Mycobacterial growth in human in vitro granulomas shows marked intra-lineage diversity and is overall increased for MTBC modern lineages.

Growth rate between days 1 and 8 p.i. stratified by (A) ancestral or modern lineage, (B) specific lineage, or (C) individual strain. Colors indicate lineage, and horizontal lines and bars represent medians. Shapes stand for (A-B) individual strains within the same lineage (LXA in circles, LXB in squares and LXC in triangles), or (C) independent donors. Statistical analyses by (A) two-tailed Mann-Whitney, (B) Kruskal-Wallis, or (C) Friedman test with post hoc Dunn’s correction. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

MTBC isolates used in this work

Propensity to enter dormancy is particularly pronounced in lineage 2 and reduced in lineage 3 strains.

Bacilli recovered on day 8 p.i. were stained with Auramine-O (Au) and Nile red (NR). The ratio between dormant (Au− NR+) and metabolically active (Au+ NR−) bacteria (dormancy ratio) was quantified by fluorescence microscopy. Data stratified by (A) individual strain, or (B) lineage. (C) Two-tailed Spearman’s correlation analysis of dormancy ratio with growth rate. Colors indicate lineage, and bars and horizontal lines represent medians. Shapes stand for (A) independent donors, or (B-C) individual strains within the same lineage (LXA in circles, LXB in squares and LXC in triangles). Statistical analyses by (A) Friedman, or (B) Kruskal-Wallis test with post hoc Dunn’s correction. ****, p < 0.0001.

MTBC strain diversity translates into a spectrum of granulomatous responses that correlates with bacterial growth.

(A) Bright field images of in vitro granulomas on day 7 p.i. from a representative donor. Scale bar = 100 μm. UI, uninfected. Number, area and aspect ratio of cell aggregates were quantified and integrated into a granuloma score (see methods). Granuloma scores stratified by (B) individual strain, and (C) ancestral or modern lineage. (D) Two-tailed Spearman’s correlation analysis of granuloma score with growth rate. Colors indicate lineage, and bars represent medians. Shapes stand for (B) independent donors, or (C-D) individual strains within the same lineage (LXA in circles, LXB in squares and LXC in triangles). Statistical analyses by (B) Friedman, or (C) two-tailed Mann-Whitney test.

Macrophage apoptosis is positively associated with mycobacterial growth rate and granuloma score.

(A) Percentage of apoptotic macrophages (CD11b+ Annexin V+ 7-AAD−) on day 6 p.i. quantified by flow cytometry. (B-C) Two-tailed Spearman’s correlation analysis of macrophage apoptosis with (B) growth rate or (C) granuloma score. Colors indicate lineage, and bars represent medians. Shapes stand for (A) independent donors, or (B-C) individual strains within the same lineage (LXA in circles, LXB in squares and LXC in triangles). (A) Statistical analysis by Friedman test with post hoc Dunn’s correction. #, p = 0.058; **, p < 0.01.

Activation and proliferation of T cells is associated with reduced mycobacterial growth across the MTBC.

Percentage of activated (expressing any of the markers) and proliferating (CFSE−) T cells on day 6 p.i. was quantified by flow cytometry. (A) Principal component (PC) analysis using raw (top panels) or scaled (lower panels) data. Data were grouped by donor (left panels), grey shades represent independent donors; or by MTBC infecting strain (right panels), colors indicate lineage and shapes stand for individual strains within the same lineage (LXA in circles, LXB in squares and LXC in triangles). (B) Heatmap representing the median scaled frequencies induced by each MTBC strain. UI, uninfected. (C) Marker co-expression on the activated/proliferating CD4 T cells induced by L1A, L2A and L5 strains. Bars represent the mean percentage of cells expressing the indicated marker combination and each filling pattern of the shapes corresponds to an individual donor. (D-E) Percentage of CD38+ CD4 T cells elicited by (D) ancestral or modern lineages, or (E) specific modern lineages. Colors and shapes same as in (A), and horizontal lines represent medians. Statistical analysis by (D) two-tailed Mann-Whitney, or (E) Kruskal-Wallis test with post hoc Dunn’s correction. (F) Heatmap of Spearman’s correlation coefficients of activated/proliferating T cell populations with MTBC growth rate (two-tailed, post hoc Benjamini-Hochberg corrected). # p = 0.09; **, p < 0.01; ****, p < 0.0001.

