Figure 3 from Cytotoxic NK Cells Impede Response to Checkpoint Immunotherapy in Melanoma with an Immune-Excluded Phenotype

<p>NK cells as modulators of ICB sensitivity in mice. <b>A,</b> Immunofluorescence of YUMM5.2 and (<b>B</b>) <i>NRAS;Ink4a</i> tumors. Tumors were stained for a melanoma marker (PDGFRa for YUMM5.2 and SOX10 for <i>NRAS;Ink4a</i>) and for CD45 and CD3ε to map the T-cell infiltration. Counterstaining was performed with DAPI. <b>C,</b> Schematic representation of NK and T-cell infiltration in the <i>NRAS;Ink4a</i> and YUMM5.2 tumors. <b>D,</b> Schematic representation of the therapeutic regimen used with YUMM5.2 and <i>NRAS;Ink4a</i> tumors. <b>E,</b> Growth curves of YUMM5.2 (top left) and <i>NRAS;Ink4a</i> tumors (bottom left) <i>in vivo</i> (<i>n</i> ≥ 5 per cohort (control, αPD-1, aNK1.1, and αPD-1 + aNK1.1). A Welch-corrected <i>t</i> test was performed on the doubling time of tumors upon exponential fitting (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01). Kaplan–Meier curves representing progression-free survival of YUMM5.2 (top right) and <i>NRAS;Ink4a</i> tumors (bottom right) <i>in vivo</i> (<i>n</i> ≥ 5 per cohort. A log-rank Mantel–Cox test was used; *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001). <b>F,</b> Cryosection immunofluorescence showing the overall immune infiltrate of <i>NRAS;Ink4a</i> tumors across cohorts. Tumor cells were identified by S100 and immune cells by CD45. Counterstaining was performed with DAPI. The dashed line marks the separation between tumor and immune areas. Quantification of overall immune infiltration in <i>NRAS;Ink4a</i> tumors (number of CD45<sup>+</sup> of all DAPI-positive cells, with <i>n</i> ≥ 6 per cohort and data analyzed with a Welch-corrected <i>t</i> test; *, <i>P</i> < 0.05; **, <i>P</i> < 0.01). <b>G,</b> Stacked bar plot showing proportions of immune cell types from the <i>NRAS;Ink4a</i> and YUMM5.2 tumors across conditions based on the scRNA-seq experiment. For each condition, at least four mice were pooled (see “Methods”). <b>H,</b> Growth curves (left) and doubling time calculation (right) of <i>NRAS;Ink4a</i> tumors <i>in vivo</i> treated with αPD-1 and/or aNK1.1 in the presence/absence of CD8<sup>+</sup> T-cell depletion (<i>n</i> = 4 per cohort and a Welch-corrected <i>t</i> test was performed on the doubling time of tumors upon exponential fitting; **, <i>P</i> < 0.01). cDC1, conventional type 1 DCs; cDC2, conventional type 2 DCs; mDC, migratory DCs, Treg, regulatory T cell. (<b>C</b> and <b>D,</b> Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>.)</p>