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Adeno-associated viral vector-mediated (AAV-mediated) expression of allogeneic major histocompatibility complex class I (MHC class I) in recipient liver induces donor-specific tolerance in mouse skin transplant models in which a class I allele (H-2Kb or H-2Kd) is mismatched between donor and recipient. Tolerance can be induced in mice primed by prior rejection of a donor-strain skin graft, as well as in naive recipients. Allogeneic MHC class I may be recognized by recipient T cells as an intact molecule (direct recognition) or may be processed and presented as an allogeneic peptide in the context of self-MHC (indirect recognition). The relative contributions of direct and indirect allorecognition to tolerance induction in this setting are unknown. Using hepatocyte-specific AAV vectors encoding WT allogeneic MHC class I molecules, or class I molecules containing a point mutation (D227K) that impedes direct recognition of intact allogeneic MHC class I by CD8+ T cells without hampering the presentation of processed peptides derived from allogeneic MHC class I, we show here that tolerance induction depends upon recognition of intact MHC class I. Indirect recognition alone yielded a modest prolongation of subsequent skin graft survival, attributable to the generation of CD4+ Tregs, but it was not sufficient to induce tolerance.

Loss of cell surface expression of CD127 on CD4(+)CD25(++) regulatory T-cells (Tregs) may be a useful marker to efficiently isolate Tregs. As FOXP3 was specifically used to identify Tregs, combining these two markers could give better identification for patient with operational tolerance (OT) after liver transplantation. To testify this mixed lymphocyte reaction (MLR), the function of circulating CD4(+)CD25(++)CD127(dim) cells (CD127(dim) cells) was examined in immunosuppression (IS)-free pediatric recipients after liver transplantation (LTx) (group operational tolerance: OT) (Gr-tol n=25) compared to recipients who could not stop IS due to clinically overt rejection (group intolerance) (Gr-intol n=18), recipients who were weaning IS (Gr-weaning n=11) and age-matched healthy volunteers (Gr-vol n=11). In addition, the frequencies of CD127(dim) cells vs CD4(+)CD25(++)CD127(dim)FOXP3(+) (CD127(dim)FOXP3(+)) cells were compared in these four groups by FACS analyses. Our results showed that The proliferation of CD4 cells to donor antigens was reduced compared to third-party antigens only in Gr-tol (P=0.022) but not in other groups (P=NS). Depletion of CD127(dim) cells resulted in a donor antigen-specific abrogation of this MLR hyporesponsiveness in Gr-tol (P<0.001) but not other groups (P=NS). This implied that CD127 efficiently isolated donor antigen-specific Tregs. The frequencies of CD127(dim) cells were significantly lower in Gr-intol (5.2%±1.9%) compared to those in Gr-tol (7.8%±1.8%) (P<0.001) as were the frequencies of CD127(dim) FOXP3(+) cells (Gr-tol: 5.4%±1.7% vs Gr-intol: 2.9%±1.0%, P<0.001). Of interest, there were fewer CD127(dim)FOXP3(+) cells in Gr-intol (2.9%±1%) than in Gr-weaning (5.1%±1.8%) (P=0.002), but no difference in CD127(dim) cells (Gr-intol: 5.2%±1.9% vs Gr-weaning: 6.7%±2.0%) (NS). Thus, combining FOXP3 with CD127 for phenotype analysis demonstrated an unequivocal difference between Gr-intol and Gr-weaning that was not detected by CD127 alone. In conclusion CD127 was a useful surface marker to isolate donor-antigen-specific-Tregs in OT after LTx. The additive effect of its combination with FOXP3 is important in phenotypical Treg analyses of OT patients., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.)

