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

Abstract

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.