Extended Data Fig. 3: Flow cytometric comparison and in vitro functional studies of stem-like and terminally differentiated CD8 T cells.

a, Flow cytometry gating scheme. FSC-A and FSC-H are used to select for singlets. Live (APC–Cy7 negative) CD3⁺ events are then selected from this population of singlets. Lymphocytes are selected from this live CD3⁺ population on the basis of FSC-A and SSC-A, and CD4⁺ and CD8⁺ T cell populations are selected from the lymphocyte population. b, Expression of various molecules by stem-like (green) and terminally differentiated (red) CD8 T cells in human tumours measured by flow cytometry. c–e, Expression of TCF1 (c), CD28 (d) and TIM3 (e) as measured by flow cytometry, by stem-like and terminally differentiated CD8 T cells isolated from patients with kidney cancer (n = 6) and cultured in vitro for 3 days with 10 U of IL-2 and with (stimulated) or without (unstimulated) anti-CD3/CD28/CD2 bead stimulation at a 1:1 ratio. Median value is shown. f, Number of live stem-like and terminally differentiated intra-tumoral CD8⁺ T cells after 3 days of in vitro culture in IL-2 supplemented media. Live/dead staining was used to determine the proportion and number of live CD8 T cells by flow cytometry. g, Composition of the CD8 T cell compartment. In 60 human kidney cancer patients, proportion of CD8 T cells that are stem-like cells (PD-1⁺CD28⁺TIM3^–) correlates with total T cell infiltration (%CD8 T cells of total cells), while proportion of terminally differentiated cells (PD-1⁺TIM3⁺) does not. h, Percentage of total CD8 T cells correlates with the percentage of total cells that are stem-like CD8 T cells.