In Vivo Function of Immune Inhibitory Molecule B7‐H4 in Alloimmune Responses

B7 ligands deliver both costimulatory and coinhibitory signals to the CD28 family of receptors on T lymphocytes, the balance between which determines the ultimate immune response. Although B7‐H4, a recently discovered member of the B7 family, is known to negatively regulate T cell immunity in autoimmunity and cancer, its role in solid organ allograft rejection and tolerance has not been established. Targeting the B7‐H4 molecule by a blocking antibody or use of B7‐H4^?/? mice as recipients of fully MHC‐mismatched cardiac allografts did not affect graft survival. However, B7‐H4 blockade resulted in accelerated allograft rejection in CD28‐deficient recipients. B7‐1/B7‐2‐double‐deficient recipients are truly independent of CD28/CTLA‐4:B7 signals and usually accept MHC‐mismatched heart allografts. Blockade of B7‐H4 in these mice also precipitated rejection, demonstrating regulatory function of this molecule independent of an intact CD28/CTLA‐4:B7 costimulatory pathway. Accelerated allograft rejection was always accompanied by increased frequencies of alloreactive IFN‐γ‐, IL‐4‐ and Granzyme B‐producing splenocytes. Finally, intact recipient, but not donor, B7‐H4 is essential for prolongation of allograft survival by blocking CD28/CTLA4:B7 pathway using CTLA4‐Ig. These data are the first to provide evidence of the regulatory effects of B7‐H4 in alloimmune responses in a murine model of solid organ transplantation.     

Deleterious Effect of CTLA4‐Ig on a Treg‐Dependent Transplant Model

Blockade of the B7:CD28 costimulatory pathway has emerged as a promising therapy to prevent allograft rejection. However, results from the belatacept phase III clinical trial demonstrated a higher rejection rate when compared to cyclosporine, raising concern about potential deleterious effects of this agent. In this study, we investigated the consequences of B7:CD28 blockade by hCTLA4Ig on regulator T cell (Treg) generation in different major histocompatibility complex (MHC) mismatch transplant models. Administration of hCTLA4Ig significantly decreased the amount of Tregs in B6 WT animals and this effect was predominant in thymus‐induced Tregs (Helios⁺). Although hCTLA4Ig prevented rejection in a fully allogeneic mismatch model, it accelerated rejection in a MHC class‐II mismatch model (MST = 26, p < 0.0001), in which long‐term allograft survival is dependent on Tregs. This accelerated rejection was associated with a marked reduction in thymus‐induced Tregs and led to a higher effector/regulatory T‐cell ratio in secondary lymphoid organs and in the allograft. This study confirms the importance of the B7:CD28 pathway in Treg homeostasis in an in vivo transplant model and suggests that hCTLA4Ig therapy may be deleterious in circumstances where engraftment is dependent on Tregs.     

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.     

LFA‐1 Antagonism Inhibits Early Infiltration of Endogenous Memory CD8 T Cells into Cardiac Allografts and Donor‐Reactive T Cell Priming

Alloreactive memory T cells are present in virtually all transplant recipients due to prior sensitization or heterologous immunity and mediate injury undermining graft outcome. In mouse models, endogenous memory CD8 T cells infiltrate MHC‐mismatched cardiac allografts and produce IFN‐γ in response to donor class I MHC within 24 h posttransplant. The current studies analyzed the efficacy of anti‐LFA‐1 mAb to inhibit early CD8 T cell cardiac allograft infiltration and activation. Anti‐LFA‐1 mAb given to C57BL/6 6 (H‐2^b) recipients of A/J (H‐2^a) heart grafts on days –1 and 0 completely inhibited CD8 T cell allograft infiltration, markedly decreased neutrophil infiltration and significantly reduced intragraft expression levels of IFN‐γ‐induced genes. Donor‐specific T cells producing IFN‐γ were at low/undetectable numbers in spleens of anti‐LFA‐1 mAb treated recipients until day 21. These effects combined to promote substantial prolongation (from day 8 to 27) in allograft survival. Delaying anti‐LFA‐1 mAb treatment until days 3 and 4 posttransplant did not inhibit early memory CD8 T cell infiltration and proliferation within the allograft. These data indicate that peritransplant anti‐LFA‐1 mAb inhibits early donor‐reactive memory CD8 T cell allograft infiltration and inflammation suggesting an effective strategy to attenuate the negative effects of heterologous immunity in transplant recipients.     

