PD‐1 expression on CD8+ T cells regulates their differentiation within lung allografts and is critical for tolerance induction

Immunological requirements for rejection and tolerance induction differ between various organs. While memory CD8⁺ T cells are considered a barrier to immunosuppression‐mediated acceptance of most tissues and organs, tolerance induction after lung transplantation is critically dependent on central memory CD8⁺ T lymphocytes. Here we demonstrate that costimulation blockade‐mediated tolerance after lung transplantation is dependent on programmed cell death 1 (PD‐1) expression on CD8⁺ T cells. In the absence of PD‐1 expression, CD8⁺ T cells form prolonged interactions with graft‐infiltrating CD11c⁺ cells; their differentiation is skewed towards an effector memory phenotype and grafts are rejected acutely. These findings extend the notion that requirements for tolerance induction after lung transplantation differ from other organs. Thus, immunosuppressive strategies for lung transplant recipients need to be tailored based on the unique immunological properties of this organ.     

B cell–derived IL‐1β and IL‐6 drive T cell reconstitution following lymphoablation

Understanding the mechanisms of T cell homeostatic expansion is crucial for clinical applications of lymphoablative therapies. We previously established that T cell recovery in mouse heart allograft recipients treated with anti‐thymocyte globulin (mATG) critically depends on B cells and is mediated by B cell–derived soluble factors. B cell production of interleukin (IL)‐1β and IL‐6 is markedly upregulated after heart allotransplantation and lymphoablation. Neutralizing IL‐1β or IL‐6 with mAb or the use of recipients lacking mature IL‐1β, IL‐6, IL‐1R, MyD88, or IL‐6R impair CD4⁺ and CD8⁺ T cell recovery and significantly enhance the graft‐prolonging efficacy of lymphoablation. Adoptive co‐transfer experiments demonstrate a direct effect of IL‐6 but not IL‐1β on T lymphocytes. Furthermore, B cells incapable of IL‐1β or IL‐6 production have diminished capacity to mediate T cell reconstitution and initiate heart allograft rejection upon adoptive transfer into mATG treated B cell deficient recipients. 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.     

Recipient Myd88 Deficiency Promotes Spontaneous Resolution of Kidney Allograft Rejection

The myeloid differentiation protein 88 (MyD88) adapter protein is an important mediator of kidney allograft rejection, yet the precise role of MyD88 signaling in directing the host immune response toward the development of kidney allograft rejection remains unclear. Using a stringent mouse model of allogeneic kidney transplantation, we demonstrated that acute allograft rejection occurred equally in MyD88-sufficient (wild-type [WT]) and MyD88^−/− recipients. However, MyD88 deficiency resulted in spontaneous diminution of graft infiltrating effector cells, including CD11b⁻Gr-1⁺ cells and activated CD8 T cells, as well as subsequent restoration of near-normal renal graft function, leading to long-term kidney allograft acceptance. Compared with T cells from WT recipients, T cells from MyD88^−/− recipients failed to mount a robust recall response upon donor antigen restimulation in mixed lymphocyte cultures ex vivo. Notably, exogenous IL-6 restored the proliferation rate of T cells, particularly CD8 T cells, from MyD88^−/− recipients to the proliferation rate of cells from WT recipients. Furthermore, MyD88^−/− T cells exhibited diminished expression of chemokine receptors, specifically CCR4 and CXCR3, and the impaired ability to accumulate in the kidney allografts despite an otherwise MyD88-sufficient environment. These results provide a mechanism linking the lack of intrinsic MyD88 signaling in T cells to the effective control of the rejection response that results in spontaneous resolution of acute rejection and long-term graft protection.     

