Fig. 2: Metformin treatment expands CD8⁺CXCR3⁺ T_M cell population in spleen and lungs of mice.

a Experimental set-up of in vivo experiments. b Visualized t-SNE map of CD8⁺ T cells from spleen of wild-type (WT) mice with automated cluster gating. t-SNE was performed on live CD45⁺CD90⁺TCRαβ⁺CD4⁻CD8⁺ cells after gating out B cells. c Normalized expression intensities of indicated marker was calculated and overlaid on the t-SNE plot, characterizing all clusters of CD8⁺ T cells in spleen. Means ± SD of four mice per group, one-tailed Mann–Whitney U test. d Manual gating analysis of CD8⁺CXCR3⁺ T_M cells in control and metformin-treated WT mice. e, f Flow cytometric analysis of splenic CD8⁺ T-cell subsets from control and metformin-treated WT mice. Pooled data from three independent experiments. Means ± SD of n = 13–15 mice per group, two-tailed Mann–Whitney U test. g Heat map displaying frequency of various surface markers in lung CD8⁺ T cells of control and metformin-treated WT mice. n = 4. *p ≤ 0.05 by two-tailed paired t test. h Flow cytometric analysis of spleen CD8⁺ T_E, T_M, and T_N cells of control and metformin-treated Cxcr3^−/− mice. Data shown one out of two experiments, mean ± SD of five mice per group. i Left—volcano plot showing DEGs from RNA sequencing data of CD8⁺ T cells from Cxcr3^−/− and WT mice. n = 5 mice/group. Right—Canonical pathway enrichment via Ingenuity pathway analysis (IPA) of 202 DEGs. j Heat map displaying the expression level of 35 FOXO1 targets in data shown in i. NS not significant.