Nanovesicle-targeted Kv1.3 knockdown in memory T cells suppresses CD40L expression and memory phenotype

Abstract

Ca2+ signaling controls activation and effector functions of T lymphocytes. Ca2+ levels also regulate NFAT activation and CD40 ligand (CD40L) expression in T cells. CD40L in activated memory T cells binds to its cognate receptor, CD40, on other cell types resulting in the production of antibodies and pro-inflammatory mediators. The CD40L/CD40 interaction is implicated in the pathogenesis of autoimmune disorders and CD40L is widely recognized as a therapeutic target. Ca2+ signaling in T cells is regulated by Kv1.3 channels. We have developed lipid nanoparticles that deliver Kv1.3 siRNAs (Kv1.3-NPs) selectively to CD45RO+ memory T cells and reduce the activation-induced Ca2+ influx. Herein we report that Kv1.3-NPs reduced NFAT activation and CD40L expression exclusively in CD45RO+ T cells. Furthermore, Kv1.3-NPs suppressed cytokine release and induced a phenotype switch of T cells from predominantly memory to naïve. These findings indicate that Kv1.3-NPs operate as targeted immune suppressive agents with promising therapeutic potentials.

Kv1.3 Channels Mark Functionally Competent CD8+ Tumor-Infiltrating Lymphocytes in Head and Neck Cancer

“…In parallel with these findings, our data indicate that the defective Ca 2+ influx in TILs can be attributed to the suppression of Kv1.3 channels. In contrast to what others have previously reported, we have conducted experiments using an experimental protocol that allows bypassing the TCR and measuring only the component of Ca 2+ influx that depends on ion channels, transporters and pumps, thus providing the mechanistic evidence that the decrease in Ca 2+ in TILs is, at least in part, is due to reduced Kv1.3 channel expression (22, 27). We cannot exclude the possibility that changes in other ionic pathways may contribute to the reduction in Ca 2+ influx in TILs, but a previous study from our laboratory showed that a 70% reduction in Kv1.3 activity results in a 40% reduction in Ca 2+ influx in PBTs, which coincides with our outcomes in TILs (23).…”

“…Ki-67 and GrB are markers of CD8 + cell functionality, which are under the control of Kv1.3-regulated Ca 2+ signaling and NFAT activity (19, 22, 23, 26). In cohorts of renal cell carcinoma, increased amounts of Ki-67 are associated with improved patient outcomes (29).…”

“…Peripheral blood mononuclear cells (PBMCs) and CD3 + T cells were isolated from whole blood as previously described (22). Briefly, PBMCs were isolated from centrifugation of whole blood using Ficoll-Paque (GE Healthcare Bio-sciences) density gradient and CD3 + cells were further isolated from the PBMCs by negative selection using EasySep™ Human T-cell enrichment kit (StemCell Technologies).…”

“…Cell viability was assessed by live labeling 0.1 × 10 6 TILs with 7-AAD viability staining solution (Biolegend) (22). For purity, cells were stained with FITC anti-CD3 and PerCP anti-CD19 antibodies.…”

“…For purity, cells were stained with FITC anti-CD3 and PerCP anti-CD19 antibodies. For phenotyping, approximately 0.5 × 10 6 TILs were stained for measuring the percentages of T reg and T EM cells using standard flow cytometry staining protocols (22). Briefly, for T reg cells, TILs were labelled live with the following antibodies: FITC anti-CD3, PE anti-CD127, PerCP anti-CD8, APC anti- CD4, APC-Cy7 anti-CD25 and PB anti-CD45.…”