is exceptionally challenging. Development of rigorous protocols to be able to remove axenic mice from their sterile isolator for surgical intervention in a class II biological safety cabinet (BSC) without compromising sterility would give many investigators access to this model and broaden possible studies. However, it is assumed that GF animals will most often become colonized with environmental microbiota on leaving the isolator. In this study, we tested whether applying sterile techniques for animal transport out of the isolator and skin transplantation in a class II BSC could maintain animal sterility. Methods Quantitative polymerase chain reaction of the bacterial 16S ribosomal RNA gene, and cultures in various aerobic and anaerobic conditions were used to probe for bacterial contamination before and after transplantation. Results Of 28 surgeries performed, only 3 mice acquired bacterial contamination coincident with a transient shutdown of the ventilation system in the BSC. Conclusions Our results indicate that skin transplantation can be successfully performed in GF mice using sterile conditions for transport and surgery in a class II BSC, but requires continuous positive airflow. Our approach paves the way to investigating the role of the microbiota in modulating immune responses to skin allografts as a first model of solid organ transplantation in GF mice.

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.

The induction of donor-specific tolerance remains a major goal in the field of transplantation immunology. Therapies that target costimulatory molecules can induce tolerance to heart and pancreatic islet allografts in mouse models, but fail to do so following transplantation of skin or intestinal allografts. We have proposed that organs colonized by commensal bacteria such as skin, lung and intestine may be resistant to such therapies as a result of bacterial translocation at the time of transplantation, which may promote antigen-presenting cell (APC) maturation and the production of pro-inflammatory cytokines, consequently enhancing responses of alloreactive T cells. Our results indicate that the inability to sense signaling by most toll-like receptors (TLRs), as well as by interleukin (IL)-1R and IL-18R, as a result of genetic ablation of myeloid differentiation factor 88 (MyD88) promotes the acceptance of skin allografts. Conversely, TLR signals and infections by a model bacterium, Listeria monocytogenes (LM), at the time of transplantation can prevent the induction of transplantation tolerance. The effects of the TLR9 agonist CpG are MyD88-dependent, while the pro-rejection capacity of LM depends on the intracellular sensing of LM and the production of type I interferon (IFN). Therefore, transiently targeting these innate, pro-inflammatory pathways may have therapeutic value to promote transplantation tolerance.

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.

BackgroundThere is considerable interest in investigating the role of the microbiota in various diseases, including transplant rejection. Germ-free (GF) and gnotobiotic mice are powerful models for this line of investigation, but performing surgery within the confines of a sterile housing isolator i

The microbiota is known to influence local and systemic immune responses. To test whether the microbiota can modulate the strength of an alloresponse, we used a minor mismatched (H-Y) skin transplant mouse model in both germ-free (GF) and antibiotic-pre-treated (Abx) mice. Abx mice showed a marked reduction in bacterial diversity of their intestinal and skin flora. Skin allograft survival more than doubled in either GF or Abx mice compared to controls and colonization with a complex microbial community from conventional mice was sufficient to restore faster rejection kinetics in GF mice. Mechanistically, CFSE-labeled CD4+ anti-H-Y Marilyn T cells displayed a decrease in proliferation following in vivo adoptive transfer into Abx hosts as well as after in vitro culture with antigen presenting cells from Abx mice. Together, these data indicate decreased priming of allogeneic T cells when the diversity of resident bacterial communities is diminished. This effect is not due to a reduction in bacterial load but rather a loss of taxon-specific host-microbe interactions. This study illustrates the profound systemic effects of the microbial environment in transplant recipients and points to the microbiota as a potential therapeutic target for enhancing graft acceptance.

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.

IL-7 and tonic TCR signaling control naïve T cell homeostasis. The transcription factor NF-κB plays a key role during T cell activation, proliferation and survival, but little is known on the role of basal NF-κB in quiescent T cells. Using mice bearing NF-κB impaired T cells (IκBαΔN), we show that basal levels of NF-κB signaling in naïve T cells are required for cell survival both in vivo and in vitro. In vitro, IκBαΔN T cells display reduced survival to IL-7, IL-7-mediated STAT5 phosphorylation and Bcl2 upregulation. Indeed, protein levels of IL-7Rα, but not common γc subunit (CD132), are markedly reduced in IκBαΔN naïve T cells. Assessing mice deficient for NF-κB1 and NF-κB2 subunits, canonical (NF-κB1) but not alternate (NF-κB2) NF-κB pathway is required for IL7Rα expression in T cells. Conversely, constitutive activation of NF-κB in T cells (Lck-Cre x IKKβCA) enhances IL-7Rα expression and IL-7 induced T cell survival. RelA/p65 and NF-κB1/p50 might control transcription through a κB binding region located 3.5 kb upstream of il7r gene, as assessed by EMSA and chromatin immunoprecipitation. Notably, using a NF-κB pharmacological inhibitor we show that NF-κB also controls IL-7Rα expression in human T cells. Taken together, our data suggest that NF-κB regulates il7r transcription both in mice and human, therefore affecting homeostasis of naive T cells.

