Lesions of T-Cell-Mediated Kidney Allograft Rejection in Mice Do Not Require Perforin or Granzymes A and B

Organ allograft rejection is strongly associated with the presence of alloreactive cytotoxic T cells but the role of cytotoxicity in the pathologic lesions is unclear. Previous studies showed that the principal lesions of kidney rejection - interstitial infiltration, tubulitis, and endothelial arteritis - are T-cell-dependent and antibody-independent. We studied the role of cytotoxic granule components perforin and granzymes A and B in the evolution of the T-cell-mediated lesions of mouse kidney transplant rejection. By real-time RT-PCR, allografts rejecting in wild-type hosts at days 5, 7, 21, and 42 showed massively elevated and persistent expression of perforin and granzymes A and B, but evolution of tubulitis and arteritis did not correlate with increasing granzyme or perforin expression. Allografts transplanted into hosts with disrupted genes for perforin or granzymes A and B showed no change in tubulitis, arteritis, or MHC induction. Thus the development of the histologic lesions diagnostic of T-cell-mediated kidney transplant rejection are associated with but not mediated by perforin or granzyme A or B. Together with previous graft survival studies, these results indicate that the granule-associated cytotoxic mechanisms of T cells are not the effectors of T-cell-mediated allograft rejection.     

IFN-γ Prevents Early Perforin-Granzyme-Mediated Destruction of Kidney Allografts by Inducing Donor Class I Products in the Kidney

Interferon-γ (Ifng) protects organ allografts: mouse kidney allografts lacking Ifng receptors rapidly fail with massive ischemic necrosis around days 5 to 7, reflecting microcirculation failure. We hypothesized that Ifng protects the graft by preventing perforin-granzyme-mediated cytotoxic damage to the microcirculation by inducing class Ia and/or Ib products. We transplanted kidney allografts lacking Ifng receptors into various knockout hosts. The necrosis/congestion phenotype did not require host B cells or IL-4 and IL-13 receptors, but required the T-cell alloresponse: it did not occur if the hosts were syngeneic or T-cell deficient. However, host perforin-granzyme mechanisms were required: no necrosis developed if hosts lacked either perforin or granzymes A and B. The ability of Ifng to protect the allograft required donor class I products: allografts lacking class I products due to Tap1 or β2 microglobulin deficiency developed a similar necrosis-congestion phenotype at day 7 despite Ifng receptors being present. Thus when host cytotoxic T cells infiltrate organ allografts, Ifng prevents their perforin-granzyme mechanism from compromising the microcirculation by a mechanism requiring donor class Ia or Ib products. We propose that donor class Ia or Ib products are needed to trigger inhibitory receptors on effector T cells.     

Interferon Gamma and Contact‐dependent Cytotoxicity Are Each Rate Limiting for Natural Killer Cell–Mediated Antibody‐dependent Chronic Rejection

Natural killer (NK) cells are key components of the innate immune system. In murine cardiac transplant models, donor‐specific antibodies (DSA), in concert with NK cells, are sufficient to inflict chronic allograft vasculopathy independently of T and B cells. In this study, we aimed to determine the effector mechanism(s) required by NK cells to trigger chronic allograft vasculopathy during antibody‐mediated rejection. Specifically, we tested the relative contribution of the proinflammatory cytokine interferon gamma (IFN‐γ) versus the contact‐dependent cytotoxic mediators of perforin and the CD95/CD95L (Fas/Fas ligand [FasL]) pathway for triggering these lesions. C3H/HeJ cardiac allografts were transplanted into immune‐deficient C57BL/6 rag^?/?γc^?/? recipients, who also received monoclonal anti–major histocompatibility complex (MHC) class I DSA. The combination of DSA and wild‐type NK cell transfer triggered aggressive chronic allograft vasculopathy. However, transfer of IFN‐γ–deficient NK cells or host IFN‐γ neutralization led to amelioration of these lesions. Use of either perforin‐deficient NK cells or CD95 (Fas)–deficient donors alone did not alter development of vasculopathy, but simultaneous disruption of NK cell–derived perforin and allograft Fas expression resulted in prevention of these abnormalities. Therefore, both NK cell IFN‐γ production and contact‐dependent cytotoxic activity are rate‐limiting effector pathways that contribute to this form of antibody‐induced chronic allograft vasculopathy.     

