Prolonged Heart Allograft Survival Research Articles

Immunomodulation of T cell-mediated alloimmunity by proximity to endothelial cells under the mammalian target of rapamycin blockade

Endothelial cells (ECs) are an initial barrier between vascularized organ allografts and the host immune system and are thus well positioned to initiate and influence alloimmune rejection. The mammalian target of rapamycin inhibitor rapamycin is known to inhibit T cell activation and attenuate acute allograft rejection. It also has numerous effects on ECs. We hypothesized that A mammalian target of rapamycin blockade might directly alter EC alloimmunogenicity and reduce alloimmune responses independent of its effects on T cell function. Here we report that rapamycin treatment modulates EC coinhibitory ligand expression and alters cytokine/chemokine production. It alters the EC transcriptome broadly associated with negative regulation of immune responses. Rapamycin-treated ECs suppress EC-specific T cell proliferation independent of programmed cell death 1/programmed death-ligand interaction and inhibit T cells responding to adjacent allogeneic cells in a contact-independent manner via secreted inhibitory mediators above 10 kDa. The T cell hyporesponsiveness induced by rapamycin-pretreated ECs was rescued by exogenous interleukin 2. Preexposing donor hearts to rapamycin improves the effect of B7 costimulation blockade in prolonging heart allograft survival in a major histocompatibility complex-mismatched mouse model. Our results indicate that rapamycin-treated ECs have reduced alloimmunogenicity and created a local, contact-independent environment that limits T cell alloreactivity via anergy induction and improves the efficacy of B7 costimulation blockade.

TIPE2 deficiency prolongs mouse heart allograft survival by facilitating immature DCs-induced Treg generation

It has been reported that deletion of tumor necrosis factor-α-induced protein-8 like 2 (TNFAIP8L2, TIPE2) facilitates the activation of T-cell receptors. However, the role of TIPE2 in T-cell-mediated acute transplant rejection remains unclear. To illustrate the underlying cellular mechanisms, we transplanted BALB/c hearts into C57BL/6 wild-type (WT) or C57BL/6 mice deficient for TIPE2 (TIPE2−/−) and found that TIPE2−/− recipient mice showed significantly prolonged survival of heart allografts and suppressed maturation of CD11c+ dendritic cells (DCs), which largely abolished the activation and proliferation of alloreactive T cells and their cytotoxic activity. TIPE2−/− DCs increased CD4+CD25+Foxp3+CD127− regulatory T cells (Tregs)generation, likely by inhibiting DCs maturation and CD80 and CD86 expression. Administration of anti-CD25 abolished the allograft survival induced by TIPE2 deficiency. Moreover, TIPE2 deficiency increased IL-10 production in T cells and in recipient serum and allografts. Mechanistic studies revealed that TIPE2−/− restrained the maturation of DCs via inhibition of PI3K/AKT phosphorylation during alloantigen stimulation. Taken together, TIPE2 deficiency in recipient mice inhibited acute rejection by increasing Tregs generated by immature DCs. Thus, TIPE2 could be a therapeutic target for suppressing rejection in organ transplantation.

Research Highlights.

Research Highlights.

Chronic CD40L blockade is required for long-term cardiac allograft survival with a clinically relevant CTLA4-Ig dosing regimen.

In de-novo kidney transplantation, the CTLA4-Ig fusion protein belatacept is associated with improved graft function but also an increased risk of acute rejection compared to calcineurin inhibitor therapy. The combination with a second costimulation blocker could potentially improve outcome while avoiding calcineurin inhibitor toxicity. The aim of this study was to define the conditions under which the combination of CTLA4-Ig and CD40L blockade leads to rejection-free permanent graft survival in a stringent murine heart transplantation model. Naïve wild-type or CD40L (CD154) knock-out mice received a fully mismatched BALB/c cardiac allograft. Selected induction and maintenance protocols for CTLA4-Ig and blocking αCD40L monoclonal antibodies (mAB) were investigated. Graft survival, rejection severity and donor-specific antibody (DSA) formation were assessed during a 100-day follow-up period. Administering αCD40L mAb as monotherapy at the time of transplantation significantly prolonged heart allograft survival but did not further improve the outcome when given in addition to chronic CTLA4-Ig therapy (which prolongs graft survival to a median of 22 days). Likewise, chronic αCD40L mAb therapy (0.5mg) combined with perioperative CTLA4-Ig led to rejection in a proportion of mice and extensive histological damage, despite abrogating DSA formation. Only the permanent interruption of CD40-CD40L signaling by using CD40L-/- recipient mice or by chronic αCD40L administration synergized with chronic CTLA4-Ig to achieve long-term allograft survival with preserved histological graft integrity in all recipients without DSA formation. The combination of α-CD40L and CTLA4-Ig works most effectively when both therapeutics are administered chronically.