Most approaches to transplant tolerance involve treatment of the recipient to prevent rejection. This study investigates donor treatment with IL-4 for its effect on subsequent rat liver allograft survival. Rat orthotopic liver transplants were performed in rejecting (PVG donor to Lewis recipient) or spontaneously tolerant (PVG to DA) strain combinations. Donors were untreated or injected intraperitoneally with IL-4 (30,000U/day) for 5days. Tissue infiltrates and gene expression were examined by immunohistochemistry and real-time quantitative PCR. IL-4 induced a marked leukocyte infiltrate in donor livers prior to transplant. Macrophages comprised the major population, although B cells, T cells and natural killer (NK) cells also increased. IL-4-induced liver macrophages had an alternatively activated phenotype with increased expression of mannose receptor but not inducible nitric oxide synthase (NOS2). IL-4 also induced IDO and IFN-gamma expression by NK cells. Donor IL-4-treatment converted rejection to acceptance in the majority of Lewis recipients (median survival time >96days) and did not prevent acceptance in DA recipients. Acceptance in Lewis recipients was associated with increased donor cell migration to recipient spleens and increased splenic IL-2, IFN-gamma and IDO expression 24h after transplantation. Donor IL-4-treatment increased leukocytes in the donor liver including potentially immunosuppressive populations of alternatively activated macrophages and IDO-expressing NK cells. Donor treatment led to long-term acceptance of most livers in association with early immune activation in recipient lymphoid tissues., (Copyright 2009 Elsevier B.V. All rights reserved.)

Previous studies have demonstrated that composite tissue transplants such as limbs reject more slowly than skin transplants. This has led to the hypothesis that a simultaneous skin graft may act as an effective marker of limb rejection. The aim of this study was to test the predictive value of a sentinel skin graft as a marker of rejection, using a hind limb transplantation model in rats. Lewis rat recipients received hind limb transplants alone from a Brown Norway donor (control; n = 15) or combined with a full-thickness 15 cm(2) sentinel skin graft (n = 45). All animals received drug therapy (tacrolimus, mycophenolate mofetil, and prednisone) for 6 weeks; then, treatment was ceased entirely. Rejection of the skin graft and limb skin was assessed both by visual and histologic grading systems. Detectable visual rejection (grade 1) was observed earlier in the sentinel skin graft than in the limb skin (P < .0005); the clearest visual rejection (grade 2) appeared earlier in the sentinel skin graft (P < .005). The average histologic grade for early rejection of the skin graft was 1.46 and 1.08 for the limb skin (P < .05). These findings confirmed a visual and histologic delay in the rejection of limb skin compared with a distant sentinel skin graft. Skin grafts transplanted simultaneously with hind limbs may be a useful marker of early rejection.

The aim of this study was to evaluate the ability of local overexpression of indoleamine dioxygenase (IDO) to abrogate rat liver transplant rejection by the use of an adeno-associated virus vector [recombinant adeno-associated virus 2/8 (rAAV2/8)] to deliver the transgene to the allograft prior to transplantation. A green fluorescent protein (GFP)-expressing vector [recombinant adeno-associated virus 2/8-liver-specific promoter 1-enhanced green fluorescent protein (rAAV2/8-LSP1-eGFP)] was used to examine the kinetics of expression and optimal dosing for transduction of Piebald Virol Glaxo (PVG) rat livers. A vector encoding the rat IDO gene (rAAV2/8-LSP1-rIDO) was constructed and tested by its ability to induce tryptophan catabolism and kynurenine production in vitro and in vivo. PVG donor rats were injected, via the portal vein, with rAAV2/8-LSP1-rIDO 2 weeks before transplantation into PVG strain isograft or Lewis (LEW) strain allograft recipients. With the enhanced GFP vector, 29.5% and 47.4% of hepatocytes were found to express GFP at 3 and 6 weeks after injection, respectively. In untransplanted PVG animals, the rAAV2/8-LSP1-rIDO vector induced, 3 weeks after administration, a 1.8-fold increase (P = 0.0161) in liver IDO activity, which was associated with a fall in serum tryptophan to 0.5 times the baseline level (P < 0.001). PVG recipients of PVG liver isografts pretreated with the IDO-expressing vector had a 45% lower level of serum tryptophan than recipients of isografts pretreated with the GFP-expressing vector (P = 0.03). LEW recipients of PVG liver allografts pretreated with the rat IDO vector had a median survival time of 12 days, whereas recipients of allografts pretreated with rAAV2/8-LSP1-eGFP had a median survival time of 13 days (P = 0.38). Both groups displayed similar histological features of acute cellular rejection. In conclusion, rAAV2/8 vectors produce highly efficient, though delayed, hepatocyte transduction in vivo and provide a useful gene delivery tool for transplantation models. However, gene delivery using IDO was unsuccessful in prolonging rat liver allograft survival., ((c) 2009 AASLD.)

