Extended Data Fig. 7: VEGF-C-dependent anti-PD-1 potentiation is specific to VEGF-C among proteins of the VEGF family, and is not caused by a direct effect on tumour or immune cells.
From: VEGF-C-driven lymphatic drainage enables immunosurveillance of brain tumours
a, b, C57BL/6 mice received an injection of AAV-CTRL or AAV-s(oluble)VEGFR-3 intracisternally through the cisterna magna. After four weeks, mice were euthanized and the dura mater was collected to image the lymphatic vasculature (LYVE1) in the confluence of sinuses (a). The relative area of lymphatic vasculature in the confluence of sinuses was quantified (b) (n = 5). c, Mice were pretreated with AAV-sVEGFR-3 four to six weeks before tumour inoculation. Seven days after tumour inoculation, mice were treated with VEGFC mRNA and anti-PD-1 (RMP1-14) antibodies (days 7, 9 and 11) (n = 5). d–f, Mice were treated with 5 µg of recombinant protein (VEGF-A, VEGF-B, VEGF-Cs or VEGF-D) in combination with anti-PD-1 (RMP1-14) antibodies (days 7, 9 and 11) and monitored for survival and tumour growth (n = 5). g–k Mice were injected with CT-2A–BFP tumours and were treated with VEGFC mRNA at day 7. On day 8, brains and lymph nodes from all mice were collected and analysed using flow cytometry. The experiment was repeated independently with similar results. g, Sample flow cytometry plots of experiments. h–k, Quantification of experiments (n = 5). l, Flow cytometry was used to evaluate the expression of VEGFR-3 in GL261 cells. A VEGFR3–GFP plasmid was transfected into HEK293T cells as a positive control. The experiment was repeated independently with similar results. m, MTT assay to measure the proliferation of GL261 cancer cells in the presence of VEGF-C after 48 h (n = 8 per group). n, Flow cytometry was used to evaluate the expression of VEGFR-3 in leukocyte compartments in the tumour. The experiment was repeated independently with similar results. o, Bone-marrow-derived dendritic cells were cultured with VEGF-C and evaluated for the expression of costimulatory molecules in the naive state (top row) or with LPS stimulation (bottom row). p, Isolated T cells were activated in vitro with CD3 or CD28 and IL-2 in the presence of VEGF-C. Data are mean ± s.d. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (two-tailed unpaired Student’s t-test or two-sided log-rank Mantel–Cox test).
