Figure 3.

Catalytic activity is required for nSMase2 to suppress melanoma growth and stimulate immune responses. A, Frequency of mutations and copy-number alterations in human melanoma samples from the indicated studies (www.cbioportal.org). B, Localization of missense (green dots) and splice (red dot) mutations on nSMase2 amino acid sequence from the studies depicted in A. The catalytic site corresponds to the green box. Mutations are predicted to be benign (green), possibly damaging (orange), or probably damaging (red; http://genetics.bwh.harvard.edu/pph2/). C, B16K1 cells expressing or not (mock) the WT or C.I. nSMase2 were intradermally injected in C57BL/6 WT mice, and tumor volumes were determined at the indicated days. Data are mean ± SEM of four mice per group (*, P < 0.05; **, P < 0.01; ***, P < 0.001). D–F, B16K1 cells expressing the WT or C.I. nSMase2 were intradermally and bilaterally injected in WT mice, and 12 days later, tumor-draining lymph nodes (TdLN) and tumors were collected. Tumors were weighed (D), and T-cell content was analyzed by flow cytometry in TdLNs (E, left plot) and tumors (E, right plot). Data are mean ± SEM of 18 mice per groups pooled from three independent experiments (D and E). F, CD8⁺ T cells specific for Trp2 peptides were quantified using dextramer technology. Representative staining and proportion of total Trp2-specific CD8⁺ T cells are depicted. Numbers are mean ± SEM of six mice per group (*, P < 0.05; **, P < 0.01).