Extended Data Fig. 3: Senescent cells induce CD4-Eomes cell differentiation in vitro.

a. Experimental setup: primary fibroblasts derived from mouse lungs were treated with etoposide to induce senescence (Methods). Both control untreated and senescent fibroblasts were then co-cultured with polyclonally activated young CD4 T cells (purified from 2-3 months old mice) for 3 or 7 days when CD4 T cells were collected for flow cytometry analysis. b. Representative images (Left panels) of senescence-associated β-Galactosidase (SA-β-Gal) staining in control (Upper) and etoposide-induced senescent fibroblasts (Lower). Scale bar represents 50 μm. Right: a graph showing the percentages of SA-β-Gal⁺ cells out of total fibroblasts under 40x magnification in control (n = 5) and etoposide-induced senescent fibroblasts (n = 5) (Etoposide). Each dot represents one culture well. c. Representative confocal images showing P16 and P21 immunostaining in control (Upper) and senescent (Lower) fibroblasts. Below to the left is a validation of the P16 antibody specificity on liver tissue sections obtained from P16 deficient mice (3 months of age) and immunostained with the P16 antibody and DAPI. Scale bars are indicated in the figures. Data from ≥4 wells d-e. Left: a representative flow cytometry histogram plot illustrating the expression of EOMES in CD4 T cells harvested from non-activated control (grey), activated with control fibroblasts (light green), or activated with senescent fibroblasts (blue), after 3 days of co-culture. Right: Fold change (FC) of median fluorescence intensity (MFI) analysis of EOMES in activated CD4 T cells co-cultured with either control (n = 9) or senescent (Sc) fibroblasts (n = 9) after 3 (d) or 7 (e) days of co-culture. Each dot represents one well. f-g. FC of GzmB MFI (Left) and percentage of GzmB⁺ cells (Right) out of activated CD4 T cells co-cultured with control (n = 9) or senescent fibroblasts (n = 9), after 3 (f) or 7 (g) days of co-culture. h-i. FC of PD1 (Left) and CD44 (Right) MFI in activated CD4 T cells co-cultured with control (PD1:n = 9, CD44:n = 6) or senescent fibroblasts (PD1:n = 9; CD44:n = 6), after 3 (h) or 7 (i) days of co-culture. Each value was normalized to the mean MFI values of the control group. j. Experimental setup: polyclonally activated young CD4 T cells were cultivated with supernatant from control or senescent fibroblasts for 3 days and then collected for flow cytometry analysis. k. FC of EOMES MFI in non-activated (NA) or activated CD4 T cells cultivated with supernatants from control (n = 6) or senescent cells (n = 6). l-m. Representative confocal images showing DAPI staining together with MHCII (l) or ICAM-1(m) immunostaining in control (Upper) and Sc (Lower) fibroblasts. Data from ≥4 well. Scale bars are indicated in the figures. Bars represent mean ± SEM from two independent experiments. Data were analyzed using a two-tailed Student’s t-test, unpaired, with exact P-values presented in the graphs. Created with BioRender.com.