Immunology and Diabetes Unit, St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia.
Diabetes Metab Res Rev. 2011 Nov;27(8):906-12. doi: 10.1002/dmrr.1271.
T-cell clones are valuable tools for investigating T-cell specificity in type 1 diabetes. Efficient methods for isolating T-cell clones have been developed, but growing enough cells to undertake a detailed analysis remains a challenge.
We optimized the conditions for isolating and growing antigen-specific human CD4+ effector T-cell clones. T-cell clones were isolated by FACS sorting antigen-responsive cells identified by carboxylfluorescein diacetate succinimidyl ester (CFSE) dilution. The cloning efficiency was compared between T cells cloned in the presence of 21 different combinations of cytokines. Following cloning, the growth of cloned T cells in the presence of seven different combinations of cytokines was compared. Finally, we sought a quicker and more sensitive assay to measure cloned T cells' responses to antigen.
IL-2+IL-4 were optimal for cloning antigen-specific CD4+ T cells. Following cloning, the most antigen-specific CD4+ T-cell clones grew in the presence of IL-15+IL-21. Antigen recognition by T cells cloned and grown under these conditions was readily detected by the increase in the expression of CD25. Induction of CD25 was a more sensitive measure of antigen recognition than 3H-thymidine incorporation assays. These findings were confirmed with two proinsulin-specific CD4+ T-cell clones isolated from an individual with type 1 diabetes.
The optimal cytokines for isolating, and growing, proinsulin-specific human, CD4+ T-cell clones are IL-2+IL-4 and IL-15+IL-21, respectively. Antigen recognition, by clones isolated and grown under these conditions is best detected by the induction of CD25.
T 细胞克隆是研究 1 型糖尿病中 T 细胞特异性的有价值工具。已经开发出了高效分离 T 细胞克隆的方法，但培养足够的细胞以进行详细分析仍然是一个挑战。
我们优化了分离和培养抗原特异性人 CD4+效应 T 细胞克隆的条件。通过 FACS 分选羧基荧光素二乙酸琥珀酰亚胺酯（CFSE）稀释鉴定的抗原反应性细胞来分离 T 细胞克隆。比较了在 21 种不同细胞因子组合存在下克隆 T 细胞的克隆效率。克隆后，比较了在 7 种不同细胞因子组合存在下克隆 T 细胞的生长情况。最后，我们寻求一种更快、更灵敏的测定法来测量克隆 T 细胞对抗原的反应。
IL-2+IL-4 是克隆抗原特异性 CD4+T 细胞的最佳选择。克隆后，在 IL-15+IL-21 存在下，生长出的最具抗原特异性的 CD4+T 细胞克隆。在这些条件下克隆和生长的 T 细胞对抗原的识别可通过 CD25 表达的增加来轻易检测到。CD25 的诱导是比 3H-胸腺嘧啶掺入测定更灵敏的抗原识别测量方法。这些发现通过从 1 型糖尿病个体中分离的两个胰岛素原特异性 CD4+T 细胞克隆得到了证实。
用于分离和培养胰岛素原特异性人 CD4+T 细胞克隆的最佳细胞因子分别是 IL-2+IL-4 和 IL-15+IL-21。在这些条件下分离和生长的克隆的抗原识别最好通过 CD25 的诱导来检测。
