IL‐17 Deficiency Attenuates Allograft Injury and Prolongs Survival in a Murine Model of Fully MHC‐Mismatched Renal Allograft Transplantation

IL‐17 is a pro‐inflammatory cytokine implicated in the pathogenesis of inflammatory and autoimmune diseases. However the role of IL‐17 in renal allograft rejection has not been fully explored. Here, we investigate the impact of IL‐17 in a fully MHC‐mismatched, life‐sustaining, murine model of kidney allograft rejection using IL‐17 deficient donors and recipients (IL‐17^?/? allografts). IL‐17^?/? allografts exhibited prolonged survival which was associated with reduced expression of the Th1 cytokine IFN‐γ and histological attenuation of acute and chronic allograft rejection, as compared to wild‐type allograft recipients. Results were confirmed in WT allograft recipients treated with an IL‐17 blocking antibody. Subsequent experiments using either donors or recipients deficient in IL‐17 showed a trend towards prolongation of survival only when recipients were IL‐17^?/?. Administration of a depleting anti‐CD25 antibody to IL‐17^?/? recipients abrogated the survival advantage conferred by IL‐17 deficiency, suggesting the involvement of a CD4⁺CD25⁺ T cell regulatory mechanism. Therefore, IL‐17 deficiency or neutralization was protective against the development of kidney allograft rejection, which may be mediated by impairment of Th1 responses and/or enhanced protection by Tregs.

     

B cells assist allograft rejection in the deficiency of protein kinase c‐theta

We have previously shown that mice deficient in protein kinase C theta (PKCθ) have the ability to reject cardiac allografts, but are susceptible to tolerance induction. Here we tested role of B cells in assisting alloimmune responses in the absence of PKCθ. Mouse cardiac allograft transplantations were performed from Balb/c (H‐2d) to PKCθ knockout (PKCθ^?/?), PKCθ and B cell double‐knockout (PBDK, H‐2b) mice and wild‐type (WT) C57BL/6 (H‐2b) mice. PBDK mice spontaneously accepted the allografts with the inhibition of NF‐κB activation in the donor cardiac allograft. Anti‐B cell antibody (rituximab) significantly delayed allograft rejection in PKCθ^?/?, but not in WT mice. Co‐transfer of PKCθ^?/? T plus PKCθ^?/? B cells or primed sera triggered allograft rejection in Rag1^?/? mice, and only major histocompatibility complex class II‐enriched B cells, but not class I‐enriched B cells, were able to promote rejection. This, together with the inability of PKCθ^?/? and CD28^?/? double‐deficient (PCDK) mice to acutely reject allografts, suggested that an effective cognate interaction between PKCθ^?/? T and B cells for acute rejection is CD28 molecule dependent. We conclude that T–B cell interactions synergize with PKCθ^?/? T cells to mediate acute allograft rejection.     

Nox2 Is a Mediator of Ischemia Reperfusion Injury

Delayed graft function (DGF) results from ischemia‐reperfusion injury (IRI) and the generation of reactive oxygen species. We hypothesized that NADPH oxidase 2 (Nox2) plays an important role in pathways leading to DGF. We tested this hypothesis in vitro, in an animal model of IRI using wild type and Nox2^?/? mice, and in patients with DGF. Under hypoxic conditions, primary tubular epithelial cells from Nox2^?/? mice had reduced expression of MMP2, vimentin, and HSP27. BUN and creatinine levels were significantly increased in both Nox2^?/? and WT mice at 4 weeks and 6 months after IRI, suggesting the development of acute and chronic kidney injury. At 4 weeks, kidney fibrosis (α‐SMA, picrosirius) and oxidative stress (dihydroethidine, HNE) were significantly reduced in Nox2^?/? mice, confirming the oxidative and pro‐fibrotic effects of Nox2. The molecular signature of IRI using genomic analyses demonstrated a significant decline in hypoxia reponse, oxidative stress, fibrosis, and inflammation in Nox2^?/? mice. Immunohistochemical analyses of pre‐implanatation kidney allograft biopsies from patients with subsequent DGF showed significantly greater Nox2 levels and vascular injury compared with patients without DGF. These studies demonstrate that Nox2 is a modulator of IRI and its absence is associated with reduced inflammation, OS, and fibrosis.

