经体外扩增的食蟹猴 CD4+ CD25 Hi Treg 细胞可抑制食蟹猴抗猪 T 和 B 细胞免疫应答。

Ex-vivo expanded baboon CD4+ CD25 Hi Treg cells suppress baboon anti-pig T and B cell immune response.

机构信息

Cardiothoracic Surgery Research Program, NHLBI, NIH, Bethesda, MD 20892, USA.

出版信息

Xenotransplantation. 2012 Mar-Apr;19(2):102-11. doi: 10.1111/j.1399-3089.2012.00697.x.

DOI:10.1111/j.1399-3089.2012.00697.x

PMID:22497512

Abstract

BACKGROUND

CD4(+) CD25(+) FoxP3(+) regulatory T (Treg) cells play an important role in regulating immune responses. A very small number of Treg cells are present in peripheral blood and lymphoid organs, but due to their ability to suppress the immune response, they have a high potential for immunotherapy in clinics. Successful ex-vivo expansion of naturally occurring CD4(+) CD25(+) T cells has been achieved after TCR stimulation in the presence of T cell growth factors. In this study, we evaluated the role of these Treg cells in suppressing proliferative response of baboon T and B cells to pig xenoantigens.

METHODS

Naturally occurring baboon CD4(+) CD25(+) regulatory T cells (nTreg) were sorted from peripheral blood and expanded in the presence of either anti-CD3/CD28 beads or irradiated pig peripheral blood mononuclear cells with IL-2. Treg cells were also enriched directly from CD4(+) T cells cultured in the presence of rapamycin (0.1-10 nm). Mixed lymphocyte culture and polyclonal B cell stimulation with ex-vivo Treg cells were performed to assess the function of ex-vivo expanded Treg cells.

RESULTS

The nTreg cells were expanded to more than 200-fold in 4 weeks and retained all the nTreg cell phenotypic characteristics, including high levels of FoxP3 expression. 2-fold increase in enrichment of CD4(+) CD25(+) FoxP3(+) Treg cells from CD4(+) cells was observed with rapamycin compared to cultures without rapamycin. The ex-vivo expanded Treg cells obtained from both methods were able to suppress the baboon anti-porcine xenogeneic T and B cell immune response in-vitro efficiently (more than 90% suppression at 1:1 ratio of T regulatory cells: T effector cells), and their suppression potential was retained even at 1:256 ratio. However, freshly isolated nTreg cells had only 70% suppression at 1:1 ratio, and their suppressive ability was reduced to ≤ 50% at 1:16 ratio. Furthermore, we have found that ex-vivo expanded Treg can also suppress the proliferation of B cells after polyclonal stimulation. Forty to 50 percent reduction in B cell proliferation was observed when ex-vivo expanded Treg cells were added to the culture at a 1:1 ratio. The addition of CD4(+) CD25(Neg) cells however induced vigorous proliferation.

CONCLUSION

Ex-vivo expanded CD4(+) CD25(+) FoxP3(+) Treg cells can be used to efficiently suppress xenogeneic immune responses by inhibiting T and B cell proliferation. These ex-vivo expanded Treg cells may also be used with other immunosuppressive agents to overcome xenograft rejection in preclinical xenotransplantation models.

摘要

背景

CD4(+) CD25(+) FoxP3(+) 调节性 T (Treg) 细胞在调节免疫反应中发挥重要作用。外周血和淋巴器官中存在极少数的 Treg 细胞，但由于其抑制免疫反应的能力，它们在临床上具有很高的免疫治疗潜力。在 T 细胞生长因子存在的情况下，经 TCR 刺激后，已成功实现天然存在的 CD4(+) CD25(+) T 细胞的体外扩增。在这项研究中，我们评估了这些 Treg 细胞在抑制狨猴 T 和 B 细胞对猪异种抗原增殖反应中的作用。

方法

从外周血中分离出天然存在的狨猴 CD4(+) CD25(+) 调节性 T 细胞 (nTreg)，并在存在抗 CD3/CD28 珠或 IL-2 照射的猪外周血单个核细胞的情况下进行扩增。还直接从用雷帕霉素（0.1-10nm）培养的 CD4(+) T 细胞中富集 Treg 细胞。进行混合淋巴细胞培养和体外 Treg 细胞多克隆 B 细胞刺激，以评估体外扩增的 Treg 细胞的功能。

结果

nTreg 细胞在 4 周内扩增超过 200 倍，并保留了所有 nTreg 细胞表型特征，包括高水平的 FoxP3 表达。与无雷帕霉素培养物相比，用雷帕霉素观察到 CD4(+) 细胞中 CD4(+) CD25(+) FoxP3(+) Treg 细胞的富集增加了 2 倍。从这两种方法获得的体外扩增的 Treg 细胞能够有效地抑制狨猴抗猪异种免疫反应（T 调节细胞与 T 效应细胞的 1:1 比例抑制超过 90%），其抑制潜力在 1:256 比例时仍保持不变。然而，新鲜分离的 nTreg 细胞在 1:1 比例时仅抑制 70%，其抑制能力在 1:16 比例时降低至≤50%。此外，我们发现体外扩增的 Treg 还可以抑制多克隆刺激后的 B 细胞增殖。当在培养物中添加 1:1 的体外扩增的 Treg 细胞时，观察到 B 细胞增殖减少 40%至 50%。然而，添加 CD4(+) CD25(neg) 细胞会诱导强烈的增殖。