CD4^(+)CD25^(+)调节性T细胞通过抑制B细胞对小鼠自身免疫性卵巢疾病发挥治疗作用

CD4^(+)CD25^(+) regulatory T cells exert therapeutic effects on autoimmune ovarian disease by suppressing B cells in mice

目的 通过建立小鼠自身免疫性卵巢疾病(autoimmune ovarian disease, AOD)模型,探索CD4^(+)CD25^(+)iTreg细胞对AOD模型诱导的卵巢功能不全(POI)小鼠的治疗作用。方法 建立出生第3天胸腺摘除Balb/c模型鼠(thymectomize on day3, D3tx),磁珠分选CD4^(+)幼稚T细胞经转化生长因子β(TGF-β)和IL-2体外诱导产生CD4^(+)CD25^(+)iTreg细胞,腹腔注射CD4^(+)CD25^(+)iTreg至5周龄的D3tx小鼠。苏木精-伊红染色(HE)和CD19免疫组化评估各级卵泡数目和B细胞浸润情况。CD4^(+)CD25^(+)iTreg与B细胞共培养,检测B细胞增殖及TGF信号通路分子表达量。结果 CD4^(+)CD25^(+)i Treg细胞回输显著增加D3tx小鼠卵巢组织中初级、次级和总卵泡数,并显著减少B细胞浸润。细胞共培养后B细胞的比例从41.4%显著降低至27.6%,共培养体系中加入TGF-β受体I特异性抑制剂ALK5i显著恢复了B细胞的增殖。结论 TGF-β和IL-2体外诱导的CD4^(+)CD25^(+)iTreg细胞可抑制D3tx小鼠卵巢中的B细胞浸润,改善POI小鼠卵巢储备功能,同时TGF-β参与了CD4^(+)CD25^(+)iTreg细胞对B细胞增殖的抑制作用。CD4^(+)CD25^(+)iTreg细胞可能通过TGF-β抑制B细胞增殖发挥对小鼠自身免疫性卵巢疾病的潜在治疗作用。

This study was designed to investigate the therapeutic effect of CD4^(+)CD25^(+)iTreg cells against premature ovarian insufficiency(POI) in a mouse autoimmune ovarian disease(AOD) model. A mouse AOD model was induced by excising the thymus of newborn Balb/c mice on day 3(D3tx). CD4^(+)CD25^(+)iTreg cells were induced in vitro with transforming growth factor β(TGF-β) and IL-2 from CD4^(+)naive T cells sorted by magnetic beads, and then they were intraperitoneally injected into 5-week-old D3tx mice. Hematoxylin-eosin staining(HE)and CD19staining experiments were conducted to assess the follicle numbers and B cell infiltration. B cells flow-sorted from the spleen of D3tx mice were co-cultured with CD4^(+)CD25^(+)i Treg cells to test B cell proliferation and the TGF signaling pathway molecules. In D3tx model mice, after treatment with CD4^(+)CD25^(+)iTregs, the infusion of CD4^(+)CD25^(+)iTreg cells significantly increased the primary, secondary and total follicle numbers in ovaries and significantly decreased the B cells infiltration. The ratio of B cells in the spleen of D3tx mice was significantly decreased from 41.4% to 27.6% after co-culture with CD4^(+)CD25^(+)iTreg cells. The TGF-β receptor I-specific inhibitor ALK5i significantly restored the proliferation of B cells co-cultured with CD4^(+)CD25^(+)iTregs. In conclusion,CD4^(+)CD25^(+)regulatory T cells induced by TGF-β and IL-2 may suppress B cell infiltration in AOD ovaries andsignificantly improve the ovarian reserve. TGF-β isinvolved in the inhibitory effect of CD4^(+)CD25^(+)iTreg cells on B cell proliferation. CD4^(+)CD25^(+)iTreg cells may have a potential therapeutic effect on POI.