Flt3L‐mobilized dendritic cells bearing H2‐Kbm1 apoptotic cells do not induce cross‐tolerance to CD8+ T cells across a class I MHC mismatched barrier

Tolerization of allogeneic CD8⁺ T cells is still a pending issue in the field of transplantation research to achieve long‐term survival. To test whether dendritic cells (DC) bearing allogeneic major histocompatibility complex (MHC) class I mismatched apoptotic cells could induce cross‐tolerance to alloreactive CD8⁺ T cells, the following experimental strategy was devised. Rag2/γ_c KO B6 mice were treated with Fms‐like tyrosine kinase 3 ligand (Flt3L)‐transduced B16 melanoma cells to drive a rapid expansion and mobilization of DC in vivo. Of all DC populations expanded, splenic CD11c⁺CD103⁺CD8α⁺ DC were selectively involved in the process of antigen clearance of X‐ray irradiated apoptotic thymocytes in vivo. Considering that CD11c⁺CD103⁺CD8α⁺ DC selectively take up apoptotic cells and that they are highly specialized in cross‐presenting antigen to CD8⁺ T cells, we investigated whether B6 mice adoptively transferred with Flt3L‐derived DC loaded with donor‐derived apoptotic thymocytes could induce tolerance to bm1 skin allografts. Our findings on host anti‐donor alloresponse, as revealed by skin allograft survival and cytotoxic T lymphocyte assays, indicated that the administration of syngeneic DC presenting K^bm1 donor‐derived allopeptides through the indirect pathway of antigen presentation was not sufficient to induce cross‐tolerance to alloreactive CD8⁺ T cells responding to bm1 alloantigens in a murine model of skin allograft transplantation across an MHC class I mismatched barrier.     

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.     

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.

     

Peritransplant VLA‐4 blockade inhibits endogenous memory CD8 T cell infiltration into high‐risk cardiac allografts and CTLA‐4Ig resistant rejection

Recipient endogenous memory CD8 T cells expressing reactivity to donor class I MHC infiltrate MHC‐mismatched cardiac allografts within 24 hours after reperfusion and express effector functions mediating graft injury. The current study tested the efficacy of Very Late Antigen‐4 (VLA‐4) blockade to inhibit endogenous memory CD8 T cell infiltration into cardiac allografts and attenuate early posttransplant inflammation. Peritransplant anti‐VLA‐4 mAb given to C57BL6 (H‐2^b) recipients of AJ (H‐2^a) heart allografts completely inhibited endogenous memory CD4 and CD8 T cell infiltration with significant decrease in macrophage, but not neutrophil, infiltration into allografts subjected to either minimal or prolonged cold ischemic storage (CIS) prior to transplant, reduced intra‐allograft IFN‐γ‐induced gene expression and prolonged survival of allografts subjected to prolonged CIS in CTLA‐4Ig treated recipients. Anti‐VLA‐4 mAb also inhibited priming of donor‐specific T cells producing IFN‐γ until at least day 7 posttransplant. Peritransplant anti‐VLA plus anti‐CD154 mAb treatment similarly prolonged survival of allografts subjected to minimal or increased CIS prior to transplant. Overall, these data indicate that peritransplant anti‐VLA‐4 mAb inhibits early infiltration memory CD8 T cell infiltration into allografts with a marked reduction in early graft inflammation suggesting an effective strategy to attenuate negative effects of heterologous alloimmunity in recipients of higher risk grafts.     

