Fig 2: Metformin reduces the secretion of IFN-γ in CD8 + senescent T cells. Representative flow histogram of IFN-γ production by CD8 + senescent T cells (CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + KLRG1 +) (a) and non- senescent T cells (CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57-KLRG1-) (e) in the control group and the 20 mM Met treatment group relative to the unstimulated controls from middle-age subjects. Analysis of IFN-γ-secreting CD8 + senescent T cells (b) and MFI of IFN-γ in CD8 + senescent T cells (c) with 20 mM metformin treatment. d Frequency of CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + KLRG1 + IFN-γ + T cells in lymphocytes with Met treatment and control. f Percentage and MFI of IFN-γ in CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57-KLRG1- T cells with Met treatment and control. g Representative flow histogram of IFN-γ secretion by CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + /CD57- population relative to the unstimulated controls from middle-age donors. h Percentage and MFI of IFN-γ in CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + / CD57- population. Expressed as the mean ± SEM. **P < 0.01, ***P < 0.001; Paired t test. Met, metformin

Fig 3: The effect of Metformin on GB secretion. Representative flow histogram of GB secretion by CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + KLRG1 + (a) and CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57-KLRG1- (d) in the control group and the 20 mM Met treatment group relative to the unstimulated controls group from middle-age donors. Analysis of GB production in CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + KLRG1 + (b) / CD57-KLRG1- (e) T cells, cultured with 0 mM or 20 mM metformin for 24 h in the middle age group. The proportion of CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + KLRG1 + GB + (c) and CD57-KLRG1-GB + (f) T cells in lymphocytes with metformin treatment compare to control group. g Representative flow histogram of GB secretion by CD3 + CD57 + and CD3 + CD57-T cells relative to the unstimulated controls group in middle-age donors. h Percentage and MFI of GB in CD3 + CD57 + and CD3 + CD57-T cells. Expressed as the mean ± SEM. *P < 0.05; Paired t test. Met, metformin

Fig 4: The highest frequency of CD8 + senescent T cells in middle-age population was analyzed by FACS. a Representative flow cytometric analyses of senescent T cells (CD3 + CD45RA + CCR7-CD27-CD28-CD57 + KLRG1 +) in CD8 and CD4 T cells (top quadrant) and representative flow cytometric analyses of CD27-CD28-CD57 + KLRG1 + subsets, gated on CD3 + CD4 + /CD8 + CD45RO + CCR7-(Tem) population (bottom quadrant). The frequency of CD8 + senescent T cells (b) and CD4 + senescent T cells (c) at young group, middle-age group and elderly group. The percentage of CD57 + KLRG1 + expression on CD3 + CD8 + CD45RO + CCR7-CD27-CD28-population (d) and CD3 + CD4 + CD45RO + CCR7-CD27-CD28-population (e) at different age groups. Expressed as the mean ± SEM. *P < 0.05, **P < 0.01; Mann– Whitney test (two-tailed) and nonpaired Student’s t-test. Tem, effector memory T cell

Fig 5: Metformin promotes the production of TNF-α in senescence T cells. Analysis of TNF-α-secreting cells from CD8 + senescent T cells (CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + KLRG1 +) (a) and CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57-KLRG1-T cells (c) in the control group and the 20 mM Met treatment group in middle-age group. b The frequency of TNF-α + CD8 + senescent T cells in lymphocytes between the control and Met treatment group. d Representative flow histogram of TNF-α in CD8 + CD45RA + CCR7-CD27-CD28-CD57 + /CD57-T cell and the percentage and MFI of TNF-α in CD8 + CD45RA + CCR7-CD27-CD28-CD57 + /CD57-T cell. Expressed as the mean ± SEM. **P < 0.01; Paired t test. Met, metformin