Fig 2: Quantitative realtime PCR analyses of Dll1 in mesometrial and anti-mesometrial uterine samples and in flow sorted uNK cells.Relative mRNA expression of Dll1 by the mesometrial side (M) of the virgin, gd4.5, gd5.5 and gd6.5 uterus of of the gd10.5 mesometrial lymphoid aggregate of pregnancy (MLAp) and decidua basalis (DB) is shown in (A). Relative mRNA expression of Dll1 by anti- mesometrial (AM) virgin uterus and by gd4.5, gd5.5 and gd6.5 anti-mesometrial decidua is shown in (B). Relative mRNA expression of Dll1 at gd10.5 by CD3-CD122+DBA- (DBA-) and CD3-CD122+DBA+ (DBA+) CD1 decidual cells normalized to Hprt1(C) is shown in (C). Data are means±SEM from all replicate analyses of two independent experiments. * P<0.05, ** P<0.01, *** P<0.001.

Fig 3: Histological analysis of DLL1 expression in mesometrial sections of gd10.5 B6 and CD1 implant sites.Photomicrographs A-L and O show gd10.5 implant site cryosections co-stained with DBA lectin-FITC (green) to identify uNK cells and anti-DLL1-PE (red). Panel M(i) is stained with only DBA lectin-FITC (green). Panels A–M(i) are B6 implant sites and panel O is a CD1 implant site. Panel M(ii) provides a diagram of the regions images were collected from for the other panels and has been marked to show the banding pattern seen for DLL1 expression. This should be compared to low power image M(i) that shows DBA lectin-stained uNK cell distribution in each banded area. These are labeled region “1” for the MLAp, region 2 for decidua basalis distal to the placenta (pdDB) and region 3 for decidua basalis proximal to the placenta (ppDB). Region 4, the AM decidua, is at the bottom of the image. “C” represents the conceptus, including the placenta and the green ring around “C” is DBA lectin-stained yolk sac endothelium surrounding the fetus. The black space between the yolk sac and the region labeled “3” is the placenta which is uNK cell deficient. Expression of DLL1 by gd10.5 DBA+ cells was stratified within the mesometrial side of the implant sites in both strains and no significant differences were noted. In B6 MLAp (A–C; labeled “1” in M(i) and diagrammed in M(ii)), DLL1 was strongly expressed only infrequently (B) and not by the smaller, immature uNK cells that proliferate in this region (arrow heads in A, C). In decidua basalis of B6 that was distal to the placenta (D–F) and CD1 (O), DBA+ uNK cells brightly expressed DLL1 (arrows in D–F; O). DBA+DLL1+ uNK cells appeared to surrounded vessels (*). Additional perivascular DLL1 staining was present that was not associated with DBA+ cells. The decidual region proximal to the placenta was devoid of DLL1+ cells but abundantly populated by DBA+ uNK cells (G). Neither DLL1+ nor DBA+ uNK cells were present in the highly regressed anti-mesometrial decidua (A-Meso; J-L). DBA-stained yolk sac endothelium was present in this region (arrows in J, L). N is a photomicrograph of the placenta distal decidua basalis in a section from an archived paraffin-embedded gd10.5 B6 implant site double stained using DBA lectin-horseradish peroxidase and Periodic Acid Schiff’s reagent [25]. The latter stain reveals all granulated uNK cells and shows cells of the DBA-PAS+ subset (yellow circle). This image shows the typical strong association of uNK cells with arterioles and with microvessels, including intravascular positions and supports interpretations of the fluorescence images. In M(ii), BV indicates entry of major blood vessel branches from the uterine artery. Bars: A, B, C, J, K, L, O: 40 µm; D, E, F, G, H, I: 20 µm; M: 200 µm.

Fig 4: Flow Cytometric and scRNAseq Analyses Confirm the Identity of the pDCs and cDC1s Generated from Human CD34+ CB Cells and Identify Heterogeneity within cDC1s(A) viSNE plots from live Lin-HLA-DR+ cells harvested from 18 day OP9+OP9_DLL1 FT7 cultures. Color codes indicate relative levels of marker cell-surface expression.(B) Overlay of the viSNE plot shown in (A) with the manually gated populations indicated in the legend. pDCs were gated as BDCA2+CD123+ and cDC1s as BDCA2-CD123neg-to-lowCADM1+CD1c+. In the remaining CADM1- cells, CD1c+ cells were subdivided into BTLA-CX3CR1+ and BTLA+ cells. Plots are representative of 4 donors from 2 experiments.(C) Lineage-HLA-DR+ single cells were index sorted for phenotypes of cDC1s (CD141+CADM1+), pDCs (CADM1-CD123+BDCA2+), putative pre-cDC2s (CADM1-BDCA2-CD1c+BTLA+), or putative cDC2s (CADM1-BDCA2-CD1c+BTLA-), as indicated by the symbols in the graphical legend. scRNAseq was performed using the SmartSeq2 protocol, followed by unsupervised dimensional reduction of the data using t-distributed stochastic neighbor embedding (t-SNE) with graph-based clustering. 7 cell clusters were identified as indicated by the color code in the graphical legend.(D) Violin plots showing mRNA expression profiles of previously known MNP-type-specific genes across all individual cells and in comparison between clusters identified in (A).(E) Enrichment analyses performed on each cell using connectivity map (cMAP), with independently generated and previously published human MNP-type-specific transcriptomic fingerprints. Results are presented as violin plots showing cell densities for the different cMAP scores (y axis) across the cell clusters (x axis) identified in (A). cMAP scores close to 1 versus -1 indicate strong enrichment of the signature of the cell type labeled on top versus bottom of the y axis.(F) Violin plots showing expression profiles of differentially expressed genes between clusters 6 and 7 (cDC1s).Individual dots represent single cells (C, D, and F). Data in (C)–(F) represent one experiment using one donor.(G) Protein expression of CLEC9A and CXCR4 on Lin-HLA-DR+CADM1+BDCA2-XCR1- and XCR1+ cDC1s from FT7 cultures analyzed by flow cytometry. Plots are representative of 3 experiments and 5 donors.(H) Protein expression of CLEC9A and XCR1 on Lin-HLA-DR+CD11c+ CD1c-CADM1+ cDC1s from adult peripheral blood analyzed by flow cytometry. Plots are representative of 3 experiments encompassing 6 different donors.(I) XCR1 protein expression on adult peripheral blood cDC1s sorted as Lin-HLA-DR+CD11c+CD1c-CD141+CADM1+CLEC9A+ and either XCR1+ or XCR1- 14 days after in vitro culture on OP9-DLL1 cells with the FT7+G cytokine cocktail. One experiment representative of three is shown. Dotted line, fluorescent minus one control; plain line, XCR1 staining.(J) Expansion of the sorted XCR1- subset of adult peripheral blood cDC1s after 14 days of in vitro culture on OP9-DLL1 cells with the FT7 cytokine cocktail supplemented with GM-CSF (1 ng/mL). Data shown are from 3 experiments, each with a different donor (symbols), with mean ± Sd shown across the three donors for each experimental condition.See also Figures S3–S6 and Data S1.