Purpose of Review: Due to the dominance of Th1 cytokines in rejection and the ability of Th2 cytokines, particularly IL-4, to inhibit Th1 responses, it has long been held that Th2 cytokines can improve transplant outcomes. Although there is some support for this, there is mounting evidence that IL-4 and Th2 cytokines can promote graft dysfunction. These disparate effects are reviewed., Recent Findings: The role of Th2 cytokines in graft dysfunction is not necessarily due to promotion of humoral immunity, but is due to their ability to drive T-cell and non-T-cell responses including alternative activation of macrophages. Alternatively, activated macrophages compete with classically activated macrophages for arginine and they are mutually exclusive, analogous to mutual competition between Th1 and Th2 cells. Recent findings also point to two subsets of regulatory T cells (Tregs), each dependent on either Th1 or Th2 cytokines. In addition to its effects on bone marrow-derived cells, IL-4 affects parenchymal cells by signalling through the type II receptor, which consists of the IL-4R alpha chain (IL-4Ralpha) and the IL-13Ralpha1, which also binds IL-13., Summary: The effects of Th2 cytokines in transplantation depend on their cellular targets, the timing and form of administration and on Th2 cytokine-dependent Tregs.

Liver transplants are not often rejected in patients weaned from immunosuppression and are spontaneously accepted in some animal models. We review past and recent findings of liver transplantation and propose a unified model in which several mechanisms act in concert to induce and maintain tolerance in both naïve and effector T cell compartments. First, passenger leukocytes migrate to lymphoid tissues and induce apoptosis of alloreactive naïve T cells. Second, antigen-specific activation and subsequent deletion of naïve and effector cells within the liver itself purge the repertoire of alloreactive T cells. Other mechanisms such as microchimerism and migration of donor dendritic cells to the thymus may play a predominant role in maintaining tolerance, and soluble major histocompatibility complex molecules, donor peptides, and regulatory T cells may participate in the induction and maintenance phases. Thus, the major challenge in liver transplantation will be to favor these tolerogenic processes while developing strategies that specifically inhibit alloreactive memory T cells.

Background: Previous studies showed that liver transplant rejection in the Piebald Virol Glaxo (PVG)-to-Lewis combination was associated with more intragraft interleukin (IL)-4 mRNA expression than in spontaneously tolerant grafts in the PVG-to-Dark Agouti (DA) combination. There was also immunoglobulin (Ig) G1 antibody deposition, suggesting an IL-4-induced IgG class switch in rejection. The aim of this study was to investigate whether IL-4 treatment converts PVG-->DA liver transplant tolerance to rejection., Methods: DA (RT1a) rats were recipients of orthotopic PVG (RT1c) liver transplants and DA liver transplants were syngeneic controls. Supernatant from IL-4-transfected Chinese hamster ovary cells (0.5 mL, 30,000 U) or from untransfected cells was injected intraperitoneally on days 3 through 7. Samples were taken for immunohistochemical staining of frozen tissue sections to analyze cellular infiltrate and antibody deposition., Results: IL-4 treatment significantly reduced survival of liver allografts from greater than 100 days in untreated animals to 9 days (P=0.004). Pathologic analysis of IL-4-treated animals showed that death was caused by liver transplant rejection, with a heavy infiltrate of mononuclear cells, disruption of portal tract areas, and infarction. Immunohistochemistry revealed an extensive infiltrate of T cells, CD25-expressing cells, and B cells that was similar to the level in PVG--> Lewis liver allograft recipients that reject the liver. There was also a more extensive monocyte-macrophage infiltrate and more major histocompatibility complex class II expression in IL-4-treated animals compared with untreated animals. There was moderate increase of IgM, little IgG1, and no IgE or IgG2a antibody deposition., Conclusions: IL-4, a T-helper type 2 cytokine that has previously been shown to inhibit delayed-type hypersensitivity responses such as rejection, was found to promote rejection of liver allografts. There was only slight evidence of a graft-specific antibody response, showing that IL-4 induces liver allograft rejection in association with some, but not all, of the changes accompanying rejection in the PVG-->Lewis strain combination.

