Aquaporin 4 blockade improves survival of murine heart allografts subjected to prolonged cold ischemia

Prolonged cold ischemia storage (CIS) is a leading risk factor for poor transplant outcome. Existing strategies strive to minimize ischemia–reperfusion injury in transplanted organs, yet there is a need for novel approaches to improve outcomes of marginal allografts and expand the pool of donor organs suitable for transplantation. Aquaporins (AQPs) are a family of water channels that facilitate homeostasis, tissue injury, and inflammation. We tested whether inhibition of AQP4 improves the survival of fully MHC‐mismatched murine cardiac allografts subjected to 8 hours of CIS. Administration of a small molecule AQP4 inhibitor during donor heart collection and storage and for a short‐time posttransplantation improves the viability of donor graft cells, diminishes donor‐reactive T cell responses, and extends allograft survival in the absence of other immunosuppression. Furthermore, AQP4 inhibition is synergistic with cytotoxic T lymphocyte–associated antigen 4–Ig in prolonging survival of 8‐hour CIS heart allografts. AQP4 blockade markedly reduced T cell proliferation and cytokine production in vitro, suggesting that the improved graft survival is at least in part mediated through direct effects on donor‐reactive T cells. These results identify AQPs as a promising target for diminishing donor‐specific alloreactivity and improving the survival of high‐risk organ transplants.     

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.     

Deletion of the activating NK cell receptor NKG2D accelerates rejection of cardiac allografts

It has already been shown that neutralization of the activating NK cell receptor NKG2D in combination with co‐stimulation blockade prolongs graft survival of vascularized transplants. In order to clarify the underlying cellular mechanisms, we transplanted complete MHC‐disparate BALB/c‐derived cardiac grafts into C57BL/6 wildtypes or mice deficient for NKG2D (Klrk1^?/?). Although median survival was 8 days for both recipient groups, we detected already at day 5 posttransplantation significantly greater intragraft frequencies of NKp46⁺ NK cells in Klrk1^?/? recipients than in wildtypes. This was followed by a significantly greater infiltration of CD4⁺, but a lesser infiltration of CD8⁺ T cell frequencies. Contrary to published observations, co‐stimulation blockade with CTLA4‐Ig resulted in a significant acceleration of cardiac rejection by Klrk1^?/? recipients, and this result was confirmed by applying a neutralizing antibody against NKG2D to wildtypes. In both experimental setups, grafts derived from Klrk1^?/? recipients were characterized by significantly higher levels of interferon‐γ mRNA, and both CD4⁺ and CD8⁺ T cells displayed a greater capacity for degranulation and interferon‐γ production. In summary, our results clearly illustrate that NKG2D expression in the recipient is important for cardiac allograft survival, thus supporting the hypothesis that impairment of NK cells prevents the establishment of graft acceptance.     

TIGIT regulates apoptosis of risky memory T cell subsets implicated in belatacept-resistant rejection

Belatacept confers increased patient and graft survival in renal transplant recipients relative to calcineurin inhibitors, but is associated with an increased rate of acute rejection. Recent immunophenotypic studies comparing pretransplant T cell phenotypes of patients who reject versus those who remain stable on belatacept identified three potential “risky” memory T cell subsets that potentially underlie belatacept-resistant rejection: CD4⁺ CD28⁺ T_EM, CD8⁺ CD28^null, and CD4⁺ CD57⁺ PD1⁻ subsets. Here, we compared key phenotypic and functional aspects of these human memory T cell subsets, with the goal of identifying additional potential targets to modulate them. Results demonstrate that TIGIT, an increasingly well-appreciated immune checkpoint receptor, was expressed on all three risky memory T cell subsets in vitro and in vivo in the presence of belatacept. Coculture of human memory CD4⁺ and CD8⁺ T cells with an agonistic anti-TIGIT mAb significantly increased apoptotic cell death of all three risky memory T cell subsets. Mechanistically, TIGIT-mediated apoptosis of risky memory T cells was dependent on FOXP3⁺ Treg, suggesting that agonism of the TIGIT pathway increases FOXP3⁺ Treg suppression of human memory T cell populations. Overall, these data suggest that TIGIT agonism could represent a new therapeutic target to inhibit belatacept-resistant rejection during transplantation.     