A Novel,Blocking, Fc‐Silent Anti‐CD40 Monoclonal Antibody Prolongs Nonhuman Primate Renal Allograft Survival in the Absence of B Cell Depletion

CD40–CD154 pathway blockade prolongs renal allograft survival in nonhuman primates (NHPs). However, antibodies targeting CD154 were associated with an increased incidence of thromboembolic complications. Antibodies targeting CD40 prolong renal allograft survival in NHPs without thromboembolic events but with accompanying B cell depletion, raising the question of the relative contribution of B cell depletion to the efficacy of anti‐CD40 blockade. Here, we investigated whether fully silencing Fc effector functions of an anti‐CD40 antibody can still promote graft survival. The parent anti‐CD40 monoclonal antibody HCD122 prolonged allograft survival in MHC‐mismatched cynomolgus monkey renal allograft transplantation (52, 22, and 24 days) with accompanying B cell depletion. Fc‐silencing yielded CFZ533, an antibody incapable of B cell depletion but still able to potently inhibit CD40 pathway activation. CFZ533 prolonged allograft survival and function up to a defined protocol endpoint of 98–100 days (100, 100, 100, 98, and 76 days) in the absence of B cell depletion and preservation of good histological graft morphology. CFZ533 was well‐tolerated, with no evidence of thromboembolic events or CD40 pathway activation and suppressed a gene signature associated with acute rejection. Thus, use of the Fc‐silent anti‐CD40 antibody CFZ533 appears to be an attractive approach for preventing solid organ transplant rejection.     

Essential Role of PDL1 Expression on Nonhematopoietic Donor Cells in Acquired Tolerance to Vascularized Cardiac Allografts

The PD1:PDL1 pathway is an essential negative costimulatory pathway that plays a key role in regulating the alloimune response. PDL1 is expressed not only on antigen‐presenting cells (APCs) but also cardiac endothelium. In this study, we investigated the importance of PDL1 expression on donor cardiac allograft in acquired transplantation tolerance in a fully MHC‐mismatched model. We generated PDL1 chimeric mice on B6 background that expressed PDL1 on either hematopoietic cells or nonhematopoietic cells of the heart. Sham animals were used as controls. These hearts were then transplanted into BALB/c recipients and treated with CTLA4‐Ig to induce tolerance. Cardiac endothelium showed significant expression of PDL1, which was upregulated upon transplantation. While the absence of PDL1 on hematopoietic cells of the heart resulted in delayed rejection and prevented long‐term tolerance in most but not all recipients, we observed an accelerated and early graft rejection of all donor allografts that lacked PDL1 on the endothelium. Moreover, PDL1‐deficient endothelium hearts had significant higher frequency of IFN‐γ‐producing alloreactive cells as well as higher frequency of CD8⁺ effector T cells. These findings demonstrate that PDL1 expression mainly on donor endothelium is functionally important in a fully allogeneic mismatched model for the induction of cardiac allograft tolerance.     

Transient Lymphopenia Breaks Costimulatory Blockade‐Based Peripheral Tolerance and Initiates Cardiac Allograft Rejection

Lymphopenia is induced by lymphoablative therapies and chronic viral infections. We assessed the impact of lymphopenia on cardiac allograft survival in recipients conditioned with peritransplant costimulatory blockade (CB) to promote long‐term graft acceptance. After vascularized MHC‐mismatched heterotopic heart grafts were stably accepted through CB, lymphopenia was induced on day 60 posttransplant by 6.5 Gy irradiation or by administration of anti‐CD4 plus anti‐CD8 mAb. Long‐term surviving allografts were gradually rejected after lymphodepletion (MST = 74 ± 5 days postirradiation). Histological analyses indicated signs of severe rejection in allografts following lymphodepletion, including mononuclear cell infiltration and obliterative vasculopathy. Lymphodepletion of CB conditioned recipients induced increases in CD44^high effector/memory T cells in lymphatic organs and strong recovery of donor‐reactive T cell responses, indicating lymphopenia‐induced proliferation (LIP) and donor alloimmune responses occurring in the host. T regulatory (CD4⁺ Foxp³⁺) cell and B cell numbers as well as donor‐specific antibody titers also increased during allograft rejection in CB conditioned recipients given lymphodepletion. These observations suggest that allograft rejection following partial lymphocyte depletion is mediated by LIP of donor‐reactive memory T cells. As lymphopenia may cause unexpected rejection of stable allografts, adequate strategies must be developed to control T cell proliferation and differentiation during lymphopenia.     

