Chymase positive MCs are predominant in TB infected human and macaque lung tissue.

Lung biopsies from healthy individuals (n = 4) or patients with PTB (n = 5) were stained for tryptase MCT (green) or chymase MCc (red). (A) Immunofluorescence microscopy shows MCTS (green) in healthy lung biopsies (HC). MCTcs (red and green merge) are located around the early granulomas, while MCCS (red) surround the late granulomas in TB infected lung biopsies. (B) Predominance of MCTS in healthy lungs transitioning to MCTCS in early granuloma and becoming MCCS in late granulomas in TB infected lungs. (C) Immunofluorescence microscopy shows MCTS (green) and MCTCS (merge) in lungs of healthy (HC), LTBI and PTB macaques. (D) Predominance of MCTS (green) and MCTCS (merge) in PTB compared to LTBI and HC. Statistical analysis was performed using unpaired, 2-tailed Student’s t test,**** p < 0.0001, *** p < 0.001, * p< 0.05.

MC signatures across disease conditions in NHPs.

Data was re-analyzed from the lungs of M. mulatta infected withMtb CDCJ551 (GSE200151). (A) Schematic of the study design across disease conditions (B) UMAP embedding of FCER1A+ mast cells, showing the distribution of these cells across the different disease conditions (PTB in pink, HC in green, and LTBI in blue). (C) Heatmap of Hallmark pathway analysis for differentially expressed genes, highlighting the top pathways with the highest FDR values for each condition. (D-G) UCell module for pathways: IFNy signaling (D), TNF-α signaling (E), Oxidative Phosphorylation (F), and Th2 signature (G) across disease conditions, shown on UMAP embeddings. (H) Violin plots of gene expression for key MC markers (CMA1, TPSG1, LOC699599) across disease conditions. (I) Cell counts of different MC subtypes (MCC, MCT, MCTC) across disease conditions (PTB, red bars, LTBI, blue bars, and HC green bars). (J) UMAP plot of the NHP lung granuloma dataset (GSE200151), showing the distribution of cells at 4 weeks (high disease burden) and 10 weeks (low disease burden) in M.fasicularis infected with Mtb Erdman. (K) Gene expression violin plots for key MC markers (CMA1, TPSG1, LOC699599) from the new dataset across time points. (L) Proportions of different MC subtypes (MCC, MCT, MCTC). (M) Violin plots of summed module scores for the key pathways (IFNγ signaling, TNF-α signaling, oxidative phosphorylation) across disease burdens, showing pathway activity. Statistical significance was assessed using Kruskal-Wallis tests with Dunn’s multiple comparison correction (**p < 0.01, ***p < 0.001, ****p < 0.0001).

MC-deficient mice are resistant to Mtb chronic infection.

(A) C57BL/6 and CgKitWsh mice were infected with a low aerosol dose (∼l00CFU) of Mtb HN878 and mice were sacrificed at 50, 100 and 150 dpi. (B) Bacterial burden was assessed in lungs and spleens by plating. (C) Lungs were harvested, fixed in formalin and embedded in paraffin. H&E staining was carried out for blinded and unbiased analysis of histopathology. (D) Representative images and the area of inflammation measured in each lobe are shown. Scale bars: 2mm. Original magnification: ×20. Data points represent the mean ± SD of two experiments (n = 8-15 per time point per group). Statistical analysis was performed using unpaired, 2-tailed Student’s t test between C57BL/6 and CgKitWSh mice, **** p < 0.0001, *** p < 0.001, * p< 0.05.

MC-deficient mice have dysregulated immune profile after Mtb infection.

C57BL/6 and CgKitWSh mice were infected with a low aerosol dose (∼l00CFU) of Mtb HN878 and mice were sacrificed at 50, 100 and 150 dpi. Number of (A) MCs, (B) DCs (C) RMs, (D) neutrophils (E)AMs, and (F) monocytes were enumerated in the lungs of Mtb-infected mice. (G) Cytokine and chemokine production in lung homogenates from mice, collected at 150 dpi, was assessed by multiplex cytokine analysis. Data points represent the mean ± SD of 1 of 2 individual experiments (n = 4-10 per time point per group). Statistical analysis was performed using unpaired, 2-tailed Student’s t test for (A) to (F) and Two-way ANOVA Sidak’s multiple comparison test for (G) between C57BL/6 and CgKitWsh mice, *** p < 0.0001, ** p < 0.001, * p< 0.05. Outliers were removed from the subsets using Grubb’s outlier test.

Wild-type mice with airway transferred MCs promote bacterial dissemination.

