Prolonged local expression of anti-CD4 antibody by adenovirally transduced allografts can promote long-term graft survival

BACKGROUND: Currently, successful transplantation of allografts requires the systemic use of immunosuppressive drugs. These can cause serious morbidity due to toxicity and increased susceptibility to cancer and infections. Local production of immunosuppressive molecules limited to the graft site would reduce the need for conventional, generalized immunosuppressive therapies and thus educe fewer side effects. This is particularly salient in a disease like type 1 diabetes, which is not immediately life-threatening yet islet allografts can effect a cure. METHODS: We studied the efficacy of locally produced anti-CD4 antibody, mediated by adenovirus (Adv-anti-CD4) transduction of islets, to enhance allograft survival. Adenovirus-transduced islets were transplanted under the kidney capsule of diabetic recipients and graft rejection determined by monitoring blood glucose levels. RESULTS: Adv-anti-CD4 transduction of mouse islets afforded protection against allogeneic rejection after transplantation into fully mismatched recipients. In some recipients, the islet allograft survival was prolonged (persisting for at least 15 weeks), corresponding to the prolonged expression of the anti-CD4 antibody. The effect was local, as absence of CD4+ T lymphocytes was observed primarily at the graft site. CONCLUSIONS: Immunosuppressive effects can be restricted locally by our strategy. Local production of a single antibody against one subset of T lymphocytes can protect mouse islets from allograft rejection during transplantation to treat diabetes. Our findings foreshadow that this strategy may be even more effective when a combination of antibodies are used and that similar strategies may prevent xenograft rejection.     

Influence of local and systemic CTLA4Ig gene transfer on corneal allograft survival

BACKGROUND: To analyse the effects of local (ex vivo) or systemic (in vivo) administration of adenovirus type 5 encoding CTLA4Ig (AdCTLA4Ig) on its influence to prolong corneal allograft survival and to study the underlying mechanisms. METHODS: A MHC class I/II mismatched rat corneal transplant model was used. Recipients were randomly assigned to receive ex vivo gene-modified corneas expressing either CTLA4Ig, CTLA4Ig/IL-10 or a single intraperitoneal (i.p.) injection (1.0 x 10(9) or 1.0 x 10(10) infectious particles) of AdCTLA4Ig 1 day before transplantation and graft survival was analysed. The immunoregulatory effect of this treatment was examined by analysing intra-graft cytokine mRNA expression pattern at day 12 post-transplant. The anti-adenovirus immunity also was investigated. RESULTS: Ex vivo gene transfer resulted in a modest but significant prolongation of graft survival (p = 0.0036 compared to no treatment). In contrast, systemic gene therapy (1.0 x 10(9) or 1.0 x 10(10) infectious particles) significantly prolonged graft survival (p = 0.0007 and 0.0001, respectively, compared to no treatment). Systemic (1.0 x 10(10) infectious particles) therapy resulted in frequent indefinite survival of allogeneic grafts which was not observed in the other therapeutic regimens. Moreover, systemic therapy prevented the intra-graft accumulation and activation of T cells and resulted in a reduced mRNA expression of both TH1 and TH2 cytokines. The generation of anti-adenovirus antibodies was also efficiently inhibited. CONCLUSIONS: CTLA4Ig gene therapy is a successful strategy for the prevention of allogeneic graft rejection in corneal transplantation. Our work has further elucidated the mechanisms of corneal allograft rejection which may lead to novel therapeutic strategies.     

IVIg Treatment Reduces Catalytic Antibody Titers of Renal Transplanted Patients

Catalytic antibodies are immunoglobulins endowed with enzymatic activity. Catalytic IgG has been reported in several human autoimmune and inflammatory diseases. In particular, low levels of catalytic IgG have been proposed as a prognostic marker for chronic allograft rejection in patients undergoing kidney transplant. Kidney allograft is a treatment of choice for patients with end-stage renal failure. Intravenous immunoglobulins, a therapeutic pool of human IgG, is used in patients with donor-specific antibodies, alone or in conjunction with other immunosuppressive treatments, to desensitize the patients and prevent the development of acute graft rejection. Here, we followed for a period of 24 months the levels of catalytic IgG towards the synthetic peptide Pro-Phe-Arg-methylcoumarinimide in a large cohort of patients undergoing kidney transplantation. Twenty-four percent of the patients received IVIg at the time of transplantation. Our results demonstrate a marked reduction in levels of catalytic antibodies in all patients three months following kidney transplant. The decrease was significantly pronounced in patients receiving adjunct IVIg therapy. The results suggests that prevention of acute graft rejection using intravenous immunoglobulins induces a transient reduction in the levels of catalytic IgG, thus potentially jeopardizing the use of levels of catalytic antibodies as a prognosis marker for chronic allograft nephropathy.     