CXCL9, granzyme B and TNF-α secretion is associated with reduced mycobacterial growth across the MTBC.

The concentration of soluble mediators in the supernantant of in vitro granulomas on days 1 and 8 p.i. was quantified by multiplex bead-based immunoassay. (A) Principal component (PC) analysis was performed using raw (top panels) or scaled (lower panels) concentrations. Data were grouped by donor (left panels), grey shades represent independent donors; or by MTBC infecting strain (right panels), colors indicate lineage and shapes stand for individual strains within the same lineage (LXA in circles, LXB in squares and LXC in triangles). (B) Heatmap of the median scaled response induced by each MTBC strain. UI, uninfected; GrzB, granzyme B. (C) IL-1β response stratified by lineage. (D) Two-tailed Spearman’s correlation analysis of IL-1β response on day 1 p.i. with granuloma score. (E) IL-13 response stratified by ancestral or modern lineages. (C-E) Colors and shapes same as in (A) and horizontal lines represent medians. Statistical analysis by (C) Kruskal-Wallis test, with post hoc Dunn’s correction, or (E) two-tailed Mann-Whitney test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. (F) Heatmap representing Spearman’s correlation coefficients with MTBC replication rate (two-tailed, post hoc Benjamini-Hochberg corrected). n.s., p > 0.1; #, p = 0.056; *, p < 0.05; **, p < 0.01.

Granulomatous response variability across MTBC strains is consistent across independent donors.

Bright field images of in vitro granulomas on day 7 p.i. from MTBC strains representative of the spectrum of granulomatous responses observed. Scale bar = 100 μm.

The number, shape and size of in vitro granulomas vary depending on the MTBC infecting strain.

(A) Total number (no.) of granulomas, as well as their individual (B) aspect ratio and (C) area were quantified on a representative bright field for each strain and donor. Colors indicate lineage, shapes correspond to individual donors and bars represent medians. Statistical analysis by Friedman test.

Flow cytometry gating strategy for the quantification of apoptosis induction (Annexin V+ 7AAD−) in CD11b+ macrophages.

Flow cytometry gating strategy for the analysis of T cell proliferation and activation.

UI, uninfected.

MTBC strain diversity results in induction of quantitatively distinct T cell responses.

Scaled frequencies of (A) proliferating and/or activated CD4 and (B) CD8 T cells. Colors indicate lineage, each filling of the diamond shapes corresponds to an independent donor and bars represent medians. Statistical analysis by Friedman test. UI, uninfected.

Induction of T cell activation and proliferation exhibits both lineage- and mycobacterial growth-associated trends.

Frequency of each population stratified by (A) ancestral or modern lineage, or (B) specific modern lineages. Statistical analysis by (A) two-tailed Mann- Whitney, or (B) Kruskal-Wallis test, with post hoc Dunn’s correction. * p < 0.05; **, p < 0.01. (C) Two- tailed Spearman’s correlation analyses of the scaled frequencies of the various proliferating/activated T cell populations with MTBC growth rate (post hoc Benjamini-Hochberg multiple testing corrected). Colors indicate lineage, shapes stand for individual strains within the same lineage (LXA in circles, LXB in squares and LXC in triangles) and horizontal lines represent medians.

MTBC diversity results in variable soluble factor immune responses.

Scaled concentrations of soluble factors induced by MTBC infection on (A) day 1 p.i. or (B) day 8 p.i. Colors indicate lineage, each filling of the diamond shapes corresponds to an independent donor and bars represent medians. Statistical analysis by Friedman test. UI, uninfected.

Several chemokine and cytokine responses associate positively or negatively with MTBC growth rate in in vitro granulomas.

Two-tailed Spearman’s correlation analyses of the indicated soluble factors (scaled concentrations) with MTBC growth rate (post hoc Benjamini-Hochberg multiple testing correction). Colors indicate lineage, and shapes stand for individual strains within the same lineage (LXA in circles, LXB in squares and LXC in triangles).