Alloreactive Memory Research Articles

Berberine Promotes Induction of Immunological Tolerance to an Allograft via Downregulating Memory CD8+ T-Cells Through Altering the Gut Microbiota.

Emerging evidence has linked the gut microbiota dysbiosis to transplant rejection while memory T-cells pose a threat to long-term transplant survival. However, it's unclear if the gut microbiome alters the formation and function of alloreactive memory T-cells. Here we studied the effects of berberine, a narrow-spectrum antibiotic that is barely absorbed when orally administered, on the gut microbiota, memory T-cells, and allograft survival. In this study, C57BL/6 mice transplanted with islets or a heart from BALB/c mice were treated orally with berberine. Allograft survival was observed, while spleen, and lymph node T-cells from recipient mice were analyzed using a flow cytometer. High-throughput sequencing and qPCR were performed to analyze the gut microbiota. CD8+ T-cells from recipients were cultured with the bacteria to determine potential T-cell memory cross-reactivity to a specific pathogen. We found that berberine suppressed islet allograft rejection, reduced effector CD8+CD44highCD62Llow and central memory CD8+CD44highCD62Lhigh T-cells (TCM), altered the gut microbiota composition and specifically lowered Bacillus cereus abundance. Further, berberine promoted long-term islet allograft survival induced by conventional costimulatory blockade and induced cardiac allograft tolerance as well. Re-colonization of B. cereus upregulated CD8+ TCM cells and reversed long-term islet allograft survival induced by berberine plus the conventional costimulatory blockade. Finally, alloantigen-experienced memory CD8+ T-cells from transplanted recipients rapidly responded to B. cereus in vitro. Thus, berberine prolonged allograft survival by repressing CD8+ TCM through regulating the gut microbiota. We have provided the first evidence that donor-specific memory T-cell generation is linked to a specific microbe and uncovered a novel mechanism underlying the therapeutic effects of berberine. This study may be implicated for suppressing human transplant rejection since berberine is already used in clinic to treat intestinal infections.

Complement-activated human endothelial cells stimulate increased polyfunctionality in alloreactive T cells.

Antibody-mediated deposition of complement membrane attack complexes (MACs) on IFN-γ-primed human endothelial cells (ECs) triggers autocrine/paracrine IL-1β-mediated EC activation and IL-15 transpresentation to alloreactive effector memory T cells (TEM ), changes that enable ECs to increase T cell proliferation and cytokine release. Here, we report the use of single-cell microchip 32-plex proteomics to more deeply assess the functionality of the activated T cells and dependence upon EC-derived signals. Compared to control ECs, MAC-activated human ECs increase both the frequency and degree of polyfunctionality among both CD4+ and CD8+ -proliferated TEM , assessed as secreted proteins. IFN-γ and TNF-α remain the predominant cytokines made by alloreactive TEM , but a few CD4+ TEM also made IL-4 while more CD8+ TEM made perforin and granzyme B. Increased polyfunctionality was attenuated by treatment of the MAC-activated ECs with anti-IL-15 blocking antibody more effectively than IL-1 receptor blockade. The increased polyfunctionality of T cells resulting from interactions with MAC-activated ECs may further link binding of donor-specific antibody to T cell-mediated allograft pathologies.

Complement-activated interferon-γ-primed human endothelium transpresents interleukin-15 to CD8+ T cells.