A Higher Risk of Acute Rejection of Human Kidney Allografts Can Be Predicted from the Level of CD45RC Expressed by the Recipients’ CD8 T Cells

Although transplantation is the common treatment for end-stage renal failure, allograft rejection and marked morbidity from the use of immunosuppressive drugs remain important limitations. A major challenge in the field is to identify easy, reliable and noninvasive biomarkers allowing the prediction of deleterious alloreactive immune responses and the tailoring of immunosuppressive therapy in individuals according to the rejection risk. In this study, we first established that the expression of the RC isoform of the CD45 molecule (CD45RC) on CD4 and CD8 T cells from healthy individuals identifies functionally distinct alloreactive T cell subsets that behave differently in terms of proliferation and cytokine secretion. We then investigated whether the frequency of the recipients CD45RC T cell subsets before transplantation would predict acute graft rejection in a cohort of 89 patients who had undergone their first kidney transplantation. We showed that patients exhibiting more than 54.7% of CD8 CD45RC^high T cells before transplantation had a 6 fold increased risk of acute kidney graft rejection. In contrast, the proportions of CD4 CD45RC T cells were not predictive. Thus, a higher risk of acute rejection of human kidney allografts can be predicted from the level of CD45RC expressed by the recipients’ CD8 T cells.     

Influence of recipient and donor IL-10, TNFA and INFG genotypes on the incidence of acute renal allograft rejection

Objective Transplantation of renal grafts is an established treatment for renal failure in a variety of medical conditions. Acute allograft rejection remains an important cause of morbidity after kidney transplantation, and has been shown to be a crucial determinant of long-term graft function. Although rejection is mediated by recipient lymphocytes, both donor and recipient factors contribute to the local environment that influences the severity of rejection response. Because cytokines are the main components of immune responses, we evaluate single nucleotide polymorphisms (SNP) of several cytokine genes that may influence the production of a given cytokine and therefore the features of immune reactions. Material and Methods The aim of this study was to determine the impact of the cytokine gene polymorphism of pro and anti-inflammatory cytokines on the development of acute allograft rejection, which could be used in pretransplant patient assessment. Three SNPs including IL-10 (−1082 G/A), TNFA (−308 G/A), and INFG (+874 T/A) were analyzed in 46 patients with acute allograft rejection, 54 patients with stable graft function and their kidney donors by PCR-ARMS method. Results We are unable to find statistically significant association between any of the studies polymorphisms and clinical outcomes. Conclusion We have found no evidence to suggest that either recipient or donor cytokines polymorphisms determine the incidence of acute rejection after renal transplantation. Our observation, however, is based on few cases, and this may mask a possible favorable effect. It is recommended that several functionally related genes should be tested in similar studies, since this approach has a higher chance to detect genetic risk factors than the screening of single genetic variants.     

A Novel Therapy to Attenuate Acute Kidney Injury and Ischemic Allograft Damage after Allogenic Kidney Transplantation in Mice

Ischemia followed by reperfusion contributes to the initial damage to allografts after kidney transplantation (ktx). In this study we tested the hypothesis that a tetrapeptide EA-230 (AQGV), might improve survival and attenuate loss of kidney function in a mouse model of renal ischemia/reperfusion injury (IRI) and ischemia-induced delayed graft function after allogenic kidney transplantation. IRI was induced in male C57Bl/6N mice by transient bilateral renal pedicle clamping for 35 min. Treatment with EA-230 (20–50mg/kg twice daily i.p. for four consecutive days) was initiated 24 hours after IRI when acute kidney injury (AKI) was already established. The treatment resulted in markedly improved survival in a dose dependent manner. Acute tubular injury two days after IRI was diminished and tubular epithelial cell proliferation was significantly enhanced by EA-230 treatment. Furthermore, CTGF up-regulation, a marker of post-ischemic fibrosis, at four weeks after IRI was significantly less in EA-230 treated renal tissue. To learn more about these effects, we measured renal blood flow (RBF) and glomerular filtration rate (GFR) at 28 hours after IRI. EA-230 improved both GFR and RBF significantly. Next, EA-230 treatment was tested in a model of ischemia-induced delayed graft function after allogenic kidney transplantation. The recipients were treated with EA-230 (50 mg/kg) twice daily i.p. which improved renal function and allograft survival by attenuating ischemic allograft damage. In conclusion, EA-230 is a novel and promising therapeutic agent for treating acute kidney injury and preventing IRI-induced post-transplant ischemic allograft injury. Its beneficial effect is associated with improved renal perfusion after IRI and enhanced regeneration of tubular epithelial cells.     

