IL-32γ potentiates tumor immunity in melanoma
Thomas Gruber, … , Antoni Ribas, Mirjam Schenk
Thomas Gruber, … , Antoni Ribas, Mirjam Schenk
Published August 25, 2020
Citation Information: JCI Insight. 2020;5(18):e138772. https://doi.org/10.1172/jci.insight.138772.
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Research Article Immunology Oncology

IL-32γ potentiates tumor immunity in melanoma

Abstract

Myeloid cells orchestrate the antitumor immune response and influence the efficacy of immune checkpoint blockade (ICB) therapies. We and others have previously shown that IL-32 mediates DC differentiation and macrophage activation. Here, we demonstrate that IL-32 expression in human melanoma positively correlates with overall survival, response to ICB, and an immune-inflamed tumor microenvironment (TME) enriched in mature DC, M1 macrophages, and CD8+ T cells. Treatment of B16F10 murine melanomas with IL-32 increased the frequencies of activated, tumor-specific CD8+ T cells, leading to the induction of systemic tumor immunity. Our mechanistic in vivo studies revealed a potentially novel role of IL-32 in activating intratumoral DC and macrophages to act in concert to prime CD8+ T cells and recruit them into the TME through CCL5. Thereby, IL-32 treatment reduced tumor growth and rendered ICB-resistant B16F10 tumors responsive to anti–PD-1 therapy without toxicity. Furthermore, increased baseline IL-32 gene expression was associated with response to nivolumab and pembrolizumab in 2 independent cohorts of patients with melanoma, implying that IL-32 is a predictive biomarker for anti–PD-1 therapy. Collectively, this study suggests IL-32 as a potent adjuvant in immunotherapy to enhance the efficacy of ICB in patients with non–T cell–inflamed TME.

Authors

Thomas Gruber, Mirela Kremenovic, Hassan Sadozai, Nives Rombini, Lukas Baeriswyl, Fabienne Maibach, Robert L. Modlin, Michel Gilliet, Diego von Werdt, Robert E. Hunger, S. Morteza Seyed Jafari, Giulia Parisi, Gabriel Abril-Rodriguez, Antoni Ribas, Mirjam Schenk

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Figure 3

Lymphocyte-derived IL-32 mediates activation of myeloid but not lymphoid or melanoma cells.

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Lymphocyte-derived IL-32 mediates activation of myeloid but not lymphoid...
(A) IL-32 expression of indicated cell types determined by single-cell RNA-Seq data obtained from GSE72056. Data are shown as box-and-whisker plots. The box extends between 25% and 75%, and the whiskers extend to the minimum and maximum. Statistical significance was determined using 1-way ANOVA followed by Tukey’s multiple comparisons test. (B) Representative histograms of IL-32 expression in indicated immune cell populations from healthy human blood assessed by flow cytometry (n = 4). (C) IL-32 mean fluorescence intensity (MFI) in each immune cell subset treated for 48 hours with anti-CD3/CD28 and IL-2 or left untreated, (n = 4). (D) Representative immunohistochemical labeling for CD8+ T cells (top) and IL-32 (bottom) in serial sections (n = 3). Original magnification, ×5; ×40 (high-magnification images). Scale bars: 200 μm; 20 μm (high-magnification images). (E) Correlation between IL-32+ and CD3+ cells, as assessed by immunofluorescence labeling of a melanoma TMA, indicated as a percentage of DAPI+ cells (n = 154; from 140 individual patients) (F) PCA from NanoString-derived gene expression profiles of purified human PBMC subsets and melanoma cell lines treated with IL-32 for 24 hours or left untreated (monocytes, n = 4; other PBMC subsets, melanoma cell lines, n = 3). Statistical significance was determined using Adonis in the vegan package in R. (G) Kinase phosphorylation levels measured by phospho-kinase array in monocytes treated with IL-32 for 20 minutes or left untreated. Two biologically independent samples were measured in duplicates. (H) RNA expression levels of the indicated DC maturation markers and chemokines in IL-32–treated (24 hours) and untreated murine BMDC (n = 6). (I) MFI of the indicated DC maturation markers assessed by flow cytometry on IL-32–treated versus untreated murine BMDC (n = 4), assessed after 48 hours. (J) Proliferation of OT-I cells cocultured with IL-32–treated or untreated BMDC after 48 hours measured using a BrdU cell proliferation assay kit (BioVision). BMDC were either pulsed with OVA protein (left) or SIINFEKL peptide (right) (n = 6). (I and J) Data shown as mean ± SEM; unpaired, 2-tailed Student’s t test. (K) Gene expression levels of the indicated macrophage activation markers and T cell–attracting chemokines in murine BMDM treated for 24 hours with IL-32 or left untreated, measured by NanoString (n = 6). (H and K) Normalized, log2-transformed mRNA counts shown as box-and-whisker plots. The box extends between 25% and 75%, and the whiskers extend to the minimum and maximum. (C, G, H, and K) Statistical significance was determined using 2-way ANOVA followed by Šidák’s multiple comparisons test. **P < 0.01, ***P < 0.001, ****P < 0.0001.