Mitochondrial permeability regulates cardiac endothelial cell necroptosis and cardiac allograft rejection

Transplantation is invariably associated with programmed cell death including apoptosis and necrosis, resulting in delayed graft function and organ rejection. We have demonstrated the contribution of necroptosis to mouse microvascular endothelial cell (MVEC) death and transplant rejection. Organ injury results in the opening of mitochondrial permeability transition pores (mPTPs), which can trigger apoptotic molecules release that ultimately results in cell death. The effect of mPTPs in the necroptotic pathway remains controversial; importantly, their role in transplant rejection is not clear. In this study, tumor necrosis factor‐α triggered MVECs to undergo receptor‐interacting protein kinase family (RIPK1/3)‐dependent necroptosis. Interestingly, inhibition of mPTP opening could also inhibit necroptotic cell death. Cyclophilin‐D (Cyp‐D) is a key regulator of the mPTPs. Both inhibition and deficiency of Cyp‐D protected MVECs from necroptosis (n = 3, P < .00001). Additionally, inhibition of Cyp‐D attenuated RIPK3‐downstream mixed‐lineage kinase domain–like protein phosphorylation. In vivo, Cyp‐D–deficient cardiac grafts showed prolonged survival in allogeneic BALB/c mice posttransplant compared with wild‐type grafts (n = 7, P < .0001). Our study results suggest that the mPTPs may be important mechanistic mediators of necroptosis in cardiac grafts. There is therapeutic potential in targeting cell death via inhibition of the mPTP‐regulating molecule Cyp‐D to prevent cardiac graft rejection.     

Regulatory T cells promote corneal endothelial cell survival following transplantation via interleukin‐10

The functional competence of corneal endothelial cells (CEnCs) is critical for survival of corneal allografts, but these cells are often targets of the immune response mediated by graft‐attacking effector T cells. Although regulatory T cells (Tregs) have been studied for their role in regulating the host’s alloimmune response towards the graft, the cytoprotective function of these cells on CEnCs has not been investigated. The aim of this study was to determine whether Tregs suppress effector T cell–mediated and inflammatory cytokine–induced CEnC death, and to elucidate the mechanism by which this cytoprotection occurs. Using 2 well‐established models of corneal transplantation (low‐risk and high‐risk models), we show that Tregs derived from low‐risk graft recipients have a superior capacity in protecting CEnCs against effector T cell–mediated and interferon‐γ and tumor necrosis factor‐α‐induced cell death compared to Tregs derived from high‐risk hosts. We further demonstrate that the cytoprotective function of Tregs derived from low‐risk hosts occurs independently of direct cell‐cell contact and is mediated by the immunoregulatory cytokine IL‐10. Our study is the first to report that Tregs provide cytoprotection for CEnCs through secretion of IL‐10, indicating potentially novel therapeutic targets for enhancing CEnC survival following corneal transplantation.     

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.     

Innate allorecognition by monocytic cells and its role in graft rejection

Innate recognition of microbial products and danger molecules by monocytes and macrophages has been well established; this is mediated primarily by pattern‐recognition receptors and is central to the activation of innate and adaptive immune cells required for productive immunity. Whether monocytes and macrophages are equipped with an allorecognition system that allows them to respond directly to allogeneic grafts is a topic of much debate. Recent studies provide compelling evidence that these cells can recognize allogeneic entities and that they mediate graft rejection via direct cytotoxicity and priming of alloreactive T cells. In addition, these studies have uncovered a mechanism of innate allorecognition based on detection of the polymorphic molecule signal regulatory protein α (SIRPα) on donor cells. Further understanding of innate allorecognition and its consequences would provide essential insight into allograft rejection and lead to better therapies for transplant patients.     

