Figure 3: Senescent stromal cells promote the development of an immunosuppressive microenvironment.

(a) MHCII^LoLy6G^Hi and MHCII^LoLy6C^Hi gating strategy for non-senescent (Non-Sen) and senescence (Sen) isografts. Cells previously gated as CD45⁺CD11b⁺MHCII^Lo (left). MHCII^Lo population is further gated for Ly6G and Ly6C (right). (b) Quantification of flow cytometry in a. Data represents CD11b⁺Ly6G^Hi or CD11b⁺Ly6C^Hi cells as % of CD45⁺ cells. *P value <0.05 by Student’s t-test. ns is not significant. Data are mean %+s.e.m. n=3. (c) Quantification of CD11b⁺Ly6G^Hi or CD11b⁺Ly6C^Hi cells represented as number of cells per mouse. *P value <0.05 by Student’s t-test. ns is not significant. Data are mean+s.e.m. n=3. (d) IF staining for Ly6G⁺ cells (red) in FASST skin. Arrows indicate Ly6G⁺ cells. Scale bar, 100 μM. (e) Quantification of Ly6G⁺ cells shown in d. *P value <0.05 by Student’s t-test. Data are mean+s.e.m. n=36–37. (f) Quantification of CD3⁺ T cells in Sen versus Non-Sen isografts. CD3⁺ cells were previously gated as CD45⁺. *P value<0.05 by Student’s t-test. Data are mean %+s.e.m. n=3–4. (g) T regulatory cell (T_reg) gating for Non-Sen and Sen isografts. Cells previously gated as CD45⁺CD3⁺CD4⁺. (h) Quantification of flow cytometry in g. *P value <0.05 by Student’s t-test. Data are mean %+s.e.m. of CD4⁺FoxP3⁺ cells in the CD45⁺ population. n=4. (i) qRT–PCR analysis of Sen versus Non-Sen isografts shows significant (P<0.05, Student’s t-test) senescence-dependent enrichment of pro-inflammatory and immunosuppressive gene expression in vivo. Data are mean fold increase+s.e.m. of the dCT normalized to GAPDH. n=11–16. (j) Schematic representation of CM assay testing the SASP's impact on naive bone marrow. Non-senescent (vehicle), senescent (TAM). (k) Flow cytometry analysis of conditioned (Non-Sen or Sen) medium-treated naive bone marrow cells from set-up in j. *P-value<0.05, Student’s t-test. Data are mean # of cells per plate+s.e.m. Representative experiment, n=4. Data are derived from experiment shown in Fig. 7b thus Sen-CM and Non-Sen-CM contain IgG control antibody. qRT–PCR, quantitative PCR with reverse transcription.