Fig 2: Engaging CD40 on Mtb-infected DCs increases NOTCH2 expression on CD4 T cells in the lungBMDCs were infected with Mtb at an MOI of 1, with or without CD40LT, for 48H. For antibody blockade studies, 60 µg/mL anti-DLL4 antibody was added during infection. DCs were then harvested and 1E6 were intratracheally (IT) transferred into the lungs of mice along with additional blocking antibody. At 4 weeks post transfer, mice were euthanized and lung suspensions were unstimulated to asses ex vivo responses using flow cytometry.(A) Frequency of NOTCH receptor-positive CD4 T cells.(B) Boolean analysis of NOTCH receptor-expressing and cytokine-positive CD4 T cells. All populations are singlets/live cells/CD3+/CD4+. Data are presented as mean ± SD. Data are representative of 2 independent experiments. Data were analyzed using a one-way ANOVA with a correction for multiple comparisons. Statistical significance p value key is the following: * = = 0.05, ** = = 0.01, *** = = 0.001, **** = = 0.0001.

Fig 3: CXCR4 expression marks rare clonal HE with HSC potential in the early murine embryo (A) Methodology for index sorting of single P-Sp-derived V+E+61+ cells to co-culture on AGM-ECs and subsequent analysis to assess HSC potential. HSC colony-forming cells (HSC-CFCs) indicate clonal V+E+61+ cells that generate colonies containing HSCs detectable by immunophenotype (VE-cadherin-/low CD45+Gr1-F4/80-Sca1hiEPCRhi, indicated in “HSC gate”), whereas HPC-CFCs lack immunophenotypically detectable HSCs (i.e., no cells detected in the HSC gate).(B) Number of HSC-CFCs and HPC-CFCs detected across seven independent experiments from E9 to E10 (19–30 somite pairs [sp]). Error bars show mean ± SEM. p values indicate unpaired Student’s t test.(C) Expression of CXCR4, CD44, and DLL4 in the VE-cadherin+ population and the V+E+61+ subset of E9.5 (29 sp) P-Sp. Blue histograms indicate isotype controls.(D) Correlation of clonal HSC-CFC and HPC-CFC potential with expression of CXCR4 on individual index-sorted V+E+61+ cells at E9.5 (26–29 sp).(E) Donor-derived peripheral blood (PB) engraftment of clonal progeny of the single HSC-CFCs in (D), including B lymphoid (CD19), T lymphoid (CD3), and myeloid contribution.(F) Correlation of clonal HSC-CFCs and HPC-CFCs with surface expression of CXCR4 and CD41, CD43, and CD45 on individual index-sorted V+E+61+ cells at E9.5 (29–30 sp).(G) Distribution of CXCR4+ and CXCR4- HSC-CFCs and HPC-CFCs detected across six independent experiments from E9 to E10 (19–30 sp). Error bars show mean ± SEM. p values indicate unpaired Student’s t test.(H) Donor-derived PB engraftment of the hematopoietic progeny of bulk-sorted CXCR4+ and CXCR4- cells within the V+E+ population at E9.5 (22–29 sp) following AGM-EC co-culture and transplantation. Error bars show mean ± SEM; n indicates number of recipient mice. All recipients of CXCR4+ cells had multilineage engraftment.See also Figure S1 and Table S1.

Fig 4: Engaging CD40 on Mtb-stimulated DCs enhances surface expression of DLL4 and Jagged1BMDCs were stimulated with either media (UI) or HK Mtb (MOI 30) with or without the addition of 1 µg/mL of CD40LT. At designated time points, cells were collected and stained for surface markers.(A) Representative flow cytometry plot of DLL4+ and Jagged1+ frequencies.(B) Frequency of DLL4+ and Jagged1+ and double-positive populations.(C) MFI of DLL4 and Jagged1 expression and representative graphs. All populations are singlets/live cells/CD11c+MHCIIhi. UI presented is 0H UI. Data are presented as mean ± SD. Data are representative of 3 independent experiments. Data were analyzed in (B) using a two-way ANOVA with a correction for multiple comparisons and (C) using an unpaired Student’s t test. Statistical significance p value key is the following: * = = 0.05, ** = = 0.01, *** = = 0.001, **** = = 0.0001.

Fig 5: Acquisition of self-renewal programs defining HSC potential(A) Violin plots of HSC self-renewal genes identified by differential gene expression between Cxcr4-positive and Cxcr4-negative HE.(B) UMAP heatmap (left) and violin plots (right) of gene-set scores for published HSC self-renewal genes. p values indicate Wilcoxon rank-sum test.(C) UMAP heatmap (left) and violin plots (right) of proliferation index. p values indicate Wilcoxon rank-sum test.(D) UMAP heatmap (left) and violin plots (right) of gene-set scores for Dll4-regulated Myc pathway genes (Luo et al., 2021). p values indicate Wilcoxon rank-sum test.(E) Model depicting distinct waves of multilineage progenitors and HSCs derived from HE distinguished by relative expression of genes driving self-renewal tied to arterial maturation and genes driving EHT. Image created with BioRender.See also Figure S4 and Tables S3 and S4.