SPLUNC1 is an allosteric modulator of the epithelial sodium channel.

Cystic fibrosis (CF) is a common genetic disease with significantly increased mortality. CF airways exhibit ion transport abnormalities, including hyperactivity of the epithelial Na+ channel (ENaC). Short-palate lung and nasal epithelial clone 1 (SPLUNC1) is a multifunctional innate defense protein that is secreted into the airway lumen. We have previously demonstrated that SPLUNC1 binds to and inhibits ENaC to maintain fluid homeostasis in airway epithelia and that this process fails in CF airways. Despite this, how SPLUNC1 actually regulates ENaC is unknown. Here, we found that SPLUNC1 caused αγ-ENaC to internalize, whereas SPLUNC1 and ß-ENaC remained at the plasma membrane. Additional studies revealed that SPLUNC1 increased neural precursor cell-expressed developmentally down-regulated protein 4-2-dependent ubiquitination of α- but not ß- or γ-ENaC. We also labeled intracellular ENaC termini with green fluorescent protein and mCherry, and found that extracellular SPLUNC1 altered intracellular ENaC Forster resonance energy transfer. Taken together, our data indicate that SPLUNC1 is an allosteric regulator of ENaC that dissociates αßγ-ENaC to generate a new SPLUNC1-ß-ENaC complex. These data indicate a novel mode for regulating ENaC at the plasma membrane.-Kim, C. S., Ahmad, S., Wu, T., Walton, W. G., Redinbo, M. R., Tarran, R. SPLUNC1 is an allosteric modulator of the epithelial sodium channel.

Novel antitumor therapeutic strategy using CD4+ T cell-derived extracellular vesicles.

Extracellular vesicles (EVs) mediate cell-cell crosstalk by carrying bioactive molecules derived from cells. Recently, immune cell-derived EVs have been reported to regulate key biological functions such as tumor progression. CD4+ T cells orchestrate overall immunity; however, the biological role of their EVs is unclear. This study reveals that EVs derived from CD4+ T cells increase the antitumor response of CD8+ T cells by enhancing their proliferation and activity without affecting regulatory T cells (Tregs). Moreover, EVs derived from interleukin-2 (IL2)-stimulated CD4+ T cells induce a more enhanced antitumor response of CD8+ T cells compared with that of IL2-unstimulated CD4+ T cell-derived EVs. Mechanistically, miR-25-3p, miR-155-5p, miR-215-5p, and miR-375 within CD4+ T cell-derived EVs are responsible for the induction of CD8+ T cell-mediated antitumor responses. In a melanoma mouse model, the EVs potently suppress tumor growth through CD8+ T cell activation. This study demonstrates that the EVs, in addition to IL2, are important mediators between CD4+ and CD8+ T cells. Furthermore, unlike IL2, clinically used as an antitumor agent, CD4+ T cell-derived EVs stimulate CD8+ T cells without activating Tregs. Therefore, CD4+ T cell-derived EVs may provide a novel direction for cancer immunotherapy by inducing a CD8+ T cell-mediated antitumor response.

Reprogramming of T cell-derived small extracellular vesicles using IL2 surface engineering induces potent anti-cancer effects through miRNA delivery.

T cell-derived small extracellular vesicles (sEVs) exhibit anti-cancer effects. However, their anti-cancer potential should be reinforced to enhance clinical applicability. Herein, we generated interleukin-2-tethered sEVs (IL2-sEVs) from engineered Jurkat T cells expressing IL2 at the plasma membrane via a flexible linker to induce an autocrine effect. IL2-sEVs increased the anti-cancer ability of CD8+ T cells without affecting regulatory T (Treg ) cells and down-regulated cellular and exosomal PD-L1 expression in melanoma cells, causing their increased sensitivity to CD8+ T cell-mediated cytotoxicity. Its effect on CD8+ T and melanoma cells was mediated by several IL2-sEV-resident microRNAs (miRNAs), whose expressions were upregulated by the autocrine effects of IL2. Among the miRNAs, miR-181a-3p and miR-223-3p notably reduced the PD-L1 protein levels in melanoma cells. Interestingly, miR-181a-3p increased the activity of CD8+ T cells while suppressing Treg cell activity. IL2-sEVs inhibited tumour progression in melanoma-bearing immunocompetent mice, but not in immunodeficient mice. The combination of IL2-sEVs and existing anti-cancer drugs significantly improved anti-cancer efficacy by decreasing PD-L1 expression in vivo. Thus, IL2-sEVs are potential cancer immunotherapeutic agents that regulate both immune and cancer cells by reprogramming miRNA levels.