Liposome-mediated combinatorial cytokine gene therapy induces localized synergistic immunosuppression and promotes long-term survival of cardiac allografts

Localized gene transfer has the potential to introduce immunosuppressive molecules only into the transplanted allograft, which would limit systemic side effects, and prolong allograft survival. However, an applicable gene transfer strategy is not available, and the feasible therapeutic gene(s) has not yet been determined. We developed an ex vivo liposome-mediated gene therapy strategy that is able to intracoronary deliver the combination of IL-4 and IL-10 cDNA expression vectors to the allograft simultaneously. We examined the efficiency, efficacy, and cardiac adverse effects of this combinatorial gene therapy protocol using a rabbit functional cervical heterotopic heart transplant model. Although the efficiency was moderate, the expression of both transgenes was long lasting and localized only in the target organ. The mean survival of cardiac allograft was prolonged from 7 to >100 days. Synergism of overexpressed IL-4 and IL-10 in the inhibition of T lymphocyte infiltration and cytoxicity, and modulation of Th1/Th2 cytokine production promote long-term survival of cardiac allografts.     

PDIA3 mRNA expression and IL-2, IL-4, IL-6, and CRP levels of acute kidney allograft rejection in rat

Kidney transplantation to treat end-stage renal disease has evolved rapidly from the first successful transplantations to the current widespread use of grafts from both cadaveric and living donors. But acute rejection is still a strong risk factor for chronic rejection in recipients of renal grafts. To investigate possible mechanisms, we describe a comparison between differentially proteins expression and immune markers profile (IL-2, IL-4, IL-6, and CRP) of acute rejection and the controls. Through quantitative real-time RT-PCR confirmation, PDIA3 mRNA and protein expression levels in serum and transplanted kidney in experiment group was significantly (P < 0.05) higher than that in control group. Immunity analysis showed that plasma IL-2, IL-4, IL-6, and CRP levels were higher in experimental rats than those in control rats. Our data thus indicate that PDIA3 might be potentially involve into the occurence and development of acute rejection response in renal transplantation and increased plasma IL-2, IL-4, IL-6, and CRP levels play an important role to prevent acute kidney allograft rejection in rats.     

Tolerance to cardiac allografts via local and systemic mechanisms after adenovirus-mediated CTLA4Ig expression

Blockade of the CD28/B7 T cell costimulatory pathway prolongs allograft survival and induces tolerance in some animal models. We analyzed the efficacy of a CTLA4Ig-expressing adenovirus in preventing cardiac allorejection in rats, the mechanisms underlying heart transplant acceptance, and whether the effects of CTLA4Ig were restricted to the graft microenvironment or were systemic. CTLA4Ig gene transfer into the myocardium allowed indefinite graft survival (>100 days vs 9 +/- 1 days for controls) in 90% of cases, whereas CTLA4Ig protein injected systemically only prolonged cardiac allograft survival (by up to 22 days). CTLA4Ig could be detected in the graft and in the serum for at least 1 year after gene transfer. CTLA4Ig gene transfer induced local intragraft immunomodulation at day 5 after transplantation, as shown by decreased expression of the IL-2R and MHC II Ags; decreased levels of mRNA encoding for IFN-gamma, inducible NO synthase, and TGF-beta; and inhibited proliferative responses of graft-infiltrating cells. Systemic immune responses were also down-modulated, as shown by the suppression of Ab production against donor alloantigens and cognate Ags, up to at least 120 days after gene transfer. Alloantigenic and mitogenic proliferative responses of graft-infiltrating cells and total splenocytes were inhibited and were not reversed by IL-2. In contrast, lymph node cells and T cells purified from splenocytes showed normal proliferation. Recipients of long-term grafts treated with adenovirus coding for CTLA4Ig showed organ and donor-specific tolerance. These data show that expression of CTLA4Ig was high and long lasting after adenovirus-mediated gene transfer. This expression resulted in down-modulation of responses against cognate Ags, efficient suppression of local and systemic allograft immune responses, and ultimate induction of donor-specific tolerance.     

