Fig 2: IL-32 treatment is synergistic with concurrent anti-PD1 in mice, and IL-32 expression is predictive for response to anti-PD1 therapy in patients with melanoma.(A) Experimental setup for B16F10 dual treatment with IL-32 and anti–PD-1 antibody used in B–D. (B) Tumor growth shown as mean ± SEM (IL-32, aPD1, IL-32 + aPD1, n = 23; PBS, n = 24). Statistical significance was determined by 2-way ANOVA followed by Šidák’s multiple comparisons test. *P = 0.0199, ****P < 0.0001. (C) Frequencies of CD45+ immune cells and (D) CD4+ T cells and CD8+ T cells as a proportion of viable cells (n = 21–23). P values were computed by 1-way ANOVA followed by Tukey’s multiple comparisons test. Data are represented as mean ± SEM. (E) Experimental setup for survival and safety assessment with additional IL-32 and anti–PD-1 treatments used for F–J. (F) Kaplan-Meier survival curves. Significance was determined by log-rank test (IL-32, IL-32 + aPD-1, n = 15; PBS, n = 17). (G) Body temperature and (H) body weight of mice upon treatment (n = 6). (I) White blood cell (WBC), lymphocyte, and red blood cell (RBC) counts as cells/µl blood (n = 4). Blood was obtained when mice were euthanized. Differences between groups were determined using 2-way ANOVA followed by Šidák’s multiple comparisons test. (J) IL32 mRNA expression levels in biopsies from patients with melanoma before anti–PD-1 (nivolumab) treatment. (K) IL32 mRNA expression in biopsies of patients with melanoma receiving neoadjuvant pembrolizumab treatment (nonrecurrence, n = 8; recurrence, n = 5). The data set was obtained from GSE123728. (J and K) P values were computed by 2-tailed, unpaired Student’s t test. Error bars show mean ± SEM. (L) Multivariate logistic regression between response to nivolumab and mRNA expression of the indicated genes or mutational load. (J and L) Patients were stratified into responders (complete response and partial response, n = 10) and nonresponders (stable disease or progressive disease, n = 39). The data set was obtained from GSE91061.