Targeting fatty acid β-oxidation impairs monocyte differentiation and prolongs heart allograft survival.

Monocytes play an important role in the regulation of alloimmune responses after heart transplantation (HTx). Recent studies have highlighted the importance of immunometabolism in the differentiation and function of myeloid cells. While the importance of glucose metabolism in monocyte differentiation and function has been reported, a role for fatty acid β-oxidation (FAO) has not been explored. Heterotopic HTx was performed using hearts from BALB/c donor mice implanted into C57BL/6 recipient mice and treated with etomoxir (eto), an irreversible inhibitor of carnitine palmitoyltransferase 1 (Cpt1), a rate-limiting step of FAO, or vehicle control. FAO inhibition prolonged HTx survival, reduced early T cell infiltration/activation, and reduced DC and macrophage infiltration to heart allografts of eto-treated recipients. ELISPOT demonstrated that splenocytes from eto-treated HTx recipients were less reactive to activated donor antigen-presenting cells. FAO inhibition reduced monocyte-to-DC and monocyte-to-macrophage differentiation in vitro and in vivo. FAO inhibition did not alter the survival of heart allografts when transplanted into Ccr2-deficient recipients, suggesting that the effects of FAO inhibition were dependent on monocyte mobilization. Finally, we confirmed the importance of FAO on monocyte differentiation in vivo using conditional deletion of Cpt1a. Our findings demonstrate that targeting FAO attenuates alloimmunity after HTx, in part through impairing monocyte differentiation.

Induction of Endotoxin Tolerance Delays Acute Rejection in a Hindlimb Transplantation Model in Rats.

Acute rejection is seen in 85 percent of composite vascular allogeneic transplants despite long-term immunosuppression. Recently, it was reported that the induction of endotoxin tolerance prolonged heart allograft survival in mice. However, it produced side effects in all the animals secondary to the inflammatory reaction. Galactomannan has shown endotoxin tolerance without this side effect in vitro. The authors hypothesized that galactomannan-induced endotoxin tolerance delays acute rejection in vascular allogeneic transplantation without the side effects produced by lipopolysaccharide. Twenty-four rat hindlimb transplants were divided into four groups according to the preconditioning received: control, lipopolysaccharide (0.16 ml/kg), galactomannan 72 hours before (galactomannan-72) (8 ml/kg), and galactomannan 24 hours before (galactomannan-24) (8 ml/kg). Median acute rejection time, weight loss, and diarrheal episodes were monitored. Blood samples were collected at 0, 7, 21, 30, 45, and 60 days. Plasma cytokines (i.e., tumor necrosis factor alpha, interferon gamma), peripheral chimerism, and lymphocyte percentages were analyzed. Median allograft survival was 40 days (range, 40 to 44 days) in the control group, 68 days (range, 61 to 71 days) in the lipopolysaccharide group, and 70 days (range, 69 to 73 days) in both galactomannan groups (p = 0.001). Weight loss was higher in the lipopolysaccharide group (p < 0.001), as was the 83.3 percent rate of diarrheal episodes (control, 0 percent, p = 0.015; galactomannan-72, 0 percent, p = 0.015; and galactomannan-24, 16.7 percent, p = 0.02). Preconditioned rats had higher peripheral blood chimerism (lipopolysaccharide, 2.30 ± 0.13 percent; galactomannan-72, 2.63 ±1.46 percent; and galactomannan-24, 2.47 ± 0.19 percent) compared to the control group (2.06 ± 0.36 percent) (lipopolysaccharide, p = 0.04; galactomannan-72, p = 0.002; and galactomannan-24, p = 0.002). Induction of endotoxin tolerance delays acute rejection in the rat hindlimb transplantation model. Galactomannan preconditioning has no lipopolysaccharide side effects and was equally effective in delaying acute rejection. The contributions of this experimental work are very incipient. Although the use of galactomannan in clinical practice requires more studies to assess its safety, there is no doubt that immunomodulation may be one of the responses that solve the problem of long-term immunosuppression.