Bone marrow derived in vitro cultured MCs (5×l04 cells/mouse) were adoptively transferred into the lung airways of C57BL/6 mice 7 days before infecting with a low aerosol dose (∼l00CFU) of Mtb HN878. MCs were replenished in these mice at 15 dpi, and mice were sacrificed at 30 dpi. Frequencies of (A) MCs, (B) neutrophils, and (C) RMs were enumerated in the lungs of Mtb-infected mice. Bacterial burden was assessed in (D) lungs and (E)spleens by plating. (F) Lungs were harvested, fixed in formalin, and embedded in paraffin. H&E staining was carried out for blinded and unbiased analysis of histopathology. (G) Immunofluorescence microscopy shows more neutrophil infiltration in the lungs of MC transferred WT mice. (H) Ly6G+ cells per area of lung granuloma measured in each lobe are shown. Scale bars: 2mm. Original magnification: ×20. Data points represent the mean± SD. Statistical analysis was performed using an unpaired, 2-tailed Student’s t test between the groups, **** p < 0.0001, *** p < 0.001, * p< 0.05.

Predominance of MCTS in human and NHPs lung interstitium, blood vessels and bronchi.

Healthy lung tissue and TB infected biopsies from human and NHP samples were stained for MCT (green), MCc (red). Accumulation and localization of (A) MCTS, (B) MCCS. and (C) MCTCS in healthy and TB infected human lung, and (D) MCTs and (E) MCTcs in LTBI and TB macaque lungs. Statistical analysis was performed using unpaired, 2-tailed Student’s t test, *** p < 0.0001, ** p < 0.001, * p< 0.05.

Immune cell marker expression and pathway analysis across disease conditions.

Data was re-analyzed from the lungs of NHPs (GSE200151). (A) UMAP embeddings of MCs showing expression of cell surface markers: FCER1A, MS4A2, CD48, and ITGAX across identified clusters. (B) Dot plot showing the expression levels of select genes across clusters, where the dot size represents the percentage of cells expressing each gene, and the color intensity represents the average expression level. (C) Violin plot of summed module scores for IFNy signaling, Oxidative Phosphorylation (D), TNF-α signaling (E), Th2 signature (F) pathway across different disease conditions: LTBI/HC (green), PTB (pink), and the respective clusters. (G) UMAP embeddings show the expression of key MC markers, including TPSG1, LOC699599, and CMA1 across identified clusters, with color intensity indicating expression levels. Statistical significance was assessed using Kruskal-Wallis tests with Dunn’s multiple comparison correction (**p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant).

MCs appear at early Mtb infection.

C57BL/6 mice were infected with a low aerosol dose (-l00CFU) of Mtb HN878 and mice were sacrificed at 5, 21 and 30 dpi. (A) The number of MCs in the lungs was enumerated in Mtb-infected mice. (B) Bacterial burden was assessed by plating. Data points represent the mean± SD (n = 4-5 per time point). Statistical analysis was performed using an unpaired, 2-tailed Student’s t test between time points, *** p < 0.0001, ** p < 0.001, * p< 0.05.

MC-deficient mice have no baseline differences in T cell numbers.

Six weeks C57BL/6 mice and CgKitWSh mice were sacrificed to enumerate baseline numbers of (A) CD4+T cells, and of (A) CD4 + T cells, and (B) CD8+ T cells. Data points represent the mean ± SD (n = 4-5 per time point).

MC-deficient mice have reduced numbers of activated CD4+ and cos+ T cells in the lung.

C57BL/6 and CgKitWsh mice were infected with a low aerosol dose (∼l00CFU) of Mtb HNS7S and mice were sacrificed at 50, 100 and 150 dpi. Number of(A) CD4+CD44+T cells, (B) CD4+CD44+IFNγ+ T cells, (C) CD4+CD44+TNF-α+T cells (D) CD4+CD44+IFNγ+ TNF--α+T cells, (E) CD8+CD44+T cells, (F) CD8+ CD44+IFNγ+T cells, (G) CD8+ CD44+ TNF-α+T cells, and (H) CD8+CD44+IFNγ+ TNF-α+T cells in the lungs of Mtb infected mice. Data points represent the mean± SD of 1 of 2 individual experiments (n = 4-10 per time point per group). Statistical analysis was performed using unpaired, 2-tailed Student’s t test between C57BL/6 and CgKitWsh mice,*** p < 0.0001, ** p < 0.001, * p< 0.05. Outliers were removed from the subsets using Grubb’s outlier test.

Lung myeloid cell accumulation did not vary in MC-transferred WT Mtb infected mice

Bone marrow derived in vitro cultured MCs (n=50,000 cells per mouse) were adoptively transferred into the lung airways of WT mice 7 days before infecting with a low aerosol dose (∼ l00CFU) of Mtb HNS78. MCs were replenished in these mice at 15 dpi, and mice were sacrificed at 30 dpi. Numbers of (A) MCs, (B) neutrophils, and (C) RMs were enumerated in the lungs of Mtb-infected mice. Data points represent the mean ± SD of 1 of 2 individual the lungs of Mtb-infected mice. Data points represent the mean ± SD of 1 of 2 individual experiments (n = 4 per group).