Single-cell analysis of human MAIT cell transcriptional, functional and clonal diversity [Exp 4]

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognize microbial metabolites through a semi-invariant T cell receptor (TCR). Major questions remain regarding the extent of human MAIT cell functional and clonal diversity. To address these, we analyzed the single-cell transcriptome and TCR repertoire of blood and liver MAIT cells and developed functional RNA sequencing, a method to integrate function and TCR clonotype at single-cell resolution. MAIT cell clonal diversity was comparable to conventional memory T cells, with private TCR repertoires shared across matched tissues. Baseline functional diversity was low and largely related to tissue site. MAIT cells showed stimulus-specific transcriptional responses in vitro, with cells positioned along gradients of activation. Clonal identity influenced resting and activated transcriptional profiles but intriguingly was not associated with the capacity to produce IL- 17. Overall, MAIT cells show phenotypic and functional diversity according to tissue localization, stimulation environment and clonotype. Isolated blood T cells were left unstimulated or stimulated for 20 h with MR1/5-OP-RU (TCR), IL-12 + IL-18 (cytokine) or both, before MAIT cell (CD3+CD26+CD161hiVα7.2+) sorting. Additionally, isolated T cells were left unstimulated or stimulated for 68 h with both MR1/5-OP-RU and IL-12 + IL-18 (TCR+cytokine), before MAIT cell sorting. Single-cell gene, surface protein and TCR repertoire analysis was performed using 10x Genomics technology.