早期凋亡的T淋巴细胞诱导生成耐受性树突状细胞的实验研究

目的： 探讨早期凋亡T淋巴细胞的抑制性免疫调节性能。方法： 采用定时紫外线照射诱导T淋巴细胞早期凋亡，深低温反复冻融获得坏死T淋巴细胞。体外诱导、纯化并培养骨髓源性不成熟树突状细胞（imDCs）,imDCs分别和早期凋亡或坏死T淋巴细胞共培养。用流式细胞仪、双夹心ELISA、［³H］掺入混合淋巴细胞反应等方法分析imDCs吞噬早期凋亡或坏死T淋巴细胞后，在不同处理条件下，MHC-Ⅱ、CD40、CD80、CD86的表达水平、分泌IL-12 p70以及刺激T淋巴细胞增殖能力的差异。结果： imDCs和坏死细胞碎片共培养后明显趋于成熟，其MHCⅡ和CD40、CD80、CD86的表达水平显著上调；分泌较高水平的IL-12 p70；和同种异体处女T淋巴细胞混合培养后显著刺激处女T淋巴细胞增殖。 imDCs和早期凋亡的T淋巴细胞共培养后,其MHC-Ⅱ、CD40、CD80和CD86的表达维持较低水平；仅分泌较低水平IL-12 p70；和同种异体处女T淋巴细胞混合培养后不能刺激淋巴细胞增殖。此外，早期凋亡的T淋巴细胞孵育上清显著抑制了吞噬坏死细胞碎片后的imDCs表达共刺激分子CD40、CD80、CD86。当TGFβ₁中和抗体和早期凋亡T淋巴细胞同加入imDCs，在表达MHC-Ⅱ、CD40、CD80、CD86，分泌IL-12 p70，刺激处女T淋巴细胞增殖等方面和吞噬坏死T淋巴细的DCs相比无显著差异。结论： 早期凋亡T淋巴细胞通过释放免疫抑制性细胞因子TGFβ₁，诱导imDCs呈现出耐受性DCs（TolDCs）的免疫表型及生物学特征，从而发挥抑制性免疫调节作用。

Early apoptotic T lymphocytes induce DCs from bone marrow to tolerogenic DCs

AIM: To study the immunosuppressive effects of early apoptotic T lymphocytes.METHODS: Early apoptotic spleen T cells were induced by ultraviolet irradiation for 5 min.After irradiation,spleen T cells were incubated at 37 ℃ with 5% CO₂ for 2 h and thus early apoptotic T lymphocytes were obtained.Three to four freeze thaw cycles resulted in disruption of the spleen T cells into fragments.imdendribic cells(DCs) were prepared from red cells and T cells depleted bone marrow cells.The imDCs were divided into five groups: group A: necrotic spleen T cells were added to imDCs;group B: early apoptotic spleen T cells were added to imDCs;group C: supernatants from early apoptotic spleen T cells alone with necrotic spleen T cells were added to imDCs;group D: TGFβ₁ neutralizing antibody along with early apoptotic T lymphocytes were added to imDCs;group E: immature dendridic cells culture in RPMI-1640 for 5 days were used as negative control.Flow cytometry was employed to analyze the expression of MHCⅡ,CD40,CD80 and CD86 on DCs in each group.ELISA was employed to assay the IL-12 p70 produced by DCs in different groups.The amounts of TGFβ₁ released by early apoptotic T lymphocytes were also determined by ELISA.T cells proliferation assay was employed to study DCs T cells stimulatory capacity.RESULTS: The DCs expressed high level of MHCⅡ,CD40,CD80 and CD86 when exposed to necrotic cells while early apoptotic cells did not.The supernatants from early apoptotic spleen T cells suppressed the expression of MHCⅡ,CD40,CD80 and CD86 on DCs exposed to necrotic spleen T cells.When TGFβ₁ neutralizing antibody along with early apoptotic spleen T were added to imDCs,the expression of MHCⅡ,CD40,CD80 and CD86 was increased significantly.The necrotic spleen T cells increased IL-12 p70 production by DCs,while apoptotic spleen T cells at early stage did not (P<0.01,group B vs group A or B;P>0.05,group B vs group E).Only the DCs that exposed to necrotic spleen T cells gained significant T lymphocytes stimulatory capacity,while DCs exposed to apoptotic cells at early stage did not.The amounts of TGFβ₁ released by early apoptotic spleen T cells were much higher than those released by viable spleen T cells.CONCLUSION: Apoptotic spleen T cells at early stage have the capacity to induce the generation of tolerogenic DCs.