The waxy cell wall of Mycobacterium tuberculosis (Mtb) contains immunogenic lipids, including phosphomycoketides (PM), recognized by Vδ1+ T cells via the CD1c antigen presentation pathway. PM is also found in the Bacillus Calmette-Guérin (BCG) vaccine, administered at birth to protect against disseminated TB in childhood. To dissect the molecular basis of CD1c-PM recognition, we analyzed γδ T cell repertoires from BCG-vaccinated South African 10-week-old infants (n=6). This cohort enabled us to characterize Vδ1 responses following in vivo PM exposure and before age-related repertoire focusing.
We used CD1c-PM tetramers to isolate CD1c-PM-specific T cells directly ex vivo and performed sort-based single-cell RNA sequencing to define paired TCRγδ chains, TCRγ constant region allele usage, and phenotypic marker expression. CD1c-PM+ cells were predominantly Vδ1+ (52 ± 21%).
Comparing CD1c-PM+ (n=458) and CD1c-PM- (n=440) γδ+ T cells revealed that CD1c-PM+ γδ T cells preferentially displayed a cytotoxic Temra phenotype (perforin+ CD45RA+ CD27-). These T cells also showed evidence of clonal expansion in three infants, with some clones comprising ~10% of the CD1c-PM+ repertoire. CD1c-PM+ cells preferentially utilized TRDV1, TRDJ1, TRGV5, and TRGJ2 gene segments, with CDR3γ regions remaining near germline, while CDR3δ regions were longer and incorporated multiple TRDD gene segments. Analysis using TCRdist3 revealed a distinct “YWGIR” CDR3δ hydrophobic motif that is partially encoded by the germline TRDD3 region. This motif was also present in published CD1c-PM+ γδ TCRs, indicating a potentially conserved mechanism of CD1c-PM recognition.
Our study provides the first in-depth characterization of CD1c-PM+ γδ T cells in BCG-vaccinated infants, revealing evidence of clonal expansion, cytotoxic phenotype, and biased TCR features. Future studies will further define the TCR determinants of CD1c-PM binding and assess their impact on vaccine-induced protection against Mtb.