| ObjectiveEstablish a stable system of expanding CD4+CD25+Foxp3+ regulatory T cells in vitro, investigate the relationship between CD4+CD25+Foxp3+ regulatory T cells and haploidentical GVHD, and explore the effect of the CD4+CD25+Foxp3+ regulatory T cells on engraftment, GVHD, GVL and long-term survival. Explore the role of HLA-Cw loci in haploidentical haemopoietic stem cell transplantation; afford a useful parameter in dornor selection.MethordsIn the first part, CD4+T cells derived from BalB/C mice spleen were selected by Mini MACS, and cultured in 24 wells plate for 3 weeks, which coated with 2ug/mlαCD3 monoclonal antibody, the medium system included 1ug/mlαCD28 monoclonal antibody, rhIL-2 100U/ml and 10nM RAPA (Rapamycin) with RPMI 1640 medium and 15% FBS, under the 37oC, 5%CO2 and saturated humidity incubator. After 3 weeks, CD4+CD25+T cells were detected by FCM, CD4+CD25+T cells were sorted by Mini MASC, the expression of Foxp3 mRNA was measured with real-time RT-PCR, MLR and proliferation inhibition were used to exam the function of CD4+CD25+T cells, IL-10 and TGF-β1 of supernatant were measured by ELISA, by comparing with CD4+CD25+T cells expanded without RAPA in vitro, to explore the effect of RAPA on expanded CD4+CD25+Foxp3+T cells.RAPA was administrated to Balb/C mice 0.4mg/day intragastrically as the dose of person, the control group mice of same age and weight were given with sterile water in the second part. After three weeks, CD4+CD25+T cells of splenocyte were detected by FCM; the relative levels of Foxp3 mRNA were determined by real-time quantitative RT-PCR in total splenocytes. CD4+CD25+T cells were sorted by Mini MACS for examination of inhibition, levers of IL-10 and TGF-β1 of blood serum were assessed by ELISA, the results were compared to the control group, to probe the effect of RAPA on CD4+CD25+ Foxp3+T cells proliferations in vivo.In the third part, the amount of CD4+CD25+ T cells and Foxp3 mRNA were examined among the GVHD group, autotransplantation and nontransplantation group, to identify the correlation of GVHD and CD4+CD25+Foxp3+T cells.The transferability EL9611 erythroleukemia mice model was established via injecting spleen cells of mice with EL9611 erythroleukemia into BalB/C and CB6F1 mice caudal vein and cavum abdominis in the fourth part, to learn the proceeding of EL9611 erythroleukemia in BalB/C and CB6F1 mice.In the fifth part, CB6F1 mice with EL9611 erythroleukemia were performed haploidentical bone marrow transplantation with or without CD4+CD25+Foxp3+T cells, The development of GVHD, engraftment, and leukemia relapse were observed to investigate the effect of CD4+CD25+Foxp3+T cells on GVHD, engraftment and GVL.In the last part, HLA-Cw of donors and recipients were detected by PCR-SSP, the effect of HLA-Cw in haploidentical hematopoietic stem cell transplantation was analyzed by collecting the clinical data, including incidence of GVHD, leukemia replase and long- term survival.ResultsCultured in these medium for 3 weeks, CD4+CD25+T cells developed from CD4+T cells rose from 8.84% to (76.05±2.73)%, higher than control group(52.17±1.36)%(P<0.001), Real-time quantitative RT-PCR showed that the levels of Foxp3 mRNA of CD4+CD25+T cells in RAPA group was 5 folds higher than the group without RAPA(P<0.001), the ability of proliferation was 0.29 folds higher than the control group(P<0.001), the ability of inhibition to CD4+T cells was 3.6 folds higher than the control group(P<0.001), the level of IL-10 and TGF-β1 of supernatant was 1.8 and 1.6 folds higher than control group, respectively (P<0.001).The CD4+CD25+T cell of experimental mice splenocytes was (24.13±10.06) %, while control group was (8.48±3.19) % (P<0.001). Real-time quantitative RT-PCR showed that the levels of foxp3 mRNA of experimental mice splenocytes was 6 folds higher than the control group (P<0.001). CD4+CD25+T cells sorted from experimental mice and control mice splenocytes inhibited CD4+T cells (57.03±3.63) % and (56.95±1.93) % respectively (P>0.05). The levels of IL-10 and TGF-β1 of blood serum were(166.28±24.07)pg/ml and (165.27±22.95)pg/ml in experimental group, while (79.50±7.68)pg/ml and (88.52±10.06) pg/ml in control group (P<0.001).CD4+CD25+T cells were detected by FCM at the point of GVHD development; the proportion of CD4+CD25+T cells was 15.4% and 24.03% in the autotransplantation group and allotransplantation group, respectively. But the proportion of CD4+CD25+T cells was 8.46% in nontransplantation group, the levels of Foxp3 mRNA of allotransplantation group mice were lower than autotransplantation or nontransplantation group significantly (P<0.001).1×106 EL9611 erythroleukemia cells were injected into BalB/C and CB6F1 mice (8 to 10 weeks) caudal vein and cavum abdominis. The time of leukemia cell appearance in peripheral blood was at day 6 and 7, and the mice were dead at day 13.3 and 21.7 respectively. The time of erythroleukemia cell appearance in peripheral blood was at day 17.2 and 24.6 when 1×106 EL9611 erythroleukemia cells were injected into BalB/C and CB6F1 mice cavum abdominis, but the mice were dead at day 21.7 and 27.5.The cumulative incidence of GVHD was 28.6% and 100% in Treg transplantation group and control group respectively. 4/7(57.14%) relapsed in autotranplantation group, but no one in Treg transplantation group.In haploidentical HSCT for person, The cumulative incidence of grades II–IV acute GVHD were 76.9% in HLA-Cw matched group and 14.3% in HLA-Cw mismatched group(P<0.05), 28 months disease-free survival probabilities was 46.2% in HLA-Cw matched group, and 85.7% in HLA-Cw mismatched group(P>0.05).ConclusionRAPA can promote CD4+CD25+Foxp3+T cells proliferation in vitro and vivo. The proporation of CD4+CD25+T cells could rise while CD4+CD25+Foxp3+T cells were reduced when GVHD happened. The CB6F1 EL9611 erythroleukemia mice model established by injected EL9611 erythroleukemia cells into caudal vein was reliable. CD4+CD25+Foxp3+T cells could reduce GVHD effectively, while not impair GVL. HLA-Cw mismatched in donor and receipt of haploidentical SCT for person was benefited to reducing II-IV aGVHD, and it was in favor of long-term survival. HLA-Cw might be one of the optimized parameter in dornor selection.
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