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

在动物模型中，CD4~+CD25~+调节性T细胞(Treg)具有广泛的免疫调节活性，可调节包括炎症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能否在体外扩增和诱导。我们用抗CD3/抗CD28的微球和IL-2刺激从UC患者的白细胞分离产品中分离的Treg。Treg在培养10天内可扩增18倍。培养的Treg维持Treg特异性标志物FOXP3的表达，并显著抑制共培养的CD4^+T细胞的增殖。接下来，我们检测了TREG能否在体外被诱导。用转化生长因子-β-1刺激和培养非TREG。因此，在体外甚至可以从非Treg诱导Treg。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CD_4~+T细胞，诱导产生Treg。在RAPA的刺激下，培养21d末CD25+FoxP3+细胞占17%。相反，在没有RAPA刺激的情况下，很少有细胞表达CD25和FoxP3。将CD4^+CD62L^+CD25^-T细胞移植至CB-17 SCID小鼠，建立慢性持续性结肠炎模型。在RAPA存在的情况下，共转移刺激的CD4^+T细胞可以抑制自然发生的Treg的结肠炎，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诱导的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，转导诱导的Treg可能对炎症性肠病有效。

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.