Rv2140c is targeted by follicular helper T cells in M.tuberculosis exposed γ Interferon Release Assay negative populations in Uganda and South Africa

Introduction/Rationale: Mycobacterium tuberculosis (Mtb) remains a leading cause of death worldwide. IFNγ Release Assay (IGRA) is widely used to diagnose Mtb infection by measuring the IFNγ response to Mtb antigens. However, healthy IGRA- individuals with high Mtb exposure (“resisters”) have shown evidence of infection and make Mtb-specific responses that differ from IGRA+ individuals. Human CD4 T cells are critical in controlling Mtb. IFNγ-expressing Th1 cells are largely believed to be the major protective subset. Resisters' negative response to IGRA suggests alternative protective mechanisms of CD4 T cells. This study aims to profile the antigen-specific CD4+ T cell responses in Mtb-exposed IGRA- individuals.

Methods: We performed TCR sequencing on Ugandan resisters and IGRA+ individuals. TCR repertoires were analyzed with GLIPH3 algorithm we recently developed to identify resister-specific TCRs. Mtb antigenic ligands were discovered by a new T cell epitope discovery platform. Antigen-specific CD4+ T cells were then isolated using peptide-MHC multimers covering the discovered antigenic peptide, and characterized by single-cell multi-omics and Flow Cytometry.

Results: We identified 24 TCR specificity groups uniquely enriched in resisters. Two ligand peptides were decoded from Mtb antigens Rv2140c and ESAT6. In a parallel South African cohort, in IGRA- individuals, we detected T cell responses to ESAT6, the antigen used in IGRA test, and a robust response to Rv2140c. Notably, Rv2140c-specific CD4 T cells were predominantly follicular helper cells (Tfh), which correlated with protection from Mtb in mice, whereas IGRA+ individuals showed primarily Th1 responses.

Conclusion: We identified a novel Mtb antigen associated with protection, highlighting its potential as vaccine candidate. ESAT6-specific responses in IGRA- individuals confirmed underlying Mtb infection, indicating the limitations of IGRA tests. The predominance of Tfh cells reveals new protective human T cell mechanism against Mtb beyond the classic Th1 response.