Alloantibodies in presensitized transplant candidates deposit complement membrane attack complexes (MACs) on graft endothelial cells (ECs), increasing risk of CD8+ T cell-mediated acute rejection. We recently showed that human ECs endocytose MACs into Rab5+ endosomes, creating a signaling platform that stabilizes NF-κB-inducing kinase (NIK) protein. Endosomal NIK activates both noncanonical NF-κB signaling to synthesize pro-IL-1β and an NLRP3 inflammasome to process and secrete active IL-1β. IL-1β activates ECs, increasing recruitment and activation of alloreactive effector memory CD4+ T (Tem) cells. Here, we report that IFN-γ priming induced nuclear expression of IL-15/IL-15Rα complexes in cultured human ECs and that MAC-induced IL-1β stimulated translocation of IL-15/IL-15Rα complexes to the EC surface in a canonical NF-κB-dependent process in which IL-15/IL-15Rα transpresentation increased activation and maturation of alloreactive CD8+ Tem cells. Blocking NLRP3 inflammasome assembly, IL-1 receptor, or IL-15 on ECs inhibited the augmented CD8+ Tem cell responses, indicating that this pathway is not redundant. Adoptively transferred alloantibody and mouse complement deposition induced IL-15/IL-15Rα expression by human ECs lining human coronary artery grafts in immunodeficient mice, and enhanced intimal CD8+ T cell infiltration, which was markedly reduced by inflammasome inhibition, linking alloantibody to acute rejection. Inhibiting MAC signaling may similarly limit other complement-mediated pathologies.

Allogeneic ADSCs Induce the Production of Alloreactive Memory-CD8 T Cells through HLA-ABC Antigens.

We investigated the immunogenicity of allogeneic human adipose-derived mesenchymal stem cells (ADSCs) through the production of alloreactive-CD8 T and -memory CD8 T cells, based on their human leukocyte antigen (HLA) expression. In surface antigen analysis, ADSCs do not express co-stimulatory molecules, but expresses HLA-ABC, which is further increased by exposure to the pro-inflammatory cytokines as well as IFN-γ alone. For immunogenicity analysis, allogeneic ADSCs cultured in xenofree medium (XF-ADSCs) were incubated with the recipient immune cells for allogeneic–antigen stimulation. As a result, XF-ADSCs induced IFN-γ and IL-17A release by alloreactive-CD8 T cells and the production of alloreactive-CD8 T cell through a direct pathway, although they have immunomodulatory activity. In the analysis of alloreactive memory CD8 T cells, XF-ADSCs also significantly induced the production of CFSE-low-CD8 TEM and -CD8 TCM cells. However, HLA-blocking antibodies significantly inhibited the production of CFSE-low memory-CD8 T cells, indicating that HLAs are the main antigens responsible for the development of allogeneic ADSCs’ immunogenicity. These results suggested that HLA surface antigens expressed in allogeneic MSCs should be solved in order to address concerns related to the immunogenicity problem.

HLA-specific memory B-cell detection in kidney transplantation: Insights and future challenges.

Humoral alloimmunity mediated by anti‐human leucocyte antigen (HLA) antibodies is a major challenge in kidney transplantation and impairs the longevity of the transplanted organ. The immunological risk of an individual patient is currently mainly assessed by detection of HLA antibodies in the serum, which are produced by long‐lived bone marrow‐residing plasma cells. However, humoral alloimmunity is complex, and alloreactive memory B cells constitute an additional factor in the interplay of immune cells. These recirculating “silent” cells are responsible for the immunological recall response by differentiating into antibody‐producing cells upon antigen re‐encounter. Historically, due to the lack of appropriate and routinely applicable assays to determine the presence and HLA specificity of alloreactive memory B cells, their contribution to the humoral alloimmune response has clinically often been suspected but could not be determined. In this review, we give an overview of recent advances in techniques to detect alloreactive memory B cells and discuss their strengths and limitations. Furthermore, we summarize experiences with these techniques in alloimmunized individuals and transplant recipients, thereby emphasizing unmet needs to be addressed in future studies.

Contrasting effects of B cell depletion on CD4+ and CD8+ memory T cell responses generated after transplantation.

