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

Zheng, M. Z. M.; Burmas, L.; Tan, H.-X.; Trieu, M.-C.; Lee, H. J.; Rawlinson, D.; Haque, A. P.; Kent, S. J.; Wheatley, A. K.; Juno, J. A.

doi:10.1101/2024.12.10.627643

bioRxiv · 10.1101/2024.12.10.627643

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

Zheng, M. Z. M.·Burmas, L.·Tan, H.-X.·Trieu, M.-C.·Lee, H. J.·Rawlinson, D.·Haque, A. P.·Kent, S. J.·Wheatley, A. K.·Juno, J. A.

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Abstract

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 CD26hi 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.

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Zheng, M. Z. M., Burmas, L., Tan, H.-X., Trieu, M.-C., Lee, H. J., Rawlinson, D., Haque, A. P., Kent, S. J., Wheatley, A. K., Juno, J. A.. 2024-12-13. Deconvoluting TCR-dependent & -independent activation is vital for reliable Ag-specific CD4+ T cell characterization by AIM assay. https://doi.org/10.1101/2024.12.10.627643

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