Immune Checkpoint Inhibitors Regulate K+ Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

Abstract

Programmed death receptor-1 (PD-1) and its ligand (PD-L1) interaction negatively regulates T cell function in head and neck squamous cell carcinoma (HNSCC). Overexpression of PD-1 reduces intracellular Ca2+ fluxes, and thereby T cell effector functions. In HNSCC patients, PD-1 blockade increases KCa3.1 and Kv1.3 activity along with Ca2+ signaling and mobility in CD8+ peripheral blood T cells (PBTs). The mechanism by which PD-L1/PD-1 interaction regulates ion channel function is not known. We investigated the effects of blocking PD-1 and PD-L1 on ion channel functions and intracellular Ca2+ signaling in CD8+ PBTs of HNSCC patients and healthy donors (HDs) using single-cell electrophysiology and live microscopy. Anti-PD-1 and anti-PD-L1 antibodies increase KCa3.1 and Kv1.3 function in CD8+ PBTs of HNSCC patients. Anti-PD-1 treatment increases Ca2+ fluxes in a subset of HSNCC patients. In CD8+ PBTs of HDs, exposure to PD-L1 reduces KCa3.1 activity and Ca2+ signaling, which were restored by anti-PD-1 treatment. The PD-L1-induced inhibition of KCa3.1 channels was rescued by the intracellular application of the PI3 kinase modulator phosphatidylinositol 3-phosphate (PI3P) in patch-clamp experiments. In HNSCC CD8+ PBTs, anti-PD-1 treatment did not affect the expression of KCa3.1, Kv1.3, Ca2+ release activated Ca2+ (CRAC) channels, and markers of cell activation (CD69) and exhaustion (LAG-3 and TIM-3). Our data show that immune checkpoint blockade improves T cell function by increasing KCa3.1 and Kv1.3 channel activity in HNSCC patients.

How the Potassium Channel Response of T Lymphocytes to the Tumor Microenvironment Shapes Antitumor Immunity

“…The effect of PD-L1 on T lymphocytes is mediated by engagement with its cognate receptor PD-1 and, consequently, inhibition of TCR-dependent effector functions, such as Ca 2+ fluxing, secretion of cytokines and cytotoxicity [ 81 ]. KCa3.1 channels provide a link between PD-1 stimulation by PD-L1 and reduced Ca 2+ -related functions [ 82 ]. PD-L1 reduces KCa3.1 activity and Ca 2+ fluxes in CD8 + T cells; this effect is rescued by treatment with anti-PD-1 blocking antibodies.…”

“…Longer exposures to PD-L1 show a role for calmodulin. Five-day exposure to PD-L1 reduced calmodulin expression in T cells by 40% while the contribution of PI3K was reduced compared to early time points [ 82 ]. The involvement of KCa3.1 in mediating the effect of the PD-L1/PD-1 response is further supported by in vitro studies showing that PD-1 and PD-L1 blocking antibodies increased KCa3.1 activity [ 82 ].…”

“…An increase in Kv1.3 activity was also reported. However, there may be a difference in sensitivity to PD-L1 between these two channels; a higher concentration of anti-PD-L1 blocking antibodies was necessary to unleash Kv1.3 activity in T cells from cancer patients compared to KCa3.1 [ 82 ]. Further studies are necessary to dissect the mechanisms mediating the effect of PD-L1 on Kv1.3 channels.…”

Regulatory role of KCa3.1 in immune cell function and its emerging association with rheumatoid arthritis

“…Interestingly, PKA-mediated phosphorylation was shown to have no regulatory effect on KCa3.1 channel in the above study ( 50 ). Moreover, a monoclonal blocking antibody against programmed death 1, pembrolizumab, has been identified that promotes KCa3.1 activity and concomitantly increases Ca 2+ flux in cytotoxic T cells of patients immediately after treatment ( 54 , 55 ). In conclusion, most KCa3.1 channel activators have low potency and poor selectivity and modulate other ion channels simultaneously.…”

“…Clinically, the antihypertensive drug nitrendipine blocks KCa3.1 channel at a dose of 100 nM ( 65 ). Previous studies have shown that in the PI3K-PI(3)P signaling pathway, LY29400259 (a phosphatidylinositol 3-kinase inhibitor) ( 55 ) and ellagic acid (a nucleoside diphosphate kinase B kinase inhibitor) ( 66 ), prevent phosphorylation of specific group amino acid and inhibit activity of the KCa3.1 channel. Recently, Licochalconer A, a chalcone compound extracted from licorice, was shown to block KCa3.1 in a concentration-dependent manner, with anti-inflammatory effects ( 67 ).…”