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Foxp3(+) regulatory T cells (Tregs) have an essential role in immune and allograft tolerance. However, in both kidney and liver transplantation in humans, FOXP3(+) Tregs have been associated with clinical rejection. Therefore, the role and function of graft infiltrating Tregs have been of great interest. In the studies outlined, we demonstrated that Foxp3(+) Tregs were expanded in tolerant kidney allografts and in draining lymph nodes in the DBA/2 (H-2(d) ) to C57BL/6 (H-2(b) ) mouse spontaneous kidney allograft tolerance model. Kidney allograft tolerance was abrogated after deletion of Foxp3(+) Tregs in DEpletion of REGulatory T cells (DEREG) mice. Kidney allograft infiltrating Foxp3(+) Tregs (K-Tregs) expressed elevated levels of TGF-β, IL-10, interferon gamma (IFN-γ), the transcriptional repressor B lymphocyte-induced maturation protein-1 (Blimp-1) and chemokine receptor 3 (Cxcr3). These K-Tregs had the capacity to transfer dominant tolerance and demonstrate donor alloantigen-specific tolerance to skin allografts. This study demonstrated the crucial role, potency and specificity of graft infiltrating Foxp3(+) Tregs in the maintenance of spontaneously induced kidney allograft tolerance., (© Copyright 2013 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Signalling through the cytokine common γ chain (γc) is crucial for survival of activated T cells. In its absence, severe combined immunodeficiency ensues and transplanted tissues are not rejected., Methods: To determine whether differences in the availability of γc signalling cytokines correlate with rejection or acceptance, we examined expression of all γc signalling components in organs transplanted between PVG donors and DA recipients. In this combination hearts or kidneys are rejected in <10 days while livers survive >100 days. Expression of the γc cytokines IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 and their receptors γc, IL-2Rα, IL-2Rβ/IL-15Rβ, IL-4Rα, IL-7Rα, IL-9Rα, IL-15Rα and IL-21Rα was determined by real-time PCR pre-transplant and on days 3, 5 and 7 after transplantation., Results: Most increased after transplantation, although there were significantly lower levels of IL-2, IL-2Rα, IL-4 and IL-15Rα in tolerant livers compared to rejecting hearts or kidneys. IL-9 was only expressed in normal kidneys and decreased during rejection. IL-15 was constitutively expressed and did not change after transplantation. IL-21 and IL-21R increased in all transplanted organs to a similar extent. IL-7Rα in liver was considerably increased compared with heart or kidney, consistent with its known inverse relationship to global levels of γc signalling., Conclusions: In transplanted livers, acceptance is associated with low levels of all γc cytokines or receptors except IL-21. This is consistent with "dilution" of γc cytokines from a finite clone size of alloreactive T cells in livers, which are ten times larger than kidneys or hearts., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Growth arrest-specific gene 6 (Gas6) and its receptors Rse, Axl and Mer have recently been found to be involved in a rat model of chronic allograft nephropathy (CAN). Thus, in this study we investigated the function of Gas6 and its receptors in human renal allograft dysfunction. Expression of Gas6 and its receptors was detected by immunohistochemical staining. Gas6 and its receptors were widely expressed in glomeruli, tubules and vessels of renal allografts. Gas6 expression was detected in normal-functioning allografts and was increased in acute rejection ( P<0.05), acute tubular necrosis ( P<0.05) and CAN ( P<0.01). Gas6 receptors were not upregulated in any of the allograft groups, except for the Axl receptor, which increased only in acute tubular necrosis ( P<0.01). Gas6 expression was also found to correspond with the expression of alpha-smooth muscle actin, a general marker of CAN ( r(2)=0.21, P<0.01). These findings suggest that Gas6, acting as a growth factor, is increased in the process of kidney allograft dysfunction and in CAN.

