Figure 4: Senescent fibroblasts reduce CD8⁺ T-cell responsiveness.

(a) Schematic representation of ex vivo CD8⁺ T-cell stimulation assay. Cells from isograft digests were stimulated with PMA. Cells were then stained for cell surface markers, fixed, permeabilized (Perm.) and stained for intracellular IFNγ. IgG antibody is used as a control. (b) Flow cytometry following ex vivo CD8⁺ T-cell stimulation from Non-Sen or Sen isografts. Experimental set-up is depicted in a. Cells were previously gated as CD45⁺CD3⁺CD8⁺ cells. % positive cells, as gated, are shown at the upper right corner of each plot. Representative plots are shown. (c) Quantification of ex vivo CD8⁺ T-cell stimulation flow cytometry analysis in b. Data are normalized based on staining in the IgG control samples, and is presented as mean % of CD8⁺IFNγ⁺ cells previously gated as CD45⁺CD3⁺ cells+s.e.m. * indicates P value <0.05 by Student’s t-test. Representative experiment. n=4. (d) Schematic representation of T-cell suppression assay using CM-treated BMDM cells. (e) T-cell suppression assay using CM (from Non-Sen or Sen MSFs) treated naive bone marrow cells. Schematic representation of experimental set-up is shown in d. Control (CTR) medium consists of RPMI+5% FBS. CFSE-labelled splenocytes and unlabelled BMDM cells were plated at indicated ratios. CD8⁺ T-cell proliferation was assessed at 72 h post CD3-stimulation by flow cytometric quantification of CFSE-dilution within the CD8⁺ population of cells. * indicates P value <0.05 by analysis of variance. Data are presented as mean+s.d. of triplicates. Representative experiment. n=6.