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 post-infection 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 (n = 4). Statistical analyses by (A) two-tailed Mann-Whitney, (B) Kruskal-Wallis or (C) Friedman tests with post hoc Dunn’s correction. #, p < 0.1; *, 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 post-infection were stained with Auramine-O (Au) and Nile red (NR) and quantified by fluorescence microscopy. Dormancy ratio was defined as the ratio between dormant (Au− NR+) and metabolically active (Au+ NR−) bacteria. 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 (n = 4) 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 tests 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 post-infection from a representative donor. Scale bar = 100 μm. UI, uninfected. Lower left corner higher magnification inserts (200 μm sides) depict granulomas of characteristic size and morphology. (B-D) Number, area and aspect ratio of cell aggregates were quantified and integrated into a granuloma score (see Methods section). 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 (n = 4), or (C-D) individual strains within the same lineage (LXA in circles, LXB in squares and LXC in triangles). Statistical analyses by (B) Friedman test with post hoc Dunn’s correction (comparisons against L1A) or (C) two-tailed Mann-Whitney test. #, p < 0.1; *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

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 post-infection 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 (n = 4) 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 < 0.1; **, 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 post-infection 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), with grey shades representing independent donors (n = 4); or by MTBC infecting strain (right panels), with colors indicating lineage and shapes standing 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 analyses by (D) two-tailed Mann-Whitney or (E) Kruskal-Wallis tests 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 < 0.1; *, p < 0.05; **, 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 post-infection (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), with grey shades representing independent donors (n = 4); or by MTBC infecting strain (right panels), with colors indicating lineage and shapes standing 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. Colors and shapes same as in (A) and horizontal lines represent medians. (D) Two-tailed Spearman’s correlation analysis of IL-1β response on day 1 p.i. with granuloma score. Colors and shapes same as in (A). (E) Effect of early blocking of IL-1β on granuloma formation using gevokizumab (GEVO). Bright field images of in vitro granulomas on day 7 p.i. from a representative donor. Iso, isotype control. Scale bar = 100 μm. Granuloma score was calculated as indicated in Figure 3. Colors indicate lineage, bars represent medians, and data from the same donor are connected through lines. (F) IL-13 response stratified by ancestral or modern lineages. Colors and shapes same as in (A) and horizontal lines represent medians. (G) Heatmap representing Spearman’s correlation coefficients of cytokine concentrations with MTBC replication rate (two-tailed, post hoc Benjamini-Hochberg corrected). (H) Effect of early blocking of CXCL9 on bacterial load on day 8 p.i. Colors indicate lineage, bars represent medians, and data from the same donor are connected through lines. Statistical analyses by (C) Kruskal-Wallis test with post hoc Dunn’s correction, (E, H) two-way ANOVA with post hoc Sidak’s correction, or (F) two-tailed Mann-Whitney tests. n.s., p > 0.1; #, p < 0.1; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Bacterial load of the selected MTBC isolates in human in vitro granulomas.

Colony forming units (CFU) were quantified on (A) day 1 and (B) day 8 post-infection. Colors indicate lineage, bars represent medians and shapes stand for independent donors (n = 4). Statistical analysis by Friedman test with post hoc Dunn’s correction. #, p < 0.1; **, p < 0.01; ***, p < 0.001.

Auramine-O/Nile red profile of the selected MTBC isolates.

Bacilli recovered on day 8 post-infection were stained with Auramine-O (Au) and Nile red (NR) and quantified by fluorescence microscopy. (A) Photomicrograph picturing representative staining phenotypes. Scale bar = 10 μm. Data stratified by (B) individual strain or (C) lineage. Bars represent medians and shapes stand for (B) independent donors (n = 4) or (C) individual strains within the same lineage (LXA in circles, LXB in squares and LXC in triangles).

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

Bright field images of in vitro granulomas on day 7 post-infection 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 (n = 4) and bars represent medians. Statistical analysis by Friedman test with post hoc Dunn’s correction (comparisons against H37Rv). #, p < 0.1; *, p < 0.05; **, p < 0.01.

Flow cytometry gating strategy for the quantification of cell death induction in CD11b+ macrophages.

MTBC strains did not induce significantly different percentages of necrotic macrophages.

Percentage of necrotic macrophages (CD11b+ Annexin V+ 7-AAD+) on day 6 post-infection quantified by flow cytometry. Colors indicate lineage, bars represent medians, and shapes stand for independent donors (n = 4). Statistical analysis by Friedman test.

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.

Frequencies of (A) proliferating and/or activated CD4 and (B) CD8 T cells quantified by flow cytometry on day 6 post-infection. UI, uninfected. Colors indicate lineage, shapes correspond to individual donors (n = 4) and bars represent medians. Statistical analysis by Friedman test with post hoc Dunn’s correction (comparisons against UI). #, p < 0.1; *, p < 0.05; **, p < 0.01. (C) Frequency of IFN-ψ+ CD4 T cells quantified by flow cytometry upon overnight PPD stimulation. Shapes same as in (A-B) and horizontal line represents the median.

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 analyses by (A) two-tailed Mann-Whitney or (B) Kruskal-Wallis test with post hoc Dunn’s correction. * p < 0.05; **, p < 0.01. 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.

Concentrations of soluble factors induced by MTBC infection on (A) day 1 or (B) day 8 post-infection (p.i). UI, uninfected. Colors indicate lineage, shapes stand for independent donors (n = 4), and bars represent medians. Statistical analysis by Friedman test with post hoc Dunn’s correction (comparisons against UI). #, p < 0.1; *, p < 0.05; **, p < 0.01; p < 0.001.