Background: Chronic Rejection (CR) is the leading cause of renal allograft dysfunction. Upregulation of growth factors has been shown in CR but the time point at which this occurs in not known. The aim of this study was to examine the time course of upregulation of growth factors and correlate this with the macrophage and myofibroblast interstitial infiltrate., Methods: Using a rat model of CR (F344 kidney donor to Lewis recipient), infiltration by ED1 + macrophages and proliferation of alpha-smooth muscle actin (alpha-SMA) and desmin-expressing cells was examined using immunohistochemistry. In addition, expression of mRNA for interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta), basic-fibroblast growth factor (b-FGF) and vascular endothelial growth factor (VEGF) was studied using a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) technique. Native Lewis rat kidney and Lewis-Lewis isografts were used as controls., Results: Immunohistochemical staining of ED1 + cells showed a marked increase in the macrophage infiltrate of allografts compared to isografts at all time periods (P = 0.0002) peaking at weeks 8-12 after transplantation. Expression of alpha-SMA was also increased in allografts (P = 0.002). RT-PCR analysis showed that mRNA for TGF-beta was maximally upregulated in allografts in comparison to isografts at week 8 after engraftment (P = 0.05) and declined thereafter, although remained at elevated levels compared to controls. IFN-gamma and b-FGF gene expression was increased in allografts late in the post-transplantation period., Conclusion: Early infiltration of macrophages and production of TGF-beta1 was followed by later upregulation of fibrogenic growth factors and myofibroblasts associated with interstitial fibrosis and organ dysfunction.

Background: Although donor leukocytes are only thought to prolong survival when administered before transplantation, recent evidence shows that they are effective at transplantation. This study aims to identify the leukocyte subset that is most active in prolonging kidney allograft survival and examine the cytokine expression in long-term acceptance., Methods: PVG rat kidneys were transplanted to completely MHC class I and class II-mismatched DA recipients. Donor B cells or T cells, purified by negative selection, were injected i.v. at the time of transplantation. Expression of interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-gamma, and transforming growth factor-beta mRNA was measured by quantitative real-time polymerase chain reaction (PCR). Immunohistochemical analysis and terminal deoxynucleotide transferase-mediated dUTP nick-end labelling (TUNEL) staining was used to identify infiltrating cells and apoptotic cells, respectively, in sections of kidney allografts., Results: Median kidney graft survival time (MST) of B cell-treated animals (n=5) was >300 days, compared with 7 days in untreated animals (n=7) (P=0.003), whereas animals treated with the same number of T cells (n=6) had a MST of 17 days (P=0.1 vs. untreated, P=0.03 vs. B cell-treated). Examination of the long-term (>300 days) accepted grafts from B cell-treated recipients showed little evidence of kidney damage but a moderate perivascular infiltrate consisting of T and B cells. This infiltrate seemed to be quiescent because there was no detectable expression of IL-2 receptors or of apoptotic cells. It produced little or no cytokine mRNA, because expression in the long-term accepted grafts was similar to levels in normal kidneys or syngeneic transplants. There was a marked increase of cytokine mRNA early after transplantation in both leukocyte-treated and untreated grafts, with more rapid appearance of IFN-gamma and IL-10 in leukocyte-treated grafts., Conclusions: Donor B cells efficiently induce long-term acceptance of transplanted kidneys in a fully MHC-mismatched rat model when administered at transplantation, by a mechanism that seems to be independent of Th2 cytokine expression within the long-term accepted graft.

Background: Growth arrest-specific gene 6 (Gas6) is involved in a number of cell functions that include proliferation of vascular smooth muscle cells and mesangial cells. The proliferation of these cells is a feature of chronic rejection (CR) after kidney transplantation. Therefore, we examined the gene expression of Gas6 and its receptors Rse, Axl, and Mer in a rat model of CR., Methods: The rat model of CR was established in Lewis rat recipients of Fisher kidney transplants. The level of mRNA was measured by real-time quantitative reverse transcription-polymerase chain reaction. The proteins were detected by immunohistochemical staining and Western blot analysis., Results: Gas6 mRNA was extensively expressed in kidney tissue of both allografts and isografts. There was significant increase in expression of Gas6 mRNA in allografts at 4 weeks posttransplantation. Immunohistochemical study showed that Gas6 and its receptor Rse proteins were highly expressed in kidney tissue. Western blot analysis has also confirmed that Gas6 and Rse proteins are expressed in kidney tissue., Conclusions: These findings suggest that Gas6 and its receptors have an as yet undefined role in kidney function and/or development and may be involved in the pathogenesis of CR. The action of Gas6 in rat kidney is mainly mediated through the Rse receptors rather than the Axl and Mer receptors.