Experimental models of orthotopic liver transplantation (OLT) have shown that the very early events post-OLT are critical in distinguishing immunogenic and tolerogenic reactions. In rodents, increased leukocyte apoptosis and cytokine expression have been demonstrated in tolerogenic strain combinations. Information from human OLT recipients is less abundant. The aim of this study was to determine the amount of early leukocyte activation and apoptosis following human OLT, and to correlate this with subsequent rejection status. Peripheral blood mononuclear cells (PBMC) were isolated from 76 patients undergoing OLT - on the day prior, 5 hrs after reperfusion (day 0), and 18-24 hrs post-OLT (day 1). The mean level of apoptotic PBMCs on post OLT day 1 was higher than healthy recipients (0.9% +/- 0.2 vs. 0.2% +/- 0.1, p=0.013). Apoptosis was greater in nonrejecting (NR) (1.1% +/- 0.3) compared with acutely-rejecting (R) (0.3% +/- 0.1, p=0.021) patients. On day 1, PBMC from NR patients had increased expression of IFN-gamma (p=0.006), IL-10 (p=0.016), and CD40 ligand (p=0.02) compared with R. Donor cell chimerism on day 1 did not differ between the groups indicating that this was unlikely to account for increased PBMC apoptosis in the NR group. Interestingly, the level of chimerism on day 0 was significantly higher in NR (3.8% +/- 0.6) compared with R (1.2% +/- 0.4, p=0.004) patients and there was a close correlation between chimerism on day 0 and cytokine expression on day 1. These results imply that similar mechanisms are occurring in the human liver to promote graft acceptance as in the experimental models of liver transplantation and suggest that strategies that promote liver transplant acceptance in rodents might be applicable to humans.

Background: Some immunosuppressive drug therapies inhibit transplant tolerance in animal models, and we have shown that treatment of recipients with methylprednisolone, but not cyclosporine, inhibits spontaneous acceptance of liver transplants. This study investigates the effects of mycophenolate mofetil (MMF) on liver acceptance and rejection., Methods: Piebald Virol Glaxo rat livers were transplanted into Dark Agouti recipients, which spontaneously tolerate (TOL) the liver, or into Lewis recipients, which reject (REJ) the liver. MMF (40 mg/kg/day subcutaneously) was given for 5 days from days 0 to 4 (early) or from days 3 to 7 (late). In separate experiments, liver grafts were collected for assessment of infiltrate and of interleukin (IL)-2, IL-4, IL-10, and interferon-gamma mRNA expression., Results: TOL liver transplants had a median survival time (MST) of more than 100 days (n=6), and neither early nor late MMF treatment of TOL transplants reduced survival (MST 85 days, P=0.19 and 78 days, P=0.08, respectively). Liver failure in most of these animals was the result of biliary problems, not rejection. There were few consistent differences between treated and untreated TOL animals in infiltrate or liver cytokine expression, although there was a moderate reduction in T-cell infiltrate in MMF-treated TOL animals (P=0.003 on day 5 TOL). In contrast, REJ transplants had an MST of 13 days (n=10), and early MMF treatment led to five of six animals surviving more than 100 days (P=0.0002), whereas late treatment was much less effective, with one of six animals surviving more than 100 days. REJ livers had significantly more IL-4 mRNA expression and immunoglobulin G1 deposition in the graft than TOL livers, and this was inhibited by early, but not late, MMF treatment., Conclusions: MMF treatment inhibited rejection but not acceptance of liver allografts. Early administration was more effective in preventing rejection and demonstrated a more marked effect on IL-4 expression and alloantibody deposition than on graft T-cell infiltrate and expression of other cytokines.