T Cell‐mediated Rejection of Kidney Transplants: A Personal Viewpoint

In kidney allografts, T cell mediated rejection (TCMR) is characterized by infiltration of the interstitium by T cells and macrophages, intense IFNG and TGFB effects, and epithelial deterioration. Recent experimental and clinical studies provide the basis for a provisional model for TCMR. The model proposes that the major unit of cognate recognition in TCMR is effector T cells engaging donor antigen on macrophages. This event creates the inflammatory compartment that recruits effector and effector memory CD4 and CD8 T cells, both cognate and noncognate, and macrophage precursors. Cognate T cells cross the donor microcirculation to enter the interstitium but spare the microcirculation. Local inflammation triggers dedifferentiation of the adjacent epithelium (e.g. loss of transporters and expression of embryonic genes) rather than cell death, via mechanisms that do not require known T‐cell cytotoxic mechanisms or direct contact of T cells with the epithelium. Local epithelial changes trigger a response of the entire nephron and a second wave of dedifferentiation. The dedifferentiated epithelium is unable to exclude T cells, which enter to produce tubulitis lesions. Thus TCMR is a cognate recognition‐based process that creates local inflammation and epithelial dedifferentiation, stereotyped nephron responses, and tubulitis, and if untreated causes irreversible nephron loss.     

Tubulitis and Epithelial Cell Alterations in Mouse Kidney Transplant Rejection Are Independent of CD103, Perforin or Granzymes A/B

One of the defining lesions of kidney allograft rejection is epithelial deterioration and invasion by inflammatory cells (tubulitis). We examined epithelial changes and their relationship to effector T cells and to CD103/E-cadherin interactions in mouse kidney allografts. Rejecting allografts showed interstitial mononuclear infiltration from day 5. Loss of epithelial mass, estimated by tubular surface area, and tubulitis were minimal through day 7 and severe by day 21. Tubules in day 21 allografts manifested severe reduction of E-cadherin and Ksp-cadherin by immunostaining with redistribution to the apical membrane, indicating loss of polarity. By flow cytometry T cells isolated from allografts were 25% CD103+. Laser capture microdissection and RT-PCR showed increased CD103 mRNA in the interstitium and tubules. However, allografts in hosts lacking CD103 developed tubulitis, cadherin loss, and epithelial deterioration similar to wild-type hosts. The loss of cadherins and epithelial mass was also independent of perforin and granzymes A and B. Thus rejection is characterized by severe tubular deterioration associated with CD103+ T cells but not mediated by CD103/cadherin interactions or granzyme-perforin cytotoxic mechanisms. We suggest that alloimmune effector T cells mediate epithelial injury by contact-independent mechanisms related to delayed type hypersensitivity, followed by invasion of the altered epithelium to produce tubulitis.     

B‐Cell‐Dependent Memory T Cells Impede Nonmyeloablative Mixed Chimerism Induction in Presensitized Mice

Presensitization to HLA antigens limits the success of organ transplantation. The achievement of donor‐specific tolerance via mixed chimerism could improve outcomes of transplantation in presensitized patients. In presensitized B‐cell‐deficient μMT B6 mice, we developed nonmyeloablative bone marrow transplantation (BMT) regimens that successfully tolerized presensitized T cells, achieving long‐term (LT) multilineage chimerism and tolerance to donor‐type skin. To apply these regimens in wild‐type (WT) animals while avoiding antibody‐mediated destruction of donor bone marrow cells, presensitized WT B6 mice were rested >2 years to allow alloantibody clearance. However, chimerism and tolerance were not reliably achieved in LT presensitized WT B6 mice in which alloantibody had declined to minimal or undetectable levels before BMT. Strong antidonor memory T‐cell responses were detected in LT presensitized WT B6 mice after rejection of donor bone marrow (BM) occurred, whereas levels of alloantibody remained consistently low. In contrast, presensitized μMT B6 mice had diminished memory T‐cell responses compared to WT B6 mice. These data implicate T‐cell memory, but not alloantibody, in rejection of donor BM in LT presensitized WT mice.     

Galectin‐1 Prolongs Survival of Mouse Liver Allografts From Flt3L‐Pretreated Donors

Liver allografts are spontaneously accepted across MHC barriers in mice. The mechanisms underlying this phenomenon remain poorly understood. Galectin‐1, an endogenous lectin expressed in lymphoid organs, plays a vital role in maintaining central and peripheral tolerance. This study was to investigate the role of galectin‐1 in spontaneous tolerance of liver allografts in mice, and to evaluate the therapeutic effects of galectin‐1 on liver allograft rejection induced by donor Flt3L pretreatment. Blockade of the galectin‐1 pathway via neutralizing antigalectin‐1 mAb did not affect survival of the liver allografts from B6 donors into C3H recipients. Administration of rGal‐1 significantly prolonged survival of liver allografts from Flt3L‐pretreated donors and ameliorated Flt3L‐triggered liver allograft rejection. This effect was associated with increased apoptosis of T cells in both allografts and spleens, decreased frequencies of Th1 and Th17 cells, decreased expression of Th1‐associated cytokines (IL‐12, IL‐2 and IFN‐γ), Th17‐associated cytokines (IL‐23 and IL‐17) and granzyme B, in parallel with selectively increased IL‐10 expression in liver allografts. In vitro, galectin‐1 inhibited Flt3L‐differentiated DC‐mediated proliferation of allo‐CD4⁺ T cells and production of IFN‐γ and IL‐17. These data provide new evidence of the potential regulatory effects of galectin‐1 in alloimmune responses in a murine model of liver transplantation.     