Memory T cell–mediated rejection is mitigated by FcγRIIB expression on CD8+ T cells

Donor‐reactive memory T cells generated via heterologous immunity represent a potent barrier to long‐term graft survival following transplantation because of their increased precursor frequency, rapid effector function, altered trafficking patterns, and reduced reliance on costimulation signals for activation. Thus, the identification of pathways that control memory T cell survival and secondary recall potential may provide new opportunities for therapeutic intervention. Here, we discovered that donor‐specific effector/memory CD8⁺ T cell populations generated via exposure to acute vs latent vs chronic infections contain differential frequencies of CD8⁺ T cells expressing the inhibitory Fc receptor FcγRIIB. Results indicated that frequencies of FcγRIIB‐expressing CD8⁺ donor‐reactive memory T cells inversely correlated with allograft rejection. Furthermore, adoptive T cell transfer of Fcgr2b^?/? CD8⁺ T cells resulted in an accumulation of donor‐specific CD8⁺ memory T cells and enhanced recall responses, indicating that FcγRIIB functions intrinsically to limit T cell CD8⁺ survival in vivo. Lastly, we show that deletion of FcγRIIB on donor‐specific CD8⁺ memory T cells precipitated costimulation blockade‐resistant rejection. These data therefore identify a novel cell‐intrinsic inhibitory pathway that functions to limit the risk of memory T cell–mediated rejection following transplantation and suggest that therapeutic manipulation of this pathway could improve outcomes in sensitized patients.     

Exercise increases skin graft resistance to rejection

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

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.     

Toll-like receptor 3 is an endogenous sensor of cell death and a potential target for induction of long-term cardiac transplant survival

Inflammation posttransplant is directly linked to cell death programs including apoptosis and necrosis. Cell death leads to the release of cellular contents which can promote inflammation. Targeting of these pathways should be an effective strategy to prevent transplant rejection. Toll-like receptor 3 (TLR3) is emerging as a major endogenous sensor of inflammation. In this study, we assessed the role of TLR3 on cell death and transplant rejection. We showed that TLR3 is highly expressed on mouse microvascular endothelial cell (ECs) and the endothelium of cardiac grafts. We demonstrated that TLR3 interacting with dsRNA or self-RNA triggered apoptosis and necroptosis in ECs. Interestingly, TLR3-induced necroptosis led mitochondrial damage. Inhibition of the mitochondrial membrane permeability molecule Cyclophilin D prevented necroptosis in ECs. In vivo, endothelium damage and activities of caspase-3 and mixed lineage kinase domain-like protein were inhibited in TLR3^−/− cardiac grafts compared with C57BL/6 grafts posttransplant (n = 5, p < .001). Importantly, TLR3^−/− cardiac grafts had prolonged survival in allogeneic BALB/c mice (mean survival = 121 ± 67 vs. 31 ± 6 days of C57BL/6 grafts, n = 7, p = .002). In summary, our study suggests that TLR3 is an important cell death inducer in ECs and cardiac grafts and thus a potential therapeutic target in preventing cardiac transplant rejection.     

A Novel Xenogeneic Graft‐Versus‐Host Disease Model for Investigating the Pathological Role of Human CD4+ or CD8+ T Cells Using Immunodeficient NOG Mice

