IL-2 and TCR stimulation induce expression and secretion of IL-32ß by human T cells.

IL-32 expression is important for pathogen clearance but detrimental in chronic inflammation, autoimmunity, and cancer. T cells are major IL-32 producers in these diseases and key mediators of pathogen and tumor elimination but also autoimmune destruction. However, their contribution to IL-32 biology during immune responses is hardly understood due to several isoforms with divergent inflammatory properties. Here, we identified IL-32ß as the predominant isoform in various T cell subsets of healthy individuals and breast cancer patients with the highest levels detected in intratumoral regulatory T cells. We show that IL-32ß is induced by IL-2 but IL-32ß release requires T Cell Receptor rather than IL2R stimulation. Using inhibitors of protein secretion pathways and serial (ultra)centrifugation of T cell supernatants, we demonstrate that T cells actively secrete IL-32ß unconventionally, as a free protein and, to a minor degree, through exosomes. Thus, our data identify activated T cells as major IL-32ß secretors in health and cancer.

Effective Reprogramming of Patient-Derived M2-Polarized Glioblastoma-Associated Microglia/Macrophages by Treatment with GW2580.

PURPOSE: Targeting immunosuppressive and pro-tumorigenic glioblastoma (GBM)-associated macrophages and microglial cells (GAM) has great potential to improve patient outcomes. Colony-stimulating factor-1 receptor (CSF1R) has emerged as a promising target for reprograming anti-inflammatory M2-like GAMs. However, treatment data on patient-derived, tumor-educated GAMs and their influence on the adaptive immunity are lacking. EXPERIMENTAL DESIGN: CD11b+-GAMs freshly isolated from patient tumors were treated with CSF1R-targeting drugs PLX3397, BLZ945, and GW2580. Phenotypical changes upon treatment were assessed using RNA sequencing, flow cytometry, and cytokine quantification. Functional analyses included inducible nitric oxide synthase activity, phagocytosis, transmigration, and autologous tumor cell killing assays. Antitumor effects and changes in GAM activation were confirmed in a complex patient-derived 3D tumor organoid model serving as a tumor avatar. RESULTS: The most effective reprogramming of GAMs was observed upon GW2580 treatment, which led to the downregulation of M2-related markers, IL6, IL10, ERK1/2, and MAPK signaling pathways, while M1-like markers, gene set enrichment indicating activated MHC-II presentation, phagocytosis, and T-cell killing were substantially increased. Moreover, treatment of patient-derived GBM organoids with GW2580 confirmed successful reprogramming, resulting in impaired tumor cell proliferation. In line with its failure in clinical trials, PLX3397 was ineffective in our analysis. CONCLUSIONS: This comparative analysis of CSF1R-targeting drugs on patient-derived GAMs and human GBM avatars identified GW2580 as the most powerful inhibitor with the ability to polarize immunosuppressive GAMs to a proinflammatory phenotype, supporting antitumor T-cell responses while also exerting a direct antitumor effect. These data indicate that GW2580 could be an important pillar in future therapies for GBM.