     

Low‐Dose Rapamycin Treatment Increases the Ability of Human Regulatory T Cells to Inhibit Transplant Arteriosclerosis In Vivo

Regulatory T cells (T_reg) are currently being tested in clinical trials as a potential therapy in cell and solid organ transplantation. The immunosuppressive drug rapamycin has been shown to preferentially promote T_reg expansion. Here, we hypothesized that adjunctive rapamycin therapy might potentiate the ability of ex vivo expanded human T_reg to inhibit vascular allograft rejection in a humanized mouse model of arterial transplantation. We studied the influence of combined treatment with low‐dose rapamycin and subtherapeutic T_reg numbers on the development of transplant arteriosclerosis (TA) in human arterial grafts transplanted into immunodeficient BALB/cRag2^?/?Il2rg^?/? mice reconstituted with allogeneic human peripheral blood mononuclear cell. In addition, we assessed the effects of the treatment on the proliferation and apoptosis of naïve/effector T cells. The combined therapy efficiently suppressed T‐cell proliferation in vivo and in vitro. Neointima formation in the human arterial allografts was potently inhibited compared with each treatment alone. Interestingly, CD4⁺ but not CD8⁺ T lymphocytes were sensitive to T_reg and rapamycin‐induced apoptosis in vitro. Our data support the concept that rapamycin can be used as an adjunctive therapy to improve efficacy of T_reg‐based immunosuppressive protocols in clinical practice. By inhibiting TA, T_reg and rapamycin may prevent chronic transplant dysfunction and improve long‐term allograft survival.     

Glutamate Receptor Interacting Protein 1 Regulates CD4+ CTLA‐4 Expression and Transplant Rejection

PDZ domains are common 80‐ to 90‐amino‐acid regions named after the first three proteins discovered to share these domains: postsynaptic density 95, discs large, and zonula occludens. PDZ domain‐containing proteins typically interact with the C‐terminus of membrane receptors. Glutamate receptor interacting protein 1 (GRIP1), a seven–PDZ domain protein scaffold, regulates glutamate receptor surface expression and trafficking in neurons. We have found that human and mouse T cells also express GRIP1. T cell–specific GRIP1^?/? mice >11 weeks old had prolonged cardiac allograft survival. Compared with wild‐type T cells, in vitro stimulated GRIP1^?/? T cells had decreased expression of activation markers and increased apoptotic surface marker expression. Surface expression of the strong T cell inhibitory molecule cytotoxic T lymphocyte antigen‐4 (CTLA‐4) was increased on GRIP1^?/? T cells from mice >11 weeks old. CTLA‐4 increases with T cell stimulation and its surface expression on GRIP1^?/? T cells remained high after stimulation was removed, indicating a possible internalization defect in GRIP1‐deficient T cells. CTLA‐4–blocking antibody treatment following heart transplantation led to complete rejection in T cell GRIP1^?/? mice, indicating that increased CTLA‐4 surface expression contributed to the extended graft survival. Our data indicate that GRIP1 regulates T cell activation by regulating CTLA‐4 surface expression.     