In vivo Treg expansion under costimulation blockade targets early rejection and improves long-term outcome

CTLA4Ig has been shown to improve kidney allograft function, but an increased frequency of early rejection episodes poses a major obstacle for more widespread clinical use. The deleterious effect of CTLA4Ig on Treg numbers provides a possible explanation for graft injury. Therefore, we aimed at improving CTLA4Ig's efficacy by therapeutically increasing the number of Tregs. Murine cardiac allograft transplantation (BALB/c to B6) was performed under CTLA4Ig therapy modeled after the clinically approved dosing regimen and Tregs were transferred early or late after transplant. Neither early nor late Treg transfer prolonged allograft survival. Transferred Tregs were traceable in various lymphoid compartments but only modestly increased overall Treg numbers. Next, we augmented Treg numbers in vivo by means of IL2 complexes. A short course of IL2/anti‐IL2‐complexes administered before transplantation reversed the CTLA4Ig‐mediated decline in Tregs. Of note, the addition of IL2/anti‐IL2‐complexes to CTLA4Ig therapy substantially prolonged heart allograft survival and significantly improved graft histology on day 100. The depletion of Tregs abrogated this effect and resulted in a significantly diminished allograft survival. The increase in Treg numbers upon IL2 treatment was associated with a decreased expression of B7 on dendritic cells. These results demonstrate that therapy with IL2 complexes improves the efficacy of CTLA4Ig by counterbalancing its unfavorable effect on Tregs.

Anti-C4d chimeric antigen receptor regulatory T cells suppressed allograft rejection in ABO-incompatible heart transplantation

Abstract Antibody-mediated rejection (ABMR) is one of the main obstacles to successful transplantation, including ABO blood group-incompatible (ABOi) transplantation. Recently, chimeric antigen receptor regulatory T cells (CAR Tregs) were developed to improve the antigen specificity, viability, and suppressive activity of Tregs. C4d deposition is a marker of ABMR and is also found in most ABOi allograft tissues. Based on these findings, we developed anti-C4d CAR Tregs to suppress ABMR in ABOi allografts. Anti-C4d CAR Tregs prepared by retroviral transduction of CAR into CD62L + CD4 + CD25 + Tregs, expressed Foxp3, CD25, CTLA-4, LAP, and GITR to similar extents as non-transduced Tregs. Anti-C4d CAR Tregs were activated by specific binding to C4d and suppressed in vitro T cell proliferation as well as nontransduced Tregs. Furthermore, adoptive transfer of anti-C4d CAR Tregs significantly prolonged mouse ABOi heart allograft survival (P

Imaging Tolerance Induction in Neonatal Mice: Hierarchical Interplay Between Allogeneic Adult and Neonatal Immune Cells.