PARP Inhibition Attenuates Acute Kidney Allograft Rejection by Suppressing Cell Death Pathways and Activating PI-3K-Akt Cascade

Background

Novel immunosuppressive therapy facilitates long term allograft survival, but acute tubular necrosis and ischemia-reperfusion during transplantation can compromise allograft function. These processes are related to oxidative stress which activates poly- (ADP-ribose) polymerase (PARP) contributing to the activation of cell death pathways. Here we raised the possibility that PARP inhibition curbs cell death pathways and shifts kinase signaling to improved graft survival.

Methods Findings

In an acute rat kidney rejection model, we provided evidence that the PARP inhibitor 4-hydroxy-quinazoline (4OHQ) attenuates rejection processes initiated oxidative/nitrosative stress, nuclear poly-ADP-ribosylation and the disintegration of the tubulo-interstitial structures. The PARP inhibitor attenuated rejection processes induced pro-apoptotic pathways by increasing Bcl-2/Bax ratio and suppressing pro-apoptotic t-Bid levels. In transplanted kidneys, the cell death inducing JNK1/2 is normally activated, but PARP inhibition suppressed this activation with having only modest effects on ERK1/2 and p38 MAP kinases. In untreated transplanted kidneys, no significant alterations were detected in the cytoprotective PI-3K-Akt pathway, but the PARP inhibitor significantly activated Akt (by S473 phosphorylation) and suppressed GSK-3β, as well as activated acute NF-kappaB activation contributing to graft protection.

Conclusion

These data show the protective role of PARP inhibition on graft survival by attenuating poly-ADP-ribosylation, oxidative stress, suppressing pro-apoptotic and increasing anti-apoptotic protein level, and by shifting MAP kinases and PI-3-K-Akt pathways to cytoprotective direction. Thus, addition of PARP inhibitors to standard immunosuppressive therapies during kidney transplantation may provide increased protection to prolong graft survival.     

Immunosuppressive effect on T cell activation by interleukin- 16-cDNA-transfected human squamous cell line

It is well known that it is difficult to induce an immunotolerance with allogeneic skin transplantation. We attempted to find the immunosuppressive protocol for prolonging skin allograft rejection by using interleukin-16 because IL-16 is considered one of the natural ligands to CD4 molecules. First we examined whether synergistic immunosuppressive effects of recombinant IL-16 plus anti-CD4 mAbs are induced in mixed lymphocyte reaction (MLR). Next we used IL-16-cDNA-transfected OSC-20 (human oral squamous cell carcinoma cell line) as an in vitro model of the epidermal keratinocyte equivalent and examined whether this transfectant could inhibit the activation of allogeneic T cells. Our data indicated that IL-16 clearly inhibited human MLR and that IL-16 increased synergistically the immunosuppressive effect of anti-CD4 mAb. We also used IL-16 transfectant and this produced more than 50 ng/ml of IL-16 in the supernatant by which human MLR was significantly inhibited. Furthermore, this transfectant also inhibited the activation of allogeneic lymphocytes stimulated directly with transfectant cells. These results indicated that the IL-16-producing allogeneic skin graft might have a local immunosuppressive action that would prolong graft survival.     

Regulatory cells potentiate the efficacy of IL-4 gene transfer by up-regulating Th2-dependent expression of protective molecules in the infectious tolerance pathway in transplant recipients