Adoptive Transfer of Tracer‐Alloreactive CD4+ T Cell Receptor Transgenic T Cells Alters the Endogenous Immune Response to an Allograft

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

Recombinant human ADAMTS13 treatment and anti‐NET strategies enhance skin allograft survival in mice

Enhancing skin allograft longevity lessens the need for new allografts before optimal intervention is available. Reduced activity of ADAMTS13 (an enzyme that cleaves the pro‐thrombotic and proinflammatory von Willebrand factor) and presence of neutrophil extracellular traps (NETs) have been implicated in liver and lung allograft failures. The effect of ADAMTS13 treatment and the impact of NETs on skin allografts, however, remain unexplored. Here, we adopted a murine model of complete mismatch full‐thickness skin transplant by grafting dorsal skin from BALB/c mice to C57BL/6J background mice. Recombinant human ADAMTS13 (rhADAMTS13) treatment of graft recipients increased allograft survival. Western blot and immunofluorescence microscopy revealed the presence of NETs in allografts of vehicle, but surprisingly, not in rhADAMTS13‐treated mice, 3 days after surgery. Recapitulating the observations in mice, NETs were also observed in all the examined allografts from burn patients. Intriguingly, knocking out peptidylarginine deiminase 4 (PAD4, a key enzyme for NET formation) or DNase 1 treatment (which cleaves NETs) also prolonged allograft survival. In summary, rhADAMTS13 lessens inflammation in allografts by reducing NET burden, resulting in enhanced allograft survival. RhADAMTS13 and anti‐NET treatments could be new therapeutic strategies to promote skin allograft longevity and, hence, the survival of patients with severe burns.     

T cell exhaustion is associated with antigen abundance and promotes transplant acceptance

Exhaustion of T cells limits their ability to clear chronic infections or eradicate tumors. Here, in the context of transplant, we investigated whether T cell exhaustion occurs and has a role in determining transplant outcome. A peptide/MHC tetramer‐based approach was used to track exhausted CD8⁺ T cells in a male‐to‐female skin transplant model. Transplant of large whole‐tail skins, but not small tail skins (0.8 cm × 0.8 cm), led to exhaustion of anti‐male tetramer⁺ CD8⁺ T cells and subsequently the acceptance of skin grafts. To study CD4⁺ T cell exhaustion, we used the TCR‐transgenic B6 TEa cells that recognize a major transplant antigen I‐Eα from Balb/c mice. TEa cells were adoptively transferred either into B6 recipients that received Balb/c donor skins or into CB6F1 mice that contained an excessive amount of I‐Eα antigen. Adoptively transferred TEa cells in skin‐graft recipients were not exhausted. By contrast, virtually all adoptively transferred TEa cells were exhausted in CB6F1 mice. Those exhausted TEa cells lost ability to reject Balb/c skins upon further transfer into lymphopenic B6.Rag1^?/? mice. Hence, T cell exhaustion develops in the presence of abundant antigen and promotes transplant acceptance. These findings are essential for better understanding the nature of transplant tolerance.     

Regulatory T Cell Infusion Can Enhance Memory T Cell and Alloantibody Responses in Lymphodepleted Nonhuman Primate Heart Allograft Recipients

The ability of regulatory T cells (Treg) to prolong allograft survival and promote transplant tolerance in lymphodepleted rodents is well established. Few studies, however, have addressed the therapeutic potential of adoptively transferred, CD4⁺CD25⁺CD127^?Foxp3⁺ (Treg) in clinically relevant large animal models. We infused ex vivo–expanded, functionally stable, nonselected Treg (up to a maximum cumulative dose of 1.87 billion cells) into antithymocyte globulin–lymphodepleted, MHC‐mismatched cynomolgus monkey heart graft recipients before homeostatic recovery of effector T cells. The monkeys also received tacrolimus, anti–interleukin‐6 receptor monoclonal antibodies and tapered rapamycin maintenance therapy. Treg administration in single or multiple doses during the early postsurgical period (up to 1 month posttransplantation), when host T cells were profoundly depleted, resulted in inferior graft function compared with controls. This was accompanied by increased incidences of effector memory T cells, enhanced interferon‐γ production by host CD8⁺ T cells, elevated levels of proinflammatory cytokines, and antidonor alloantibodies. The findings caution against infusion of Treg during the early posttransplantation period after lymphodepletion. Despite marked but transient increases in Treg relative to endogenous effector T cells and use of reputed “Treg‐friendly” agents, the host environment/immune effector mechanisms instigated under these conditions can perturb rather than favor the potential therapeutic efficacy of adoptively transferred Treg.     