Alloreactive memory T cells play a key role in transplantation by accelerating allograft rejection and preventing tolerance induction. Some studies using µMT mice, which are constitutionally devoid of B cells, showed that B cells were required for the generation of memory T cells after allotransplantation. However, whether B cell depletion in normal adult mice has the same effect on memory responses by CD4+ and CD8+ T cells activated after transplantation has not been thoroughly investigated. In this study, we tested the effect of anti-CD20 antibody-mediated B cell depletion on CD4+ and CD8+ memory T cell alloresponses after skin transplantation in wild-type mice. We found that B cell depletion prevented the development of memory alloresponses by CD4+ T cells but enhanced that of CD8+ memory T cells. Next, we tested the influence of B cell depletion on hematopoietic chimerism. In OT-II CD4+ anti-OVA TCR transgenic mice sensitized to ovalbumin antigen, B cell depletion also impaired allospecific memory T cell responses and thereby enhanced donor hematopoietic chimerism and T cell deletion after bone marrow transplantation. This study underscores the complexity of the relationships between B and T cells in the generation and reactivation of different memory T cell subsets after transplantation.

Memory T Cells in Transplantation: Old Challenges Define New Directions

Immunologic memory is the ability of adaptive immune system to quickly and specifically recognize previously encountered antigens and initiate an effector response. Alloreactive memory cells can mount rapid and robust responses to the transplanted organ resulting in allograft injury. Thus preexisting humoral or cellular memory alloresponses are typically associated with poor graft outcomes in experimental and clinical transplantation. While both B and T lymphocytes exhibit memory responses, this review discusses recent updates on the biology of memory T cells and their relevance to the field of transplantation. Three major areas of focus are the emergence and characterization of tissue resident memory T cells, manipulation of T cell metabolic pathways, and the latest promising approaches to targeting detrimental T cell memory in the settings of organ transplantation.

Pretransplant Donor-specific IFNγ ELISPOT as a Predictor of Graft Rejection: A Diagnostic Test Accuracy Meta-analysis.

Pretransplant interferon-γ enzyme-linked immunospot (IFN-γ ELISPOT) has been proposed as a tool to quantify alloreactive memory T cells and estimate the risk of acute rejection (AR) after kidney transplantation, but studies have been inconclusive so far. We performed a meta-analysis to evaluate the association between pretransplant IFN-γ ELISPOT and AR and assess its predictive accuracy at the individual level. We estimated the pooled summary of odds ratio for AR and the joined sensitivity and specificity for predicting AR using random-effects and hierarchical summary receiver-operating characteristic models. We used meta-regression models with the Monte Carlo permutation method to adjust for multiple tests to explain sensitivity and specificity heterogeneity across studies. The meta-analytic estimates of sensitivity and specificity were used to calculate positive and negative predictive values across studies. The analysis included 12 studies and 1181 patients. IFN-γ ELISPOT was significantly associated with increased AR risk (odds ratio: 3.29; 95% confidence interval (CI), 2.34-4.60); hierarchical summary receiver operating characteristic jointly estimated sensitivity and specificity values were 64.9% (95% CI, 53.7%-74.6%) and 65.8% (95% CI, 57.4%-73.5%), respectively, with moderate heterogeneity across studies. After adjusting for multiple testing, meta-regression models showed that thymoglobulin induction, recipient black ethnicity, living versus deceased donors, and geographical location did not affect sensitivity or specificity. Because of the varying AR incidence of the studies, positive and negative predictive values ranged between 16%-60% and 70%-95%, respectively. Pretransplant IFN-γ ELISPOT is significantly associated with increased risk of AR but provides suboptimal predictive ability at an individual level. Prospective randomized clinical trials are warranted.

Divergent TCR-Initiated Calcium Signals Govern Recruitment versus Activation of Human Alloreactive Effector Memory T Cells by Endothelial Cells.