In Medawar's murine neonatal tolerance model, injection of adult semiallogeneic lymphohematopoietic cells (spleen cells [SC] and bone marrow cells [BMC]) tolerizes the neonatal immune system. An eventual clinical application would require fully allogeneic (allo) cells, yet little is known about the complex in vivo/in situ interplay between those cells and the nonconditioned neonatal immune system. To this end, labeled adult SC and BMC were injected into allogeneic neonates; interactions between donor and host cells were analyzed and modulated by systematic depletion/inactivation of specific donor and host immune effector cell types. Consistent with effector cell compositions, allo-SC and allo-SC/BMC each induced lethal acute graft-versus-host disease, whereas allo-BMC alone did so infrequently. CD8 T cells from SC inoculum appeared naïve, while those of BMC were more memory-like. Age-dependent, cell-type dominance defined the interplay between adult donor cells and the neonatal host immune system such that if the dominant adult effector type was removed, then the equivalent neonatal one became dominant. Depletion of donor/host peripheral T cells protected against acute graft-versus-host disease and prolonged heart allograft survival; peripheral CD8 T-cell depletion together with CD4 T cell-costimulation blockade induced more robust tolerance. This comprehensive study provides direct observation of the cellular interplay between allogeneic donor and host immune systems, adds to our previous work with semiallogeneic donor cells, and provides important insights for robust tolerance induction. Induction of transplant tolerance in neonates will likely require "crowd sourcing" of multiple tolerizing cell types and involve depletion of immune effector cells with costimulation blockade.

ACTH treatment promotes murine cardiac allograft acceptance.

Heart transplantation is the optimal therapy for patients with end-stage heart disease, but its long-term outcome remains inadequate. Recent studies have highlighted the importance of the melanocortin receptors (MCRs) in inflammation, but how MCRs regulate the balance between alloreactive T cells and Tregs, and whether they impact chronic heart transplant rejection, is unknown. Here, we found that Tregs express MC2R, and MC2R expression was highest among all MCRs by Tregs. Our data indicate that adrenocorticotropic hormone (ACTH), the sole ligand for MC2R, promoted the formation of Tregs by increasing the expression of IL-2Rα (CD25) in CD4+ T cells and activation of STAT5 in CD4+CD25+ T cells. ACTH treatment also improved the survival of heart allografts and increased the formation of Tregs in CD28KO mice. ACTH treatment synergized with the tolerogenic effect of CTLA-4–Ig, resulting in long-term survival of heart allografts and an increase in intragraft Tregs. ACTH administration also demonstrated higher prolongation of heart allograft survival in transgenic mouse recipients with both complete KO and conditional KO of PI3Kγ in T cells. Finally, ACTH treatment reduced chronic rejection markedly. These data demonstrate that ACTH treatment improved heart transplant outcomes, and this effect correlated with an increase in Tregs.

Berberine Prolongs Mouse Heart Allograft Survival by Activating T Cell Apoptosis via the Mitochondrial Pathway.

Berberine, which is a traditional Chinese medicine can inhibit tumorigenesis by inducing tumor cell apoptosis. However, the immunoregulatory of effects berberine on T cells remains poorly understood. Here, we first examined whether berberine can prolong allograft survival by regulating the recruitment and function of T cells. Using a major histocompatibility complex complete mismatch mouse heterotopic cardiac transplantation model, we found that the administration of moderate doses (5 mg/kg) of berberine significantly prolonged heart allograft survival to 19 days and elicited no obvious berberine-related toxicity. Compared to that with normal saline treatment, berberine treatment decreased alloreactive T cells in recipient splenocytes and lymph node cells. It also inhibited the activation, proliferation, and function of alloreactive T cells. Most importantly, berberine treatment protected myocardial cells by decreasing CD4+ and CD8+ T cell infiltration and by inhibiting T cell function in allografts. In vivo and in vitro assays revealed that berberine treatment eliminated alloreactive T lymphocytes via the mitochondrial apoptosis pathway, which was validated by transcriptome sequencing. Taken together, we demonstrated that berberine prolongs allograft survival by inducing apoptosis of alloreactive T cells. Thus, our study provides more evidence supporting the potential use of berberine in translational medicine.

Suppressive effects of vitamin C-treated induced-regulatory T cells on heart allograft rejection under vitamin C-deficient or -sufficient conditions.

