An improved method for growing and analysing human antigen‐specific CD4 + T‐cell clones

Background 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. Methods 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. Results 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 3 H‐thymidine incorporation assays. These findings were confirmed with two proinsulin‐specific CD4 + T‐cell clones isolated from an individual with type 1 diabetes. Conclusion 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. Copyright © 2011 John Wiley & Sons, Ltd.