Apoptotic Cells Enhance Islet Engraftment By Induction Of Regulatory T Cells And Tolerogenic Dendritic Cells

Immune tolerance is maintained by several mechanisms, in addition to central tolerance, clonal ignorance to self-antigen and clonal anergy or deletions of antigen specific T or B cells are also contributed to the local homeostasis,and as a regulatory T cell subset, CD4~+CD25~+Foxp3+ regulatory T cells (Treg) have also been firmly confirmed to be specialized for immune suppression. Donor specific transplant tolerance involves suppression of graft-reactive T cells while sparing responses against other antigens. How to achieve donor specific tolerance is all we pursued these years. Apoptotic lymphocytes are always thought to be endowed with immunomodulatory properties and can be used to induce donor specific tolerance, but the mechanism hide behind this phenomenon is still unknown.Apoptosis is a normal physiological process without causing autoimmunity. The effective clearance of apoptotic cells is essential for development, homeostasis, and response to injury. Several mechanisms explaining the absence of immune responses against apoptotic cell-derived antigens have been proposed. First, professional phagocytes can quickly capture apoptotic bodies and prevent the release of harmful products by the dying cells. On the other hand, apoptotic cells can release anti-inflammatory cytokines like interleulin-10(IL-10) and transforming growth factor-β(TGF-β).Various types of regulatory T (Treg) cells have been described on the basis of their origin, generation and the mechanism of action: thymically derived Foxp3+Treg cells and inducible Treg cells, which are comprised of IL-10 producing T regulatory (Tr1) cells, TGF-βproducing T helper (Th3) cells and inducible Foxp3+Treg cells. The mechanism by which Treg cells induced immune tolerance is rather complex. (1) upon antigen stimulation, antigen–specific Tregs are swiftly recruited in response to chemokines to antigen presenting cells (APC) and compete with the na?ve T cells in aggregating around the APCs; (2) antigen-activated Tregs contacting APCs then down modulate their functions, thereby hindering the activation of the other T cells that are recruited to the APCs; (3) Tregs may secrete granzyme, perforin, IL-10 or other immunosuppressive cytokines; (4) Tregs can induce reactive T cells to apoptosis by exhausting IL-2.We wonder if Tregs are involved in the apoptosis induced tolerance as an antigen specific suppressor. And if so, how does Tregs generate and work in this process. In order to answer all these questions, we try to figure out more underlying mechanism in the apoptosis induced tolerance.PartⅠ.Apoptotic cells induced transplantation toleranceDiabetic mice model was established by interperitoneal injection of streptozocin (STZ) 200mg/kg, Mice were considered to be diabetic and can be used as recipients when non-fasting blood glucose levels were >18mmol/L on 3 consecutive days. Islets were isolated by distend the pancreas with collagenaseⅤ(0.5mg/ml) and then incubated at 37℃for 20 minutes . Islets with diameters between 75-250μm were purified by OptiPrep? gradients centrifuge. The isolated islets can be stained by dithizone (DTZ) in red colour. Usually, more than 150 islets can be got from one mouse.Donor (C57BL/6) splenocyte cell suspensions were treated with UVB irradiation to induce apoptosis. About 50-80% splenocyte were confirmed to be apoptic after irradiation by FITC-conjugated AnnexinⅤand PI. The recipient mice were divided into four groups: transplant with or without apoptotic cells, apoptotic cell infusion only group and non-treatment group. Apoptotic splenocytes (1×106) were injected via the tail vein 7 days before islets transplantation. About 200 freshly isolated islets were transplanted under renal capsules. The survival rate of mice and the blood glucose levels were observed after transplantation for more than 90 days. As compared with the transplantation mice without apoptotic cell administration, transplantation mice with apoptotic cell administration have a much longer survival period and lower blood glucose. Mice in the other two groups without transplantation are both have a shorter survival period than the apoptotic cell administration group. Our present results demonstrated that the apoptotic cell pre-infusion can promote islets survival in an allogenic transplantation model and can lenitive the diabetic symptom. In conclusion, transfusion of apoptotic cell can induce donor specific transplantation tolerance.PartⅡ. The mechanism of apoptotic cell induced toleranceRecipients with grafts surviving more than 60days were defined as tolerant and used in our experiment. First, we evaluate the amount of CD4~+CD25~+Treg cells in spleen of tolerant mice or the control mice by FACS analysis. Significant increasement of the CD4~+CD25~+Treg cell population was observed in recipient mice engrafting after infusion compared with that in the wide type mice (7.5±0.6% versus 3.3±0.4%). Then we sorted the CD4CD25 double positive T cells in tolerant mice spleen by MACS sorting, the Foxp3 positive T cells were analyzed by flow cytometry. A higher percentage of Foxp3 positive cell populations (14.67±0.5%) was also detected in tolerant mice as compared with that of wide type control (9.65±0.3%). All these results indicate that increased number of Treg cells in tolerant mice may play an important role in the maintenance of transplantation tolerance.In order to convince the suppression function of Treg cells, we isolated CD4CD25 double positive T cells in spleen of tolerant mice and wide type mice. CD4~+CD25~-T cells from recipient were used as responders and mature bone marrow derived DCs from donor mice were used as stimulators. The MLR reaction taken place in the presence of either tolerant mice derived Tregs or wide type ones. According to the FACS counters, the Tregs from tolerant mice have stronger suppression ability. Tregs from tolerant mice also have stronger ability to suppress the carboxykfluoroscein succinimidyl ester (CFSE) labeled CD4~+CD25~-T cells to division.Consistent with other reporter, here we showed that promotion of Tregs with stronger suppressive ability may play an important role in transplant tolerance induced by apoptic cell pre-infusion. But how did this Treg group generate is still under investigation. Early neonatal thymectomy experiments in mice strongly suggest that Treg cells are generated in the thymus. Studies with Foxp3 reporter mice have also traced the development of Foxp3 positive cells to the thymus. Aside from evidence that natural Foxp3 positive Treg cells arise and mature in the thymus, there is mounting evidence that Tregs can develop extra-thymically under certain conditions. Na?ve T cells been shown to express Foxp3 in vitro after T cell receptor stimulation in the presence of TGF-β. There is a rapidly expanding literature which revealed that DC appears to be important cells in the normal control of CD4~+CD25~+ Treg cell function and expansion. Thus, in our subsequent experiment, the phenotype and function property of DCs in tolerant mice were analyzed.Splenic DCs of tolerant recipients were isolated by anti-mouse CD11c mAb-conjugated magnetic micro beads from spleen single cell suspensions. As compared with the wide type control, DCs from tolerant mice showed lower level of MHC class II, CD40, CD80/86 and higher level of PD-L1. It may be work as a tolerogenic DC (Tol-DC) group. Tol-DC purified from tolerant recipients incubated with CD4~+CD25~- cells could induce the expansion of CD4~+CD25~+ T cells. Tol-DC mediated Foxp3 induction was impaired in the presence of antibodies against PD-L1.Vice versa, incubation of Treg cells isolated from tolerant recipients with bone marrow derived dendritic cells (BMDC) progenitors could result in the generation of DC with Tol-DC phenotype.Taking together, Apoptotic cells could significantly favor the islets engraftment in allogenic transplantation. The number of Treg and Tol-DCs is upregulated in the tolerant mice. Treg cells from tolerant recipients can induce generation of Tol-DCs while Tol-DCs from tolerant recipient could also promote Treg'generation. This crosstalk between DCs and Treg should be involved in the apoptotic cells induced transplantation tolerance.