Background: Orthotopic liver transplants in many animal models are spontaneously accepted without requiring immunosuppression. Liver transplant acceptance is associated with early immune activation, and immunosuppressive drugs such as methylprednisolone inhibit acceptance. We investigated whether cyclosporine (CsA) inhibits rat liver transplant acceptance. We also examined the effects of CsA on infiltration and cytokine gene expression., Methods: Orthotopic liver transplantation was performed in the PVG donor to Dark Agouti recipient rat strain combination, which accepts the graft (tolerance; TOL), and in the PVG-to-Lewis combination, which rejects the graft in 9 to 16 days (rejection; REJ). CsA (1.5 mg/kg per day subcutaneously) was given to recipients for 5 days, starting from the day of transplantation to day 4 or from day 3 to day 7. In a separate experiment, transplanted livers were collected at days 1, 3, 5, and 7 after transplantation and examined for infiltration by immunohistochemistry and for expression of interleukin (IL)-2, IL-4, IL-10, and interferon-gamma mRNA by quantitative reverse transcriptase-polymerase chain reaction., Results: Both early and delayed treatment with CsA significantly increased survival in the REJ strain combination, with a median survival time of 81 days and more than 100 days, respectively, compared with 13 days in the untreated group. Neither treatment affected survival of TOL animals, and all TOL groups had a median survival time of more than 100 days. Delayed treatment did not reduce survival; more animals survived for greater than 100 days after delayed treatment, although this did not reach significance ( P=0.08). T-cell infiltrate was inhibited in CsA-treated TOL animals compared with untreated animals at all times after treatment, whereas CD25 cells were only inhibited on day 3. CsA treatment of TOL grafts markedly reduced expression of IL-2, IL-4, and interferon-gamma compared with untreated recipients., Conclusions: CsA did not significantly inhibit liver transplant acceptance and allowed some activation of T cells and CD25 expression but almost completely inhibited IL-2 and IL-4, which are required for survival of activated T cells.

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: 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.

Background: The effects of immunosuppressive drugs on transplant tolerance have not been extensively studied, although their effect on rejection is well established., Methods: We examined the effects of a short course of treatment with the immunosuppressive drug methylprednisolone (MP) on the survival of PVG liver allografts in Dark Agouti (DA) recipients that accepted the livers and in Lewis recipients that rejected the livers. Infiltration of liver allografts was examined by immunohistochemical staining of liver sections, and apoptosis was measured by terminal deoxynucleotide transferase-mediated dUTP nick end labeling., Results: A 5-day course of MP (days 0 to 4) led to rejection of four of six livers (mean survival time [MST] 99 days) in DA recipients compared with long-term survival (MST >100 days) in untreated animals. Delayed administration of MP (days 3 to 7) exacerbated rejection in DA recipients, and all eight animals rejected the graft (MST 68.5 days). Treatment of Lewis recipients with MP did not significantly prolong survival when administered from days 0 to 4 (MST 13 days), although delay of administration improved the outcome. Treatment from days 3 to 7 resulted in an MST of 21 days, whereas treatment from days 7 to 11 resulted in an MST of 41.5 days. MP treatment from day 3 to day 7 reduced T cells and interleukin 2 receptor-expressing cells but increased the numbers of apoptotic cells infiltrating both DA and Lewis strain allografts., Conclusions: These results show that immunosuppression with MP inhibits both spontaneous tolerance and rejection of liver allografts in a rat model and question the efficacy of administering MP to all liver allograft recipients from the time of transplantation.

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.