Foxp3 stability of vitamin C-treated induced-regulatory T cells (V-iTregs) is superior to that of conventional iTregs (C-iTregs). However, the role of V-iTregs in allograft rejection under vitamin C-deficient conditions, such as those seen in humans, remains unclear. We aimed to elucidate the role of vitamin C treatment on generation and maintenance of iTregs from gulo knockout (Gulo-KO) mice as well as wild type (WT) mice, and in vitro and in vivo suppressive effects of V-iTregs on heart allograft rejection in either Gulo-KO or WT recipient mice. Conversion efficiency of iTregs was similar between C- and V-iTregs in both WT and Gulo-KO mice. V-iTregs from WT or Gulo-KO mice showed better in vitro Foxp3 stability than C-iTregs, although there was no difference between WT V-iTregs and Gulo-KO V-iTregs. Furthermore, V-iTregs from WT or Gulo-KO mice suppressed in vitro T cell proliferation better than C-iTregs. Heterotrophic heart transplantation from BALB/c mice to WT or vitamin C-deficient Gulo-KO C57BL/6J mice was performed following adoptive transfer of C- or V-iTregs. V-iTregs as well as C-iTregs prolonged heart allograft survival in WT and Gulo-KO mice. However, there was no difference between the C- and V-iTreg groups. Supplementation of low- or high-dose vitamin C did not induce significant changes in heart allograft survival in Gulo-KO recipients that had received V-iTregs. In conclusion, V-iTregs do not exert better suppressive effects on heart allograft survival than C-iTregs in either WT or vitamin C-deficient recipients.

Senescence of fibroblastic reticular cells in draining lymph nodes: immunoregulation following transplantation

The lymph node (LN) is an intriguing site not only for inducing protective effector immunity but also for inducing tolerance against peripherally encountered antigens such as tissue-specific self-antigens that are regionally drained and through draining lymph nodes (DLNs). The dual functions of DLNs in immunity are attributable at least in part to fibroblastic reticular cells (FRCs), which are a major population of the nonhematopoietic compartment in the LN. In this issue of the JCI, Li, Zhao, and colleagues investigated DLNs in the transplantation setting. The authors demonstrated that, following skin transplantation, the donor mast cell-mediated senescence in FRCs was associated with collagen 1 deposition in DLNs. Systemic administration to mice of FRCs that were expanded ex vivo decreased DLN fibrosis and strengthened the effect of anti-CD40L in prolonging heart allograft survival. These data implicate the DLN as a target for immunomodulatory therapy of transplant rejection.

Lymph node fibroblastic reticular cells deposit fibrosis-associated collagen following organ transplantation.

Although the immune response within draining lymph nodes (DLNs) has been studied for decades, how their stromal compartment contributes to this process remains to be fully explored. Here, we show that donor mast cells were prominent activators of collagen I deposition by fibroblastic reticular cells (FRCs) in DLNs shortly following transplantation. Serial analysis of the DLN indicated that the LN stroma did not return to its baseline microarchitecture following organ rejection and that the DLN contained significant fibrosis following repetitive organ transplants. Using several FRC conditional-knockout mice, we show that induction of senescence in the FRCs of the DLN resulted in massive production of collagen I and a proinflammatory milieu within the DLN. Stimulation of herpes virus entry mediator (HVEM) on FRCs by its ligand LIGHT contributed chiefly to the induction of senescence in FRCs and overproduction of collagen I. Systemic administration of ex vivo-expanded FRCs to mice decreased DLN fibrosis and strengthened the effect of anti-CD40L in prolonging heart allograft survival. These data demonstrate that the transformation of FRCs into proinflammatory myofibroblasts is critically important for the maintenance of a proinflammatory milieu within a fibrotic DLN.

B cell-derived IL-1β and IL-6 drive homeostatic T cell recovery following lymphoablation