Antibody‐Mediated Rejection of Single Class I MHC‐Disparate Cardiac Allografts

Murine CCR5^?/? recipients produce high titers of antibody to complete MHC‐mismatched heart and renal allografts. To study mechanisms of class I MHC antibody‐mediated allograft injury, we tested the rejection of heart allografts transgenically expressing a single class I MHC disparity in wild‐type C57BL/6 (H‐2^b) and B6.CCR5^?/? recipients. Donor‐specific antibody titers in CCR5^?/? recipients were 30‐fold higher than in wild‐type recipients. B6.K^d allografts survived longer than 60 days in wild‐type recipients whereas CCR5^?/? recipients rejected all allografts within 14 days. Rejection was accompanied by infiltration of CD8 T cells, neutrophils and macrophages, and C4d deposition in the graft capillaries. B6.K^d allografts were rejected by CD8^?/?/CCR5^?/?, but not μMT^?/?/CCR5^?/?, recipients indicating the need for antibody but not CD8 T cells. Grafts recovered at day 10 from CCR5^?/? and CD8^?/?/CCR5^?/? recipients and from RAG‐1^?/? allograft recipients injected with anti‐K^d antibodies expressed high levels of perforin, myeloperoxidase and CCL5 mRNA. These studies indicate that the continual production of antidonor class I MHC antibody can mediate allograft rejection, that donor‐reactive CD8 T cells synergize with the antibody to contribute to rejection, and that expression of three biomarkers during rejection can occur in the absence of this CD8 T cell activity.     

Differential Effects of Pharmacological HIF Preconditioning of Donors Versus Recipients in Rat Cardiac Allografts

Ischemia‐reperfusion injury (IRI) induces hypoxia‐inducible factor‐1 (HIF‐1) in the myocardium, but the consequences remain elusive. We investigated HIF‐1 activation during cold and warm ischemia and IRI in rat hearts and cardiac syngrafts. We also tested the effect of HIF‐α stabilizing prolyl hydroxylase inhibitor (FG‐4497) on IRI or allograft survival. Ex vivo ischemia of the heart increased HIF‐1α expression in a time‐ and temperature‐dependent fashion. Immunohistochemistry localized HIF‐1α to all cardiac cell types. After reperfusion, HIF‐1α immunoreactivity persisted in smooth muscle cells and cardiomyocytes in the areas with IRI. This was accompanied with a transient induction of protective HIF‐1 downstream genes. Donor FG‐4497 pretreatment for 4 h enhanced IRI in cardiac allografts as evidenced by an increase in cardiac troponin T release, cardiomyocyte apoptosis, and activation of innate immunity. Recipient FG‐4497 pretreatment for 4 h decreased infiltration of ED1⁺ macrophages, and mildly improved the long‐term allograft survival. In syngrafts donor FG‐4497 pretreatment increased activation of innate immunity, but did not induce myocardial damage. We conclude that the HIF‐1 pathway is activated in heart transplants. We suggest that pharmacological HIF‐α preconditioning of cardiac allografts donors would not lead to clinical benefit, while in recipients it may result in antiinflammatory effects and prolonged allograft survival.     

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.     

Blockade of P‐Selectin Is Sufficient to Reduce MHC I Antibody‐Elicited Monocyte Recruitment In Vitro and In Vivo

Donor‐specific HLA antibodies significantly lower allograft survival, but as yet there are no satisfactory therapies for prevention of antibody‐mediated rejection. Intracapillary macrophage infiltration is a hallmark of antibody‐mediated rejection, and macrophages are important in both acute and chronic rejection. The purpose of this study was to investigate the Fc‐independent effect of HLA I antibodies on endothelial cell activation, leading to monocyte recruitment. We used an in vitro model to assess monocyte binding to endothelial cells in response to HLA I antibodies. We confirmed our results in a mouse model of antibody‐mediated rejection, in which B6.RAG1^?/? recipients of BALB/c cardiac allografts were passively transferred with donor‐specific MHC I antibodies. Our findings demonstrate that HLA I antibodies rapidly increase intracellular calcium and endothelial presentation of P‐selectin, which supports monocyte binding. In the experimental model, donor‐specific MHC I antibodies significantly increased macrophage accumulation in the allograft. Concurrent administration of rPSGL‐1‐Ig abolished antibody‐induced monocyte infiltration in the allograft, but had little effect on antibody‐induced endothelial injury. Our data suggest that antagonism of P‐selectin may ameliorate accumulation of macrophages in the allograft during antibody‐mediated rejection.     

