Fig 2: Comparative analysis of peripheral blood mononuclear cell (PBMC) DCs and uterine DCs(A) UMAP visualization of DC from the uterine tissues showing seven clusters and cluster 1 identified as PB-uDCs.(B) UMAP visualization of DC from the PBMC dataset showing eight clusters, with cluster 3 highlighted, which was identified as plasmacytoid DCs.(C) Heatmap showing number of genes shared from comparing the top 50 markers from clusters 0–7 from PBMC and clusters 0–6 from uDCs. Cluster 1 from uDC has the highest number of shared genes with cluster 3 of PBMC DCs highlighted in black, and the list of 22 shared genes is shown on the side. The number of genes shared with each cluster of uDCs with each cluster of PB-DCs are shown in the box.(D) UMAP showing expressions of marker genes such as GZMB, JCHAIN, and IRF8 within cluster 1. Violin plots below show the quantification of expression levels of these marker genes in different clusters. GZMB and JCHAIN were specifically expressed in cluster 1, whereas IRF8 was found in clusters 1, 2, 3, and 4.(E) Circos plots showing the biological processes for the differentially expressed genes in the PB-uDC cluster along with the top 10 genes associated with each pathway. Red color indicates the genes were upregulated; blue color indicates that the genes were downregulated.(F) UMAP showing co-expression of the marker genes of the cluster—GZMB, JCHAIN, and IRF8. Yellow color indicates genes are co-expressed.(G) UMAP showing the expression of the marker gene GZMB across 10 phases of the menstrual cycle, from the menstrual stage through to decidualization (Decp and Decb), indicating dynamic changes in gene expression. Alongside is shown a line graph combined with a scatterplot overlay depicting the expression level of GZMB marker gene (1st y axis) and number of cells expressing the gene (2nd y axis) across different stages of the menstrual cycle (x axis). Each point represents the expression level of a gene in a particular cluster, while the line graph emphasizes the overall expression trend, aiding in the identification of patterns or anomalies in gene expression related to specific menstrual phases.

Fig 3: Comprehensive analysis of uterine dendritic cell (uDC) subsets and their gene expression profiles(A) Schematic representation of the sample collection process and subsequent single-cell RNA sequencing (scRNA-seq), CITE-seq, and multiplex immunohistochemistry (IHC) workflow. Uterine tissue samples were enzymatically digested to obtain single-cell suspensions, which were then sorted via FACS to isolate CD45+ immune cells. The isolated cells underwent scRNA-seq analysis using the involving MARS-seq2.0 protocol. Subsequent bioinformatic analysis enabled the identification and characterization of distinct dendritic cell subpopulations. The findings were further validated through wet-lab techniques, including flow cytometry and IHC, to confirm the presence of these dendritic cell subpopulations.(B) UMAP plot showing all immune cells in gray and cells expressing LYZ and IRF8 in blue. Small subsets (indicated in boxes) were identified as dendritic cells, which was selected and used for downstream analysis.(C) UMAP plot visualizing the clustering of uDCs based on their gene expression profiles. Each point represents a single cell, colored according to the identified cluster (0–6), illustrating the distinct transcriptional landscapes within the uDC population.(D) Heatmap showing the expression of top 10 marker genes (shown on y axis) across different uDC clusters (shown on x axis). Each row represents a gene, and each column corresponds to a single cell, organized by cluster identity. Yellow color denotes upregulated genes, whereas pink denotes expression of the downregulated genes.(E) Spatial distribution for key markers (LYZ, IRF8, and CD14) across the uDC clusters shown on UMAP.(F) Expression level for key marker genes (LYZ, IRF8, and CD14) was quantified using violin plots. X-axis shows the clusters and y axis shows the expression level of the genes indicated on top of each plot.

Fig 4: Identification and characterization of conventional uDCs (C-uDCs) on the UMAP(A) UMAP visualization uDCs with cluster 2 being identified as C-uDCs.(B) UMAP projection displays the distribution of all analyzed cells (depicted in gray) alongside the subpopulation expressing cDC markers CLEC9A, XCR1, LYZ, and IRF8 (highlighted in blue).(C) Illustrates the subset of cells that co-express CLEC9A and XCR1, indicative of cDC identity (top panel). Middle panel depicts a distinct group of cells characterized by the co-expression of both progenitor (AXL) and cDC markers (CLEC9A), representing a potential DC subset arising from the resident progenitor DCs. Bottom panel shows co-expression of LYZ and IRF8 in the cluster 2, both the markers used for isolation of uDCs from all immune cells.(D) Violin plots quantifying the expression level and cluster specificity for the C-uDC marker genes.(E) Circos plots showing the biological processes for the C-uDC cluster along with the genes associated with each pathway.(F) UMAP showing the expression of the marker gene XCR1 across 10 phases of the menstrual cycle, from the menstrual stage through to decidualization (Decp and Decb), indicating dynamic changes in gene expression. Alongside is shown a line graph combined with a scatterplot overlay depicting the expression level of XCR1 (1st y axis) and number of cells expressing the gene (2nd y axis) across different stages of the menstrual cycle (x axis). Each point represents the expression level of a gene in a particular cluster, while the line graph emphasizes the overall expression trend, and the expression peaks in late-secretory (WOI) period.

Fig 5: Multimodal validation and characterization of uterine dendritic cells (uDCs) in human endometrium(A) CITE-seq analysis of DCs in the human endometrium. (i) UMAP showing all cell types identified in the endometrial CITE-seq dataset. (ii) DCs were isolated using antibody-derived tags (CD11c+HLA-DR+CD83+), forming distinct clusters. (iii and iv) Violin plots showing surface protein expression of canonical DC markers (CD11c, HLA-DP, HLA-DQ, and HLA-DR) across identified clusters.(B) UMAP feature plots depicting the expression of specific genes: IRF8, LYZ, GZMB, CD1C, CLEC9A, LAMP3, CLEC10A, and CX3CR1.(C) Flow cytometry plots in (C) show the expression of CX3CR1 on P-uDCs.(D) C-uDCs displaying expression of XCR1.(E) M-uDCs displaying the expression of CCR7, identifying their surface marker profile.(F) Illustrates the expression of CD11C and granzyme B in PB-uDCs, indicating the proportion of cells with cytotoxic potential.(G) Depicts the expression of CCR5 in combination with CD1C, CX3CR1, and CD103 on uDCs.(H) shows the expression of CXCR4 in combination with CD1C, CX3CR1, and CD103, indicating the presence and distribution of this chemokine receptor on uDCs.