Figure S1.

Gating Strategy for HSCs and Progenitors and Their Kinetics Post-infection, Related to Figure 1

(A) Cells were gated for FSC-A against SSC-A. Doublets were excluded using FSC-H against FSC-A. Viable cells were gated and lineage-committed cells were excluded. Within the lineage-negative population, cells were gated as LKS-defined as double positive for cKit and Sca-1. Gated on the LKS population, cells were divided into LT-HSC, ST-HSC and MPP based on CD150 and CD48 expression. MPPs were characterized as MPP3 or MPP4 by their surface expression of CD34 and Flt3. In a second strategy, lineage negative cells were gated based on CD127⁺ and CD127^-. Lin^- CD127^- population was further defined by Sca-1 and c-Kit. c-Kit⁺ Sca-1^- cells were further gated based on CD34 and CD16/32 to define CMP, GMP and MEP. Lineage^- and CD127⁺ cells are defined as CLPs based on Sca-1^lo and c-Kit^lo expression. Finally, in another set of experiments, Lineage⁺ cells and then Sca-1⁺ cells were excluded. The remaining cells were further subdivided into cKit⁺ CD16/32⁺ and cKit⁺ CD16/32^- groups. In the cKit⁺ CD16/32^- group, CD34⁺ Flt3⁺ cells were denoted as MDP by being CD115⁺ but Ly6C^-. cKit⁺CD16/32⁺ cells were further gated on CD34⁺ Flt3^- cells. Within this fraction, Ly6C⁺ CD115^- cells were the GP and Ly6C⁺ CD115⁺ were cMoP. (B-D) Kinetics of the frequency among single viable BM cells (top panel) and total cell counts (bottom panel) of LT-HSC, ST-HSC, MPP3/MPP4 in the BM of BCG-iv vaccinated or Mtb-iv infected mice. (E) BM cells from WT mice were infected with BCG-GFP or H37Rv-GFP for 4 hours in vitro (MOI 3). ImageStream analysis of H37Rv-GFP infection (top panel) and BCG-GFP (bottom panel) in Lin^- cKit⁺ Sca-1^- progenitors. (F-H) Mice were intravenously infected with 1x10⁶ CFU of BCG, Mtb or Mtb-ΔRD-1 for 28 days. Frequency of LKS in each group (F) and HSC/progenitor subsets of BCG versus Mtb-ΔRD-1 (G), or Mtb versus Mtb-ΔRD-1 (H; left panel frequency, right panel total cell counts). (I-O) 1x10⁶ CFU of BCG or Mtb were delivered intravenously for 28 days. Percentage of MDP (I), cMoP (J), GP (K) and Ly6C^hi monocytes (L) and frequency and total numbers of neutrophils (M) in the BM. (N-O) Frequency and total LKS cells (N), as well as the frequency and total cell number of the HSC/progenitor fractions in BCG versus Mtb-infected mice in the spleen after 28 days (O). Differences assessed by Two-tailed Student’s T-Test for each individual cell population in B-D and G-O or One-way ANOVA followed by Tukey’s Multiple Comparisons Test in F.