Early human allograft rejection can be initiated when circulating human host versus graft Ag-specific CD8 and CD4 effector memory T cells directly recognize MHC class I and II, respectively, expressed on the luminal surface by endothelium lining graft blood vessels. TCR engagement triggers both graft entry (TCR-driven transendothelial migration or TEM) and production of proinflammatory cytokines. Both TCR-driven TEM and cytokine expression are known to depend on T cell enzymes, myosin L chain kinase, and calcineurin, respectively, that are activated by cytoplasmic calcium and calmodulin, but whether the sources of calcium that control these enzymes are the same or different is unknown. Using superantigen or anti-CD3 Ab presented by cultured human dermal microvascular cells to freshly isolated peripheral blood human effector memory T cells under conditions of flow (models of alloantigen recognition in a vascularized graft), we tested the effects of pharmacological inhibitors of TCR-activated calcium signaling pathways on TCR-driven TEM and cytokine expression. We report that extracellular calcium entry via CRAC channels is the dominant contributor to cytokine expression, but paradoxically these same inhibitors potentiate TEM. Instead, calcium entry via TRPV1, L-Type Cav, and pannexin-1/P2X receptors appear to control TCR-driven TEM. These data reveal new therapeutic targets for immunosuppression.

Histological picture of antibody-mediated rejection without donor-specific anti-HLA antibodies: Clinical presentation and implications for outcome.

In this cohort study (n=935 transplantations), we investigated the phenotype and risk of graft failure in patients with histological criteria for antibody-mediated rejection (ABMR) in the absence of circulating donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA), and compared this to patients with definite ABMR and HLA-DSA-positivity. The histological picture did not differ between HLA-DSA-positive (n=85) and HLA-DSA-negative (n=123) cases of ABMR histology, apart from increased complement split product 4d (C4d) deposition in the peritubular capillaries in HLA-DSA-positive cases. Histology of ABMR without HLA-DSA was more transient than DSA-positive ABMR, and patients with ABMR histology without HLA-DSA had graft survival superior to that of HLA-DSA-positive patients, independent of concomitant T cell-mediated rejection (38.2%) or borderline changes (17.9%). Multivariate analysis showed that the risk of graft failure was not higher in patients with histological picture of ABMR (ABMRh ) in the absence of HLA-DSA, compared to patients without ABMRh . Despite an association between C4d deposition and HLA-DSA-positivity, using C4d deposition as alternative for the DSA criterion in the diagnosis of ABMR, as proposed in Banff 2017, did not contribute to the prognosis of graft function and graft failure. We concluded that biopsies with ABMRh but without detectable HLA-DSA represent a distinct, often transient phenotype with superior allograft survival.

C5aR1 regulates migration of suppressive myeloid cells required for costimulatory blockade-induced murine allograft survival.

Costimulatory blockade-induced murine cardiac allograft survival requires intragraft accumulation of CD11b+ Ly6Clo Ly6G- regulatory myeloid cells (Mregs) that expand regulatory T cells (Tregs) and suppress effector T cells (Teffs). We previously showed that C5a receptor (C5aR1) signaling on T cells activates Teffs and inhibits Tregs, but whether and/or how C5aR1 affects Mregs required for transplant survival is unknown. Although BALB/c hearts survived >60days in anti-CD154 (MR1)-treated or cytotoxic T-lymphocyte associated protein 4 (CTLA4)-Ig-treated wild-type (WT) recipients, they were rejected at ~30days in MR1-treated or CTLA4-Ig-treated recipients selectively deficient in C5aR1 restricted to myeloid cells (C5ar1fl/fl xLysM-Cre). This accelerated rejection was associated with ~2-fold more donor-reactive T cells and ~40% less expansion of donor-reactive Tregs. Analysis of graft-infiltrating mononuclear cells on posttransplant day 6 revealed fewer Ly6Clo monocytes in C5ar1fl/fl xLysM-Cre recipients. Expression profiling of intragraft Ly6Clo monocytes showed that C5aR1 deficiency downregulated genes related to migration/locomotion without changes in genes associated with suppressive function. Cotransfer of C5ar1fl/fl and C5ar1fl/fl xLysM-Cre myeloid cells into MR1-treated allograft recipients resulted in less accumulation of C5ar1-/- cells within the allografts, and in vitro assays confirmed that Ly6Chi myeloid cells migrate to C5a/C5aR1-initiated signals. Together, our results newly link myeloid cell-expressed C5aR1 to intragraft accumulation of myeloid cells required for prolongation of heart transplant survival induced by costimulatory blockade.