Role of CD8+ lymphocytes in chronic rejection of transplanted hearts

BACKGROUND: The contribution of CD8(+) lymphocytes to the pathogenesis of cardiac allograft vasculopathy, or chronic rejection in heart transplants, remains undefined. We used both major histocompatibility complex class I mismatched and major histocompatibility complex class II mismatched models of cardiac allograft vasculopathy to characterize the role of CD8(+) lymphocytes in the development of cardiac allograft vasculopathy. METHODS: Donor hearts from B10.A mice were transplanted into B10.BR recipients (major histocompatibility complex class I mismatched). Donor hearts were harvested at 1, 7, 14, and 30 days after transplantation and (1) quantitated morphometrically for lesion development, (2) stained immunohistochemically, or (3) digested for isolation of graft-infiltrating cells. The cytotoxic phenotype of graft-infiltrating CD8(+) lymphocytes was determined with flow cytometry. Intracellular cytokine staining of CD8(+) and CD4(+) lymphocytes for interleukin 2, interferon g, interleukin 4, and interleukin 10 was performed with 2-color flow cytometry. Finally, B6.C-H2(bm12) donor hearts were transplanted into either C57BL/6 wild-type (major histocompatibility complex class II mismatched) or CD8 -/- knockout recipients and examined for the development of cardiac allograft vasculopathy. RESULTS: In the major histocompatibility complex class I mismatched model, CD8(+) lymphocytes were the predominant T-lymphocyte subset that infiltrated the allografts and demonstrated markers of activation. The intracellular cytokine-staining assay demonstrated that CD8(+) lymphocytes were the primary sources of allograft interleukin 2 and interferon gamma. Intimal lesions developed in the allografts by day 14 (12.0% +/- 4.0%) and further increased by day 30 (44.0% +/- 5.0%). In the major histocompatibility complex class II mismatched model, the donor hearts in the CD8 -/- knockout recipients had substantially less severe intimal lesions when compared with the donor hearts in wild-type recipients (19.0% +/- 6.0% vs 50.0% +/- 7.0%, respectively; P <.05). CONCLUSIONS: In both major histocompatibility complex class I and II mismatched models, CD8(+) lymphocytes contribute significantly to chronic rejection. The findings of this study suggest that control of chronic rejection requires interventions directed at CD8(+) lymphocytes.     

Analysis of Human Biologics With a Mouse Skin Transplant Model in Humanized Mice

Preclinical testing of human therapeutic monoclonal antibodies has been limited in murine models due to species differences in pharmacokinetics and biologic responses. To overcome these constraints we developed a murine skin transplant model in humanized mice and used it to test human monoclonal antibody therapy. Neonatal NOD/SCID/IL2Rγc^null mice (NSG) were reconstituted with human CD34⁺ hematopoietic stem cells (hNSG). When adult, these mice rejected MHC mismatched murine C57BL/6J skin grafts. Rejection required adequate reconstitution with human cells. There was diffuse infiltration of the epidermis and dermis with hCD8 and hCD4 cells in rejected grafts by immunohistochemistry. Studies with B6/MHC class I and II knockout mice donors indicated that neither is required for rejection. Graft rejection was associated with the development of effector and central memory T cells and an increase in serum immunoglobulins. We also tested the effects of teplizumab (anti‐CD3 mAb) and found it could delay skin graft rejection, whereas ipilimumab (anti‐CTLA‐4 [cytotoxic T‐lymphocyte antigen‐4] mAb) treatment accelerated rejection. These findings demonstrate that hNSG mice reliably and predictably reject a xenogenic mouse skin graft by a human T cell mediated mechanism. The model can be utilized to investigate the ability of human immunotherapies to enhance or suppress functional human immune responses.     

A major role for host MHC class I antigen presentation for promoting islet allograft survival

