A subset of individuals with a high probability of exposure to M. tuberculosis (M.tb) appears to ‘resist’ established M.tb infection, as demonstrated by serially negative tuberculin skin test (TST) or IFN-γ release assay (IGRA) results. While these ‘resisters’ (RSTR) display IFN-γ-independent T cell responses to the M.tb-specific antigens ESAT-6 and CFP-10, it is currently unknown whether unique T cell functional programs are associated with this clinical outcome. We used multi-modal single-cell RNA, TCR sequencing, multi-parameter flow cytometry, and cytokine analysis in a discovery and validation format to compare the phenotypes and functions of M.tb-specific T cells between RSTRs and matched controls with ‘latent’ M.tb infection (LTBI). M.tb-specific T cells were clonally expanded in both RSTRs and LTBIs, confirming the priming of adaptive immune responses after M.tb exposure. However, M.tb-specific T cells derived from RSTRs showed enrichment of T regulatory as well as Th17-like functional programs compared to LTBIs, which were characterized by Th1*-like effector programs. Th17-like functional programs were also associated with a lack of progression to active TB among South African adolescents with LTBI, as well as bacterial control in published non-human primate studies. Together, these data suggest that ‘resisters’ may successfully control M.tb after exposure and immune priming and establish a set of T cell biomarkers to facilitate further study of this important clinical phenotype.
Overall design: We conducted the SELECT-seq protocol, which includes stimulation and sorting procedures, as described in a recent publication: Huang et al, PNAS, 2019, Select sequencing of clonally expanded CD8+ T cells reveals limits to clonal expansion. Briefly, PBMCs were stimulated by the overlapping peptide pools targeting ESAT-6 and CFP-10. Notably, before the stimulation, the cells were cultured in 1 μg/mL anti-CD154 antibody for 30 mins to prevent CD154 downregulation. After stimulation, cells were first stained and index-sorted on live CD3+/TCRαβ+ cells positive for CD69 and CD154 and/or CD137 on a BD FACSAria™ Fusion. These antigen-specific T cells were FACS-sorted into individual wells of a 96-well PCR plate with lysis buffers. To assess the technical variability, we mixed the lysis buffers with the external RNA control – ERCC RNA Spike-In (a final estimate of ~5000 molecules per well, Thermo Fisher Scientific, Waltham, MA). We used the modified Smart-Seq2 protocol (Clontech Laboratories, Mountain View, CA) to generate the cDNA library. From the library, we took a small aliquot (1 μL) for the nested PCR to amplify and sequence 15 targeted RNAs (TBX21, RORC, GATA3, FOXP3, RUNX1, RUNX3, IFNG, IL2, IL17A, GZMB, PERF, IL4, IL5, IL13, TGFB) and the CDR3 regions of both TCRα and TCRβ chains. Based on the TCR sequences, the Smart-seq2 generated cDNAs of the clonally expanded activated T cells were manually selected only on the ESAT-6/CFP-10-reactive cells for the high-coverage in-depth single-cell full transcriptomic sequencing. We applied the tagmentation and indexing protocol as in the SMART-seq2 protocol and amplified the tagmented DNA. The final pooled library was prepared using the Nextera XT library prep kit (96 index primers; Illumina, San Diego, CA) protocol and was sequenced on an Illumina HiSeq 2500.
Less...