Graft‐versus‐host disease (GVHD) is a major complication of allogenic bone marrow transplantation and involves the infiltration of donor CD4⁺ and/or CD8⁺ T cells into various organs of the recipient. The pathological role of human CD4⁺ and CD8⁺ T cells in GVHD remains controversial. In this study, we established two novel xenogeneic (xeno)‐GVHD models. Human CD4⁺ or CD8⁺ T cells were purified from peripheral blood and were transplanted into immunodeficient NOD/Shi‐scid IL2rg^null (NOG) mice. Human CD8⁺ T cells did not induce major GVHD symptoms in conventional NOG mice. However, CD8⁺ T cells immediately proliferated and induced severe GVHD when transferred into NOG mice together with at least 0.5 × 10⁶ CD4⁺ T cells or into NOG human interleukin (IL)‐2 transgenic mice. Human CD4⁺ T cell–transplanted NOG mice developed skin inflammations including alopecia, epidermal hyperplasia, and neutrophilia. Pathogenic T helper (Th)17 cells accumulated in the skin of CD4⁺ T cell–transplanted NOG mice. Further, an anti‐human IL‐17 antibody (secukinumab) significantly suppressed these skin pathologies. These results indicate that pathogenic human Th17 cells induce cutaneous GVHD via IL‐17–dependent pathways. This study provides fundamental insights into the pathogenesis of xeno‐GVHD, and these humanized mouse models may be useful as preclinical tools for the prevention of GVHD.     

Superior inhibition of alloantibody responses with selective CD28 blockade is CTLA-4 dependent and T follicular helper cell specific

Anti-donor antibodies cause immunologic injury in transplantation. CD28 blockade with CTLA-4-Ig has the ability to reduce the incidence of these donor-specific antibodies (DSA), but its mechanism is suboptimal for the inhibition of alloimmunity in that CTLA-4-Ig blocks both CD28 costimulation and CTLA-4 coinhibition. Thus selective CD28 blockade that spares CTLA-4 has potential to result in improved inhibition of humoral alloimmunity. To test this possibility, we utilized a full allogeneic mismatch murine transplant model and T follicular helper (Tfh):B cell co-culture system. We observed that selective blockade with an anti-CD28 domain antibody (dAb) compared to CTLA-4-Ig led to superior inhibition of Tfh cell, germinal center, and DSA responses in vivo and better control of B cell responses in vitro. CTLA-4 blockade enhanced the humoral alloresponse and, in combination with anti-CD28 dAb, abrogated the effects of selective blockade. This CTLA-4-dependent inhibition was Tfh cell specific in that CTLA-4 expression by Tfh cells was necessary and sufficient for the improved humoral inhibition observed with selective CD28 blockade. As CD28 blockade attracts interest for control of alloantibodies in the clinic, these data support selective CD28 blockade as a superior strategy to address DSA via the sparing of CTLA-4 and more potent targeting of Tfh cells.     

IL‐22 is required for the induction of bronchus‐associated lymphoid tissue in tolerant lung allografts

Long‐term survival after lung transplantation remains profoundly limited by graft rejection. Recent work has shown that bronchus‐associated lymphoid tissue (BALT), characterized by the development of peripheral nodal addressin (PNAd)‐expressing high endothelial venules and enriched in B and Foxp3⁺ T cells, is important for the maintenance of allograft tolerance. Mechanisms underlying BALT induction in tolerant pulmonary allografts, however, remain poorly understood. Here, we show that the development of PNAd‐expressing high endothelial venules within intragraft lymphoid follicles and the recruitment of B cells, but not Foxp3⁺ cells depends on IL‐22. We identify graft‐infiltrating gamma‐delta (γδ) T cells and Type 3 innate lymphoid cells (ILC3s) as important producers of IL‐22. Reconstitution of IL‐22 at late time points through retransplantation into wildtype hosts mediates B cell recruitment into lymphoid follicles within the allograft, resulting in a significant increase in their size, but does not induce PNAd expression. Our work has identified cellular and molecular requirements for the induction of BALT in pulmonary allografts during tolerance induction and may provide a platform for the development of new therapies for lung transplant patients.     