Isolated vascular “v” lesions in liver allografts: How to approach this unusual finding

According to the Banff criteria for kidney allografts, isolated vascular or “v” lesions are defined as intimal inflammation, age‐inappropriate fibro‐intimal hyperplasia, or both, without the presence of associated interstitial T cell‐mediated rejection (TCMR). In general, these lesions portend a worse outcome for kidney allografts, particularly in those where the “v” lesions are identified in patients with coexistent donor specific antibodies (DSA) or later after transplantation. Although affected arteries are rarely sampled in liver allograft biopsies, we identified nine patients at a mean of 1805 days posttransplantation and compared these to matched controls. Almost half (4 of 9) of the study patient biopsies showed inflammatory arteritis associated with focal or diffuse C4d positivity, which was not observed in matched controls. One “v” lesion patient progressed to rejection‐related graft failure and two developed moderate/severe TCMR in subsequent biopsies, whereas only one rejection episode occurred in follow‐up biopsies, and no rejection‐related deaths or graft failures were detected in controls. In conclusion, patients with liver allograft isolated “v” lesions should undergo further evaluation and closer follow‐up for impending TCMR and/or underlying co‐existent chronic antibody‐mediated rejection (AMR).     

A critical role for granzyme B, in addition to perforin and TNFalpha, in alloreactive CTL-induced mouse pancreatic beta cell death

BACKGROUND: The precise effector mechanisms and molecular mediators used by alloreactive cytotoxic T lymphocytes to kill transplanted pancreatic beta cells are poorly defined. We have used mouse (H2b-anti-d) CTLs raised in strains deficient in various key cytotoxic effector molecules to assess the importance of the various signaling pathways mobilized to kill primary mouse pancreatic islet cells, the beta cell line NIT-1, and NIT-1 cells overexpressing dominant-negative FADD and Bcl-2. METHODS: Death of target cells was assessed using 51Cr release assays. RESULTS: In short-term assays (<5 hours) beta cell death did not require a functional FasL/Fas pathway, and was not inhibited by Bcl-2. However, the absence of either perforin or granzyme B resulted in cell survival. By contrast, a crucial role for granzyme B was not seen when hematopoietic P815 cells were used as targets, indicating differential regulation of apoptosis. Interestingly, coincubation with CTL for 24 hours revealed an additional but less potent "late phase" of beta cell death that did not require perforin. This delayed death was blocked by dominant-negative FADD, but not by Bcl-2, and was likely to be due to TNFalpha secretion. CONCLUSIONS: This study suggests that strategies to protect beta cells from allogeneic CTL attack will need to inhibit the perforin/granzyme and probably also the TNFalpha pathway. As there are no known pharmacological approaches to blocking perforin, therapeutic approaches based on overexpressing both dominant negative FADD and an inhibitor of granzyme B may hold promise in prolonging beta cell survival in the allogeneic setting.     

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.     

Role of CD4+ and CD8+ T cells in murine skin and heart allograft rejection across different antigenic desparities

The factors that influence the relative contribution of the T cell subsets to allograft rejection remain unclear. We compared skin and heart rejection in CD4 Knockout (KO), and CD8 KO mice across full-, minor-, and class II histocompatibility antigen (HA) mismatches. Skin allografts were rejected by either CD4+ or CD8+ T cells alone at any degree of antigenic mismatch. However, either the absence of CD4+ cells or a lesser degree of HA mismatch resulted in prolongation of graft survival. In contrast, fully allogeneic heart grafts were accepted in CD4 KO recipients, and minor HA mismatched heart grafts were accepted by both CD4 KO and CD8 KO mice. Thus, the T cell subsets required for allograft rejection are determined by the immunogenicity of the tissue transplanted. In the absence of CD8+ T cells, perforin and Fas ligand (FasL) but not granzyme B mRNA were detected in rejecting grafts. Thus, granzyme B is a CD8+ cytotoxic T lymphocyte (CTL)-specific effector molecule.     