Bacterial products in donor airways prevent the induction of lung transplant tolerance

Although postoperative bacterial infections can trigger rejection of pulmonary allografts, the impact of bacterial colonization of donor grafts on alloimmune responses to transplanted lungs remains unknown. Here, we tested the hypothesis that bacterial products present within donor grafts at the time of implantation promote lung allograft rejection. Administration of the toll-like receptor 2 (TLR2) agonist Pam₃Cys₄ to Balb/c wild-type grafts triggered acute cellular rejection after transplantation into B6 wild-type recipients that received perioperative costimulatory blockade. Pam₃Cys₄-triggered rejection was associated with an expansion of CD8⁺ T lymphocytes and CD11c⁺CD11b^hiMHC (major histocompatibility complex) class II⁺ antigen-presenting cells within the transplanted lungs. Rejection was prevented when lungs were transplanted into TLR2-deficient recipients but not when MyD88-deficient donors were used. Adoptive transfer of B6 wild-type monocytes, but not T cells, following transplantation into B6 TLR2-deficient recipients restored the ability of Pam₃Cys₄ to trigger acute cellular rejection. Thus, we have demonstrated that activation of TLR2 by a bacterial lipopeptide within the donor airways prevents the induction of lung allograft tolerance through a process mediated by recipient-derived monocytes. Our work suggests that donor lungs harboring bacteria may precipitate an inflammatory response that can facilitate allograft rejection.     

Age‐Dependent Metabolic and Immunosuppressive Effects of Tacrolimus

Immunosuppression in elderly recipients has been underappreciated in clinical trials. Here, we assessed age‐specific effects of the calcineurin inhibitor tacrolimus (TAC) in a murine transplant model and assessed its clinical relevance on human T cells. Old recipient mice exhibited prolonged skin graft survival compared with young animals after TAC administration. More important, half of the TAC dose was sufficient in old mice to achieve comparable systemic trough levels. TAC administration was able to reduce proinflammatory interferon‐γ cytokine production and promote interleukin‐10 production in old CD4⁺ T cells. In addition, TAC administration decreased interleukin‐2 secretion in old CD4⁺ T cells more effectively while inhibiting the proliferation of CD4⁺ T cells in old mice. Both TAC‐treated murine and human CD4⁺ T cells demonstrated an age‐specific suppression of intracellular calcineurin levels and Ca²⁺ influx, two critical pathways in T cell activation. Of note, depletion of CD8⁺ T cells did not alter allograft survival outcome in old TAC‐treated mice, suggesting that TAC age‐specific effects were mainly CD4⁺ T cell mediated. Collectively, our study demonstrates age‐specific immunosuppressive capacities of TAC that are CD4⁺ T cell mediated. The suppression of calcineurin levels and Ca²⁺ influx in both old murine and human T cells emphasizes the clinical relevance of age‐specific effects when using TAC.     

On the impact of hepatitis C virus and heterologous immunity on alloimmune responses following liver transplantation

Virus-induced heterologous immunity is considered a barrier to transplantation tolerance. Yet, hepatitis C (HCV)-infected liver transplant (LT) patients occasionally achieve operational tolerance. We investigated the mechanisms through which HCV infection modulates donor-specific T cell responses following LT and the influence of HCV eradication. We generated T cell lines from HCV-infected LT and non-LT patients before and after HCV eradication and quantified alloreactive responses using cell lines expressing single-HLA class-I antigens in the presence/absence of PD-1/CTLA-4 blockade. HCV-specific CD8⁺ T cells cross-reacted with allogeneic class-I HLA molecules. HCV-positive LT recipients exhibited a higher proportion of CD8⁺ T cells coexpressing inhibitory receptors (PD-1/CTLA4) than HCV-negative LT, and their expression correlated with CXCL10 plasma levels. This resulted in decreased antidonor and third-party proliferative responses, which were significantly reversed by HCV eradication. PD-1/CTLA-4 blockade increased the proportion of HCV-specific CD8⁺ T cells reacting against donor only before viral clearance. In conclusion, HCV infection results in the generation of HCV-specific CD8⁺ T cells capable of reacting against allogeneic HLA molecules. Following LT, this results in a PD-1/CTLA4-dependent decrease in alloimmune responses. Our findings challenge the notion that heterologous immunity is necessarily detrimental in LT and provide an explanation for the association between HCV eradication and immune-mediated allograft damage.     