We have previously shown that the tolerant state in allograft recipients can be maintained and perpetuated by an "infectious" T cell-dependent regulatory mechanism. Hence, 1) treatment of LEW rats with RIB-5/2, a CD4 nondepleting mAb, produces indefinite survival of LBNF1 cardiac allografts; 2) donor-specific tolerance can be then transferred by spleen cells into new cohorts of test allograft recipients; and 3) putative regulatory CD4+ Th2-like cells are instrumental in this tolerance model. We now report on studies aimed at exposing mechanisms underlying the infectious tolerance pathway, with emphasis on the interactions between intragraft adenovirus-IL-4 gene transfer and systemic infusion of regulatory cells from tolerant hosts. Unlike individual treatment regimens, adjunctive therapy with adenovirus-IL-4 and suboptimal doses of regulatory spleen cells was strongly synergistic and extended donor-type test cardiac allograft survival to about 2 mo. RT-PCR-based expression of intragraft mRNA coding for IL-2 and IFN-gamma remained depressed, whereas that of IL-4 and IL-10 reciprocally increased selectively in the combined treatment group, data supported by ELISA studies. In parallel, only adjunctive treatment triggered intragraft induction of molecules with anti-oxidant (HO-1) and anti-apoptotic (Bcl-xL/Bag-1) but not with pro-apoptotic (CPP-32) functions, both in the early and late posttransplant phases. Hence, systemic infusion of regulatory cells potentiates the effects of local adenovirus-IL-4 gene transfer in transplant recipients. Th2-driven up-regulation of protective molecule programs at the graft site, such as of anti-oxidant HO-1 and/or anti-apoptotic Bcl-xL and Bag-1, may contribute, at least in part, to the maintenance of the infectious tolerance pathway in transplant recipients.     

Immunosuppressive properties of sera and urine dialysates from kidney-graft recipients treated with azathioprine,prednisolone, and niridazole

Niridazole, an antischistosomal agent, was given to renal transplant recipients in addition to azathioprine and prednisolone, as there is experimental evidence that this combination of drugs is highly immunosuppressive. Sera obtained from kidney-graft recipients during the first two weeks after transplantation were examined for their ability to inhibit the one-way mixed lymphocyte reaction (MLR). Sera from seven patients receiving azathioprine, prednisolone, and niridazole (triple-drug treatment), five patients receiving azathioprine and prednisolone, and two other patients treated with niridazole alone for schistosomiasis produced MLR inhibition by comparison with pretreatment (control) sera.A mean of 78% inhibition was observed with sera taken after one day''s treatment with the three-drug combination, whereas this level of in-vitro immunosuppression occurred only after eight days of treatment with azathioprine and prednisolone. Niridazole alone produced an effect similar to azathioprine and prednisolone. Concentrated dialysate of urine from a patient receiving triple-drug treatment not only inhibited the MLR but also significantly prolonged the survival of heterotopic heart allografts in rats, whereas dialysate from the same patient after niridazole had been stopped gave less MLR inhibition and failed to prolong heart allograft survival.Since niridazole thus increased the in-vitro and in-vivo immunosuppressive action of azathioprine and prednisolone, we suggest that this triple-drug combination might be useful for preventing early acute kidney graft rejection.     

Evaluation of biomarker discovery approaches to detect protein biomarkers of acute renal allograft rejection

Management of host responses to allografts by immunosuppressive therapy is the cornerstone of transplantation medicine, but it is still deficient in one important element: biomarkers that are readily accessible and predict the fate of the transplant early, specifically, and reliably. Using a Brown Norway (BN)-to-Lewis rat renal allograft model of kidney transplantation, this study aims at evaluating two proteomic approaches to discover biomarkers for acute rejection: SELDI-MS technology and 2D gel electrophoresis combined with mass spectrometry. Several novel potential serum biomarkers have been identified for follow up. Overall, the conclusion is that apparently at the serum protein level, dramatic changes only occur at a stage where kidney function is already severely affected. Multivariate analysis of serum profiles suggests that there is an ensemble of subtle changes, comprising a proteomic signature of acute rejection at an early stage, a more detailed evaluation of which might provide novel opportunities for the diagnosis of acute rejection. Profiling of the excreted proteins indicates that urine might even present the earliest signs of the rejection process.     

Gene therapy strategies to facilitate organ transplantation.

Organ transplantation is now the definitive therapy for many forms of end-organ disease, but chronic allograft rejection, the side effects of chronic immunosuppressive therapy and the severe donor organ shortage continue to limit its success. Gene therapy has the potential to prevent graft rejection by manipulating the immune response in the microenvironment of the graft or by facilitating the induction of tolerance. Genetic manipulation of stem cells to create transgenic and/or knockout animals that could serve as organ or cell donors could be combined with gene therapy approaches to overcome the problem of limited allogeneic donor organ supply.     