Belatacept and Autoimmune Adverse Events

Belatacept is widely used to prevent rejection in kidney transplants with its excellent tolerability profile. Here, we report a severe psoriasis manifesting in a kidney transplant recipient raising the possibility of belatacept-induced autoimmunity. Our patient was a 42-year-old man with an end-stage renal disease secondary to anti neutrophil cytoplasmic antibody-associated vasculitis. In January 2014, more than 2 years after receiving rituximab as treatment, he underwent primary renal transplantation (with 1 B mismatch). Immunosuppressive induction therapy consisted of anti-IL2R (basiliximab), whereas maintenance therapy consisted of mycophenolic acid and tacrolimus delivered at their standard regular dosages. On day 10 postoperation, a graft biopsy revealed marked thrombotic microangiopathy. Tacrolimus toxicity was suspected and tacrolimus was substituted with belatacept (5 mg/kg monthly) with an excellent result (serum creatinine, 100 μmol/L). In September 2014, physical examination revealed psoriatic plaques, confirmed by skin biopsy. Topical treatment with calcipotriol was introduced. In July 2016, because of a deterioration in his skin condition, we decided to stop mycophenolic acid and commence ciclosporin. Despite an initial improvement, the psoriasis progressively deteriorated (Figure 1). Therapeutic strategies were constrained, because in this transplant recipient, phototherapy and most biologic therapies were contraindicated. In February 2017, we decided to stop belatacept and reintroduce mycophenolic acid. A few weeks after its discontinuation, the plaques progressively disappeared, and complete resolution was finally observed.FIGURE 1: Diffuse well-demarcated psoriasis form plaques.Several factors suggest a casual association between this exposure and the development of the lesions: (i) our patient had neither a personal nor familial history of psoriasis; (ii) its onset at 7 months of costimulation blockade could be plausible (see below); (iii) rapid improvement was observed after its removal; (iv) a biological plausibility to explain this effect can be found. Psoriasis has been reported in patients with rheumatoid arthritis treated with abatacept.1 Regarding the pooled safety data of the BENEFIT trials, a similar frequency of autoimmune-related adverse events (AEs) between belatacept-treated recipients and ciclosporin-treated controls has been observed. However, psoriasis was the most common autoimmune disorder to manifest in belatacept-treated patients, whereas hyperthyroidism was the most frequent to manifest in controls. In the susceptible patient, belatacept could dysregulate immunity in different ways. First, by inhibiting regulatory T (Treg) cells either quantitatively or qualitatively.2 Indeed, abatacept and belatacept are both CTLA4-Ig fusion proteins antagonizing CD80-86, the ligand for CD28 (essential to Treg generation and maintenance) and CTLA-4 (crucial for Treg functions). Second, belatacept blocks the CTLA-4 inhibitory pathway on memory T cells. Belatacept-resistant rejection is thought to be due to alloreactive memory T cells, which are less dependent on CD28 signaling for their activation and are regulated by CTLA-4 intrinsic mechanisms.3 Krummey and Cheeseman4 demonstrated that interleukin 17-secreting T helper (Th17) cells are especially sensitive to CTLA-4 coinhibition and resistant to costimulation blockade. Psoriasis and inflammatory bowel disease, for which a belatacept-induced case has been recently reported,5 are both Th17 mediated. Pharmacovigilance in the “real life” transplant setting often yields new safety concerns about rare drug-related adverse events. Transplant community physicians should be aware that the use of belatacept after kidney transplantation may be implicated in psoriasis occurrence. Patient data were obtained from the DIVAT clinical cohort (divat.fr) ethically approved: N8CNIL 891735 version 2, August 2004.