Vendor‐specific microbiome controls both acute and chronic murine lung allograft rejection by altering CD4+Foxp3+ regulatory T cell levels

Despite standardized postoperative care, some lung transplant patients suffer multiple episodes of acute and chronic rejection while others avoid graft problems for reasons that are poorly understood. Using an established model of C57BL/10 to C57BL/6 minor antigen mismatched single lung transplantation, we now demonstrate that the recipient microbiota contributes to variability in the alloimmune response. Specifically, mice from the Envigo facility in Frederick, Maryland contain nearly double the number of CD4⁺Foxp3⁺ regulatory T cells (T_regs) than mice from the Jackson facility in Bar Harbor, Maine or the Envigo facility in Indianapolis, Indiana (18 vs 9 vs 7%). Lung graft recipients from the Maryland facility thus do not develop acute or chronic rejection. Treatment with broad‐spectrum antibiotics decreases T_regs and increases both acute and chronic graft rejection in otherwise tolerant strains of mice. Constitutive depletion of regulatory T cells, using Foxp3‐driven expression of diphtheria toxin receptor, leads to the development of chronic rejection and supports the role of T_regs in both acute and chronic alloimmunity. Taken together, our data demonstrate that the microbiota of certain individuals may contribute to tolerance through T_reg‐dependent mechanisms and challenges the practice of indiscriminate broad‐spectrum antibiotic use in the perioperative period.     

Impact of selective CD28 blockade on virus‐specific immunity to a murine Epstein‐Barr virus homolog

CTLA‐4Ig (belatacept) blocks the CD80/CD86 ligands for both CD28 and CTLA‐4; thus, in addition to the intended effect of blocking CD28‐mediated costimulation, belatacept also has the unintended effect of blocking CTLA‐4–mediated coinhibition. Recently, anti‐CD28 domain antibodies (dAb) that selectively target CD28 while leaving CTLA‐4 intact were shown to more effectively inhibit alloimmune responses and prolong graft survival. However, the impact of selective CD28 blockade on protective immunity has not been extensively investigated. Here, we sought to compare the impact of CTLA‐4Ig vs anti‐CD28dAb on CD8⁺ T cell immunity to a transplant‐relevant pathogen, a murine homolog of Epstein‐Barr virus. Mice were infected with murine gammaherpesvirus‐68 (MHV) and treated with vehicle, CTLA‐4Ig, or anti‐CD28dAb. Although anti‐CD28dAb resulted in a decrease in virus‐specific CD8⁺ T cell numbers as compared to CTLA‐4Ig, cytolytic function and the expression of markers of high‐quality effectors were not different from CTLA‐4Ig treated animals. Importantly, MHV‐68 viral load was not different between the treatment groups. These results suggest that preserved CTLA‐4 coinhibition limits MHV‐specific CD8⁺ T cell accumulation, but the population that remains retains cytolytic function and migratory capacity and is not inferior in its ability to control viral burden relative to T cell responses in CTLA‐4Ig‐treated animals.     

Tracking of TCR‐Transgenic T Cells Reveals That Multiple Mechanisms Maintain Cardiac Transplant Tolerance in Mice

Solid organ transplantation tolerance can be achieved following select transient immunosuppressive regimens that result in long‐lasting restraint of alloimmunity without affecting responses to other antigens. Transplantation tolerance has been observed in animal models following costimulation or coreceptor blockade therapies, and in a subset of patients through induction protocols that include donor bone marrow transplantation, or following withdrawal of immunosuppression. Previous data from our lab and others have shown that proinflammatory interventions that successfully prevent the induction of transplantation tolerance in mice often fail to break tolerance once it has been stably established. This suggests that established tolerance acquires resilience to proinflammatory insults, and prompted us to investigate the mechanisms that maintain a stable state of robust tolerance. Our results demonstrate that only a triple intervention of depleting CD25⁺ regulatory T cells (Tregs), blocking programmed death ligand‐1 (PD‐L1) signals, and transferring low numbers of alloreactive T cells was sufficient to break established tolerance. We infer from these observations that Tregs and PD‐1/PD‐L1 signals cooperate to preserve a low alloreactive T cell frequency to maintain tolerance. Thus, therapeutic protocols designed to induce multiple parallel mechanisms of peripheral tolerance may be necessary to achieve robust transplantation tolerance capable of maintaining one allograft for life in the clinic.