Granzyme B Induces Endothelial Cell Apoptosis and Contributes to the Development of Transplant Vascular Disease

Endothelial cell death induced by cytotoxic T cells is a key initiating event in the development of transplant vascular disease (TVD), the leading cause of late solid organ transplant failure. We studied the role of the granzyme B (GrB) pathwaye, which is one of the main mechanisms by which T cells induce apoptosis of allogeneic targets, in the pathogenesis of TVD. Granzyme B, in combination with perforin (pfn), induced apoptosis of cultured endothelial cells. In hearts transplanted into GrB knockout (GrB-KO) mice, there was a similar level of vasculitis as compared to WT mice, indicating that GrB does not affect immune infiltration into allograft arteries. However, there was a significant reduction in luminal narrowing of allograft arteries from GrB-KO mice as compared to WT recipients. These results indicate that GrB plays a role in endothelial cell death in allograft arteries and in the resultant development of TVD.     

Prolonged survival of pig islets xenograft by adenovirus‐mediated expression of either the membrane‐bound human FasL or the human decoy Fas antigen gene

Abstract: Background: Pig islets are considered an attractive alternative treatment for patients with Type 1 diabetes. However, pig islet xenografts, transplanted into non‐human primates, are directly rejected by cell‐mediated processes. We have previously reported that cell‐mediated xenograft‐rejections, and especially human CD8⁺ cytotoxic T lymphocytes (CTL)‐mediated cytotoxicity, are highly detrimental to pig xenograft cells. Moreover, we have explored novel strategies for the prevention of CTL killing by overexpression of either human decoy Fas antigen or membrane‐bound human FasL in pig endothelial cells. In this study, we assessed the cytoprotective effects of these molecules for pig islets both in vitro and in vivo. Materials and methods: Pig islets were freshly isolated by modified Ricordi’s methods. Subsequently, these islets were transfected with an adenoviral expression vector containing the DNA fragments of either membrane‐bound human FasL or human decoy Fas. Transfected islets were transplanted into preimmunized diabetic rats under the kidney capsule. Control pig islets (i.e., MOCK), which were transfected with an adenoviral expression vector containing only the enhanced green fluorescent protein gene, were also transplanted. Results: Efficiency of adenoviral expressions of these molecules in pig islets was approximately 80% at a multiplicity of infection of 100. In an in vitro assay, approximately 80% suppression of cytotoxicity was observed in membrane‐bound human FasL‐expressing pig islets and 60% inhibition of CTL killing was displayed in decoy Fas expression pig islets. In an in vivo transplant model, prolonged survival of pig islets xenografts, expressing either membrane‐bound human FasL or human decoy Fas genes, was elicited in comparison with that of control islets xenografts. Conclusion: The extracellular remodeling of either death receptor or death ligand genes by adenoviral expression was effective for the prevention of CTL‐mediated xenocytotoxicity in pig islets.     

Mechanism of T cell-mediated endothelial apoptosis

BACKGROUND: Cytotoxic T lymphocyte (CTL)-mediated destruction of allogeneic vascular endothelium is important in the pathogenesis of both acute and chronic allograft rejection. Despite the importance of this phenomenon, the effector mechanisms responsible for endothelial cell killing are not well defined, and conflicting conclusions have been reached based on variation in experimental methodology. METHODS: We used a recently described method for isolating mouse vascular endothelium to evaluate endothelial cell lysis by CTLs. Endothelial cell destruction was assessed in vitro both by 51Cr release and DNA fragmentation using wild-type and lpr (Fas deficient) endothelium of C3H/HeJ (H2(k)) mice by MHC alloantigen-specific T cells of wild-type, gld (Fas ligand deficient), and perforin-deficient mice on a C57BL/6 (H2(b)) background. RESULTS: Although maximal lysis of 56.6+/-0.8% was seen when using wild-type targets and effectors, only a moderate decrease in apoptosis to 37.6+/-4.0% was detected when the Fas/Fas ligand death receptor pathway was eliminated. This decrease in cytotoxicity occurred despite the preserved functional capacity of this pathway. Alternatively, a significant decrease in cytotoxicity to 17.4+/-4.7% was seen when the perforin/granzyme exocytosis pathway was eliminated. CONCLUSIONS: These data indicate that CTLs destroy vascular endothelium primarily by the perforin/granzyme exocytosis pathway with only a minor contribution to apoptosis by the Fas/Fas ligand death receptor pathway. These data are critical for the proper interpretation of studies evaluating acute and chronic allograft rejection and for the design of rational strategies to ameliorate vascular injury concomitant to the rejection process.