Pyroptosis activates conventional type I dendritic cells to mediate the priming of highly functional anticancer T cells.

BACKGROUND: Initiation of antitumor immunity is reliant on the stimulation of dendritic cells (DCs) to present tumor antigens to naïve T cells and generate effector T cells that can kill cancer cells. Induction of immunogenic cell death after certain types of cytotoxic anticancer therapies can stimulate T cell-mediated immunity. However, cytotoxic therapies simultaneously activate multiple types of cellular stress and programmed cell death; hence, it remains unknown what types of cancer cell death confer superior antitumor immunity. METHODS: Murine cancer cells were engineered to activate apoptotic or pyroptotic cell death after Dox-induced expression of procell death proteins. Cell-free supernatants were collected to measure secreted danger signals, cytokines, and chemokines. Tumors were formed by transplanting engineered tumor cells to specifically activate apoptosis or pyroptosis in established tumors and the magnitude of immune response measured by flow cytometry. Tumor growth was measured using calipers to estimate end point tumor volumes for Kaplan-Meier survival analysis. RESULTS: We demonstrated that, unlike apoptosis, pyroptosis induces an immunostimulatory secretome signature. In established tumors pyroptosis preferentially activated CD103+ and XCR1+ type I conventional DCs (cDC1) along with a higher magnitude and functionality of tumor-specific CD8+ T cells and reduced number of regulatory T cells within the tumor. Depletion of cDC1 or CD4+ and CD8+ T cells ablated the antitumor response leaving mice susceptible to a tumor rechallenge. CONCLUSION: Our study highlights that distinct types of cell death yield varying immunotherapeutic effect and selective activation of pyroptosis can be used to potentiate multiple aspects of the anticancer immunity cycle.

Complementary Nck1/2 Signaling in Podocytes Controls α Actinin-4-Mediated Actin Organization, Adhesion, and Basement Membrane Composition.

BACKGROUND: Maintenance of the kidney filtration barrier requires coordinated interactions between podocytes and the underlying glomerular basement membrane (GBM). GBM ligands bind podocyte integrins, which triggers actin-based signaling events critical for adhesion. Nck1/2 adaptors have emerged as essential regulators of podocyte cytoskeletal dynamics. However, the precise signaling mechanisms mediated by Nck1/2 adaptors in podocytes remain to be fully elucidated. METHODS: We generated podocytes deficient in Nck1 and Nck2 and used transcriptomic approaches to profile expression differences. Proteomic techniques identified specific binding partners for Nck1 and Nck2 in podocytes. We used cultured podocytes and mice deficient in Nck1 and/or Nck2, along with podocyte injury models, to comprehensively verify our findings. RESULTS: Compound loss of Nck1/2 altered expression of genes involved in actin binding, cell adhesion, and extracellular matrix composition. Accordingly, Nck1/2-deficient podocytes showed defects in actin organization and cell adhesion in vitro, with podocyte detachment and altered GBM morphology present in vivo. We identified distinct interactomes for Nck1 and Nck2 and uncovered a mechanism by which Nck1 and Nck2 cooperate to regulate actin bundling at focal adhesions via α actinin-4. Furthermore, loss of Nck1 or Nck2 resulted in increased matrix deposition in vivo, with more prominent defects in Nck2-deficient mice, consistent with enhanced susceptibility to podocyte injury. CONCLUSION: These findings reveal distinct, yet complementary, roles for Nck proteins in regulating podocyte adhesion, controlling GBM composition, and sustaining filtration barrier integrity.

Multivalent nephrin-Nck interactions define a threshold for clustering and tyrosine-dependent nephrin endocytosis.

Assembly of signaling molecules into micrometer-sized clusters is driven by multivalent protein-protein interactions, such as those found within the nephrin-Nck (Nck1 or Nck2) complex. Phosphorylation on multiple tyrosine residues within the tail of the nephrin transmembrane receptor induces recruitment of the cytoplasmic adaptor protein Nck, which binds via its triple SH3 domains to various effectors, leading to actin assembly. The physiological consequences of nephrin clustering are not well understood. Here, we demonstrate that nephrin phosphorylation regulates the formation of membrane clusters in podocytes. We also reveal a connection between clustering and endocytosis, which appears to be driven by threshold levels of nephrin tyrosine phosphorylation and Nck SH3 domain signaling. Finally, we expose an in vivo correlation between transient changes in nephrin tyrosine phosphorylation, nephrin localization and integrity of the glomerular filtration barrier during podocyte injury. Altogether, our results suggest that nephrin phosphorylation determines the composition of effector proteins within clusters to dynamically regulate nephrin turnover and podocyte health.