The purpose of this study was to determine the role for CD8 T cells versus generalized MHC class I-restricted antigen presentation in islet allograft rejection and tolerance. Diabetic C57BI/6 (B6, H-2(b)) controls, C57BI/6 CD8-deficient (CD8 KO), or MHC class I-deficient C57BI/6 (beta 2m KO) recipients were grafted with allogeneic BALB/c (H-2(d)) islets. Islet allografts were acutely rejected in untreated B6, CD8 KO, and in beta 2m KO mice, indicating that neither CD8 T cells nor host MHC class I is required for allograft rejection. We then determined the efficacy of costimulation blockade in these same strains. Costimulation blockade with anti-CD154 therapy facilitated long-term islet allograft survival in both B6 and in CD8 KO recipients. However, anti-CD154 treated beta 2m KO recipients were completely refractory to anti-CD154 therapy; all treated animals acutely rejected islet allografts with or without therapy. Also, anti-NK1.1 treatment of wild-type B6 mice abrogated graft prolongation following anti-CD154 therapy. Taken together, results show a dramatic distinction between two forms of MHC class I-restricted pathways in allograft prolongation. Although anti-CD154-induced allograft survival was CD8 T-cell independent, an intact host MHC class I-restricted (beta 2m-dependent) pathway is nevertheless necessary for allograft survival. This pathway required NK1.1+ cells, implicating NK and/or NKT cells in promoting allograft prolongation in vivo.     

CD154 Deficiency Uncouples Allograft CD8+ T‐Cell Effector Function from Proliferation and Inhibits Murine Airway Obliteration

Obliterative bronchiolitis (OB) limits the long‐term success of lung transplantation, while T‐cell effector mechanisms in this process remain incompletely understood. Using the murine heterotopic tracheal transplant model of obliterative airway disease (OAD) to characterize airway allograft rejection, we previously reported an important role for CD8⁺ T cells in OAD. Herein, we studied the role of CD154/CD40 costimulation in the regulation of allospecific CD8⁺ T cells, as airway rejection has been reported to be CD154‐dependent. Airway allografts from CD154^−/− recipients had significantly lower day 28 OAD scores compared to wild‐type (WT) recipients, and adoptive transfer of CD8⁺ T cells from WT recipients, but not CD154^−/− recipients, were capable of airway rejection in fresh CD154^−/− allograft recipients. Intragraft CD8⁺ T cells from CD154^−/− mice showed similar expression of the surface markers CD69, CD62L^low CD44^high and PD‐1, but markedly impaired IFN‐γ and TNF‐α secretion and granzyme B expression versus WT controls. Unexpectedly, intragraft and systemic CD8⁺ T cells from CD154^−/− recipients demonstrated robust in vivo expansion similar to WT recipients, consistent with an uncoupling of proliferation from effector function. Together, these data suggest that a lack of CD154/CD40 costimulation results in ineffective allospecific priming of CD8⁺ T cells required for murine OAD.     

Immunosuppression With CD40 Costimulatory Blockade Plus Rapamycin for Simultaneous Islet–Kidney Transplantation in Nonhuman Primates

The lack of a reliable immunosuppressive regimen that effectively suppresses both renal and islet allograft rejection without islet toxicity hampers a wider clinical application of simultaneous islet–kidney transplantation (SIK). Seven MHC‐mismatched SIKs were performed in diabetic cynomolgus monkeys. Two recipients received rabbit antithymocyte globulin (ATG) induction followed by daily tacrolimus and rapamycin (ATG/Tac/Rapa), and five recipients were treated with anti‐CD40 monoclonal antibody (mAb) and rapamycin (aCD40/Rapa). Anti‐inflammatory therapy, including anti–interleukin‐6 receptor mAb and anti–tumor necrosis factor‐α mAb, was given in both groups. The ATG/Tac/Rapa recipients failed to achieve long‐term islet allograft survival (19 and 26 days) due to poor islet engraftment and cytomegalovirus pneumonia. In contrast, the aCD40/Rapa regimen provided long‐term islet and kidney allograft survival (90, 94, >120, >120, and >120 days), with only one recipient developing evidence of allograft rejection. The aCD40/Rapa regimen was also tested in four kidney‐alone transplant recipients. All four recipients achieved long‐term renal allograft survival (100% at day 120), which was superior to renal allograft survival (62.9% at day 120) with triple immunosuppressive regimen (tacrolimus, mycophenolate mofetil, and steroids). The combination of anti‐CD40 mAb and rapamycin is an effective and nontoxic immunosuppressive regimen that uses only clinically available agents for kidney and islet recipients.     

