Mycobacterium tuberculosis peptide-specific T cells in pulmonary granulomas display broad effector functions

Abstract

Mycobacterium tuberculosis (Mtb) peptide-specific T cell responses in granulomas are essential for host protection. Here we profiled macaque and human granuloma Mtb-specific T cells with single cell RNA sequencing after stimulation with peptide pools. Mtb peptide-specific T cells are primarily Th1^∗ cells and express pro-inflammatory cytokines, mutiple chemokines, TNF superfamily molecules, granzyme B and perforin, SLAM family molecules, semaphorins, growth factors, proteases, and collagen. Peptide recognition by Mtb-specific T cells also drives responses in bystander and unconventional T cells including expression of FLT3LG, LTA, and XCL1. In granulomas from a patient that underwent a pneumonectomy to remove tuberculosis-destroyed lung, we identify a population of peptide-specific T cells similar to macaque peptide-stimulated T cells. Thus, Mtb peptide-specific T cells may mediate protection against Mtb infection through the combined effects of many functions as well as the induction of bystander responses by neighboring T cells.

Keywords: immunology; microbiology; transcriptomics.

Graphical abstract

Figure 1

Mtb peptide-specific T cells in granulomas co-express many effector molecules after stimulation by cognate antigen (A) Granuloma cells from rhesus macaques were stimulated with Mtb peptide megapools for 3 h and combinatorial indexing scRNAseq analysis was performed. (B) TRAC expression by granuloma T cells. (C) T cells were identified as TRAC+ or CD3E+ and negative for CD19, MS4A, CD68, IDO1, FLT3, FCRL5, CD20, and CIITA, to remove a significant number of presumptive T cell: APC conjugates we observed with this technique. Plot shows IFNG expression on T cells cultured without or with the Mtb MTB300 and CD8 Mtb peptide megapools. Cluster 10 is highlighted as a distinct population of IFNG^high cells that appears after peptide stimulation. (D) Percentage of cluster 10 cells among T cells without or with peptide megapool stimulation. (E) Plot shows selected genes, primarily with putative effector or regulatory function, that were differentially expressed higher by cluster 10 cells compared to all other T cells. All genes shown were adjP <0.05. (F–K) Bivariate plots of SEMA7A (F), SLAMF1 (G), SLAMF7 (H), TNFSF14 (I), IL26 (J), and CCL20 (K) each versus IFNG. Cluster 10 cells are highlighted in pink and all other T cells are plotted in gray. To facilitate visualization of gene co-expression and cells with expression levels of 0, the axes have been bi-exponentially transformed and jitter added. (L) Cluster 10 cells were reclustered, yielding three subsets. Numbers represent the percentage of each subcluster among all cluster 10 cells. (M) Violin plot of selected genes separated by the three subsets of cluster 10 cells.

Figure 2

Peptide recognition by Mtb-specific granuloma T cells drives effector responses in bystander and innate-like T cells (A) Expression of selected early and immediate-early response genes that were differentially expressed higher in cluster 10 cells compared to other T cells. Gene names highlighted in green were selected to create a recent TCR stim score. (B) Plot shows this recent TCR stim score in macaque granuloma T cells that cultured without or with Mtb peptide megapools. (C) Granuloma T cells were subclustered and different subsets of T cells were identified by differential gene expression analysis. Well recognized cell populations (e.g., Tregs and Th1∗/Tc1∗ cells) are referred to as common names. Other populations are referred to by their distinguishing features. (D) Dotplot showing selected makers used to describe the clusters of T cells show in C. (E–F) Plots showing gene expression levels of CISH (E), SOCS3 (F), BCL2, (G), FLT3LG (H), LTA (I), and XCL1 (J) in granuloma T cells cultured either without or with Mtb peptide megapools. Cell clusters are outlined to depict statistically significant comparisons (adjP <0.05) between unstimulated and +megapool cells.

Figure 3

Mtb peptide-specific T cells express GZMB, while other populations of bystander and unconventional T cells selectively express GZMK, GZMM, PRF1, and GNLY (A–F) Bivariate plots of showing co-expression of IFNG with GZMB (A), GZMA (B), GZMK (C), GZMM (D), PRF1 (E), and GLNY (F). Cluster 10 cells are highlighted in pink and all other T cells are plotted in gray. To facilitate visualization of gene co-expression and cells with express levels of 0, the axes have been bi-exponentially transformed and jitter added. Numbers represent the percentages of either cluster 10 cells (pink) or all other cells (gray) in each quadrant. (G) Dotplot showing expression levels of selected genes encoding granzymes, cytotoxic, and pro-apoptotic molecules. (H–M) Histogram density plots of the expression levels of the transcripts for the transcription factors TBX21 (H), EOMES (I), PRDM1 (Blimp) (J), RORC (K), RORA (L), and AHR (aryl hydrocarbon receptor) (M). Cluster 10 cells are shown in pink histograms and all other T cells in gray. Numbers represent the percentage of cluster 10 or other T cells expressing the indicated transcription factor.

Figure 4

Human and rhesus macaque Mtb-specific T cells in granulomas are functionally similar (A) Individually resected granulomas were obtained from a patient that underwent a right pneumonectomy surgery as a result of tuberculosis. Cells were cultured with or without MTB300 and CD8 Mtb peptide megapools for 3 h. Stimulation was performed alongside the macaque granuloma cells described in the previous figures. Cells were then analyzed by scRNAseq. Cells were sequenced in same run as the rhesus macaque cells. (B) T cells were identified as TRAC+ or CD3E+ and negative for CD19, MS4A, CD68, IDO1, FLT3, FCRL5, CD20, and CIITA. Plots show expression levels of transcripts for CD4, CD8A, and FOXP3. Numbers represent the percentage of outlined clusters among all T cells. (C) IFNG expression on human granuloma T cells cultured without or with Mtb peptide megapools. Cluster 3 is noted due to is elevated expression of IFNG. (D) Percentage of cluster 3 among T cells that were cultured without or with peptide Mtb megapools. (E) Plots of human granuloma T cells showing the expression levels of the recent TCR stim score developed from peptide stimulation of rhesus macaque T cells as shown in Figures 2A and 2B. (F) Human cluster 3 cells were clustered, yielding 3 new subclusters. (G) Percentage of cluster 3-derived subclusters with and without megapool stimulation. (H–J) Featureplots of IFNG (H), CD40LG (I), and NR4A3 (J) expression in cluster 3-derived subclusters. (K) All statistically significant (adjP <0.05) DEGs between human cluster 3->2 vs. all other human T cells were plotted against all statistically significant (adjP <0.05) DEGs between macaque cluster 10 and all other T cells. Venn diagram represents overlap between the two DEG lists. Bivariate plot shows the correlation between genes that were shared between the two lists shown in purple. DEGs only found in the human list are shown in pink and DEGs only found in the macaque list are shown in light blue. (L) Dotplot shows expression of genes encoding selected effector genes that were statistically significant DEGs in both lists. (M) Dotplot shows expression of genes encoding selected early response genes that were statistically significant DEGs in both lists. (N) Dotplot shows expression of genes encoding selected surface molecule genes that were statistically significant DEGs in both lists.