Extended Data Fig. 6: Quantitative immunofluorescence analysis of tumour immune infiltration. | Nature
a, Flow cytometry data illustrating the number of naive cells present intra-tumorally. Left, representative patient. Right, summary data. b, Comparative amounts of CD45RO expression on naive and stem-like intra-tumoral CD8 T cells. c, Workflow for immunofluorescence imaging analysis and immuno-map creation. Single channel immunofluorescence images are imported into CellProfiler. CD8+ and MHC-II+ objects are identified in the respective channel images. The XY location of each CD8+ and MHC-II+ object is exported. The TCF1 staining intensity is measured inside the CD8+ objects. These parameters are used to calculate MHC-II+ density, measure the distance from each CD8+ object to its nearest MHC-II+ neighbour, and to finally create immuno-maps for immunofluorescence images. d, Histo-cytometric analysis of tumour infiltrating immune populations. Location and fluorescence intensity of CD8+ and MHC-II+ cells were determined using CellProfiler. After image compensation, CD8+ and MHC-II+ cells were gated. TCF1 intensity of each cell is shown on histograms for each population below. Comparison of flow cytometry data from the same patient sample is also shown. e, Patients with kidney cancer with high CD8 infiltration determined by flow cytometry. Patients that were determined to have high CD8 infiltration by flow cytometry were selected for analysis by immunofluorescence. f, Haematoxylin and eosin stains of human kidney tumour. Selected slides from human kidney tumour shown in part e, to be highly infiltrated by T cells. Regions of tumour tissue are highlighted in yellow. g, Immunofluorescence imaging of kidney tumour. Selected tumours shown to be highly infiltrated by T cells. Tumour section was stained for MHC-II to identify antigen-presenting cells, and CD8 and TCF1 to identify stem-like and terminally differentiated CD8 T cell populations. Insets shows zoomed regions highlighted in the larger image. h, Dendritic cells populations, stem-like, and terminally differentiated CD8 T cells in three representative kidney cancer patients. i, Cellular spatial relationship map (middle) analysis and construction conducted as in Fig. 3e. j, CD8 expression of TCF1 preferentially occurs in dense APC zones. Amount of TCF1 expressed in each CD8 T cell graphed against the density of MHC-II around each T cell (MHC-II+ cells per 10,000 µm2). k, l, TCF1+ CD8 T cells are localized near dense MHC-II regions in other cancers. Prostate and bladder tumours were imaged for CD8, MHCII and TCF1. Regions of dense MHC-II aggregates are shown in grey and the location of TCF1+ CD8 T cells in green (l).