Identification of CD1b-restricted human T cells recognizing a mycobacterial adjuvant, trehalose monomycolate (TMM)

Mycobacterium tuberculosis causes tuberculosis (TB) which remains one of the world’s deadliest diseases. As mycobacteria are protected by its thick lipid cell wall, the host has developed immune systems targeting their unique lipids. Among them is an unconventional T cell subset called mycolipid-specific T cells restricted by common antigen-presenting molecules, such as CD1. Here, we identified novel mycolipid-specific T cells using a method combining bulk T cell stimulation with crude lipids and single cell-TCR-RNA-sequencing-based clonotypic analysis. In human PBMCs, a CD4+ T cell clone with cytotoxic signature, Y-50, was proliferated in response to mycobacterial crude lipid extract. Activity-based purification of using reporter cells reconstituted with Y-50 TCR identified trehalose monomycolate (TMM) as an antigen. CD1b was necessary and sufficient for the presentation of TMM to be recognized by Y-50. Y-50 TCRab chains displayed characteristic features, such as positively-charged CDR3a and extra-long CDR3b. Mutagenesis and structural analysis of TCRab suggested that these unique CDR3 properties were required for the recognition of TMM presented by CD1b. CD1b tetramer loaded with TMM could detect T cells with similar characteristics to Y-50 in uninfected healthy donors, but their frequency was significantly higher in PBMCs from TB patients. In this study, we identified pre-set unconventional T cells specific for a mycobacterial adjuvant, TMM.