Mycobacterium tuberculosis resisters despite HIV exhibit activated T cells and macrophages in their pulmonary alveoli [scRNA-seq]

Natural resistance to Mycobacterium tuberculosis (Mtb) infection in some people living with HIV (PLWH) is unexplained. We performed single cell RNA-sequencing of bronchoalveolar lavage cells, unstimulated or ex vivo stimulated with Mtb, for 7 PLWH who were Tuberculin skin test (TST) & Interferon Gamma Release Assay (IGRA) positive (called LTBI) and 6 who were persistently TST & IGRA negative (called resisters). Alveolar macrophages (AM) from resisters displayed a stronger baseline M1 phenotype than LTBI AM. Resisters displayed alveolar lymphocytosis (10%-60%), with enrichment of all T cell subpopulations including CD4+ and CD8+ IFNG-expressing cells. Alveolar lymphocytosis was strongly associated with most of the AM transcriptomic features of resisters. In both groups, mycobactericidal granulysin was expressed almost exclusively by a CD8+ T cell subtype that co-expressed granzyme B, perforin and NK cell receptors. These poly-cytotoxic T lymphocytes (CTL) over-expressed activating NK cell receptors and were increased in resisters. Following challenge with Mtb, only a subpopulation of Intraepithelial Lymphocytes (IEL)-like cells in LTBI participants responded with increased transcription of IFNG. AM from resisters responded with stronger TNF signature at 6h post-infection (p.i.) while at 24h p.i. AM from LTBI displayed stronger IFN-? signature. Conversely, at 24h p.i. only AM from resisters displayed a significant upregulation of MICA transcripts which encode an activating ligand for the CD8+ poly-CTL. These results suggest that CD8+ CTL and AM mediate the resister phenotype in PLWH. Overall design: Our study was restricted to people living with HIV (PLWH) on long-term anti-retroviral therapy (ART) with no history of tuberculosis (TB) despite long-term exposure to Mycobacterium tuberculosis (Mtb). The 14 participants belonged to two phenotypic groups of equal size: participants classified as “LTBI (latent TB infection)” who tested Interferon Gamma Release Assay (IGRA) positive and displayed a Tuberculin skin test (TST) = 10 mm, and participants coined “resisters” who persistently tested IGRA negative with a TST = 0 mm. Bronchoalveolar lavage (BAL) were collected from these participants and the recovered cells were kept uninfected (MtbNEG) or challenged with Mtb (MtbINF) for approximatly 6h and 24h. Single-cell RNA-sequencing (scRNA-seq) was performed to investigate the BAL cellular composition, gene expression levels in the absence of Mtb and the transcriptomic responses to Mtb challenge. BAL cells from one resister participant were lost due to high proportion of dead cells. CITE-seq was performed in two libraries, one LTBI and one resister, to aid in the cell-type annotation.