Extended Data Fig. 6: Exosomal PD-L1 secreted by mouse melanoma B16-F10 cells inhibits the proliferation and cytotoxicity of mouse splenic CD8 T cells.
From: Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response
a, Representative contour plots showing the general gating strategy used to identify the purified CD8 T cells (CD3+CD8+CD4−) from mouse splenocytes. b, Representative histogram of CFSE-labelled mouse splenic CD8 T cells (stimulated with anti-CD3/CD28 antibodies) after treatment with B16-F10 cell-derived exosomes with or without blocking by IgG isotype or the anti-PD-L1 antibodies (left). The proportion of cells with diluted CFSE dye is shown at the right panel. c, Representative contour plots of mouse splenic CD8 T cells (stimulated with anti-CD3/CD28 antibodies) examined for the expression of Ki-67 and granzyme B (GzmB) after treatment with B16-F10 cell-derived exosomes with or without blocking by IgG isotype or the anti-PD-L1 antibodies (left). The percentage of Ki-67+GzmB+ CD8 T cells stimulated with anti-CD3/CD28 antibodies is shown (right). d, Representative contour plots of mouse splenic CD8 T cells (stimulated with anti-CD3/CD28 antibodies) examined for the expression of Ki-67 and GzmB after treatment with B16-F10 cell-derived exosomes in the presence or absence of anti-PD-1 blocking antibodies (left). The percentage of Ki-67+GzmB+ CD8 T cells stimulated with anti-CD3/CD28 antibodies is shown at the right panel. e, OT-I CD8 T cell-meditated tumour cell killing assay was performed in B16-OVA cells with PD-L1 knockdown, or B16-F10 cells with PD-L1 knockdown (negative control). Apoptosis of tumour cells was evaluated by flow cytometric analysis of intracellular cleaved caspase-3 (left), and the relative cytotoxicity was calculated (right). f, OT-I CD8 T cells, activated by OVA-pulsed bone marrow-derived dendritic cells and treated with PBS (as a control), exosomes derived from B16-F10 cells with or without IgG isotype or PD-L1 antibody blocking, were co-cultured with PD-L1 knockdown B16-OVA cells for 48 h. Tumour cell apoptosis was evaluated by flow cytometric analysis of intracellular cleaved caspase-3 (left), and the relative cytotoxicity was calculated (right). Data represent mean ± s.d. of three (b–f) independent biological replicates. Statistical analyses are performed using two-sided unpaired t-test (b–f).
