Intraperitoneal BCG results in heterologous S.Tm protection and a distinct myeloid subsets with signatures driven by STAT1.

(A) Experimental schema of in-vivo training. (B) Splenic S.Tm CFU 24 hours post infection between control (n=11) and trained mice (n=11). (C-D) Flow cytometry plots of myeloid populations (C) and mean percentage fold change of BCG over control for each given gated population percent from the Lin- population (D). (E) K-nearest neighbors (KNN) plot for total CD11b+ single cells sorted from control and BCG mice. Color is based on conditions, or cluster identity. (F) Cell markers and training induced genes for each subset. Size and color intensity indicates percentage of cells within a given cluster expressing the gene and average expression. (G) Proportions of monocyte subsets based on classifications in E. (H-I) Number of DEGs in each cell subset (H), and their corresponding gene set enrichment analysis (I). (J) Volcano plot of DEGs in CM-T subset. Data in bar graphs are presented as mean±SEM, with each individual point in B a biological repeat. Two-tailed t-test used for data in B and D (*P<0.05, **P<0.01).

Dynamics of TI-associated subsets and signatures indicates early and delayed kinetics.

(A) Experimental schema tracking TI kinetics over a two-month interval. (B) Splenic S.Tm CFU at 24 hours post infection for control and BCG mice at 14- and 60-days after inoculation (n=5-6). (C) BCG CFU from spleen and BM of BCG inoculated mice (n=4) across time points. Red-dotted line indicates limit of detection. (D) MP populations from control (PBS) and BCG mice (days post injection). Percentage of CM, NCM, and dendritic cells calculated from flow cytometry analysis of CD11b+ population. Percentage of RPM calculated from Lin- population (control: n=3, BCG: n=4 in each time point). PBS values are the mean of all time points. (E) Heatmap of upregulated genes due to training and relative gene expression ordered according to peak expression time. (F) Gene set enrichment analysis of DEGs in days 14 and 30. (G-H) Heatmap of IFNγ response genes (G) and their average expression dynamics (H). Data in bar and line graphs are presented as mean±SEM. For bar graph B and C, each individual point is a biological repeat. For line graph H, significance represents comparison between day 60 control and BCG. Heatmap rows in E and G indicate biological replicates. Two-tailed t-test used for data in B, C, and H (*P<0.05, **P<0.01, ***P<0.005, ****P<0.001).

RPM niche is replenished by recruited trained monocytes and by local training of tissue-resident populations.

(A) Scheme representing known myeloid differentiation pathways and potential trans-differentiation of trained CM to RPM. (B) Experimental schema for lineage tracing of TI-associated MP populations. (C-D) Flow cytometry analysis of TdTm+ or Tdtm- Ly6C+ MPs and RPM and quantification of RPM TdTm+ subset (n=3). (D-F) Number of DEGs of each sorted population (D), heatmap of normalized log2 expression from TI-associated DEGs specific to trained RPM populations and gene set enrichment analysis of DEGs in each sorted population (F). Data in bar graphs are presented as mean±SEM. Heatmap rows in E indicate biological replicates. Two-tailed t-test used for data in C (*P<0.05).

Transient IFNγ-STAT1 inhibition prevents TI signatures and splenic infection resistance.

(A) Experimental schema of training with early treatment of Fedratinib inhibitor. (B) Splenic S.Tm CFU for control and BCG mice, with and without Fedratinib inhibitor, 24h post infection (n=2-6).(C) MP populations from control (gray) and BCG (black) mice, with or without Fedratinib inhibition. Percentage of CM and NCM cells calculated from CD11b+ population. Percentage of RPM calculated from Lin- population. (D) Percentage of CM, NCM, and RPM populations expressing CXCL9 from control (gray) and BCG (black) mice, with or without Fedratinib inhibition (control: n=3, BCG: n=4, control+Fedratinib: n=3, BCG+Fedratinib: n=6). (E) Heatmap of normalized log2 expression of DEGs across naïve and training conditions. (F) Gene set enrichment analysis of DEGs from E. Data in bar graphs are presented as mean±SEM. For bar graph B each individual point is a biological repeat. Heatmap rows in E indicate biological replicates. Two-tailed t-test used for data in B, C, and D (*P<0.05, **P<0.01, ***P<0.005, ****P<0.001).