Fig. 3.

Attenuated tumor growth in Camkk2 KO host is mediated by CD8+ T cells. Murine E0771 (4 × 10⁵) cells were orthotopically grafted in WT and Camkk2^−/− mice, and subsequently tumors of comparable sizes (500–700 mm³) were harvested and digested with both collagenase and DNase I. Single-cell suspensions were then stained for lymphoid and myeloid markers, and cell subsets were identified according with gating strategy reported in Supplementary Fig. 3A–C. a Increased accumulation of CD8⁺ T cells in E0771 cell-derived tumors propagated in Camkk2^−/− mice compared with WT. Bar graph reports the mean ± SEM from two combined independent experiments. b Percentages of tumor-infiltrating lymphocyte subsets in E0771 tumors growing in Camkk2^−/− mice compared with WT. (upper left panel) Intracellular expression of Granzyme B⁺ (GzmB⁺). CD69⁺, PD-1⁺, and LAG-3⁺ expression was detected on an independent set of eight and four tumors removed from WT and Camkk2^−/− mice, respectively. Bar graph reports the mean ± SEM. A t test was used to calculate p-values. c Representative FACS dot plots of tumor single-cell suspensions stained with myeloid markers (left). Bar graph reports the mean ± SEM of CD11b⁺ MHC II⁺ subset from combined independent experiments. A t test was used to calculate p-values. N = 13 and 15 tumors removed from WT and Camkk2^−/− mice, respectively. d Mammary tumor growth in Camkk2^−/− mice depleted of CD8⁺ T cells. Camkk2^−/− mice were treated with anti-CD8 antibody or control isotype every 3 days starting a week before tumor cell inoculation. At day 0, E0771 cells were orthotopically grafted, and mice were treated with antibodies every 4 days. Graph depicts tumor size (mean ± SEM; N = 6 in each group). e Mammary tumors display retarded growth in mice devoid of Camkk2 in myeloid cells. E0771 cells were orthotopically grafted into LysMCre⁺ Camkk2^wt/wt and LysMCre⁺ Camkk2^fl/fl mice. Tumor volume was measured (mean ± SEM; N = 4 for each group). This experiment was replicated with similar results. A two-way ANOVA test was used to calculate p-values. *p < 0.05, ***p < 0.005