Fig. 4.

Grail ^−/− CD8⁺ T cells exhibit a higher response to IL-21 than WT cells. a Real-time (RT)-PCR analysis of IL-21R expression in FACS-sorted and CD8⁺ T cells from TILs after anti-CD3 restimulation. Results for target genes are presented after normalising to β-actin and shown as mean ± SEM. (n = 3). b, c Naive WT and Grail ^−/− CD8⁺ T cells were activated with anti-CD3 alone or with IL-21. b Proliferation of WT and Grail ^−/− CD8⁺ T cells was assayed 72 h after activation by adding [³H]-thymidine to the culture for the last 8 h. c Cells were harvested for RT-PCR analysis 24 h after activation. Results for target genes are presented after normalising to β-actin and shown as mean ± SEM. The mRNA expression level in WT CD8⁺ T cells activated by anti-CD3 alone was set as 1. d, e Naive WT and Grail−/− CD8⁺ T cells were stimulated with anti-CD3 in the presence or absence of IL-21 for 3 days. d IFN-γ and granzyme B (GzmB) protein production after determined by ELISA and flow cytometry. e Flow cytometry staining was performed for CD132 and IL-21Rα expression. f–h WT, Grail ^−/− and Grail ^−/− Il21 ^−/− mice were inoculated with EG-7 tumour cells and analysed 17 days later. f Tumour size was measured and calculated as in Fig. 1a. g, h TILs were stained and analysed as described in Fig. 1. Results are shown on bar graphs as mean ± SEM as well as individual mice per group (n = 10 mice for WT and Grail ^−/− Il21 ^−/− groups and n = 9 mice for Grail ^−/− group). All experiments were independently performed twice. *p < 0.05, **p < 0.01, ***p < 0.001 as determined using a Student’s t-test. CPM count per minute