Elimination of donor CD47 protects against vascularized allograft rejection in mice

CD47 is a ubiquitously expressed transmembrane glycoprotein that plays a complex role in regulation of cell survival and function. We have previously shown that the interspecies incompatibility of CD47 plays an important role in triggering rejection of cellular xenografts by macrophages. However, the role of CD47 in solid organ transplantation remains undetermined. Here, we explored this question in mouse models of heart allotransplantation. We observed that the lack of CD47 in donor hearts had no deleterious effect on graft survival in syngeneic or single MHC class I‐mismatched recipients, in which both wild‐type (WT) and CD47 knockout (CD47 KO) mouse hearts survived long term with no sign of rejection. Paradoxically, elimination of donor CD47 was beneficial for graft survival in signal MHC class II‐ and class I‐ plus class II‐mismatched combinations, in which CD47 KO donor hearts showed significantly improved survival compared to WT donor hearts. Similarly, CD47 KO donor hearts were more resistant than WT hearts to humoral rejection in α1,3‐galactosyltransferase‐deficient mice. Moreover, a significant prolongation of WT allografts was observed in recipient mice treated with antibodies against a CD47 ligand thrombospondin‐1 (TSP1) or with TSP1 deficiency, indicating that TSP1‐CD47 signaling may stimulate vascularized allograft rejection. Thus, unlike cellular transplantation, donor CD47 expression may accelerate the rejection of vascularized allografts.     

Endogenous Memory CD8 T Cells Are Activated Within Cardiac Allografts Without Mediating Rejection

Endogenous memory CD8 T cells infiltrate MHC‐mismatched cardiac allografts within 12–24 h posttransplant in mice and are activated to proliferate and produce IFN‐γ. To more accurately assess the graft injury directly imposed by these endogenous memory CD8 T cells, we took advantage of the ability of anti‐LFA‐1 mAb given to allograft recipients on days 3 and 4 posttransplant to inhibit the generation of primary effector T cells. When compared to grafts from IgG‐treated recipients on day 7 posttransplant, allografts from anti‐LFA‐1 mAb‐treated recipients had increased numbers of CD8 T cells but these grafts had marked decreases in expression levels of mRNA encoding effector mediators associated with graft injury and decreases in donor‐reactive CD8 T cells producing IFN‐γ. Despite this decreased activity within the allograft, CD8 T cells in allografts from recipients treated with anti‐LFA‐1 mAb continued to proliferate up to day 7 posttransplant and did not upregulate expression of the exhaustion marker LAG‐3 but did have decreased expression of ICOS. These results indicate that endogenous memory CD8 T cells infiltrate and proliferate in cardiac allografts in mice but do not express sufficient levels of functions to mediate overt graft injury and acute rejection.     

Interleukin‐33 prolongs allograft survival during chronic cardiac rejection

Interleukin‐33 (IL‐33) stimulates the generation of cells and cytokines characteristic of a Th2 immune response. We examined the effects of IL‐33 on allografted heart tissue in a chronic cardiac rejection model, including analysis of the peripheral myeloid and lymphoid compartments. B6.C‐H2bm12/KhEg hearts were transplanted into MHC class II‐mismatched C57Bl/6J mice; IL‐33 was administered daily. Cells from allografts and spleens were isolated for flow cytometry and cultured for cytokine production; some tissues were used for immunohistochemistry. Animals treated with IL‐33 showed significantly longer allograft survival, which was associated with a distinct cytokine profile produced by graft‐infiltrating cells. Proinflammatory IL‐17A production was decreased with IL‐33 treatment, while increased levels of IL‐5, IL‐10, and IL‐13 were observed. After IL‐33 therapy, flow cytometry showed a direct induction of CD4⁺ Foxp3⁺ Treg, whereas the number of B220⁺ CD19⁺ B cells, and circulating, as well as allograft deposited, alloantibodies was reduced. Following IL‐33 treatment, a significant decrease in graft‐infiltrating CD11b^highGr1^high granulocytes coincided with a significant increase in CD11b^highGr1^intermediate myeloid‐derived suppressor cells (MDSC). In conclusion, IL‐33 treatment in the setting of chronic rejection promotes the development of a Th2‐type immune response that favors MDSC and Treg expansion, reduces antibody‐mediated rejection (AMR), and ultimately, prolongs allograft survival.