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目的探讨吞噬体外光化学法(PUVA)处理的同种异基因淋巴细胞的树突细胞(DC)对抗原特异性CD4+ CD25 +Foxp3+调节性T细胞的体外诱导作用。方法分离LEW大鼠骨髓细胞,经大鼠重组IL-4和GM-CSF共同诱导培养制备骨髓来源的LEW大鼠DC。分离DA大鼠脾淋巴细胞(SP),制备经PUVA处理的DA大鼠脾淋巴细胞(PUVA-SP)并用流式细胞仪检测其凋亡率。在体外将DA大鼠的PUVA-SP或SP与LEW大鼠骨髓来源的DC共同培养,得到PUVA-SPDC和SPDC,以Luminex液相芯片法检测PUVA-SPDC和SPDC培养上清中IL-10、IL-12的含量。以CFSE标记PUVA-SP,流式细胞仪检测LEW大鼠DC对DA大鼠PUVA-SP的摄取情况。将LEW大鼠CD4+25-T细胞与PUVA-SPDC或SPDC混合培养5d,流式细胞仪检测诱导形成的CD4+ CD25+ Foxp3+ T细胞,同时分离培养体系中的CD4+ CD25+ T细胞,通过混合淋巴细胞反应(MLR)检测PUVA-SPDC诱生的CD4+ CD25+ T细胞对LEW大鼠CD4+ CD25- T细胞(效应性T细胞)体外增殖的抑制作用。结果DA大鼠脾淋巴细胞经PUVA处理后细胞凋亡率为81.93%。LEW大鼠DC可有效摄取PUVA-SP,吞噬率达70.72%。SPDC培养上清中IL-12和IL-10浓度分别为700±18、39±3pg/ml;与SPDC相比,PUVA-SPDC培养上清中的IL-12水平(148±9pg/ml)明显降低(P<0.01),而IL-10水平(75±4pg/ml)则明显增加(P<0.01)。与PUVA-SPDC混合培养后,LEW大鼠CD4+T细胞中CD4+ CD25+ T细胞的比例为15.4%,其中Foxp3阳性率为44.1%;而与SPDC混合培养后LEW大鼠CD4+T细胞中CD4+ CD25+ T细胞的比例只有6.5%,其中Foxp3阳性率仅为8.2%。PUVA-SPDC诱生的CD4+ CD25+ T细胞对体外增殖反应有明显的抗原特异性抑制作用。结论PUVA-SPDC在体外能够有效诱生抗原特异性CD4+ CD25+ Foxp3+调节性T细胞。
Objective To investigate the in vitro induction of antigen-specific CD4 + CD25 + Foxp3 + regulatory T cells by dendritic cells (DCs) of allogeneic lymphocytes phagocytosed with PUVA. Methods Bone marrow cells of LEW rats were isolated and cultured. The bone marrow-derived LEW rat DCs were prepared by co-culture of rat recombinant IL-4 and GM-CSF. Splenic lymphocytes (SPs) of DA rats were isolated and splenic lymphocytes (PUVA-SP) of DA rats treated with PUVA were prepared and their apoptotic rates were determined by flow cytometry. PUVA-SP or SP in DA rats were co-cultured with LEW rat bone marrow-derived DCs in vitro to obtain PUVA-SPDC and SPDC. Luminex LC-MS was used to detect IL-10 in PUVA-SPDC and SPDC culture supernatants, IL-12 content. CFU-labeled PUVA-SP and flow cytometry were used to detect the uptake of PUVA-SP by LEW rat DC. CD4 + CD25 + T cells from LEW rats were mixed with PUVA-SPDC or SPDC for 5 days. CD4 + CD25 + Foxp3 + T cells induced by induction were detected by flow cytometry. CD4 + CD25 + T cells were isolated from culture system and mixed lymphocyte reaction (MLR) was used to detect the inhibitory effect of CD4 + CD25 + T cells induced by PUVA-SPDC on the proliferation of CD4 + CD25- T cells (effector T cells) in vitro in LEW rats. Results The apoptotic rate of DA rats spleen lymphocytes treated with PUVA was 81.93%. LEW rat DC can be effectively uptake PUVA-SP, phagocytosis rate of 70.72%. The concentration of IL-12 and IL-10 in the culture supernatant of SPDC were 700 ± 18,39 ± 3pg / ml, respectively. Compared with SPDC, the level of IL-12 in culture supernatant of PUVA-SPDC (148 ± 9pg / ml) (P <0.01), while IL-10 level (75 ± 4pg / ml) increased significantly (P <0.01). After incubation with PUVA-SPDC, the percentage of CD4 + CD25 + T cells in CD4 + T cells of LEW rats was 15.4%, and the positive rate of Foxp3 was 44.1%. After incubation with SPDC, CD4 + CD25 + T cells only 6.5%, of which Foxp3 positive rate was only 8.2%. The CD4 + CD25 + T cells induced by PUVA-SPDC had obvious antigen-specific inhibitory effect on the proliferation in vitro. Conclusion PUVA-SPDC can effectively induce antigen-specific CD4 + CD25 + Foxp3 + regulatory T cells in vitro.