In Vivo Development of Transplant Arteriosclerosis in Humanized Mice Reflects Alloantigen Recognition and Peripheral Treg Phenotype of Lung Transplant Recipients

Experimentally, regulatory T cells inhibit rejection. In clinical transplantations, however, it is not known whether T cell regulation is the cause for, or an epiphenomenon of, long‐term allograft survival. Here, we study naïve and alloantigen‐primed T cell responses of clinical lung transplant recipients in humanized mice. The pericardiophrenic artery procured from human lung grafts was implanted into the aorta of NODrag^?/?/IL‐2rγc^?/? mice reconstituted with peripheral blood mononuclear cells (PBMCs) from the respective lung recipient. Naïve or primed allogeneic PBMCs procured 21 days post–lung transplantation with or without enriching for CD4⁺CD25^high T cells were used. Transplant arteriosclerosis was assessed 28 days later by histology. Mice reconstituted with alloantigen‐primed PBMCs showed significantly more severe transplant arteriosclerosis than did mice with naïve PBMCs (p = 0.005). Transplant arteriosclerosis was equally suppressed by enriching for autologous naïve (p = 0.012) or alloantigen‐primed regulatory T cells (Tregs) (p = 0.009). Alloantigen priming in clinical lung recipients can be adoptively transferred into a humanized mouse model. Transplant arteriosclerosis elicited by naïve or alloantigen‐primed PBMCs can be similarly controlled by potent autologous Tregs. Cellular therapy with expanded autologous Tregs in lung transplantation might be a promising future strategy.     

Primary Cytomegalovirus Infection Significantly Impacts Circulating T Cells in Kidney Transplant Recipients

Cytomegalovirus (CMV) infection profoundly affects the T cell compartment and is associated with alterations in T cell aging parameters and generation of cytotoxic CD4⁺CD28null T cells. Hence, the effect of a primary CMV infection post–kidney transplantation (KT) on the peripheral T cell compartment was examined. As aging parameters, we determined the T cell differentiation status, T cell receptor excision circle (TREC) content, CD31⁺ naïve T cell numbers and relative telomere length (RTL) pre‐KT and 12 months post‐KT. CMV‐seronegative KT recipients, receiving a kidney from a CMV‐seropositive donor (D+/R?) were compared to D+/R+ KT recipients. Eleven out of the 22 D+/R? KT recipients had CMV viremia post‐KT. They developed CMV‐specific CD4⁺ and CD8⁺ T cells and their T cell compartment shifted towards a more differentiated memory phenotype with expansion of CD4⁺CD28null and CD8⁺CD28null cells. One year post‐KT, the CD8⁺ T cell count was almost doubled compared to nonviremic D+/R? and D+/R+ KT recipients. In addition, the RTL of the CD8⁺ T cell was significantly lower and both the TREC content and CD31⁺ naïve T cell numbers significantly decreased. Moreover, primary CMV infection was associated with a negative impact on glomerular filtration rate. In conclusion, primary CMV infection has a substantial impact on the number and phenotype of peripheral T cells and may negatively affect renal allograft function.

     

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.     

Induction of Alloimmune Tolerance in Heart Transplantation Through Gene Silencing of TLR Adaptors

Toll‐like receptors (TLRs) activate biochemical pathways that evoke activation of innate immunity, which leads to dendritic cell (DC) maturation and initiation of adaptive immune responses that provoke allograft rejection. We aimed to prolong allograft survival by selectively inhibiting expression of the common adaptors of TLR signaling, namely MyD88 and TRIF, using siRNA. In vitro we demonstrated that blocking expression of MyD88 and TRIF led to reduced DC maturation. In vivo treatment of recipients with MyD88 and TRIF siRNA significantly prolonged allograft survival in the BALB/c > C57BL6 cardiac transplant model. Moreover, the combination of MyD88 and TRIF siRNA along with a low dose of rapamycin further extended the allograft survival (88.8 ± 7.1 days). Tissue histopathology demonstrated an overall reduction in lymphocyte interstitium infiltration, vascular obstruction and hemorrhage in mice treated with MyD88 and TRIF siRNA vector plus rapamycin. Furthermore, treatment was associated with an increase in the numbers of CD4⁺CD25⁺FoxP3⁺ regulatory T cells and Th2 deviation. To our knowledge, this study is the first demonstration of prolonging the survival of allogeneic heart grafts through gene silencing of TLR signaling adaptors, highlighting the therapeutic potential of siRNA in clinical transplantation.     

