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Background: Dying tumor cells release intracellular potassium (K +), raising extracellular K + ([K + ] e) in the tumor microenvironment (TME) to 40-50 mM (high-[K + ] e). Here, we investigated the effect of high-[K + ] e on T cell functions. Materials and Methods: Functional impacts of high-[K + ] e on human T cells were determined by cellular, molecular, and imaging assays. Results: Exposure to high-[K + ] e suppressed the proliferation of central memory and effector memory T cells, while T memory stem cells were unaffected. High-[K + ] e inhibited T cell cytokine production and dampened antitumor cytotoxicity, by modulating the Akt signaling pathway. High-[K + ] e caused significant upregulation of the immune checkpoint protein PD-1 in activated T cells. Although the number of K Ca 3.1 calcium-activated potassium channels expressed in T cells remained unaffected under high-[K + ] e , a novel K Ca 3.1 activator, SKA-346, rescued T cells from high-[K + ] e-mediated suppression. Conclusion: High-[K + ] e represents a so far overlooked secondary checkpoint in cancer. K Ca 3.1 activators could overcome such ''ionic-checkpoint''-mediated immunosuppression in the TME, and be administered together with known PD-1 inhibitors and other cancer therapeutics to improve outcomes.

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The steroidal lactone withaferin A (WFA) is a dietary phytochemical, derived from Withania somnifera . It exhibits a wide range of biological properties, including immunomodulatory, anti-inflammatory, antistress, and anticancer activities. Here we investigated the effect of WFA on T-cell motility, which is crucial for adaptive immune responses as well as autoimmune reactions. We found that WFA dose-dependently (within the concentration range of 0.3–1.25 μM) inhibited the ability of human T-cells to migrate via cross-linking of the lymphocyte function-associated antigen-1 (LFA-1) integrin with its ligand, intercellular adhesion molecule 1 (ICAM-1). Coimmunoprecipitation of WFA interacting proteins and subsequent tandem mass spectrometry identified a WFA-interactome consisting of 273 proteins in motile T-cells. In particular, our data revealed significant enrichment of the zeta-chain-associated protein kinase 70 (ZAP70) and cytoskeletal actin protein interaction networks upon stimulation. Phospho-peptide mapping and kinome analysis substantiated kinase signaling downstream of ZAP70 as a key WFA target, which was further confirmed by bait-pulldown and Western immunoblotting assays. The WFA-ZAP70 interaction was disrupted by a disulfide reducing agent dithiothreitol, suggesting an involvement of cysteine covalent binding interface. In silico docking predicted WFA binding to ZAP70 at cystine 560 and 564 residues. These findings provide a mechanistic insight whereby WFA binds to and inhibits the ZAP70 kinase and impedes T-cell motility. We therefore conclude that WFA may be exploited to pharmacologically control host immune responses and potentially prevent autoimmune-mediated pathologies.