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.     

Effects of Donor Age and Cell Senescence on Kidney Allograft Survival

The biological processes responsible for somatic cell senescence contribute to organ aging and progression of chronic diseases, and this may contribute to kidney transplant outcomes. We examined the effect of pre-existing donor aging on the performance of kidney transplants, comparing mouse kidney isografts and allografts from old versus young donors. Before transplantation, old kidneys were histologically normal, but displayed an increased expression of senescence marker p16 ^INK4a . Old allografts at day 7 showed a more rapid emergence of epithelial changes and a further increase in the expression of p16 ^INK4a . Similar but much milder changes occurred in old isografts. These changes were absent in young allografts at day 7, but emerged by day 21. The expression of p16 ^INK4a remained low in young kidney allografts at day 7, but increased with severe rejection at day 21. Isografts from young donors showed no epithelial changes and no increase in p16 ^INK4a . The measurements of the alloimmune response—infiltrate, cytology, expression of perforin, granzyme B, IFN-γ and MHC—were not increased in old allografts. Thus, old donor kidneys display abnormal parenchymal susceptibility to transplant stresses and enhanced induction of senescence marker p16 ^INK4a , but were not more immunogenic. These data are compatible with a key role of somatic cell senescence mechanisms in kidney transplant outcomes by contributing to donor aging, being accelerated by transplant stresses, and imposing limits on the capacity of the tissue to proliferate.     

Spontaneous acceptance of mouse kidney allografts is associated with increased Foxp3 expression and differences in the B and T cell compartments

Spontaneous acceptance of organ allografts can identify novel mechanisms of drug-free transplantation tolerance. Spontaneous acceptance occurs in both mouse kidney transplants and rat liver transplants however the early immune processes of mouse kidney acceptance have not been studied. Acceptance of C57BL/6 strain kidney allografts in fully MHC-incompatible B10.BR recipients was compared with rejection (REJ) of heart allografts in the same strain combination. Graft infiltrate and antibody deposition were examined by immunohistochemical staining. Expression of mRNA was measured by quantitative real-time PCR. Apoptosis was examined by TUNEL staining. The majority of kidney allografts were accepted long-term and induced tolerance (TOL) of donor-strain skin grafts, showing that acceptance was not due to immune ignorance. There was an extensive infiltrate of T cells in the TOL kidney that exceeded the level in REJ hearts but subsequently declined. The main differences were deposition of IgG2a antibody in REJ that was absent in TOL, more B cells infiltrating TOL kidneys and a progressive increase in the ratio of CD8:CD4 cells during rejection. There was also significantly greater Foxp3 mRNA expression in TOL. Kidneys from RAG-/- donors were accepted, showing that donor lymphocytes were not necessary for acceptance. Neutralising antibodies to TGF-β administered from day 0 to day 6 did not prevent TOL. On the basis of cytokine expression and apoptosis there was no evidence for immune deviation or deletion as mechanisms of acceptance. In accord with the findings of spontaneous acceptance of liver allografts in rats, the main difference between mouse kidney TOL and heart REJ was in the B cell compartment. The major difference to rat liver allograft acceptance was that apoptosis of infiltrate did not appear to play a role. Instead, increased Foxp3 expression in TOL kidneys implies that regulatory T cells might be important.     

Long-term outcome and alloantibody production in a non-myeloablative regimen for induction of renal allograft tolerance

BACKGROUND: Multilineage chimerism and long-term acceptance of renal allografts has been produced in non-human primates conditioned with a nonmyeloablative regimen. Our study was undertaken to evaluate the immunological and pathological status of long-term survivors and to define the role of splenectomy and of the primarily vascularized kidney in the regimen. METHOD: Monkeys were treated with the basic regimen, including: total body irradiation, thymic irradiation, antithymocyte globulin, donor bone marrow transplantation, and a 4-week course of cyclosporine after which no further immunosuppression was given. They were divided into four groups according to the timing of kidney transplantation (KTx) and splenectomy as follows; group A (n=13): KTx and splenectomy on the day of donor bone marrow transplantation (day 0); group B (n=3): KTx on day 0 without splenectomy; group C (n=7): splenectomy on day 0 but delayed KTx until 3 to 16 weeks post-donor bone marrow transplantation; group D (n=3): both splenectomy and KTx delayed until day 120 post-donor bone marrow transplantation. RESULTS: In group A, 11 of 13 monkeys developed chimerism and 9 monkeys achieved long-term survival of 4 to 70 months without evidence of chronic vascular rejection. Alloantibodies were detected in only one long-term survivor. In contrast, all three monkeys in group B developed alloantibodies and rejected their allografts. In group C, long-term survival without alloantibody production was observed in two of three monkeys that had developed chimerism. In group D, all three recipients were sensitized and rejected the kidney allografts rapidly after transplantation. CONCLUSIONS: 1) Production of anti-donor antibody was prevented in most recipients that developed mixed chimerism in the regimens with splenectomy at the time of donor bone marrow transplantation. 2) If splenectomy is not included in the initial conditioning regimen, induction of B cell tolerance is less likely and the result is late onset of alloantibody production and allograft rejection. 3) Immediate transplantation of the kidney at the time of recipient conditioning is not essential for induction of donor specific hyporesponsiveness by bone marrow transplantation.     