Differential Requirement of CD27 Costimulatory Signaling for Naïve Versus Alloantigen‐Primed Effector/Memory CD8+ T Cells

CD8⁺ memory T cells endanger allograft survival by causing acute and chronic rejection and prevent tolerance induction. We explored the role of CD27:CD70 T‐cell costimulatory pathway in alloreactive CD8⁺/CD4⁺ T‐cell activation. CD27‐deficient (CD27^?/?) and wild‐type (WT) B6 mice rejected BALB/c cardiac allografts at similar tempo, with or without depletion of CD4⁺ or CD8⁺ T cells, suggesting that CD27 is not essential during primary T‐cell alloimmune responses. To dissect the role of CD27 in primed effector and memory alloreactive T cells, CD27^?/? or WT mice were challenged with BALB/c hearts either 10 or 40 days after sensitization with donor‐type skin grafts. Compared to WT controls, allograft survival was prolonged in day 40‐ but not day 10‐sensitized CD27^?/? recipients. Improved allograft survival was accompanied by diminished secondary responsiveness of memory CD8⁺ T cells, which resulted from deficiency in memory formation rather than their lack of secondary expansion. Chronic allograft vasculopathy and fibrosis were diminished in CD27^?/? recipients of class I‐ but not class II‐mismatched hearts as compared to WT controls. These data establish a novel role for CD27 as an important costimulatory molecule for alloreactive CD8⁺ memory T cells in acute and chronic allograft rejection.     

Complement Dependence of Murine Costimulatory Blockade‐Resistant Cellular Cardiac Allograft Rejection

Building on studies showing that ischemia–reperfusion‐(I/R)‐injury is complement dependent, we tested links among complement activation, transplantation‐associated I/R injury, and murine cardiac allograft rejection. We transplanted BALB/c hearts subjected to 8‐h cold ischemic storage (CIS) into cytotoxic T‐lymphocyte associated protein 4 (CTLA4)Ig‐treated wild‐type (WT) or c3^?/? B6 recipients. Whereas allografts subjected to 8‐h CIS rejected in WT recipients with a median survival time (MST) of 37 days, identically treated hearts survived >60 days in c3^?/? mice (p < 0.05, n = 4–6/group). Mechanistic studies showed recipient C3 deficiency prevented induction of intragraft and serum chemokines/cytokines and blunted the priming, expansion, and graft infiltration of interferon‐γ–producing, donor‐reactive T cells. MST of hearts subjected to 8‐h CIS was >60 days in mannose binding lectin (mbl1^?/?mbl2^?/?) recipients and 42 days in factor B (cfb^?/?) recipients (n = 4–6/group, p < 0.05, mbl1^?/?mbl2^?/? vs. cfb^?/?), implicating the MBL (not alternative) pathway. To pharmacologically target MBL‐initiated complement activation, we transplanted BALB/c hearts subjected to 8‐h CIS into CTLA4Ig‐treated WT B6 recipients with or without C1 inhibitor (C1‐INH). Remarkably, peritransplantation administration of C1‐INH prolonged graft survival (MST >60 days, p < 0.05 vs. controls, n = 6) and prevented CI‐induced increases in donor‐reactive, IFNγ‐producing spleen cells (p < 0.05). These new findings link donor I/R injury to T cell–mediated rejection through MBL‐initiated, complement activation and support testing C1‐INH administration to prevent CTLA4Ig‐resistant rejection of deceased donor allografts in human transplant patients.     

Noninvasive Imaging of Activated Complement in Ischemia‐Reperfusion Injury Post–Cardiac Transplant

Ischemia‐reperfusion injury (IRI) is inevitable in solid organ transplantation, due to the transplanted organ being ischemic for prolonged periods prior to transplantation followed by reperfusion. The complement molecule C3 is present in the circulation and is also synthesized by tissue parenchyma in early response to IRI and the final stable fragment of activated C3, C3d, can be detected on injured tissue for several days post‐IRI. Complement activation post‐IRI was monitored noninvasively by single photon emission computed tomography (SPECT) and CT using ^99mTc‐recombinant complement receptor 2 (^99mTc‐rCR2) in murine models of cardiac transplantation following the induction of IRI and compared to ^99mTc‐rCR2 in C3^?/? mice or with the irrelevant protein ^99mTc‐prostate–specific membrane antigen antibody fragment (PSMA). Significant uptake with ^99mTc‐rCR2 was observed as compared to C3^?/? or ^99mTc‐PSMA. In addition, the transplanted heart to muscle ratio of ^99mTc‐rCR2 was significantly higher than ^99mTc‐PSMA or C3^?/?. The results were confirmed by histology and autoradiography. ^99mTc‐rCR2 can be used for noninvasive detection of activated complement and in future may be used to quantify the severity of transplant damage due to complement activation postreperfusion.