Contribution of renal secreted complement C3 to the circulating pool in humans

Complement C3 produced within the kidney may be an important mediator of local inflammatory and immunological injury. The overall level of renal C3 production and consequently its contribution to the total circulating C3 level are, however, unknown. This was investigated by using the conversion of C3 from recipient to donor allotype following renal transplantation. The C3 F and S allotypes of 80 consecutive renal donor-recipient pairs (148 individuals) were determined by amplification refractory mutation system analysis. The extent of allotype conversion in C3 F/S mismatched recipients was quantified at different stages after transplantation, using an enzyme-linked immunosorbent assay specific for the HAV 4-1 polymorphism of C3 that is strongly associated with C3F. Twenty-one of the eighty recipients were potentially informative, i.e., were C3 SS recipients of C3 FF or FS donor kidneys. In the early postoperative period, donor-derived C3 (HAV 4-1-positive) was undetectable, increasing to 9.6% of the total circulating C3 at times of acute allograft rejection. When graft dysfunction occurred from causes other than rejection, donor C3 remained undetectable. After stable graft function was attained (3-13 mo after transplantation), donor C3 made up 4.5% of the total circulating C3 pool. Our findings demonstrate that human transplant kidney in the resting state is a significant source of extrahepatic C3. Its heightened local synthesis during rejection episodes suggests a possible pathogenic role for C3 in this immunological process.     

Use of EQUORAL in de novo renal transplant recipients

This paper presents our experience to date with using a cyclosporine formulation Equoral (IVAX Pharmaceuticals) together with mycophenolate mofetil plus a steroid immunosuppressive regimen in the treatment of de novo renal transplant recipients. Ten cadaveric donor renal transplant recipients of mean age 51.6 years (range 37-66) were followed up over 6 months for the development of rejection attacks and side effects. All patients received prednisolone, mycophenolate mofetil (1 g/day during the first 5 days posttransplant and then 20 mg/kg/day) plus cyclosporine (3 mg/kg/day). Biopsy proven acute rejection episodes were observed in 2 out of 10 patients (20%). Six months patient as well as renal graft survival rate was 100%. The development of graft function was immediate after transplantation. The mean serum creatinine levels were gradually decreased. Over the 6-month posttransplant period, the function of the graft was satisfactory and stable. The majority of observed adverse events were those commonly reported with the use of cyclosporine and they resolved with a reduction in cyclosporine dose. Equoral treatment demonstrated an acceptable safety profile with maintenance of adequate renal function without incidence of malignancy/lymphoproliferative disease or serious infections. In conclusion, Equoral plus mycophenolate mofetil immunosuppression seems effective and safe on terms acute rejection rates, patient and renal graft survival rates and side profiles.     

Interleukin (IL)-10 induced by CD11b(+) cells and IL-10-activated regulatory T cells play a role in immune modulation of mesenchymal stem cells in rat islet allografts

Mesenchymal stem cells (MSCs) are suggested to be immune modulators because of their therapeutic potential in transplantation. In the present study, we evaluated the therapeutic potential of autologous MSCs for preventing graft rejection after allogeneic rat islet transplantation. We assessed the ability of MSCs to elicit an antiproliferative response in alloreactive lymphocytes and tested the immunosuppressive effect of MSCs in allogeneic islet transplantation. In islet allotransplantation, injection of autologous MSCs or a subtherapeutic dose of cyclosporine A (CsA; 5 mg/kg) alone did not prolong allograft survival. However, graft survival was attained for >100 d in 33% of autologous MSC-plus-CsA-treated recipients, indicating that graft acceptance was achieved in a subgroup of allograft recipients. Splenocytes from autologous MSC-plus-CsA-treated rats exhibited a reduced mixed lymphocyte reaction (MLR)-proliferative response to donor stimulators and increased interleukin (IL)-10 release. Interestingly, after excluding host CD11b(+) cells, splenic T cells from autologous MSC-plus-CsA-treated rats did not produce IL-10 or did not inhibit proliferative responses under the same conditions. The use of autologous MSC-plus-CsA downregulated immune responses, inducing donor-specific T-cell hyporesponsiveness by reducing the production of proinflammatory cytokines and inducing antiinflammatory cytokine production, especially that of IL-10, during the early posttransplantation period. T-regulatory cells made a contribution at a later phase. In conclusion, the combined use of autologous MSCs and low-dose CsA exerted a synergistic immunosuppressive effect in an islet allograft model, suggesting a role for autologous MSCs as an immune modulator.