Figure 5. Functional loss of APLNR reduces efficacy of cancer immunotherapy.

a, Non-synonymous mutations detected in APLNR in human melanoma tumours refractory to indicated immunotherapies. Ipi, ipilimumab; Nivo, nivolumab; Pembro, pembrolizumab; EC, extracellular domain; CS, cytoplasmic signalling domain. b, Mutations encoding G349E and T44S in APLNR resist the restoration of T-cell mediated cytolysis in APLNR-perturbed tumour cells. n = 3 biological replicates. c, Second most enriched sgRNA score in CRISPR screens for 96 APLNR-interacting proteins from the BioGrid database³¹. d, Western blot probed for APLNR after immunoprecipitation pull-down of JAK1 from A375 cell lysates. Results were similar in an independent replicate experiment (not shown). e, Quantitative PCR analysis of JAK1-STAT1 pathway induced genes in wild-type and APLNR-edited cells at 0, 8 and 24 h post treatment with 1 μg/ml IFNγ. n = 3 biological replicates. f, Induction of surface expression of β2M on APLNR-edited cells upon co-culture with ESO T cells as measured by FACS. n = 3 biological replicates. g, IFNγ secretion from ESO T cells after overnight co-culture with CRISPR edited A375 cells determined using ELISA. n = 3 biological replicates. h, Schematic of in vivo experiments to test the response of B2m and Aplnr knock-out tumours to ACT in immunocompetent mice. i, j, Subcutaneous tumour growth in mice receiving ACT of Pmel-1 T cells. Tumour area (i) and overall survival (j) are shown. Significance for tumour growth kinetics were calculated by Wilcoxon rank sum test. Survival significance was assessed by a log-rank Mantel-Cox test. n = 5 mice per ‘No treatment’ groups. For ‘Pmel ACT’ groups, n = 9 mice in control group, n = 10 mice per B2m-sg and Aplnr-sg groups. All values are mean ± s.e.m. ****P < 0.0001, **P < 0.01, *P < 0.05. Data are representative of two independent experiments.