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Deconvoluting TCR-dependent and -independent activation is vital for reliable Ag-specific CD4+ T cell
Ming Z M Zheng1, Lauren Burmas1, Hyon-Xhi Tan1
1Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia.
Activation-induced marker (AIM) assays identify antigen (Ag)-specific T cells, but recent studies revealed AIM+ T helper cell 17 (TH17)-like (CCR6+) and circulating T follicular helper cells (cTfh) were not associated with peptide/HLA tetramer staining. We show that CD39+ regulatory T cell (Treg)-like and CD26hi TH22-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 Treg and TH22 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.
Activation-induced marker (AIM) assays identify antigen (Ag)-specific T cells, but recent studies revealed AIM+ T helper cell 17 (TH17)-like (CCR6+) and circulating T follicular helper cells (cTfh) were not associated with peptide/HLA tetramer staining. We show that CD39+ regulatory T cell (Treg)-like and CD26hi TH22-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 Treg and TH22 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.