T Cell Repertoire Maturation Induced by Persistent and Latent Viral Infection Is Insufficient to Induce Costimulation Blockade Resistant Organ Allograft Rejection in Mice.

CD28:CD80/86 pathway costimulation blockade (CoB) with the CD80/86-specific fusion protein CTLA4-Ig prevents T cell-mediated allograft rejection in mice. However, in humans, transplantation with CoB has been hampered by CoB-resistant rejection (CoBRR). CoBRR has been attributed in part to pathogen-driven T cell repertoire maturation and resultant heterologous alloreactive memory. This has been demonstrated experimentally in mice. However, prior murine models have used viral pathogens, CoB regimens, graft types, and/or antigen systems atypically encountered clinically. We therefore sought to explore whether CoBRR would emerge in a model of virus-induced memory differentiation designed to more closely mimic clinical conditions. Specifically, we examined mouse homologs of clinically prevalent viruses including murine polyomavirus, cytomegalovirus, and gammaherpesvirus 68 in the presence of clinically relevant maintenance CoB regimens using a fully MHC-mismatched, vascularized allograft model. Infected mice developed a significant, sustained increase in effector memory T cells consistent with that seen in humans, but neither developed heterologous alloreactivity nor rejected primarily vascularized heterotopic heart transplants at an increased rate compared with uninfected mice. These results indicate that memory acquisition alone is insufficient to provoke CoBRR and suggest that knowledge of prior latent or persistent viral infection may have limited utility in anticipating heterologous CoB-resistant alloimmunity.

Interferon-γ converts human microvascular pericytes into negative regulators of alloimmunity through induction of indoleamine 2,3-dioxygenase 1.

Early acute rejection of human allografts is mediated by circulating alloreactive host effector memory T cells (TEM). TEM infiltration typically occurs across graft postcapillary venules and involves sequential interactions with graft-derived endothelial cells (ECs) and pericytes (PCs). While the role of ECs in allograft rejection has been extensively studied, contributions of PCs to this process are largely unknown. This study aimed to characterize the effects and mechanisms of interactions between human PCs and allogeneic TEM. We report that unstimulated PCs, like ECs, can directly present alloantigen to TEM, but while IFN-γ-activated ECs (γ-ECs) show increased ability to stimulate alloreactive T cells, IFN-γ-activated PCs (γ-PCs) instead suppress TEM proliferation but not cytokine production or signaling. RNA sequencing analysis of PCs, γ-PCs, ECs, and γ-ECs reveal induction of indoleamine 2,3-dioxygenase 1 (IDO1) in γ-PCs to significantly higher levels than in γ-ECs that correlates with tryptophan depletion in vitro. Consistently, shRNA knockdown of IDO1 markedly reduces γ-PC-mediated immunoregulatory effects. Furthermore, human PCs express IDO1 in a skin allograft rejection humanized mouse model and in human renal allografts with acute T cell-mediated rejection. We conclude that immunosuppressive properties of human PCs are not intrinsic but instead result from IFN-γ-induced IDO1-mediated tryptophan depletion.

Refinement of humoral immune monitoring in kidney transplantation: the role of "hidden" alloreactive memory B cells.

The advent of novel sensitive assays assessing circulating anti-human leukocyte antigen (HLA) antibodies has allowed recognizing humoral alloimmunity as the main immune-mediated mechanism responsible for allograft rejection and graft loss in kidney transplantation. However, current immune-monitoring techniques, exclusively focusing on circulating anti-HLA antibodies, may underestimate the magnitude of humoral immune response as they exclude the memory B-cell (mBC) pool. Different biological compartments are involved in the intricate mechanisms triggering humoral alloimmune responses even in absence of detectable circulating alloantibodies. Recent studies in animal models as well as in clinical kidney transplantation have shown the key role of this B-cell subset triggering allograft rejection, thus emphasizing the value of recognizing antidonor mBC both as a biomarker of allosensitization and as therapeutic targets. Therefore, considerable efforts are being made among the transplant research community to better understand the role, hierarchy, and impact of mBC in the context of organ transplantation. In this review article, we provide a deep insight into the biology of mBC as well as main evidence of their role orchestrating allograft rejection. Also, we provide a thorough description of main immune-monitoring tools aiming at tracking mBC and a rational for their potential use to refine current humoral immune-risk assessment in kidney transplantation.