IFN-γ is an Absolute Requirement for Spontaneous Acceptance of Liver Allografts

Experimental liver allografts undergo spontaneous acceptance despite undergoing rejection during the first few weeks post transplant. We explored the role of interferon-gamma (IFN-gamma) in the spontaneous acceptance of mouse liver allografts. Strain of mouse (CBA) liver allografts transplanted into normal BALB/c mice developed histologic changes typical of rejection that spontaneously regressed, permitting long-term survival of these allografts similar to that of syngeneic grafts. In contrast, CBA liver allografts in IFN-gamma-deficient hosts manifested not only infiltration but also hemorrhage and necrosis, with no survival beyond 14 days. Despite differences in survival, local expression of cytotoxic T-cell genes in the transplant was not increased in IFN-gamma-deficient hosts, but livers in interferon-gamma-deficient mice (GKO) hosts displayed much less induction of major histocompatibility complex (MHC) class I and II expression. To determine whether the difference in survival was secondary to the direct effects of IFN-gamma on the liver, we transplanted livers from IFN-gamma-receptor-deficient mice into normal hosts. Liver allografts lacking IFN-gamma receptors also developed hemorrhage and necrosis with minimal induction of MHC expression. Thus IFN-gamma mediates a direct effect on rejecting liver allografts that reduces hemorrhage and necrosis, induces MHC expression, and is absolutely required for spontaneous acceptance.     

Detailed analysis and demonstration of differences in the kinetics of induction of class I and class II major histocompatibility complex antigens in rejecting cardiac and kidney allografts in the rat

In this paper, we analyze in detail donor class I and class II major histocompatibility complex (MHC) antigen induction in heart and kidney allografts in the DA-to-PVG rat strain combination. The immunohistological techniques and quantitative absorption analyses utilize monoclonal antibodies and assay systems specific for donor class I and class II MHC antigens, to enable precise interpretation of the results in terms of the MHC antigens of the graft. Quantitative absorption analyses were performed on homogenates comprising 4-6 allografts pooled at each interval examined (days 1-5 for kidneys, days 3-7 for hearts). In the heart allografts, donor class I antigen induction begins at day 3 after transplantation and proceeds rapidly on the 4th and 5th postoperative days. The maximum level (a 10-fold increase in comparison with normal heart) occurs at day 6, and thereafter the level declines. Donor class II antigen induction in the heart allografts follows a similar pattern. In kidney allografts, it was of particular interest that donor class I induction occurred much more rapidly, being already evident on the first postoperative day, and reaching levels 20-fold greater than normal kidney by day 3. Maximum levels (approximately 30-fold that of normal kidney) of donor class I antigens were reached on days 4 and 5. Donor class II induction, by contrast, developed in kidney grafts with kinetics similar to that seen for class II induction in heart grafts (beginning at day 3 and reaching a maximum of 7-fold over normal kidney at day 5). Immunohistological studies were performed at days 1, 3, 5, and 7 after transplantation. These confirmed the early induction of donor class I antigen in the kidney allografts. In kidney, by the fifth postoperative day, all tubules in the cortex and medulla, and the arteriolar vascular endothelium, were strongly positive for class II antigens. However, the glomerulus, including the glomerular capillary endothelium, remained donor-class-II-negative, except for induction of class II antigens on Bowman's capsule. The endothelium of interstitial capillaries also probably remained class-II-negative. These results have potentially important implications for understanding the development of the rejection response.     

