Fig 2: Defining the Transcriptome of the First Functional HSCs(A) Plot of hscScore against CD27 MFI for 85 CD31hiSSClocKithiGata2medCD27+N cells. Majority of ICGS2 cells (black) found in the CD27med and higher hscScore range and ICGS1 cells (red) within the full CD27 MFI and mostly low hscScore range. ICGS3 cells are indicated in gray.(B) Ly6a expression was plotted on the SPRING visualization of (Bi) 85 CD31hiSSClocKithiGata2medCD27+N cells, (Bii) 71 CD27med cells (CD27hi in green), and (Biii) 14 CD27hi cells (CD27med in green) within HC1 (gray).(C) Replotting of 85-cell heatmap in Figure S4 (maintaining the same order and position) with subset of ICGS output (Figure S5) for the 23 Ly6a+CD27med cells belonging to HC1 (L2.7+L2.8). 3 gene clusters (RowCluster) and 2 cell clusters (ICGS1 and ICGS2) can be defined. HscScore, log10 of the CD27 index-sorting (protein) levels and Louvain identity (L2.7, L2.8) are also depicted.

Fig 3: All Functional AGM HSCs Are within the HC1 CD27med Fraction(A) Violin plots of Tnfrsf7 (CD27) expression in the 5 major transcriptomic clusters. EC, endothelial-like cluster; HC1, hematopoietic cluster 1; HC2, hematopoietic cluster 2; UC1, unknown cluster 1; UC2, unknown cluster 2.(B) Enrichment sorting parameters for CD27+ HC1 cell isolation. Side scatter (SSC-A), CD31, cKit, Gata2 (G2), and CD27 sorting gates and percentage (±SEM) of cells. CD27+ cells (%) are indicated within a wide (+W) and a narrow (+N) mean fluorescent intensity (MFI) gate.(C) Analysis of HSC function within CD27 fractions (CD27+W, CD27+N, and CD27-/lo) of HC1 cells by in vivo transplantation. Open squares denote CD27+W HC1 cell recipients (n = 2) of 442 cells (2.5ee) and 479 cells (3.3 ee) from 15 and 9 (42–50 somite pairs, sp) embryos, respectively. Closed squares (n = 4; 42–49 sp; embryos/experiment: 25, 11, 22, 28) denote recipients injected with CD27+N HC1 cells (238–957; 3.7–9.8 ee), EC cells (899–2,333; 3.9–8.6 ee), CD27-/lo HC1 cells (1,042–4,325; 4.1-9.8 ee), and UC1+2+HC2 cells (1,361–3,741; 4.2–9.9 ee). Percentage of donor cell chimerism in peripheral blood of recipients at 4 months post-injection. Black line indicates mean.(D) Single-cell RNA-seq of 119 CD31hiSSClocKithiGata2medCD27+N sorted cells (all passed quality control) from 6 embryos (41–47 sp). These data combined with the previous dataset (gray) resulted in an extended dataset SPRING plot of 1,087 cells in total. Overlay of CD27+N cells (red) showing localization to one HC1 subcluster (circled).(E) Single CD27+N HC1 cells from E11 G2V IAHCs (50 embryos; 42–49 sp) deposited into methylcellulose and colony-forming unit-culture (CFU-C) counts at 8–10 days (138 total CFU-Cs). Colony type (individual dot) plotted against log10-normalized mean fluorescence intensities (MFIs) for (Ei) CD27, (Eii) G2, (Eiii) cKit, and (Eiv) CD31 expression. GEMM, granulocyte-erythroid-macrophage-megakaryocyte; GM, granulocyte-macrophage. Red lines indicate mean ± SEM.(F) Representative FACS plot showing newly defined CD27med MFI range (red dotted line) within the CD27+N fraction. Percentage ±SEM.(G) Summary of enrichment strategy. The HC1 fraction containing all HSCs is 10.5-fold enriched compared to previous sorting strategies (Eich et al., 2018). Further enrichments are based on gating of specific CD27+N and CD27med fractions to give an additional 6.5-fold enrichment. After iterative analyses and refinement of sorting gates, a total combined HSC enrichment of 68.3-fold is achieved.

Fig 4: CXCR5-PD-1+ICOS+ T Cells in HIV-Infected LNs Express TFH-Cell-Related Proteins and Function(A) Histograms showing fluorescent intensity of BCL6, Maf, and CD84 staining in naive (1), CXCR5-PD-1-ICOS- (2), CXCR5-PD-1+ICOS+ (3), and TFH cells (4). Plots summarize data from 8 HIV LNs.(B) Plasma cell phenotype of B cells cocultured with the indicated T cell subsets without or with SEB.(C-E) Quantification of total IgG (n = 13) (C), plasma cells (n = 15) (D), or CD4+ T cells (n = 15) (E) on the 7th day of each coculture condition.(F) IFN-? level in the supernatant of wells containing TFH cells or CXCR5-PD-1+ICOS+ T cells (n = 9).(G) Example gate for T-bet+ B cells. Naive IgD+CD27- B cells were excluded. Scatterplot quantifies T-bet+ B cells in 7 HC and 25 HIV-infected LNs.(H) Correlation between IFN-? expression in CXCR5-PD-1+ICOS+ T cells and T-bet+ B cell frequency in HIV-infected LN samples.For (A) and (C)–(E), Friedman test was performed and corrected using Dunn’s multiple comparisons test. For (F), Wilcoxon matched-pairs signed rank test was used. For (G), Mann-Whitney test was used. For (H), association was measured by Spearman correlation. Data are represented as mean ± SEM.Also see Figures S4D and S5A.