Methicillin-resistant Staphylococcus aureus (MRSA) causes skin infections in epidemic proportions among otherwise healthy individuals. Previous evidence shows that Th17 cells may be crucial to prevent localized S. aureus infections, as they occur in patients deficient in STAT3 or mice lacking IL-17A and F. In this study, we hypothesized that patients presenting to the emergency room (ER) with skin infections but no known genetic deficiency may have a reduction in Th17 differentiation, either globally or specifically to S. aureus antigens because of inhibitory factors produced by MRSA strains. Sera from 72 patients and 143 controls were collected and cytokines were measured by MultiPlex beads assay. It showed elevated cytokines in patients with skin abscesses on day 0. PBMCs from the same groups were stimulated with MRSA lysates, or anti-CD3, and supernatants were collected at 24h for detection of cytokines by ELISA. The production of IFNγ and IL-17 in response to anti-CD3 was similar in patients and controls, but MRSA stimulation triggered greater secretion of IL-6 and IFNγ in select groups of patients than in controls. These data suggest that our skin infected patients do not have a global defect in Th17 differentiation but may have a specific increase in IFNγ production in response to S. aureus antigens. Future experiments in mice will investigate whether this response may be detrimental to prevent S. aureus infections.

Methicillin-resistant S. aureus (MRSA) causes skin infections in epidemic proportions among otherwise healthy individuals. Several lines of evidence suggest that Th17 cells may be crucial to prevent localized S. aureus infections, as they occur in patients deficient in STAT3 or mice lacking IL-17A and F. In this study, we tested the hypothesis that patients presenting to the emergency room (ER) with skin infections have a reduction in Th17 differentiation, either globally, or specifically to S. aureus antigens because of inhibitory factors produced by MRSA strains. Blood samples from 71 patients presenting to the ER with skin infections and 142 controls were stimulated with MRSA lysates, or with the T cell mitogen OKT3, and supernatants were collected at 24h for detection of cytokines by ELISA. Upon stimulation with MRSA lysates, PBMCs of infected patients displayed increased production of IL-1β and IL-6, innate cytokines that can drive Th17 differentiation. In contrast, production of IFNγ and IL-17 in response to OKT3 was similar in patients and controls. These data suggest that unlike immunodeficient patients with recurrent S. aureus infections, our otherwise healthy ER patients with skin infections do not have a global intrinsic defect in Th17 differentiation. The next set of experiments is addressing the specific T cell recall responses to S. aureus antigens in patients and controls to determine whether patients can mount an anti-S.aureus T cell response in vivo.

NF-κB is an essential transcription factor for the survival of T cells following antigen stimulation, but whether it plays a role in maintaining the survival of unstimulated naïve T cells is not known. We have detected basal NF-κB activity in naïve quiescent T cells and investigated whether it is important in cell survival in the absence of T cell activation. NF-κB-impaired naïve CD4+ T cells showed decreased lifespan in vivo. IL-7 has been shown to be important for naïve T cell survival prompting investigation into whether basal NF-κB activity could regulate IL-7/IL-7R signaling. Our results indicate that NF-κB-impaired naïve T cells are resistant to the pro-survival effects of IL-7 in vitro, and that these T cells express less surface IL-7Rα than wild type T cells. To determine whether NF-κB could regulate IL-7Rα expression by transcription, a putative NF-κB-binding site 3.5 kB upstream of the IL7r translational start site was tested. Nuclear NF-κB from naïve T cells was capable of binding an oligonucleotide probe containing this sequence, as determined by EMSA. A ChIP assay confirmed that the p50 and p65 subunits of NF-κB bound this region in live wild type but not NF-κB-impaired naïve T cells. Finally, re-expression of IL-7Rα after IL-7-mediated degradation was RNA polymerase- and NF-κB-dependent. Together, these results support the idea that basal NF-κB activity plays a role in the survival of quiescent naïve T cells by controlling transcription of the IL7r gene.

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.