In vitroexpansion or induction of regulatory T cells from olcerative colitis patients

CD4^+CD25^+调节性T细胞（Treg）具有广泛的免疫调节活性，在动物模型中调节包括炎性肠病在内的自身免疫性疾病。针对Treg在溃疡性结肠炎（UC）治疗中的临床应用，探讨Treg在体外是否可以扩增或诱导。我们用抗cd3 /抗cd28珠和IL-2刺激UC患者白细胞分离产物中分离的Tregs。培养10 d， Treg可增加18倍。培养的Treg维持Treg特异性标志物FOXP3的表达，并显著抑制CD4^+ T细胞共培养的增殖。因此，Treg可以在体外扩增。接下来，我们在体外测试是否可以诱导Treg， CD4^+CD255non-Treg在TGF-β1存在下被刺激培养。培养细胞表达FOXP3，共培养抑制CD4^+ T细胞增殖。因此，Treg甚至可以在体外由非Treg诱导产生。然后，我们研究诱导的Treg是否能够抑制肠道炎症。我们在雷帕霉素（RAPA）存在下用抗cd3 /抗cd28和IL-2刺激CD4^+ T细胞诱导Treg。在RAPA作用下刺激CD4^+ T细胞，培养21 d时，17%为CD25^+FoxP3^+。相比之下，没有RAPA刺激的细胞很少表达CD25和FoxP3。用CD4^+CD62L^+CD25^-T细胞转染CB-17 Scid小鼠，小鼠发生慢性持续性结肠炎。在RAPA存在的情况下，CD4^+ T细胞的共同转移作为自然发生的Treg抑制结肠炎，而在没有RAPA的情况下培养的CD4^+ T细胞未能改善肠道炎症。因此，RAPA诱导Treg，具有抑制结肠炎的作用。这些结果表明，Treg可以在体外扩增或诱导，诱导Treg的转移可能对炎症性肠病的治疗有效。

CD4^+CD25^+ regulatory T cells (Treg) possess broad range of immunoregulatory activity which regulates autoimmune disorders including inflammatory bowel diseases in animal models. Aiming the clinical application of Treg for the treatment of ulcerative colitis (UC), we investigated whether Treg can be expanded or induced in vitro. We stimulated Tregs isolated from the leukapheresis products from UC patients with anti-CD3/anti-CD28-bearing beads and IL-2. Treg could be increased 18 times in 10 days culture. Cultured Treg maintained the expression of a Treg-specific marker, FOXP3 and significantly suppressed the CD4^+ T cell-proliferation upon coculture. Thus, Treg can be expanded in vitro.Next, we tested whether Treg can be induced in vitro CD4^+CD255non-Treg were stimulated and cultured in the presence of TGF-β1. Cultured cells expressed FOXP3 and suppressed the CD4^+ T cell-proliferation upon coculture. Therefore, Treg can be even induced from non-Treg in vitro.Then, we investigate whether induced Treg are able to suppress intestinal inflammation. We induced Treg from CD4^+ T cells stimulating with anti-CD3/anti-CD28 and IL-2 in the presence of rapamycin (RAPA). When CD4^+ T cells were stimulated in the presence of RAPA, 17% was CD25^+FoxP3^+ at the end of 21-day culture. In contrast, few cells stimulated without RAPA expressed CD25 and FoxP3. CB-17 Scid mice were transferred with CD4^+CD62L^+CD25^-T cells, which developed chronic persistent colitis. Co-transfer of CD4^+ T cells stimulated in the presence of RAPA suppressed colitis as naturally-occurring Treg, while CD4^+ T cells cultured in the absence of RAPA failed to ameliorate intestinal inflammation. Therefore, RAPA induced Treg, which are capable of suppressing colitis.These results indicate that Treg can be expanded or induced in vitro and transfer of induced Treg may be efficacious for the treatment of inflammatory bowel diseases.