High PD-L1/CD86 MFI ratio and IL-10 secretion characterize human regulatory dendritic cells generated for clinical testing in organ transplantation

Human regulatory dendritic cells (DCreg) were generated from CD14 immunobead-purified or elutriated monocytes in the presence of vitamin D3 and IL-10. They exhibited similar, low levels of costimulatory CD80 and CD86, but comparatively high levels of co-inhibitory programed death ligand-1 (PD-L1) and IL-10 production compared to control immature DC (iDC). Following Toll-like receptor 4 ligation, unlike control iDC, DCreg resisted phenotypic and functional maturation and further upregulated PD-L1:CD86 expression. Whereas LPS-stimulated control iDC (mature DC; matDC) secreted pro-inflammatory tumor necrosis factor but no IL-10, the converse was observed for LPS-stimulated DCreg. DCreg weakly stimulated naïve and memory allogeneic CD4+ and CD8+ T cell proliferation and IFNγ, IL-17A and perforin/granzyme B production in MLR. Their stimulatory function was enhanced however, by blocking PD-1 ligation. High-throughput T cell receptor (TCR) sequencing revealed that, among circulating T cell subsets, memory CD8+ T cells contained the most alloreactive TCR clonotypes and that, while matDC expanded these alloreactive memory CD8 TCR clonotypes, DCreg induced more attenuated responses. These findings demonstrate the feasibility of generating highly-purified GMP-grade DCreg for systemic infusion, their influence on the alloreactive T cell response, and a key mechanistic role of the PD1 pathway.

High Eomesodermin Expression Correlates With Human and Non-Human Primate Alloreactive Effector CD8 + T Cells

Introduction: The transcription factors Eomesodermin (Eomes) and T-bet are thought to play critical roles in CD8+ effector memory T cell development. [1][2] However, the role of Eomes in alloreactive CD8+T cells is not well known. We have previously shown that reduced Eomes expression and upregulation of CTLA4 by CD8+T cells in renal allograft recipient monkeys is associated with attenuation of donor-reactive memory T cell responses and prolonged graft survival.[3] We hypothesized that Eomes expression might be associated with enhanced effector/cytotoxic function by human and non-human primate (NHP) alloreactive CD8+T cells. Methods: Human and NHP Tcells were purified from peripheral blood mononuclear cells (PBMC) and then co-cultured with irradiated T-cell depleted allogeneic PBMC. Allo-activated CD8+T cells were evaluated by flow cytometry for expression of Eomes, T-bet and CTLA4. Proliferation, intracellular cytokines (IL2, INFγ and TNFα), and Granzyme B following allo-activation were also assessed. Results: Non-activated human Eomeshi CD8+ T cells, exhibited higher T-bet expression and comprised more IFNγ+, TNFα+ and granzyme-B+, but less IL-2+ cells than Eomes-/lo cells. Non-activated rhesus Eomeshi CD8+T cells, although exhibited low T-bet expression, comprised more IFNγ+ and IL-2+, equivalent TNFα+, but less granzyme-B+ cells than Eomes-/lo cells. After allo-stimulation human Eomeshi population comprised more IFNγ+, TNFα+ and granzyme-B+ cells and a variable proportion of IL-2+ cells. Rhesus Eomeshi cells comprised more IFNγ+ and TNFα+, but less IL-2+, and a variable proportion of granzyme-B+ cells. Human EomeshiT-bethi CD8+T cells exhibited the highest incidence of IFNγ+TNFα+ cells, while rhesus EomeshiT-betlo CD8+T cells exhibited the highest incidence of IFNγ+TNFα+ cells. Following allo-activation there was an inverse correlation between CTLA4 and Eomes expression in NHP but not in human CD8+ T cells.FIGURE 1: Cytokine production by Eomeshi and Eomes-/lo CD8+ T cells after allo-activation.FIGURE 2: T-bet expression by alloactivated Eomeshi and Eomes-/lo human and rhesus CD8+T cells.Conclusion: These data suggest that alloreactive EomeshiCD8+T cells may potentially correlate with anti-donor effector Tmem after transplantation and that phenotypic and functional differences between human and NHP EomeshiCD8+T cells need to be taken into consideration. Eomes may be an important novel biomarker for distinguishing potent alloreactive effector memory T cells. Yoshihiro Ono. Alan F. Zahorchak. Bala Ramaswami.