     

RIPK3‐Mediated Necroptosis Promotes Donor Kidney Inflammatory Injury and Reduces Allograft Survival

Kidney transplant injury occurs with ischemia and alloimmunity. Members of the receptor interacting protein kinase family (RIPK1,3) are key regulators of “necroptosis,” a newly recognized, regulated form of necrosis. Necroptosis and apoptosis death appear to be counterbalanced as caspase‐8 inhibition can divert death from apoptosis to necrosis. Inhibition of necroptosis in donor organs to limit injury has not been studied in transplant models. In this study, necroptosis was triggered in caspase inhibited tubular epithelial cells (TEC) exposed to tumor necrosis factor alpha in vitro, while RIPK1 inhibition with necrostatin‐1 or use of RIPK3^?/? TEC, prevented necroptosis. In vivo, short hairpin RNA silencing of caspase‐8 in donor B6 mouse kidneys increased necroptosis, enhanced high‐mobility group box 1 release, reduced renal function and accelerated rejection when transplanted into BALB/c recipients. Using ethidium homodimer perfusion to assess necrosis in vivo, necrosis was abrogated in RIPK3^?/? kidneys postischemia. Following transplantation, recipients receiving RIPK3^?/? kidneys had longer survival (p = 0.002) and improved renal function (p = 0.03) when compared to controls. In summary, we show for the first time that RIPK3‐mediated necroptosis in donor kidneys can promote inflammatory injury, and has a major impact on renal ischemia–reperfusion injury and transplant survival. We suggest inhibition of necroptosis in donor organs may similarly provide a major clinical benefit.

     

Prolongation of Cardiac Allograft Survival by a Novel Population of Autologous CD117+ Bone Marrow‐Derived Progenitor Cells

Autologous CD117⁺ progenitor cells (PC) have been successfully utilized in myocardial infarction and ischemic injury, potentially through the replacement/repair of damaged vascular endothelium. To date, such cells have not been used to enhance solid organ transplant outcome. In this study, we determined whether autologous bone marrow‐derived CD117⁺PC could benefit cardiac allograft survival, possibly by replacing donor vascular cells. Autologous, positively selected CD117⁺PC were administered posttransplantation and allografts were assessed for acute rejection. Although significant generation of recipient vascular cell chimerism was not observed, transferred PC disseminated both to the allograft and to peripheral lymphoid tissues and facilitated a significant, dose‐dependent prolongation of allograft survival. While CD117⁺PC dramatically inhibited alloreactive T cell proliferation in vitro, this property did not differ from nonprotective CD117^? bone marrow populations. In vivo, CD117⁺ PC did not significantly inhibit T cell alloreactivity or increase peripheral regulatory T cell numbers. Thus, rather than inhibiting adaptive immunity to the allograft, CD117⁺ PC may play a cytoprotective role in prolonging graft survival. Importantly, autologous CD117⁺PC appear to be distinct from bone marrow‐derived mesenchymal stem cells (MSC) previously used to prolong allograft survival. As such, autologous CD117⁺PC represent a novel cellular therapy for promoting allograft survival.     

