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. 2020 Apr 13;130(5):2570–2586. doi: 10.1172/JCI133055

Figure 1. PMN-MDSC accumulation contributes to tumor progression following anti–PD-1 Ab immunotherapy.

Figure 1

(A) Schematic overview of the adaptive resistance pathway. (B) RNA-Seq differential gene expression analysis of tumor tissues following treatment of the autochthonous BRAFV600E PTEN–/– melanoma model with anti–PD-1 Ab therapy versus IgG isotype control (Ctrl) (n = 3). (C) qRT-PCR analysis of target genes of interest in serial tumor fine-needle aspiration (FNA) biopsy specimens harvested from the transgenic BRAFV600E PTEN–/– melanoma model treated with anti–PD-1 Ab versus IgG isotype control (n = 5). (D) Gr-1 immunohistochemical analysis of transgenic BRAFV600E PTEN–/– melanoma tissues following treatment with anti–PD-1 Ab versus IgG isotype control. Original magnification, ×40. Gr-1 staining is shown in red. Images are representative of 3 tumors per group. (E) PMN-MDSC flow cytometric analysis of transgenic BRAFV600E PTEN–/– melanoma tissues following treatment with anti–PD-1 Ab versus IgG isotype control. PMN-MDSCs were defined as live+CD45+CD11b+Ly6G+Ly6CintF4/80– cells. Shown are a representative flow dot plot and quantification graph of PMN-MDSC flow cytometric data (n = 5). (F) qRT-PCR analysis of CXCR2 ligands in BRAFV600E PTEN–/– melanoma tissues treated with anti–PD-1 Ab following CD8+ T cell ablation in vivo (n = 3). (G) In vivo tumor study of BRAFV600E PTEN–/– melanoma genetically silenced for CXCL5. Quantitation of tumor-infiltrating PMN-MDSCs by flow cytometry is shown along with an in vivo tumor growth curve of CXCL5-silenced BRAFV600E PTEN–/– melanoma versus BRAFV600E PTEN–/– NTC melanoma control tumors treated with anti–PD-1 Ab. Data were normalized to tumors treated with IgG isotype control (n = 5). (H) Combination treatment with anti–PD-1 Ab and CXCR2 inhibitor (CXCR2i) in an in vivo BRAFV600E PTEN–/– melanoma study (n = 5). Graphs show flow cytometric analysis of tumor-infiltrating PMN-MDSCs and live+CD45+CD3+CD8+ T cells. *P < 0.05, **P < 0.005, and ***P < 0.0005, by Student’s t test with Holm-Sidak post hoc correction for multiple comparisons (B, C, and F), Student’s t test (E and G), or 1-way ANOVA with Sidak’s post hoc multiple comparisons test (H). See also Supplemental Figures 1, 2, and 5C.