Liver allografts in many animal models are often spontaneously accepted across a complete histocompatibility barrier without requirement for immunosuppression. In contrast, skin allografts are usually rejected, even across minor histocompatibility barriers. To identify the mechanism of liver allograft acceptance we have compared skin rejection with liver acceptance in DA rat strain recipients of PVG donors, a major histocompatibility complex (MHC) class I plus II mismatch. In spite of the established role of draining lymph nodes (LN) in induction of rejection of skin allografts, there was much greater involvement of LN after liver than after skin transplantation. Few donor cells migrated to these organs from transplanted skin but many cells migrated from transplanted liver. There was also a paradoxical increase in interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) mRNA in LN and spleen of liver allograft recipients that greatly exceeded their expression in skin allograft recipients. For example, there were 2. 7+/-1.6x104 molecules of IFN-gamma per 106 molecules of beta-actin mRNA in the LN draining liver allografts 1 day after transplantation compared with 2.0+/-0.3x103 molecules/106 beta-actin in LN draining skin allografts and 8.1+/-1.8x102 molecules/106 beta-actin in LN draining skin isografts. Examination of the graft showed that infiltration and cytokine mRNA up-regulation occurred more slowly in the transplanted skin than in liver but progressed inexorably in skin grafts until rejection. These results show that liver acceptance is associated with a paradoxical marked early activation then subsequent decline of the immune response.

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.

Liver allografts in the fully allogenic combination of LEW donor liver to DA recipient (LEW-->DA) are spontaneously tolerated (TOL) with no requirement for immunosuppression, while DA-->LEW allografts are rejected in 12-15 days (REJ). We investigated the mechanism of tolerance induction by identifying differences between TOL and REJ grafts from day 1 to day 9 after transplantation and in normal livers and syngeneic liver graft controls. Infiltrating cell populations were counted after immunohistochemical staining of liver graft sections. There were occasional minor differences between TOL and REJ grafts in the T cell or CD11b/c+ (monocyte/macrophage/granulocyte) infiltrate. In contrast, there was a major difference in B cell infiltrate between TOL and REJ liver grafts. Membrane IgD+ cells were significantly greater in TOL (1796 +/- 225) versus REJ (569 +/- 281) (P = 0.004) portal tracts, as were B220+ cells (1086 +/- 100 vs. 181 +/- 105, P = 0.0004) and CD45RC+ cells (2317 +/- 456 vs. 597 +/- 194, P = 0.004). IgG1, IgG2a, IgM, and IgD deposition in liver allografts, identified by immunohistochemical staining of tissue sections, revealed no IgG or IgD in normal rat liver and low levels of IgM. Deposition of IgG1 was observed in REJ but not in TOL liver on days 7 and 9. IgM was increased in both TOL and REJ liver and appeared to be associated mainly with hepatocytes in REJ and with infiltrate in TOL liver. There was a parallel increase in IgG1-expressing plasma cells in the spleen and lymph nodes of REJ but not TOL animals. Cytokine mRNA was analyzed by reverse transcription and semiquantitative polymerase chain reaction amplification of liver RNA. Increased levels of IL-2, IL-4, IL-6, IL-10, TNF-alpha, transforming growth factor-beta, and IFN-gamma were observed, with similar levels of expression in TOL compared with REJ liver. Cytokine mRNA in syngeneic grafts was not different from normal except for IL-6 and transforming growth factor-beta, which were increased. There is no major difference in the T cell component of the infiltrate or in the extent of upregulation of cytokine mRNA between TOL and REJ grafts. There is a major difference in the B cell compartment, with more B cells in TOL livers and deposition of IgG1 in REJ grafts.

In liver allograft rejection, interlobular bile ducts are thought to be the main target of rejection. In contrast, in other organ allografts the capillary bed of the graft appears to be the primary target. To determine whether portal tract microvasculature is destroyed in liver allografts during rejection, we have identified portal tract microvasculature in 11 normal livers and 38 liver allograft biopsy specimens using monoclonal antibodies to capillary endothelium in immunohistochemical staining. E1.5, CD31 and EL-4 antibodies identified portal microvascular endothelium in normal liver that had the morphology of capillaries. In allograft biopsies the number of microvascular structures per portal tract was reduced markedly in acute cellular rejection to 1.1 +/- 0.6 (n = 25) and to 0.65 +/- 0.9 (n = 15) in chronic ductopenic rejection compared with nonrejecting allografts (2.8 +/- 0.6) (n = 4) or normal liver (3.8 +/- 0.7) (n = 11). To determine whether loss of microvascular structures preceded bile duct destruction in rejection, sections were double-stained to identify both microvasculature and bile ducts. The number of microvascular structures per bile duct was significantly lower in acute cellular rejection (0.5 +/- 0.4) (n = 18) or chronic rejection (0.3 +/- 0.4) (n = 8) compared with normal liver (2.3 +/- 0.6) (n = 7) (P < 0.0001), demonstrating that components of the portal vasculature are destroyed prior to bile ducts. There was a correlation between the severity of rejection and the loss of microvascular structures per bile duct (P < 0.001). In conclusion, in common with other allografted organs, the microvasculature of liver allografts appears to be an early target of rejection.