Donor leukocytes play a dual role in rejection and acceptance of transplanted organs. They provide the major stimulus for rejection, and their removal from the transplanted organ prolongs its survival. Paradoxically, administration of donor leukocytes also prolongs allograft survival provided that they are administered 1 wk or more before transplantation. Here we show that administration of donor leukocytes immediately after transplantation induced long-term acceptance of completely MHC-mismatched rat kidney or liver transplants. The majority of long-term recipients of kidney transplants were tolerant of donor-strain skin grafts. Acceptance was associated with early activation of recipient T cells in the spleen, demonstrated by a rapid increase in IL-2 and IFN-gamma at that site followed by an early diffuse infiltrate of activated T cells and apoptosis within the tolerant grafts. In contrast, IL-2 and IFN-gamma mRNA were not increased in the spleens of rejecting animals, and the diffuse infiltrate of activated T cells appeared later but resulted in rapid graft destruction. These results define a mechanism of allograft acceptance induced by donor leukocytes that is associated with activation-induced cell death of recipient T cells. They demonstrate for the first time that posttransplant administration of donor leukocytes leads to organ allograft tolerance across a complete MHC class I plus class II barrier, a finding with direct clinical application.

Background: Increased numbers of macrophages and myofibroblasts are observed to occur in chronic renal allograft rejection (CR). The aim of this study was to examine the expression of cellular markers for the macrophage and myofibroblast in early renal transplant biopsy specimens and correlate these findings with allograft outcome., Methods: The first postengraftment biopsy specimens from 53 patients who underwent renal transplantation between January 1993 and December 1995 were studied using immunohistochemistry with antibodies to alpha-smooth muscle actin, which identifies myofibroblasts and CD68, a marker for monocytes and macrophages. Patients were followed until December 1998 (mean follow-up 4.7+/-1.2 years)., Results: Nine patients had progressed to CR by the time of the study, whereas 44 patients continued to have stable renal function. A marked increase in both macrophages (P=0.02) and myofibroblasts (P=0.04) was noted in the first biopsy specimen obtained after engraftment in the patients who developed CR compared with those with stable allograft function. There was a positive correlation between alpha-smooth muscle actin and collagen expression (P=0.0001)., Conclusion: Significant increases in macrophages and myofibroblasts occur in the first renal biopsy specimen in those patients who later develop CR.

Background: Chronic renal allograft rejection is characterized by interstitial fibrosis and vasculopathy. Vascular endothelial growth factor (VEGF) is an endothelial mitogen with increased expression in inflammation and vasculopathy., Methods: Renal tissue from 17 patients with chronic rejection was examined for VEGF protein and the presence of CD 68-positive macrophages, and compared to biopsies from patients with temporary allograft dysfunction, acute rejection, and native kidneys with thin membrane disease., Results: In the chronic rejection group, there was markedly increased expression of VEGF protein in the interstitium (P<0.0001). In serial sections, VEGF colocalized with the expression of CD 68-positive macrophages. Significantly more macrophages were in the tubulointerstitium in tissue with chronic rejection than in those with temporary allograft dysfunction (P<0.005). Additionally, VEGF protein expression in the glomeruli and the vascular compartment of patients with chronic rejection was increased., Conclusion: The up-regulation of VEGF in chronic renal allograft rejection may be important in inflammation and development of fibrosis.

Background: Liver transplants in the rat strain combination PVG-to-Dark Agouti are spontaneously tolerated, whereas kidney transplants in the same strain combination are rejected in 7-9 days., Methods: To identify organ-specific differences that might yield further information about the mechanism of tolerance induction in this strain combination, liver or kidney grafts, spleen, and draining lymph nodes were harvested at days 1, 3, 5, and 7, and examined by immunohistochemistry, terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay, and reverse transcriptase-polymerase chain reaction for interferon-gamma, interleukin (IL)-2, IL-4, and IL-10., Results: Renal allograft rejection was associated with the progressive development of an intense mononuclear cell infiltrate. Markers of lymphocyte activation and cytokine up-regulation appeared from day 3, and many apoptotic parenchymal cells were noted on days 5-7, at the peak of rejection. Conversely, liver allograft tolerance was associated with more rapid infiltration by activated T cells and earlier increases in cytokine expression, but with a more limited degree of cellular infiltration. Concurrent with the early activation, high levels of apoptosis were found in areas of leukocyte infiltrate, paralleling the disappearance of activated T cells from the graft between days 3 and 5., Conclusions: Apoptosis of infiltrating leukocytes in liver allografts may represent an important process in the induction of spontaneous liver transplant tolerance and may underlie the abortive nature of the effector response observed within tolerated livers. In contrast, activated cells in renal allografts in the same strain combination survive and proliferate, express high levels of cytokines, and are efficient in bringing about graft destruction.