Abstract T cell reconstitution after lymphoablation in allograft recipients may lead to acute rejection and poor graft outcome. Using a mouse model of heart transplantation and murine anti-thymocyte globulin (mATG) we showed that while T cell recovery depends on B cells, B cell MHC expression is dispensable. The goal of this study was to investigate the role of B cell-derived IL-1β and IL-6 in T cell homeostatic reconstitution after mATG lymphoablation. The NanoString analysis showed that mATG upregulated IL-1β and IL-6 gene expression in recipient B cells. To validate NanoString findings, IL-1β and IL-6 expression was measured by intracellular flow cytometry. Following heart allograft placement and mATG treatment, B cells are the main source of IL-1β and IL-6, and these cytokines mutually regulate each other’s production. Either treatment with anti-IL-1β mAb or the use of IL-1R1 KO, caspase-1 KO and MyD88 KO recipients diminished both CD4+ and CD8+ T cell recovery along with improved heart allograft survival and decreased donor-reactive IFN-γ producing cells after mATG treatment. Similarly, in vivo IL-6 neutralization with mAb or using IL-6 KO recipients delayed CD4+ and CD8+ T cell recovery, markedly prolonged heart allograft survival and decreased donor-specific T cell responses suggesting that IL-1β and IL-6 are essential for optimal T cell recovery. Adoptive B cell transfers into μMT mice showed that B cell-derived IL-1β and IL-6 supported CD8+ T cells recovery. The absence of either IL-1R or IL-6R on CD8+ T cells markedly impaired their homeostatic recovery following mATG depletion. These findings reveal the essential role of B cell-derived IL-1β and IL-6 during homeostatic T cell expansion in a clinically relevant model of lymphoablation.

Marginal zone B cells induce T cell homeostatic proliferation following antibody-mediated lymphoablation

Abstract Antibody-mediated lymphoablation is commonly used in clinical transplantation. Using mouse model of heart transplantation and murine anti-thymocyte globulin (mATG) lymphoablation, we previously showed that B cell-derived IL-1β and IL-6 are critical mediators of homeostatic T cell proliferation. While marginal zone (MZ) B cells are best known as important sensors and amplifiers of inflammation, their role in depletion-induced lymphopenia has not been previously addressed. The goal of this study was to test the contribution of distinct B cell subsets to proinflammatory cytokine production and T cell homeostatic reconstitution. B6 (H-2b) recipients of BALB/c (H-2d) heart allografts were treated with mATG (d. 0, 4). At d. 8 posttransplant, the proportion of IL-1β, IL-6 and TNF-α secreting cells was significantly higher within MZ vs follicular (FO) B cell subset. Depletion of both FO and MZ B cells with anti-CD20 mAb in addition to mATG (d. -3, 11) significantly diminished T cell reconstitution and prolonged heart allograft survival compared to mATG treatment alone. Notably, specific MZ B cell depletion with anti-VLA4 and anti-LFA1α mAbs (d. -4, -2) resulted in similarly impaired T cells recovery along with improved heart allograft survival. Consistent with these findings, mATG treated Notch2flox/flox CD19Cre/+ mice, which lack MZ B cells but contain normal numbers of FO B cells, had delayed T cell recovery and prolonged heart allograft survival compared to WT recipients. These results show the critical role of MZ B cells in stimulating homeostatic T cell proliferation in lymphopenic heart allograft recipients and suggest that transient removal of MZ B cells may improve the efficacy of lymphoablative induction therapies.

Targeted delivery of immune therapeutics to lymph nodes prolongs cardiac allograft survival.

The targeted delivery of therapeutic drugs to lymph nodes (LNs) provides an unprecedented opportunity to improve the outcomes of transplantation and immune-mediated diseases. The high endothelial venule is a specialized segment of LN vasculature that uniquely expresses peripheral node addressin (PNAd) molecules. PNAd is recognized by MECA79 mAb. We previously generated a MECA79 mAb-coated microparticle (MP) that carries tacrolimus. Although this MP trafficked to LNs, it demonstrated limited therapeutic efficacy in our transplant model. Here, we have synthesized a nanoparticle (NP) as a carrier of anti-CD3, and optimized the conjugation strategy to coat the NP surface with MECA79 mAb (MECA79-anti-CD3-NP) to enhance LN accumulation. As compared with nonconjugated NPs, a significantly higher quantity of MECA79-NPs accumulated in the draining lymph node (DLN). Many MECA79-NPs underwent internalization by T cells and dendritic cells within the LNs. Short-term treatment of murine cardiac allograft recipients with MECA79-anti-CD3-NP resulted in significantly prolonged allograft survival in comparison with the control groups. Prolonged graft survival following treatment with MECA79-anti-CD3-NP was characterized by a significant increase in intragraft and DLN Treg populations. Treg depletion abrogated the prolongation of heart allograft survival. We believe this targeted approach of drug delivery could redefine the methods of administering immune therapeutics in transplantation.