Inhibition of Innate Co‐Receptor TREM‐1 Signaling Reduces CD4+ T Cell Activation and Prolongs Cardiac Allograft Survival

The innate receptor “triggering‐receptor‐expressed‐on‐myeloid‐cells‐1” (TREM‐1) enhances downstream signaling of “pattern recognition receptor” (PRR) molecules implicated in inflammatory responses. However the mechanistic role of TREM‐1 in chronic heart rejection has yet to be elucidated. We examined the effect of TREM‐1⁺ antigen‐presenting cells (APC) on alloreactive CD4⁺ lymphocytes. Bm12 donor hearts were transplanted into wild‐type MHC‐class‐II‐mismatched C57BL/6J recipient mice. Progressive allograft rejection of bm12‐donor hearts with decreased organ function, severe vasculopathy and allograft fibrosis was evident within 4 weeks. TREM‐1⁺CD11b⁺MHC‐II⁺F4/80⁺CCR2⁺ APC and IFNγ‐producing CD4⁺ cells were detected during chronic rejection. Peptide inhibition of TREM‐1 attenuated graft vasculopathy, reduced graft‐infiltrating leukocytes and prolonged allograft survival, while being accompanied by sustained low levels of CD4⁺ and CD8⁺ cell infiltration. Remarkably, temporary inhibition of TREM‐1 during early immune activation was sufficient for long‐term allograft survival. Mechanistically, TREM‐1 inhibition leads to reduced differentiation and proliferation of IFNγ‐producing Th1 cells. In conclusion, TREM‐1 influences chronic heart rejection by regulating the infiltration and differentiation of CD4⁺ lymphocytes.     

Ex vivo allotransplantation engineering: Delivery of mesenchymal stem cells prolongs rejection‐free allograft survival

Current pharmacologic regimens in transplantation prevent allograft rejection through systemic recipient immunosuppression but are associated with severe morbidity and mortality. The ultimate goal of transplantation is the prevention of allograft rejection while maintaining recipient immunocompetence. We hypothesized that allografts could be engineered ex vivo (after allotransplant procurement but before transplantation) by using mesenchymal stem cell–based therapy to generate localized immunomodulation without affecting systemic recipient immunocompetence. To this end, we evaluated the therapeutic efficacy of bone marrow–derived mesenchymal stem cells in vitro and activated them toward an immunomodulatory fate by priming in inflammatory or hypoxic microenvironments. Using an established rat hindlimb model for allotransplantation, we were able to significantly prolong rejection‐free allograft survival with a single perioperative ex vivo infusion of bone marrow–derived mesenchymal stem cells through the allograft vasculature, in the absence of long‐term pharmacologic immunosuppression. Critically, transplanted rats rejected a second, nonengineered skin graft from the same donor species to the contralateral limb at a later date, demonstrating that recipient systemic immunocompetence remained intact. This study represents a novel approach in transplant immunology and highlights the significant therapeutic opportunity of the ex vivo period in transplant engineering.     

Donor kidney injury molecule‐1 promotes graft recovery by regulating systemic necroinflammation

Ischemia‐reperfusion injury during kidney transplantation predisposes to delayed graft function, rejection, and premature graft failure. Exacerbation of tissue damage and alloimmune responses may be explained by necroinflammation: an autoamplification loop of cell death and inflammation, which is mediated by the release of damage‐associated molecular patterns (eg, high‐mobility group box‐1; HMGB1) from necrotic cells that activate both innate and adaptive immune pathways. Kidney injury molecule‐1 (KIM‐1) is a phosphatidylserine receptor that is upregulated on injured proximal tubular epithelial cells and enables them to clear apoptotic and necrotic cells. Here we show a pivotal role for clearance of dying cells in regulating necroinflammation in a syngeneic murine kidney transplant model. We found persistent KIM‐1 expression in KIM‐1^+/+ kidney grafts posttransplantation. Compared to recipients of KIM‐1^+/+ kidneys, recipients of KIM‐1^?/? kidneys exhibited significantly more renal dysfunction, apoptosis and necrosis, tubular obstruction, and graft failure. KIM‐1^?/? grafts also had more inflammatory cytokines, infiltrating neutrophils, and macrophages compared to KIM‐1^+/+ grafts. Most significantly, passive release of HMGB1 from apoptotic and necrotic cells led to dramatically higher serum HMGB1 levels and increased proinflammatory macrophages in recipients of KIM‐1^?/? grafts. Our data identify an endogenous protective mechanism against necroinflammation in kidney grafts that may be of therapeutic relevance in transplantation.