Identification of human T cells that recognize a mycobacterial adjuvant, trehalose monomycolate (TMM)

Mycobacterium tuberculosis (M. tb) causes tuberculosis (TB) which remains one of the world's deadliest diseases. As M.tb are protected by its thick lipid cell wall, the host has developed specific immune receptors that recognize their unique lipids as antigens. Among them is a mycolipid-specific T cells restricted by common antigen-presenting molecules, such as CD1. However, complete understanding of their subsets, clonotypes and antigen-specificity has not been fully achieved. Here, we identified novel mycolipid-specific T cells by comprehensive screening using lipid biochemical ´ single cell-TCR-RNA-sequencing-based clonotypic analysis. When human PBMCs were co-cultured with mycobacterial crude lipid extract, a CD4+ CTL-like clone, Y-50, was highly proliferated. Indeed, NFAT-GFP reporter cells reconstituted TCRaß chains expressed by Y-50 strongly reacted with crude lipids in the presence of monocyte-derived dendritic cells from syngenic/allogenic donors. Activity-based purification of crude lipids identified trehalose monomycolate (TMM) as an antigen. CD1b was necessary and sufficient for the presentation of TMM to be recognized by Y-50. TMM-loaded CD1b tetramer-positive T cells were detected in PBMCs and cord blood cells. The frequency of tetramer+ T cells in PBMCs was higher in active TB patients than uninfected donors. These results demonstrate the presence of novel unconventional T cells in humans that recognize TMM. Overall design: Reconstituted CDR3 mutant library cells were stimulated with TMM for 20 h and then sorted by GFP negative/positive populations. Unstimulated library cells were uesed as a control group. Each sorted cells was analyzed by bulk TCR-sequencing