In normal kidneys, peritubular and glomerular capillaries can be readily identified by their intense expression of HLA class I and class II compared to other cells within the graft. This high density of expression of MHC, plus their exposure to activated circulating lymphocytes, makes these cells the likely early and primary target of rejection responses. The fate of these capillaries during renal allograft rejection was examined using an indirect immunoperoxidase staining technique and monoclonal antibodies to class I and class II MHC antigens as well as other antigens on capillary endothelium including ICAM-1, LFA-3, and a novel antigen identified by E1.5. Expression of HLA-DR by peritubular capillaries was decreased during rejection, and this disappearance of peritubular capillaries with severe rejection was confirmed by loss of other markers of microvascular endothelium. These studies suggest peritubular capillaries may be the major target of the acute rejection response, and the techniques described allow assessment of degree of damage to these structures in renal allograft biopsies.

Leucocyte interaction with other cells is facilitated by the adhesion molecules, leucocyte function-associated antigen-1 (LFA-1)-binding to intercellular adhesion molecule-1 (ICAM-1) and for T cells and natural killer (NK) cells the binding of LFA-2 (CD2) to LFA-3. As these interactions are critical for the mediation of graft destruction by effector T cells, we examined whether there was a change in the expression of these molecules during rejection compared to normal kidneys. In normal kidneys, peritubular and glomerular capillaries and large vessel endothelium expressed ICAM-1 and LFA-3, but tubular cells expressed only low levels of LFA-3. LFA-1-expressing cells, which were probably macrophages, were observed in the glomerulus. A few scattered LFA-1-expressing cells in the interstitium were probably tissue macrophages or dendritic cells, and only occasional interstitial mononuclear cells expressed LFA-2. During rejection, there was an infiltrate of mononuclear cells expressing LFA-1 and the T cell and NK cell component of the infiltrate expressed LFA-2. Neither of these markers was expressed by kidney parenchymal cells except for one allograft with severe rejection which showed LFA-1 beta chain expression by some tubular cells. Tubular cells had increased expression of ICAM-1 during rejection but there was no increase in LFA-3. The importance of LFA-2 and ICAM-1 expression on kidney tubular cells for adhesion of activated T cells was also examined in an in vitro system.(ABSTRACT TRUNCATED AT 250 WORDS)

Whether the expression of DR antigens is altered in cadaver renal transplants was examined by the use of monoclonal antibodies to the non-polymorphic region of the DR molecule and an indirect immunoperoxidase stain. Expression of DR antigens increased considerably on renal tubular cells in all 25 biopsy specimens which showed severe cellular rejection, but in only 4 of 14 biopsy specimens with no or minimum evidence of rejection. 2 of these 4 specimens were from patients recently treated for severe rejection and the other 2 subsequently lost their grafts from chronic rejection. DR antigens were also expressed on the cell surface of isolated tubular cells aspirated from transplanted kidneys with acute cellular rejection but not on tubular cells in normal kidneys or aspirates from kidneys without rejection. Biopsy specimens with increased DR expression in tubules usually had an interstitial T cell infiltrate. Expression of DR antigens on tubular cells was not related to HLA-DR incompatibility between donor and host, or to the type of immunosuppressive therapy given. The expression of DR antigens on renal tubular cells may be induced by the infiltrating activated T cells or be a consequence of tubular regeneration following rejection or ischaemic damage. The increased expression of DR antigens on renal tubular cells during rejection makes these cells potential targets for delayed type hypersensitivity responses, which are only effective against DR antigen bearing cells.

