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.