Research Highlights

Ablation of Transcription Factor IRF4 Promotes Transplant Acceptance by Driving Allogenic CD4+ T Cell Dysfunction Jie Wu, Hedong Zhang, Xiamomin Shi, et al. Immunity. 2017;47(6):1114–1128.e6. The transcriptional programs regulating allogenic T-cell responses have not been adequately documented thus far. Here, the authors used a knockout mouse model to demonstrate the importance of interferon regulatory factor 4 (IRF4) in controlling graft rejection. IRF4 is a key transcriptional factor running T-cell responses during and after transplantation. The authors performed several experiments on IRF4 deficient (IRF4−/−) as well as wild type mice by transplanting them with mismatched heart allografts. First, they demonstrated that graft-infiltrating T cells expressed a consistently elevated mean fluorescence intensity of IRF4. Notably, they were able to show that IRF4−/− mice did not reject their allografts. Moreover, all IRF−/− mice survived the observation period of 100 days with intact myocytes and only minimal cellular infiltrates into the graft. The authors then went on transferring either 2 million wild type CD4+ or CD8+ T cells or 20 million IRF4−/− CD4+ or CD8+ T cells into IRF4−/− mice and found that the adaptive transfer of unmanipulated T cells facilitated rejection, whereas the transfer of IRF−/− T cells did not. Furthermore, IRF4−/− mice failed to show increased T cells in their spleen after transplantation, indicating that expansion of alloreactive T cells in IRF4−/− was impaired. Mechanistically, the authors demonstrated the critical role of PD-1 expression by alloantigen-specific T cells for the long-term acceptance of cardiac allografts in IRF−/− mice. Finally, the authors showed that trametinib, a MEK1/2 inhibitor most potently reduced IRF4 expression in activated T cells, inhibited Th1 and Th17 cell differentiation and prolonged heart allograft survival. This intriguing data show the critical role of IRF4 expression on T cells in alloimmunity and indicate the translational potential of trametinib to in improving transplant outcomes. Cutting Edge: Allograft Rejection Is Associated With Weak T-Cell Responses to Many Different Graft Leukocyte-Derived Peptides Burrack AL, Malhotra D, Dileepan T, Osum KC, Swanson LA, Fife BT, Jenkins MK. J Immunol. 2018; 200: 477–482. Allografts are rejected by T lymphocytes that recognize foreign MHC class I and II antigens. The robustness of this T-cell response, however, is not understood. Previous concepts have attributed the rejection process largely on the frequency of T cells that recognize either allogeneic MHC molecules alone or MHC plus-bound peptides. The authors explored the specificity of directly alloreactive T cells, using a peptide-MHC II tetramer-based approach. Tail skin from H2-DM- and CD74-deficient mice were grafted onto the flanks of recipient laboratory-bred strain house mouse. These grafts were rejected after 10 to 12 days. Spleen, lymph node, or blood cells were harvested and stained 7 to 14 days after transplantation. The total number of tetramer-positive cells in the enriched fraction from each mouse was determined, using AccuCheck counting beads. Differences in T-cell expansion and differentiations after transplantation between and across groups were established. CD4+ cells were identified as viable lymphocyte-sized single cells that expressed CD3 or CD90.2. Graft-responsive CD4 T cells expanded an average of 28-fold after transplantation. Mice transplanted with B6 skin also demonstrated a clonal T-cell expansion, indicating that this phenomenon was not limited to autoimmunity-prone NOD mice. In the transplanted mice, tetramer binding sites were examined and sites increased fivefold in NOD mice that had been transplanted with B6 skin. These results demonstrate that directly alloreactive T cells recognize an allogeneic MHC II molecule and a graft leukocyte peptide in the same way that microbe-reactive T cells recognize a syngeneic MHC II molecule and a microbial peptide. The data suggest that the potency of direct alloreactivity is not due to a high frequency of T cells that recognize MHC molecules alone, but rather the consequence of an additional weak activation of T cells, specific for hundreds of different graft leukocyte peptide-allogeneic MHC complexes. These data have important conceptual relevance in understanding basic mechanisms that drive alloimmunity and inflammation.

