Figure 2: Bladder BCG directly colonizes the bone marrow and reprograms HSPCs.

A) Cultured BCG in mouse bone marrow after weekly doses of bladder BCG. The proportion of culture positive versus negative mice according to number of weekly BCG treatments received is displayed on the right.

B) Experimental schematic: Bladder PBS, Bladder BCG (5 doses), or intravenous BCG (single dose).

C) Bone marrow HSPC subsets from mice in in Figure 2B were quantified by flow cytometry. Intravenous BCG samples were from an independent reference experiment and were not compared statistically with bladder PBS or bladder BCG samples.

D) Morphologic quantification of colonies of the indicated types was performed from single cell suspensions of bone marrow plated into methocult for 14 days. E – erythroid; G – granulocyte; M – macrophage.

E) Experimental schematic (left panel). Mice were implanted with MB49 administered 3 weekly doses of bladder PBS or bladder BCG. Bone marrow was harvested 7 days after the final administration, sorted for lineage^- cells, and paired snMulti-ome was performed. In right panel, UMAP(right) demonstrates cellular lineages.

F) Density of PBS- or BCG-treated cells projected onto the UMAP.

G) Predicted transcription factor (TF) activity (snATAC-seq): HSC/MPP, monocyte, and neutrophil populations after BCG.

H) Conserved TF activity changes (human vs. mouse): HSPC/HSC-MPP and monocyte populations.

I) Mouse ATAC-seq comparison: bulk LSK vs pseudobulk (snATAC-seq) HSC/MPP for CD74 (top) and H2-Eb1(bottom).

J) Serum Cytokine levels after bladder BCG: IFNγ (top panel), G-CSF (middle panel), and TNF (bottom panel).

Data shown in panels C, D and J represent mean with standard deviation P-values were derived by Student’s t-test. P > 0.05 = ns, P ≤ 0.05 = *, P ≤ 0.01 = **, P ≤ 0.001 = ***, P ≤ 0.0001 = ****.

See also Figures S2 and S3.