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

AIM assays are thought to detect antigen (Ag)-specific T cell responses in an HLA- and cytokine-independent manner. Recent studies using AIM assays identified prominent Th17-like (CCR6 ⁺ ) CD4 ⁺ T cells and circulating follicular T helper cells (cTfh) in anti-viral contexts, but were not observed with peptide/HLA tetramer staining. We demonstrate that CD39 ⁺ Treg-like and CD26 ^hi Th22-like cells can be activated by cytokines in a TCR-independent manner during in vitro Ag stimulation, leading to non-specific upregulation of prototypical AIM readouts. Transcriptional analysis of memory CD4 ⁺ T cells that underwent TCR-dependent or -independent activation enabled discrimination of bona fide Ag-specific T cells from cytokine-activated Treg and Th22 cells. CXCR4 downregulation emerged as a hallmark of clonotypic expansion and TCR-dependent activation in memory CD4 ⁺ T cells and cTfh. Tracking tetramer-binding cells during re-stimulation showed that CXCR4 ^- CD137 ⁺ cells provide a more accurate measure of the Ag-specific population than standard AIM readouts. This modified assay excludes the predominately CCR6 ⁺ cytokine-activated T cells that contribute to an average 12-fold overestimation of the Ag-specific population. As AIM assays enable the rapid study of T cell responses against emerging pathogens, our findings provide a highly accurate approach to characterize genuine Ag-specific T cell responses that contribute to protective immunity.