Cytokine gene expression was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of RNA from 27 human liver allograft specimens diagnosed as acute (n = 19) or chronic (n = 8) rejection and from 12 normal human livers. In initial screening experiments, mRNA for cytokines interleukin (IL)-1 beta, IL-6, IL-10 and gamma-interferon (IFN-gamma) was expressed in all normal livers and almost all allograft specimens tested. IL-2 mRNA was expressed at barely detectable levels in four of 12 normal livers screened and in 20 of 26 liver allograft specimens with rejection. This constitutive expression of cytokine mRNA required semiquantitative PCR analysis to differentiate levels of cytokine mRNA expression between specimens. Titration of cDNA prior to PCR amplification was initially used and showed significantly more IL-2 (p = 0.02) and IFN-gamma (p = 0.03) in acute rejection compared to normal liver. There was also significantly less IL-10 in chronic rejection compared to acute rejection (p = 0.02) or normal liver (p = 0.01) and less IL-6 in acute rejection compared to chronically rejecting liver (p = 0.05). IL-1 beta (p = 0.04) and IL-6 (p = 0.01) were reduced in acute rejection compared to normal liver. The slight increase of IL-2 in acute rejection and the slight decrease of IL-10 in chronic rejection was confirmed by a second semiquantitative analysis which involved removal of aliquots of PCR reaction at successive cycles followed by dot-blotting and hybridization.(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of this study was to quantitate T-cell populations infiltrating portal tracts, bile ducts and hepatic lobules in 82 biopsy specimens from 25 patients after orthotopic liver transplantation. Biopsy specimens taken immediately after revascularization of the graft were used as controls. Patients studied include 18 with initial rejection episodes, 11 with unresolved rejection, five with vanishing bile duct syndrome and eight patients with other forms of liver injury. Quantitation was done in a blinded fashion for the first 20 biopsy specimens. A double immunolabeling technique was used to simultaneously immunolabel bile duct structures (with anti-major histocompatibility complex class II or antikeratins) and lymphoid populations (with anti-CD2, anti-CD4 or anti-CD8). This facilitated the accurate quantitation of intraepithelial lymphocytes within bile ducts. This technique also enabled simultaneous detection of CD4 and CD8 antigens on lymphocytes in portal tracts. The predominant lymphocyte subtype within biliary epithelium during acute and chronic rejection was of the CD2+/CD8+ phenotype. CD8+/CD4+ ratio in bile ducts was approximately 5:1 in acute, unresolved and chronic rejection. In vanishing bile duct syndrome, double immunolabeling enabled the detection of destroyed interlobular bile duct remnants that were not apparent on routine hematoxylin and eosin staining. Attached to some of these structures were CD8+ lymphocytes. Lobular CD8+ cells were not prominent in acute rejection but increased significantly in biopsy specimens from patients with unresolved and chronic rejection. In chronic rejection, a selective increase was seen in these CD8+ cells in centrizonal regions.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