In Vivo Mobilization and Functional Characterization of Nonhuman Primate Monocytic Myeloid‐Derived Suppressor Cells

Increasing evidence from small animal models shows that myeloid‐derived suppressor cells (MDSCs) can play a crucial role in inhibiting allograft rejection and promoting transplant tolerance. We identified CD3^?CD20^?HLA‐DR^?CD14⁺CD33⁺CD11b⁺ cells in peripheral blood of healthy rhesus macaques. These putative monocytic MDSCs constituted 2.1% ± 1.7% of lin^?HLA‐DR^? peripheral blood mononuclear cells. Administration of granulocyte‐macrophage colony‐stimulating factor (CSF) and granulocyte CSF increased their incidence to 5.3% ± 3.4%. The total number of MDSCs that could be flow sorted from a single whole rhesus leukapheresis product was 38 ± 13 × 10⁶ (n = 10 monkeys). Freshly isolated or cryopreserved MDSCs from mobilized monkeys incorporated in cultures of anti‐CD3– and anti‐CD28–stimulated autologous T cells markedly suppressed CD4⁺ and CD8⁺ T cell proliferation and cytokine secretion (interferon γ, IL‐17A). Moreover, these MDSCs enhanced CD4⁺CD25^hiFoxp3⁺ regulatory T cell (Treg) expansion while inhibiting proliferation of activated memory T cells and increasing Treg relative to effector and terminally differentiated memory T cells. Inhibition of arginase‐1, but not inducible nitric oxide synthase activity, partially reversed the inhibitory effect of the MDSCs on CD8⁺ T cell proliferation. Consequently, functional MDSCs can be isolated from nonhuman primates for prospective use as therapeutic cellular vaccines in transplantation.

     

Regulatory T Cells Are Critical to Tolerance Induction in Presensitized Mouse Transplant Recipients Through Targeting Memory T Cells

Memory T cells are a significant barrier to induction of transplant tolerance. However, reliable means to target alloreactive memory T cells have remained elusive. In this study, presensitization of BALB/c mice with C57BL/6 skin grafts generated a large number of OX40⁺CD44^hieffector/memory T cells and resulted in rapid rejection of donor heart allografts. Recognizing that anti‐OX40L monoclonal antibody (mAb) (α‐OX40L) monotherapy prolonged graft survival through inhibition and apoptosis of memory T cells in presensitized recipients, α‐OX40L was added to the combined treatment protocol of LF15–0195 (LF) and anti‐CD45RB (α‐CD45RB) mAb—a protocol that induced heart allograft tolerance in non‐presensitized recipients but failed to induce tolerance in presensitized recipients. Interestingly, this triple therapy restored donor‐specific heart allograft tolerance in our presensitized model that was associated with induction of tolerogenic dendritic cells and CD4⁺CD25⁺Foxp3⁺ T regulatory cells (Tregs). Of note, CD25⁺ T cell depletion in triple therapy recipients prevented establishment of allograft tolerance. In addition, adoptive transfer of donor‐primed effector/memory T cells into tolerant recipients markedly reduced levels of Tregs and broke tolerance. Our findings indicated that targeting memory T cells, by blocking OX40 costimulation in presensitized recipients was very important to expansion of Tregs, which proved critical to development of tolerance.     

