Figure 5: T-cell activation in lymph nodes and tumour.

(a) Lymph nodes: number of proliferating cells within the tumour-draining lymph nodes (LNs) of 6- to 12-week-old C57BL/6 WT and Tlr3/7/9^−/− mice was assessed at day 6 and day 10 by immunofluorescence double staining of frozen sections using αCD4 and αKi67 antibodies. Quantification of positive cells was performed by observer-assisted counting on four randomly selected × 200 magnified images per sample (n=3). (b,c) Tumour: flow cytometric analysis of T-cell activation of tumour-infiltrating lymphocytes of 6- to 12-week-old C57BL/6 WT and Tlr3/7/9^−/− mice at day 6 and day 10. (b) Activation status of CD4⁺ cells was analysed by staining of CD62L⁻/CD154⁺ cells. Activated CD8⁺ cells are determined by showing Granzyme B⁺ (GzmB) and CD107a⁺ expression. (c) Cytokine production of tumour-infiltrating CD4⁺ and CD8⁺ cells was assessed after ex vivo stimulation with αCD3/CD28 antibodies and subsequent flow cytometry staining using αIFN-γ and αTNF-α antibodies. Single values and mean (horizontal line) are shown. Results were considered significant at *P⩽0.05 and ** P⩽0.005 (t-test).