Induction of Cardiac Allograft Tolerance Across a Full MHC Barrier in Miniature Swine by Donor Kidney Cotransplantation

We have previously shown that tolerance of kidney allografts across a full major histocompatibility complex (MHC) barrier can be induced in miniature swine by a 12‐day course of high‐dose tacrolimus. However, that treatment did not prolong survival of heart allografts across the same barrier. We have now tested the effect of cotransplanting an allogeneic heart and kidney from the same MHC‐mismatched donor using the same treatment regimen. Heart allografts (n = 3) or heart plus kidney allografts (n = 5) were transplanted into MHC‐mismatched recipients treated with high‐dose tacrolimus for 12 days. As expected, all isolated heart allografts rejected by postoperative day 40. In contrast, heart and kidney allografts survived for >200 days with no evidence of rejection on serial cardiac biopsies. Heart/kidney recipients lost donor‐specific responsiveness in cell‐mediated lympholysis and mixed‐lymphocyte reaction assays, were free of alloantibody and exhibited prolonged survival of donor, but not third‐party skin grafts. Late (>100 days) removal of the kidney allografts did not cause acute rejection of the heart allografts (n = 2) and did not abrogate donor‐specific unresponsiveness in vitro. While kidney‐induced cardiac allograft tolerance (KICAT) has previously been demonstrated across a Class I disparity, these data demonstrate that this phenomenon can also be observed across the more clinically relevant full MHC mismatch. Elucidating the renal element(s) responsible for KICAT could provide mechanistic information relevant to the induction of tolerance in recipients of isolated heart allografts as well as other tolerance‐resistant organs.     

The effects of cyclosporine on the induction of donor class I and class II MHC antigens in heart and kidney allografts in the rat

We have previously reported 5-30-fold increases in the expression of class I and class II major histocompatibility complex (MHC) antigens in rejecting heart and kidney allografts in the DA-to-PVG rat strain combination. We examine here the effects of immunosuppression with cyclosporine on the induction of donor class I and class II MHC antigens in heart and kidney allografts in this strain combination. Immunohistological studies and quantitative absorption analyses using monoclonal antibodies and assay systems specific for donor class I and class II MHC antigens were used throughout. Heart allografts in cyclosporine-treated rats were examined on day 3,5,7,9,11, and 14 after transplantation, and kidney allografts in cyclosporine-treated rats were examined at day 7. In addition, untreated heart and kidney isografts were studied at days 1,3,5, and 7 after grafting. Immunohistological studies on frozen sections showed that cyclosporine-treated heart and kidney allografts showed no induction of class II MHC antigens, in contrast to untreated heart and kidney allografts. Class I MHC antigen induction did occur in spite of cyclosporine-therapy, but at levels lower than those seen in untreated allografts. Moreover, the pattern and degree of class I induction in the cyclosporine-treated allografts resembled very closely those seen in isografts, and so this induction was, in all probability, a consequence of the transplantation procedure rather than of specific immune responses. We also noted, in the cyclosporine-treated heart allografts, that all donor interstitial dendritic cells had disappeared and been replaced by recipient interstitial dendritic cells by the end of the second week after grafting. In addition, there was no reduction in the class II antigen content of kidney allografts treated for 7 days with cyclosporine. The absence of class II antigen induction in allografts where rejection is effectively suppressed with cyclosporine might be of clinical value in the differential diagnosis between rejection and cyclosporine toxicity in renal transplantation, and between active and inactive cellular infiltrates in heart transplantation.     

Absence of donor MHC antigen expression ameliorates chronic kidney allograft rejection

