Deconvoluting TCR-dependent and -independent activation is vital for reliable Ag-specific CD4(+) T cell characterization by AIM assay.

Activation-induced marker (AIM) assays identify antigen (Ag)-specific T cells, but recent studies revealed AIM(+) T helper cell 17 (T(H)17)-like (CCR6(+)) and circulating T follicular helper cells (cTfh) were not associated with peptide/HLA tetramer staining. We show that CD39(+) regulatory T cell (T(reg))-like and CD26(hi) T(H)22-like cells undergo T cell receptor (TCR)-independent activation by cytokines during Ag stimulation, leading to nonspecific up-regulation of AIM readouts. Transcriptional analysis enabled discrimination of bona fide Ag-specific T cells from cytokine-activated T(reg) and T(H)22 cells. CXCR4 down-regulation emerged as a hallmark of clonotypic expansion and TCR-dependent activation in memory CD4(+) T cells and cTfh. By tracking tetramer-binding cells upon Ag restimulation, we demonstrated that CXCR4-CD137(+) cells provided a more accurate measure of Ag-specificity than standard AIM readouts. This modified assay excluded the predominantly CCR6(+) cytokine-activated T cells that contributed to an average 12-fold overestimation of the Ag-specific population. Our findings provide an accurate approach to characterize genuine Ag-specific T cells.