Fig 2: Cross-Tissue and Cross-Samples Distribution of Interacting Cell Pairs for Selected Types of Cell-to-Cell Interactions, Related to Figure 5CInteracting cell pairs are marked with white and cyan circles on the montage of IgD CD90 (B cell and T cell markers) staining of every sample of the dataset. Due to cell proximity in most cases cyan circles practically completely overlay white.(A and B) Interaction of CD4 and CD8 T cells with ERTR stroma (change in odds ratio score correlates with change in interaction count).(C–E) Interaction of granulocytes with CD4 T cells, dendritic cells and erythroblasts.(F and G) Interaction of erythroblasts with stromal and B220(+) DN T cells. Interactions in (C-G) scored as increased in early MRL/lpr (-4,-5,-6) as compared to BALBc spleens (FDR of t test on normalized interaction counts between conditions < 0.05, difference in interaction counts > 0).(H and I) Interactions of B220(+) DN T cells with CD8 T cells and stromal cells. These interactions scored as increased in intermediate and late MRL/lpr (-7,-8,-9) as compared to early MRL/lpr spleens (FDR of t test on normalized interaction counts between conditions < 0.05, difference in interaction counts > 0). More examples of cell type pairs with change in interactions across dataset can be found in associated online repository (see STAR Methods).

Fig 3: Interleukin (IL)-17 does not originate from Th17 cells and T cells after high tidal volume (HTV) ventilation. (A) Number of CD4+IL-17+ cells in mouse lung tissue. (B) Co-localization of IL-17 with CD4 and CD90 in the lung tissues. Data are shown as mean ± SD (n = 6). CON, non-ventilated mice; NTV, ventilated for 4 h with normal tidal volume; HTV, ventilated for 4 h with high tidal volume.

Fig 4: CTSK is expressed in JBMMSC and osteoblasts during the process of alveolar bone filling. A, Immunohistochemistry staining of CTSK in the extraction socket filling process. B, CTSK-positive cells per mm2 was assessed. C, Distribution of osteoclasts was analysed by TRAP staining. Red arrowheads indicate osteoclasts. D, Number of TRAP-positive osteoclast per alveolar bone surface was analysed. E, Immunofluorescent staining of CTSK (green) in extraction socket (7d post-extraction). CD44 (red) and CD90 (red) were stained as the bone marrow mesenchymal stem cells marker. Nuclei were stained with the DAPI (blue). White arrowheads indicate CTSK+/CD90+ or CTSK+/CD44+ cells. F, Immunofluorescent staining of CTSK (green) and Osterix (red) in extraction socket (7d post-extraction). Nuclei were stained with the DAPI (blue). White arrowheads indicate CTSK+/OSX+cells. The statistical analysis was shown: **P <.01; ***P <.001. AB, alveolar bone; BC, bone marrow cavity; ES, extraction socket; TB, trabecular bone

Fig 5: Unbiased Identification of i-niches in Multidimensional CODEX Data(A) On the left diagram explaining the terminology used for defining i-niche (a ring of first tier neighbors for central cell). On the right Delaunay triangulation graph used for identification of first tier of neighbors for every cell.(B) Heatmap depicting frequency of cell types in 100 types of i-niches identified by K-means (K = 100) clustering of all index cells in the dataset (each cell is an index cell for its i-niche) based on frequency of different cell types in the first tier of neighbors. The color indicates the average fraction of corresponding cell type in the the i-niche.(C) An example of marginal zone and follicular (B-zone) B cells defined by residence in distinct i-niches (e.g., marginal zone i-niche includes a marginal zone macrophage marked by letter H and green color). Positions of B cells in each i-niche is marked with red circles over the schematic of BALBc spleen.(D and E) Two heatmaps from top to bottom show average expression of selected surface markers measured in a central cell across 100 i-niches (same left to right order as in B) when central cell is B cells (D) or CD4 T cell (E) accordingly. The color indicates the relative level of surface-marker expression as measured across dataset. Gray columns indicate absence of cells in corresponding niches. Two orange rectangles over top heatmap indicates position of i-niches with high CD35 (containing FDCs and marginal zone macrophages). Cyan rectangle shows location of family of i-niches with high content of F4/80 macrophages and low B220 and CD19 in central B cells. Purple rectangle indicates family of i-niches enriched with ERTR-7 positive stroma. Below top heatmap, location of selected i-niches shown in (E) are indicated. Over bottom heatmap, yellow rectangle indicates the family of i-niches with dominating presence of B cells. Two green rectangles indicate family of niches with high levels of CD90 and CD27 in the index CD4 T cells.(F and G) Abundance of 100 i-niches in normal spleen (top bar graph) (F) and relative distribution of i-niches (G) between splenic histological subdivisions (PALS, red pulp, marginal zone, and B-zone) shown as a heatmap. To illustrate a variety of tissue distribution pattern by i-niches an overlay of selected i-niches over a schematic of normal spleen (BALBc-1) is shown. Heatmap color indicates fraction of corresponding i-niche per splenic anatomic subdivision.(H) Top right shows a biaxial plot of flow data for CD79b and B220 measured in isolated splenocytes. Top left shows levels of CD79b and B220 in central B cells as measured across all 100 i-niches. To illustrate i-niche-dependent variability of surface-marker expression, images of central cells (marked with red cross) with levels of surface marker indicated in pseudocolor palette are shown for selected exemplary i-niches in the bottom panels.See also Figures S4K and S4L.