Interruption of OX40L signaling prevents costimulation blockade-resistant allograft rejection.

The potential of costimulation blockade to serve as a novel transplant immunosuppression strategy has been explored for over 20 years, culminating in the recent clinical approval of belatacept for renal transplant patients. Despite improving long-term graft function and survival compared with calcineurin inhibitors, clinical acceptance of belatacept has been hindered by elevated rates of acute rejection. We examined the signaling pathways required to activate costimulation blockade-resistant alloreactive T cells and identified the OX40/OX40L secondary costimulatory pathway as a promising target. We next sought to improve the clinical efficacy of traditional costimulation blockade using belatacept by coupling it with anti-OX40L. Using a murine transplant model, we demonstrate that combined blockade enhances the suppression of alloreactive T cell proliferation and effector functions including both cytokine release and cytotoxic degranulation. We also show that anti-OX40L may be particularly useful in targeting alloreactive memory T cell responses that are relatively unaffected by traditional costimulation blockade regimens. Finally, we translated this therapy to a clinically relevant nonhuman primate renal transplant model, validating the efficacy of this regimen in a potentially novel steroid- and calcineurin inhibitor-free immunosuppression regimen.

Role of Memory T Cells in Allograft Rejection and Tolerance.

Memory T cells are characterized by their low activation threshold, robust effector functions, and resistance to conventional immunosuppression and costimulation blockade. Unlike their naïve counterparts, memory T cells reside in and recirculate through peripheral non-lymphoid tissues. Alloreactive memory T cells are subdivided into different categories based on their origins, phenotypes, and functions. Recipients whose immune systems have been directly exposed to allogeneic major histocompatibility complex (MHC) molecules display high affinity alloreactive memory T cells. In the absence of any prior exposure to allogeneic MHC molecules, endogenous alloreactive memory T cells are regularly generated through microbial infections (heterologous immunity). Regardless of their origin, alloreactive memory T cells represent an essential element of the allograft rejection process and a major barrier to tolerance induction in clinical transplantation. This article describes the different subsets of alloreactive memory T cells involved in transplant rejection and examine their generation, functional properties, and mechanisms of action. In addition, we discuss strategies developed to target deleterious allospecific memory T cells in experimental animal models and clinical settings.

What's Hot, What's New From the 2016 American Transplant Congress.

From June 11-15, 2016 the American Transplant Congress, the joint meeting of the American Society of Transplantation and the American Society of Transplant Surgeons, held its annual meeting in Boston, MA. The meeting, attended by 5200 registrants, included pre-meeting conferences, focused topic sessions, and hundreds of high-quality presentations from the transplant field. This meeting report highlights key findings from specific basic science, translational, and clinical research presentations deemed to have notable impact in thematic areas. In particular, there were a number of transformative studies indicating important advances in the understanding of alloimmunity, chronic rejection, tolerance, and organ-specific outcomes. Many of these results are discussed in the context of the published literature to showcase rapid advances in the transplant field.