BACKGROUND: In previous studies, we have demonstrated that a subset of mouse kidney allografts has prolonged survival without any immunosuppressive treatment. Chronic rejection (CR) develops in these long surviving grafts. The pathologic features of CR in this model are similar to CR in human kidney grafts. METHODS: To explore the role of donor major histocompatibility complex (MHC) antigens in the development of CR, we performed vascularized kidney transplants using kidneys from donor mice that lack expression of both MHC class I and II antigens (MHC-/-). RESULTS: Survival was significantly improved in recipients of MHC-/- allografts. This enhanced survival was associated with higher glomerular filtration rate (GFR) in MHC-/- allografts (4.92 +/- 0.54 cc/min/kg) compared to controls (2.19 +/- 0.63 cc/min/kg; P = 0.004). The typical histologic features of CR were markedly reduced in MHC-/- allografts. Semiquantitative histopathological scores for MHC-/- grafts (13.3 +/- 2.1) were significantly lower than in control allografts (19.0 +/- 1.0; P = 0.04). Along with this improvement in structural abnormalities, significantly fewer CD4+ T (38.3 cells/mm(2) vs. 75.0 cells/mm(2); P = 0.008), CD8+ T cells (38.7 vs. 96 cells/mm(2), respectively; P = 0.008) and macrophages (60 vs. 134 cells/mm(2), respectively; P = 0.04) infiltrated MHC-/- allografts compared to controls. The levels of intragraft cytokine mRNA expression also were reduced in MHC-/- allografts compared to control allografts. Finally, serum alloantibodies were virtually undetectable in recipients of MHC-/- kidney allografts. CONCLUSIONS: Cell surface expression of donor MHC antigens promotes the development of CR. Donor antigen expression promotes the accumulation of infiltrating cells in the graft and the development of donor specific alloantibodies. Abrogation of these responses is associated with improved graft survival and reduced CR in MHC-/- grafts.     

Donor Fas Is Not Necessary for T‐Cell‐Mediated Rejection of Mouse Kidney Allografts

It is important to resolve whether T‐cell‐mediated rejection (TCMR) is mediated by contact‐dependent cytotoxicity or by contact‐independent inflammatory mechanisms. We recently showed that the cytotoxic molecules perforin and granzymes A and B are not required for TCMR of mouse kidney transplants. Nevertheless, TCMR could still be mediated by cytotoxicity via Fas on donor cells engaging Fas ligand on host T cells. We examined whether the diagnostic TCMR lesions would be abrogated if donor Fas was absent, particularly in hosts deficient in perforin or granzymes A and B. Kidneys from Fas‐deficient donors transplanted into major histocompatibility complex (MHC)‐ mismatched hosts developed tubulitis and diffuse interstitial infiltration indistinguishable from wild‐type (WT) allografts, even in hosts deficient in perforin and granzymes A and B. Gene expression analysis revealed similar molecular disturbances in Fas‐deficient and WT allografts at day 21 transplanted into WT, perforin and granzyme A/B‐deficient hosts, indicating epithelial injury and dedifferentiation. Thus, donor Fas is not necessary for TCMR diagnostic lesions or molecular changes, even in the absence of perforin–granzyme mechanisms. We propose that in TCMR, interstitial effector T cells mediate parenchymal injury by inflammatory mechanisms that require neither the perforin–granzyme nor the Fas–Fas ligand cytotoxic mechanisms.     

Cold ischemia augments allogeneic-mediated injury in rat kidney allografts

BACKGROUND: Some clinical studies demonstrate that kidney grafts with prolonged cold ischemia experience early acute rejection more often than those with minimal ischemia. The mechanism, however, is putative. Therefore, the aim of this study was to unravel the impact of ischemia on the immune response in rat kidney allografts compared with that in isografts. METHODS: To induce ischemic injury, donor kidneys were preserved for 24 hours in 4 degrees C University of Wisconsin solution before transplantation. No immunosuppression was administered. The histomorphology according to the BANFF criteria for acute rejection and infiltrating cells were assessed at days 1, 2, 3, 4, 6, and 8 post-transplantation. RESULTS: In allografts, exposure of the kidney to ischemia led to a significantly earlier onset of interstitial cell infiltration and tubulitis compared with nonischemic allografts. The BANFF score of interstitial cell infiltration was 1 +/- 0 vs. 0.25 +/- 0.29 at day 3 and 2 +/- 0 vs. 1.25 +/- 0.25 at day 4. In contrast, in isografts, the effect of ischemia on the histology was not significant. From day 6, the histologic differences between ischemic and nonischemic grafts disappeared. Ischemia led to a more intense expression of P-selectin (day 1), intercellular adhesion molecule-1 (ICAM-1; day 2), and major histocompatibility complex (MHC) class II on endothelium and proximal tubular cells (day 2) in both allografts and isografts. Concurrently with the up-regulated ICAM-1 and MHC expression, significantly more CD4(+) cells and macrophages infiltrated the ischemic allografts at days 2 and 3 and the ischemic isografts at day 4. Importantly, the influx of these cells after ischemia was significantly greater in allografts than in isografts. CONCLUSIONS: Cold ischemia augments allogeneic-mediated cell infiltration in rat kidney allografts. The earlier onset of acute rejection in 24-hour cold preserved allografts may be prevented by better preservation or treatment using tailored immunosuppression.