Study On The Regulation And Its Mechanisms Of CD19⁺CD5⁺CD1d^hiB Cells In Immune Response Of Islet Transplantation

Part I Detection of B10 cells of C57BL/6 mouse and establishment of B10 cells activation program in vitroObjective To assess the frequency of B10 cells and the expression of IL-10 in different tissues of naive wild-type mice, explore and establish B10 cells activation program in vitro.Methods The frequency of B10 cells and the expression of IL-10 in different tissues of naive wild-type mice were detected by flow cytometry; the subsets of B cells were sorted by FACS (Fluorescence-activated cell sorting) and the expression of cytoplasmic IL-10 was observed; the impact of LPS (Lipopolysaccharide) on the activation of B10 cells was observed and B10 cells activation program was established in vitro.Results ① The phenotypically CD 19+CD5+CD1dhi subset of regulatory B cells was found within the spleens, peripheral blood and lymph nodes of naive wild-type mice. The frequencies of CD19+CD5+CD1dhiB cells within the spleens were 3.95%±0.79%, which were the highest between the groups (P<0.05). B cells were induced to express cytoplasmic IL-10 following 5-h in vitro stimulation with LPS, PMA (Phorbol 12-myristate 13-acetate), ionomycin, plus monensin (L+PIM), and the expression of CD19+IL-10+ B cells was also the highest in spleens (2.02%±0.16%, P<0.05); ② IL-10 was mainly secreted in the phenotypically CD5+CDldhl subset, while was significantly less expressed in CD5-CDldhi、CD5-CD1dlo and CD5+CD1dlo subsets (P<0.01); ③ B10 cells were induced to express cytoplasmic IL-10 with LPS, PMA, plus ionomycin stimulation in vitro for 5h. The expression of CD19+CD5+CD1dhi B cells (11.24%±1.26%, P<0.01) and the secretion of IL-10 (6.71%±0.64%, P<0.01) were significantly increased with prolonged LPS stimulation (48h).Conclusions B10 cells subset was highly expressed in spleen of naive wild-type mice and was activated through TLR (Toll-like receptor) signaling pathway, the immunosuppressive factor IL-10 was futher highly expressed by activated B10 cells.Part Ⅱ Study on functional characteristics and immune regulatory mechanisms of B10 cellsObjective To isolate B10 cells and identify their biological functions, explore immunoregulatory mechanisms of B10 cells to the proliferation of effector T cells (CD4+CD25-T) and Tregs (Regulatory T cells).Methods CD19+B cells sorted by MACS (Magnetic-activated cell sorting) were activated 48h in vitro and CD19+CD5+CD1dhi Bcells were seperated by FACS; MACS was used to purify CD4+CD25- T cells and Tregs, CFSE (5,6-carboxyfluorescein diacetateN-succinimidyl ester)-labeled CD4+CD25- T cells and Tregs were stimulated and cultured alone or with B10 cells. Their proliferative response was determined 72 hours later based on the CFSE staining.Results ① CD19+CD5+CD1dhiB and CD19+CD5T-D1dlo B cells were successfully sorted by MACS combined with FACS, the purity was more than 95%; ② The co-culture of B10 cells decreased significantly (in a dose-dependent way) the proliferative capacity of CD4+CD25- T cells (13.75%±0.96% vs.32.76%±0.81%, P<0.01), increased the IL-10 secretion of CD4+ T cells (0.47%±0.25% vs.0.21%±0.05%, P<0.01); ③ The proliferation of Tregs was increased with the co-culture of B10 cells (26.60%±1.14% vs.21.52%±0.81%, P<0.01).Conclusions The method of MACS combined with FACS was used to highly purify CD19+CD5+CD1dhi B cells which have the quality of immunosuppression, B10 cells subset can regulate immune response of CD4+CD25- T cells and Tregs through the production of IL-10.Part Ⅲ Study on the regulation and its mechanisms of B10 cells in immune response of islet transplantationObjective To study the changes of immune cells and cytokines in allogeneic islet transplantation immunity and further explore the regulatory role and its mechanisms of B10 cells to allograft immune rejection.Methods STZ (Streptozotocin)-induced diabetic BALB/c-mice were used as recipients, while C57BL/6-mice as donors, CD19+CD5+CD1dhi B cells seperated by FACS were intravenous transfused combined with islet transplantation under the renal capsule. The survival time of grafts was observed; HE staining was used to evaluate pathological characteristics of the grafts; flow cytometry was used to detect the expression of B10 cells、IL-10、CD4+/CD8+、Tregs and IFN-γ+/IL-17+ in mice spleens, kidney draining lymph nodes and peripheral blood before and after islet transplantation; ELISA was used to detect the production of serum IL-10 and IFN-γ before and after transplant.Results ① The separated and purificated islets of donors were round or oval-shaped with different sizes and complete envelope, the survival rate was more than 95% with AO/PI staining; ② The average survival time of islet grafts was (9.00±0.82) days in the group of 1 million CD19+CD5+CDldhi B cells transfusion combined with islet transplantation, compared with the survival time (7.43±0.79 days) in the group of islet transplantation, the difference was not statistically significant (P=0.877); the survival time of islet grafts was (9.57±0.79) days after 2 million CD19+CD5+CDldhi B cells further transfused, compared with the group of islet transplantation, the difference was still not statistically significant (P=0.848); ③ At posttransplant day 7, recipients of islet tansplantation with CD19+CD5+CD1dhi B cells histopathological showed complete islets;④ The expression of B10 cells、IL-10 and Tregs in spleens, kidney draining lymph nodes and peripheral blood of islet transplant recipients was increased, while the ratio of CD4+/CD8+ and IFN-γ+/IL-17+ was decreased by CD19+CD5+CD1dhi B cells; ⑤ The production of serum IL-10 of islet transplant recipients was up-regulated by CD19+CD5+CD1dhiB cells, while serum IFN-y was down-regulated.Conclusions The production of negative immunoregulatory cells and immune suppressive cytokines was significantly increased by 1 million CD19+CD5+CD1dhi B cells in islet transplant recipients, while the expression of positive immunoregulatory cells and pro-inflammatory cytokines was decreased and the survival time of grafts was not significantly prolonged.