CD1 family proteins are specialized for presenting lipid antigens. Among them, the most well-known antigens are mycobacteria-derived lipids (mycolipids), such as mycolic acid (MA), glucose monomycolate (GMM), sulfoglycolipid (SGL), mannosyl phosphomycoketide (MPM) or dideoxymycobactin (DDM). However, as mycobacteria possess a large variety of lipids on their cell wall, we hypothesized that other unappreciated mycolipids may also act as antigens to activate T cells for protective immunity.
We therefore searched for novel mycolipid-reactive T cells by comprehensive screening using unfractionated whole mycobacterial lipids × human peripheral T cells from various donors. Expanded T cells upon lipid stimulation were sorted and subjected to single cell-TCR-RNA sequencing (sc-TCR-RNA-seq). Among expanded T cell clonotypes, the CD4+ clone, Y-50, strongly reacted with crude lipids when its TCRαβ were reconstituted into a reporter T cell line in the presence of monocyte-derived dendritic cells. This activation was blocked by anti-CD1b, whereas it was induced by the addition of a mouse cell line expressing human CD1b. Activity-based purification of crude lipids identified a glycolipid adjuvant as an antigen. Determination of the crystal structure and mutagenesis analysis of Y-50 identified the characteristic of the TCRαβ suitable for the antigen recognition. By generating CD1b tetramers loaded with the antigen, we could detect tetramer+ T cells in all donors tested. The frequency of tetramer+ T cells per donor was higher in active TB patients than in uninfected donors. scTCR-RNA-sequencing of tetramer+ T cells revealed that cytotoxicity-related gene signature was enriched.
These results show the presence of novel universal unconventional T cells recognizing a mycobacterial adjuvant.