In rat models, CD4+CD25+ T regulatory cells (Treg) play a key role in the induction and maintenance of antigen-specific transplant tolerance, especially in DA rats with PVG cardiac allografts (1, 2).We have previously described generation of alloantigen-specific Treg (Ts1), by culture of naïve natural CD4+CD25+ Treg (nTreg) with specific alloantigen and IL-2 for 4 days. These cells express mRNA for IFN-γ receptor (ifngr ) and suppress donor but not third party cardiac allograft rejection mediated by alloreactive CD4+ T cells at ratios of <1:10. Here, we show that Ts1 also expressed the IL-12p70 specific receptor (il-12rβ2) and that rIL-12p70 can induce their proliferation. Ts1 cells re-cultured with rIL-12p70 alone or rIL-12p70 and recombinant interleukin-2 (rIL-2), suppressed proliferation of CD4+ T cells in mixed lymphocyte culture at <1:1024, whereas Ts1 cells re-cultured with rIL-2 and alloantigen only suppressed at 1:32–64. The rIL-12p70 alloactivated Ts1 cells markedly delayed PVG, but not third party Lewis, cardiac allograft rejection in normal DA recipients.Ts1 cells re-cultured for 4 days with rIL-12p70 alone, but not those re-cultured with rIL-12p70 and rIL-2, expressed more il-12rβ2, t-bet, and ifn-γ, and continued to express the markers of Ts1 cells, foxp3, ifngr, and il-5 indicating Th1-like Treg were induced. Ts1 cells re-cultured with rIL-2 and alloantigen remained of the Ts1 phenotype and did not suppress cardiac graft rejection in normal DA rats.We induced highly suppressive Th1-like Treg from naïve nTreg in 7 days by culture with alloantigen, first with rIL-2 then with rIL-12p70. These Th1-like Treg delayed specific donor allograft rejection demonstrating therapeutic potential. [ABSTRACT FROM AUTHOR]

Summary In many animal models transplanted livers are not rejected, even when there is a complete MHC mismatch between the donor and recipient and the recipient is not immunosuppressed. This distinguishes liver transplants from other organs, such as kidneys and hearts, which are rapidly rejected in mismatched individuals. Acceptance of transplanted livers in a rat model is not due to the absence of an immune response to the liver and there is a rapid, abortive response that is ultimately exhausted. Donor leucocytes transferred with the liver appear to be responsible for both liver acceptance and the abortive activation of the recipient’s T cells. The immune mechanism of liver transplant acceptance appears to be due to ‘death by neglect’ in which T cells are activated to express IL-2 and IFN-γ mRNA in the recipient lymphoid tissues, but not at adequate levels within the graft. Subsequently the activated T cells die leading to specific clonal deletion of liver donor-reactive T cells. These findings have important implications for liver transplant patients as immunosuppressive drugs that are given to prevent rejection can also interfere with this form of tolerance. In addition, it might be possible to modify the immunosuppressive drug treatment of transplant patients to promote the process of death by neglect of recipient alloreactive T cells. [ABSTRACT FROM AUTHOR]

Most approaches to transplant tolerance involve treatment of the recipient to prevent rejection. This study investigates donor treatment with IL-4 for its effect on subsequent rat liver allograft survival. Rat orthotopic liver transplants were performed in rejecting (PVC donor to Lewis recipient) or spontaneously tolerant (PVG to DA) strain combinations. Donors were untreated or injected intraperitoneally with IL-4 (30,000 U/day) for 5 days. Tissue infiltrates and gene expression were examined by immunohistochemistry and real-time quantitative PCR. IL-4 induced a marked leukocyte infiltrate in donor livers prior to transplant. Macrophages comprised the major population, although 13 cells, T cells and natural killer (NK) cells also increased. lL-4-induced liver macrophages had an alternatively activated phenotype with increased expression of mannose receptor but not inducible nitric oxide synthase (NOS2). lL-4 also induced lDO and IFN-gamma expression by NK cells. Donor IL-4-treatment converted rejection to acceptance in the majority of Lewis recipients (median survival time >96 days) and did not prevent acceptance in DA recipients. Acceptance in Lewis recipients was associated with increased donor cell migration to recipient spleens and increased splenic lL-2. IFN-gamma and IDO expression 24 h after transplantation. Donor IL-4-treatment increased leukocytes in the donor liver including potentially immuno-suppressive populations of alternatively activated macrophages and IDO-expressing NK cells. Donor treatment led to long-term acceptance of most livers in association with early immune activation in recipient lymphoid tissues. [ABSTRACT FROM AUTHOR]