Fig. 3

CAFs reduce CD8⁺ T cell viability in an antigen-specific death ligand dependent manner. a-c Quantification of T cell viability after conditioning in culture (a) or in the presence of normal fibroblasts (b), CAFs (c) and presence/absence of antigen and target tumour cells as indicated on graphs. d Representative micrographs of T cells (green) co-cultured with normal fibroblasts (red, f-actin) and CAF (red, f-actin) showing intact T cells (yellow arrows), T cells surrounded by actin bundles (blue arrows), and fragmented T cells (white arrows). Nuclei stained with DAPI (blue). Quantification of PD-1 expression (e-g) and FAS expression (h-j) on T cells following culture with normal fibroblasts (f, i), CAFs (g, j). k-m Quantification of cognate ligand expression by co-cultured fibroblasts by flow cytometry; FASL (k), PD-L2 (l) and PD-L1 (m) expression by NORM (yellow bars) and CAFs (red bars) with and without antigen and antigen-specific OT-I T cells as indicated. n B16.OVA tumour cell viability following culture with CAFs, antigen and OT-1 T cells following CAF-specific neutralisation of PD-L2 and/or FASL. Data shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Tukey’s or Dunnett’s post hoc analysis. NS, not significant. Comparisons indicated by horizontal lines. Scale bar 40 μm. Assays performed in or quadruplet from two experiments (a-c) or duplicate from three (e-m) or two (n) independent experiments