Dexamethasone-Induced Myeloid-Derived Suppressor Cells Prolong Allo Cardiac Graft Survival through iNOS- and Glucocorticoid Receptor-Dependent Mechanism

How to induce immune tolerance without long-term need for immunosuppressive drugs has always been a central problem in solid organ transplantation. Modulating immunoregulatory cells represents a potential target to resolve this problem. Myeloid-derived suppressor cells (MDSCs) are novel key immunoregulatory cells in the context of tumor development or transplantation, and can be generated in vitro. However, none of current systems for in vitro differentiation of MDSCs have successfully achieved long-term immune tolerance. Herein, we combined dexamethasone (Dex), which is a classic immune regulatory drug in the clinic, with common MDSCs inducing cytokine granulocyte macrophage colony stimulating factor (GM-CSF) to generate MDSCs in vitro. Addition of Dex into GM-CSF system specifically increased the number of CD11b+ Gr-1int/low MDSCs with an enhanced immunosuppressive function in vitro. Adoptive transfer of these MDSCs significantly prolonged heart allograft survival and also favored the expansion of regulatory T cells in vivo. Mechanistic studies showed that inducible nitric oxide sythase (iNOS) signaling was required for MDSCs in the control of T-cell response and glucocorticoid receptor (GR) signaling played a critical role in the recruitment of transferred MDSCs into allograft through upregulating CXCR2 expression on MDSCs. Blockade of GR signaling with its specific inhibitor or genetic deletion of iNOS reversed the protective effect of Dex-induced MDSCs on allograft rejection. Together, our results indicated that co-application of Dex and GM-CSF may be a new and important strategy for the induction of potent MDSCs to achieve immune tolerance in organ transplantation.

Influence of the Novel ATP-Competitive Dual mTORC1/2 Inhibitor AZD2014 on Immune Cell Populations and Heart Allograft Rejection.

Little is known about how new-generation adenosine triphosphate-competitive mechanistic target of rapamycin (mTOR) kinase inhibitors affect immunity and allograft rejection. mTOR complex (C) 1 and 2 signaling in dendritic cells and T cells was analyzed by Western blotting, whereas immune cell populations in normal and heart allograft recipient mice were analyzed by flow cytometry. Alloreactive T cell proliferation was quantified in mixed leukocyte reaction; intracellular cytokine production and serum antidonor IgG levels were determined by flow analysis and immunofluorescence staining used to detect IgG in allografts. The novel target of rapamycin kinase inhibitor AZD2014 impaired dendritic cell differentiation and T cell proliferation in vitro and depressed immune cells and allospecific T cell responses in vivo. A 9-day course of AZD2014 (10 mg/kg, intraperitoneally, twice daily) or rapamycin (RAPA) (1 mg/kg, intraperitoneally, daily) prolonged median heart allograft survival time significantly (25 days for AZD2014, 100 days for RAPA, 9.5 days for control). Like RAPA, AZD2014 suppressed graft mononuclear cell infiltration, increased regulatory T cell to effector memory T cell ratios and reduced T follicular helper and B cells 7 days posttransplant. By 21 days (10 days after drug withdrawal), however, T follicular helper and B cells and donor-specific IgG1 and IgG2c antibody titers were significantly lower in RAPA-treated compared with AZD2014-treated mice. Elevated regulatory T cell to effector memory T cell ratios were maintained after RAPA, but not AZD2014 withdrawal. Immunomodulatory effects of AZD2014, unlike those of RAPA, were not sustained after drug withdrawal, possibly reflecting distinct pharmacokinetics or/and inhibitory effects of AZD2014 on mTORC2.