Fig. 6: Cell–cell interactions and multivalent immune cell niches.

a, A single image plane of a PD1⁺CD8⁺ T cell interacting with two MART1⁺ tumor cells. The white arrow denotes the juxtacrine PD1–PDL1 interaction. b, The membrane intensity profile perpendicular to axis of interaction of membranes from cells 1 and 3 in a and demonstrating a type I interaction. c,d, A dendritic cell interacting with a CD8⁺ T cell as a maximum projection (c) and membrane intensity profile (type I interaction) (d). The box indicates the region of line membrane intensity profile shown in d. e,f, Representations of a T_PEX cell from the invasive margin as a maximum projection (e) and membrane intensity profile (type I interaction) (f), for the region marked by the white dashed box in I. g,h, A CD4⁺ T_helper cell (magenta) interacting with a dendritic cell (yellow), as a maximum intensity projection (g). Membrane intensity profiles are representative of type II interactions (h). The selected fields of view and intensity profiles for selected membrane–membrane interactions in dataset 1 (LSP13626) are shown for a–h. Scale bars, 5 µm. i–k, Examples of type III interactions (lymphonets), involving CD4⁺CD8⁺ dendritic cells in the MIS. These networks are characterized by loosely packed cells with cell–cell interactions via relatively small membrane domains. l, The stroma in the vicinity of the VGP melanoma showing neighborhoods rich in CD20⁺ B cells, CD11C⁺ dendritic cells and CD3E⁺ T cells but without the clusters of proliferating Ki67⁺ cells that are characteristic of mature germinal centers. m–o, A total of 11 cells from the MIS lying in proximity to the DEJ, shown as primary data (m), 3D surface renderings (n) and as a schematic representation of three type I, four type II and two type III interactions inferred from membrane intensity profiles in single-plane images (o). Community in m and n, also shown in Supplementary Video 5. Scale bars, 10 µm. p, A cross-sectional slice of two CD8⁺ and two CD4⁺ T cells depicting the direct engagement of cell membranes (Supplementary Video 4). Scale bar, 3 µm. q,r, A membrane intensity profile depicting CD4 and CD8 average expression within the bottom boxed region of p (q) or right boxed region of r (s). s–u, A type II interaction between a MART1⁺ tumor cell and CD4⁺ T cells, as a representative image (s) and membrane intensity profiles for the interaction between cells 1–2 at region A (t) and B (u). Scale bars, 3 µm. For membrane intensity profiles, the solid lines are from raw data, dashed lines from polynomial curve fitting. The red ‘X’s mark the maximum intensity along membrane intensity profile and thus, the centroid of the cell membrane for each channel. See Supplementary Fig. 13 for a detailed diagram of which markers were used to define each cell type.