Combined Anti‐CD154/CTLA4Ig Costimulation Blockade‐Based Therapy Induces Donor‐Specific Tolerance to Vascularized Osteomyocutaneous Allografts

Tolerance induction by means of costimulation blockade has been successfully applied in solid organ transplantation; however, its efficacy in vascularized composite allotransplantation, containing a vascularized bone marrow component and thus a constant source of donor‐derived stem cells, remains poorly explored. In this study, osteomyocutaneous allografts (alloOMCs) from Balb/c (H2^d) mice were transplanted into C57BL/6 (H2^b) recipients. Immunosuppression consisted of 1 mg anti‐CD154 on day 0, 0.5 mg CTLA4Ig on day 2 and rapamycin (RPM; 3 mg/kg per day from days 0–7, then every other day for 3 weeks). Long‐term allograft survival, donor‐specific tolerance and donor–recipient cell trafficking were evaluated. Treatment with costimulation blockade plus RPM resulted in long‐term graft survival (>120 days) of alloOMC in 12 of 15 recipients compared with untreated controls (median survival time [MST] ≈10.2 ± 0.8 days), RPM alone (MST ≈33 ± 5.5 days) and costimulation blockade alone (MST ≈45.8 ± 7.1 days). Donor‐specific hyporesponsiveness in recipients with viable grafts was demonstrated in vitro. Evidence of donor‐specific tolerance was further assessed in vivo by secondary donor‐specific skin graft survival and third‐party graft rejection. A significant increase of Foxp3⁺ regulatory T cells was evident in tolerant animals. Donor cells populated peripheral blood, thymus, and both donor and recipient bone marrow. Consequently, combined anti‐CD154/CTLA4Ig costimulation blockade‐based therapy induces donor‐specific tolerance in a stringent murine alloOMC transplant model.     

Depletion‐Resistant CD4 T Cells Enhance Thymopoiesis During Lymphopenia

Lymphoablation is routinely used in transplantation, and its success is defined by the balance of pathogenic versus protective T cells within reconstituted repertoire. While homeostatic proliferation and thymopoiesis may both cause T cell recovery during lymphopenia, the relative contributions of these mechanisms remain unclear. The goal of this study was to investigate the role of the thymus during T cell reconstitution in adult allograft recipients subjected to lymphoablative induction therapy. Compared with euthymic mice, thymectomized heart allograft recipients demonstrated severely impaired CD4 and CD8 T cell recovery and prolonged heart allograft survival after lymphoablation with murine anti‐thymocyte globulin (mATG). The injection with agonistic anti‐CD40 mAb or thymus transplantation only partially restored T cell reconstitution in mATG‐treated thymectomized mice. After mATG depletion, residual CD4 T cells migrated into the thymus and enhanced thymopoiesis. Conversely, depletion of CD4 T cells before lymphoablation inhibited thymopoiesis at the stage of CD4^?CD8^?CD44^hiCD25⁺ immature thymocytes. This is the first demonstration that the thymus and peripheral CD4 T cells cooperate to ensure optimal T cell reconstitution after lymphoablation. Targeting thymopoiesis through manipulating functions of depletion‐resistant helper T cells may thus improve therapeutic benefits and minimize the risks of lymphoablation in clinical settings.     

DEPTOR modulates activation responses in CD4+ T cells and enhances immunoregulation following transplantation

DEPTOR is an evolutionarily conserved cell‐intrinsic binding partner of mTOR that functions as a negative regulator of signaling responses. In this study, we show that DEPTOR is expressed within CD4⁺ T cells, and we observed that its relative level of expression modulates differentiation as well as glucose utilization within CD4⁺ T effectors in vitro. Using knock‐in mice, we also find that induced expression of DEPTOR within CD4⁺ T regulatory cells stabilizes Foxp3 expression, shifts metabolism toward oxidative phosphorylation, and increases survival and suppressive function. In vivo, fully MHC mismatched cardiac allograft survival is significantly prolonged in knock‐in recipients and sustained recipient expression of DEPTOR in combination with costimulatory blockade induces long‐term graft survival. Furthermore, we show that the induced expression of DEPTOR in CD4⁺ T effectors fails to inhibit acute allograft rejection. Rather, prolonged survival is dominantly mediated via induced expression and function of DEPTOR within recipient CD4⁺ T regulatory cells. These collective findings identify DEPTOR as a novel protein that functions in CD4⁺ T cells to augment immunoregulation in vitro and in vivo.