Fig. 3: Visualizing functional tumor-immune interactions in native tissue.

a,b, Two CD8⁺ T cells (labelled A and B) expressing PD1 and PDL1 interacting within the VGP, shown as a maximum intensity projection (a). White arrow indicates contact surface between cells A and B with high colocalization of PD1 and PDL1. Transparent surface mesh showing contact area and colocalized PD1 and PDL1 as spheres (b). c, CD8⁺ T cell and dendritic cell interacting with a tumor cell with filopodia. d, The same cells as c with GZMB, PD1 and LAG3 are shown; the markers show that the T cell is activated and cytotoxic. e, A surface rendering of interactions in c and d, showing filopodia in greater detail. f, A multicellular interaction in metastatic melanoma. A dendritic cell interacting with a tumor cell (with surface rendering overlaid onto immunofluorescence) and a CD4⁺ T_helper cell interacting with a CD8⁺ T cell through filopodia (inset and Supplementary Video 6). g, Same field of view in f, showing PD1 and PDL1 colocalization between CD8⁺ T cell and dendritic cell (blue arrow). In the CD8 cell, GZMB is polarized toward the tumor cell (green arrow and inset). The location of the inset is shown by the box with the dotted line. Scale bars are as indicated. h–j, The interaction of a MART1⁺ tumor cell with CD3⁺ T cell, shown as single optical planes (CD3, MART1 (h) and MART1, PD1, PDL1, GZMB (i) and as a surface rendering (j). The inset in h depicts computation of a membrane intensity profile by integrating the fluorescence intensity parallel to the cell membrane (y) and plotting it as a function of distance perpendicular to the membrane (x). j, The arrows indicate region of active PD1–PDL1 interaction. k, The membrane intensity profile of region indicated in h, spanning a point of tumor-immune cell contact demonstrating a type I interaction. l–n, The interaction of a MART1⁺ tumor cell with a CD3⁺ T cell, shown as single optical plane (CD3, MART1 (l) and MART1, PD1, PDL1, GZMB (m) and as a surface rendering (n) and highlighting PD1–PDL1 interactions and proximity to GZMB granules. o, The membrane intensity profile shown in l demonstrating a type II interaction. For the plots in k and o, the solid lines are from raw data, and the dashed lines are from a polynomial curve fitting. The red ‘X’s mark the maximum intensity along the intensity profile, which we defined as the midpoint of the cell membrane for each channel.