Fig. 5: anti-PD-L1 promotes activating CCR7⁺ DC-cytotoxic CD8⁺ T cell interactions in the TME.

a UMAP of scRNA-seq of CD8⁺ T cells, from FACS-sorted CD45⁺ tumour-infiltrating lymphocytes 48 h after photoconversion of subcutaneous MC38-Ova tumours. b Proportion of T_EX_CD8T cells by Kaede fluorescence and treatment group. c Number of significant DEGs in anti-PD-L1 versus isotype control-treated tumours by cluster. DEGs calculated using Wilcoxon rank-sum test with Benjamini-Hochberg multiple-testing correction. d CellPhoneDB ligand-receptor analysis between tumour DCs and CD8⁺ T_EX cells. Only significant interactions (p < 0.05) shown. e Representative confocal microscopy images of MC38 tumours, showing co-localisation of CCR7⁺MHC-II⁺ DCs and CD3⁺CD8⁺ 4-1BB⁺/Ki-67⁺ T cells (arrows). Scale bar, 20 μm. (f–k) In vitro and ex vivo cultures. f Phenotype of bone marrow-derived DCs (BMDC) following culture with UV-irradiated MC38-Ova tumour cells. g Experiment set-up of DC:OT-I co-culture. h Representative flow cytometry of CD44⁺ OT-I cell activation and proliferation following co-culture with MC38-Ova-experienced CCR7⁺ BMDC; +/– anti-PD-L1, OX40L-expressing (+, OX40L^fl/fl) or OX40L-deficient (–, CD11c^creOX40L^fl/fl) BMDCs. i Quantification of h and granzyme B (GzmB) expression. Values shown are relative to cultures with isotype control antibodies and OX40L-expressing DCs (baseline), to facilitate comparisons across experiments. j Flow cytometry of CCR7⁺ DCs from subcutaneous MC38-Ova tumours following 8 h culture ex vivo with antibodies or recombinant IFNγ (rIFNg). k Flow cytometry of OT-I cells following co-culture with MC38-Ova-experienced CCR7⁺ BMDC; CCR7⁺ BMDCs pre-treated with rIFNg (+) or PBS (–), OX40L-expressing (+) or OX40L-deficient (–) BMDCs. One-way analysis of variance (ANOVA) and Šidák’s multiple comparisons test was used, data are shown as means ± s.d. (f, i–k). The results shown in (e) are representative of two independent experiments (n = 5 animals); f from one experiment (n = 4 biological samples) representative of three independent experiments; h, i from two independent experiments (n = 7 biological samples); j from one experiment (n = 5 animals), representative of two independent experiments; and (k) from one experiment (n = 4 biological samples), representative of two independent experiments.