Patients receiving renal allografts from relatively incompatible donors were randomly assigned to one of two immunosuppressive regimens: azathioprine and prednisone with or without a 2-week course of horse antihuman thymocyte globulin (ATG). The number of circulating lymphocytes fell to about one-third of pretreatment values in both groups of patients. In patients given ATG, the proportion of sheep red blood cell-rosetting lymphocytes (SRBC-R) fell promptly to less than 10% of pretreatment values. In contrast, the percentage of SRBC-R remained at about 70% in patients receiving only prednisone and azathioprine. The addition of ATG reduced the number of SRBC-R/mm3 to one-tenth to one-thirtieth the number seen in non-ATG-treated patients. Plasma of patients undergoing therapy did not inhibit rosetting in vitro. It is proposed that the monitoring of circulating rosetting cells may be a useful clinical guide to the degree of T cell immunosuppression. It is also suggested that the regimen of azathioprine, prednisone, and ATG results in a more effective suppression of circulating T cells than that produced by azathioprine and prednisone alone.

The composition of the mononuclear cell infiltrate in rejecting renal allografts was determined on 96 renal biopsies and 22 nephrectomy specimens by the use of monoclonal antibodies to mononuclear cell surface markers and an indirect immunoperoxidase staining technique. During rejection the composition of the infiltrate was heterogeneous, with T cells (T11), monocytes (OKM1) and HLA-DR expressing mononuclear cells the most frequent sub-populations. B cells (B1) and activated T cells, identified by OKT10, were always in the minority. The T cells infiltrate usually included the helper/inducer (T4) and cytotoxic (T8) subclasses, which suggests that both may contribute to the mediation of rejection. Whether T4 or T8 predominated in the graft did not relate to the ratio of T4:T8 in blood, the HLA A, B or DR incompatibilities of the graft, or the immunosuppressive used. The frequency of T11, T4, T8, HLA-DR positive cells and monocytes, but not B cells, increased with the severity of rejection and was similar in biopsies from patients immunosuppressed with Cyclosporine (CSA) to those given a combination of azathioprine, prednisone and antilymphocyte globulin (AZA). Severe rejection episodes which did not respond to treatment with corticosteroids were more often characterized by a predominance of T8 over T4 cells and T cells infiltrating the glomeruli. In grafts with evidence of cellular rejection, renal tubular cells were shown to have a marked increase in their expression of HLA-DR antigens compared to normal kidneys or grafts with minimal rejection. The expression of HLA-DR antigens on graft tubular cells correlated with the presence of T cells in the interstitium and the severity of rejection, except for moderate rejection in CSA treated biopsies, in which HLA-DR expression was lower than in AZA biopsies. These immunopathological studies have demonstrated that a variety of potential effector cells exist within the graft, and several features have been identified which may assist in assessing the prognosis of the rejection episode.

In the rejection response against renal allografts, the relative importance of helper/inducer T cells mediating a delayed-type hypersensitivity response and of T cells with direct cytotoxicity has not been defined. These subpopulations were identified with commercially available monoclonal antibodies and an indirect immunoperoxidase technique in 31 renal biopsies from patients undergoing acute rejection episodes and in 9 rejected nephrectomy specimens. T lymphocytes were the predominant cell population in all biopsies and in 8 of 9 nephrectomies. The T4 helper/inducer subset was equal to, or greater than, the T8 cytotoxic/suppressor subset in 28 of the 31 biopsies and in the 8 nephrectomy specimens that had histological evidence of cellular rejection. T4 lymphocytes were found predominantly in large areas of cellular infiltrate. T8 lymphocytes had a more diffuse interstitial distribution and were a minority of the cells in the large areas of cellular infiltration. These results show that helper/inducer T lymphocytes are often more frequent than cytotoxic/suppressor cells in acute renal allograft rejection in humans and they suggest that helper/inducer T cells may play an important role in the mediation of graft destruction.

Fine-needle aspiration biopsy specimens of renal transplants were analysed by means of commercially available monoclonal antibodies and an immunoperoxidase stain. Three cellular features associated with acute cellular rejection were identified--heavy infiltrates of activated T cells or large mononuclear cells strongly expressing HLA-DR antigens, and HLA-DR expression by renal tubular cells. A combination of semiquantitative scores for these features correctly identified rejection in 32 of 34 cases, with no false positives in cases of cyclosporin nephrotoxicity or stable graft function.