PD1 blockade enhances K⁺channel activity, Ca²⁺signaling, and migratory ability in cytotoxic T lymphocytes of patients with head and neck cancer

Abstract

BackgroundImmunotherapy has emerged as a promising treatment modality for head and neck squamous cell carcinoma (HNSCC). Pembrolizumab, an anti-programmed death 1 antibody, is an immunotherapy agent currently approved for metastatic HNSCC and curative intent clinical trials. Although clinical responses to pembrolizumab are promising, many patients fail to respond. However, it is well known that T cell cytotoxicity and chemotaxis are critically important in the elimination of HNSCC tumors. These functions depend on ion channel activity and downstream Ca2+ fluxing abilities, which are defective in patients with HNSCC. The purpose of this study was to elucidate the effects of pembrolizumab on potassium (K+) channel (KCa3.1 and Kv1.3) activity, Ca2+ fluxes, and chemotaxis in the cytotoxic T cells of patients with HNSCC and to determine their correlation with treatment response.MethodsFunctional studies were conducted in CD8+ peripheral blood T cells (PBTs) and tumor infiltrating lymphocytes (TILs) from patients with HNSCC treated with pembrolizumab. Untreated patients with HNSCC were used as controls. The ion channel activity of CD8+ T cells was measured by patch-clamp electrophysiology; single-cell Ca2+ fluxing abilities were measured by live microscopy. Chemotaxis experiments were conducted in a three-dimensional collagen matrix. Pembrolizumab patients were stratified as responders or non-responders based on pathological response (percent of viable tumor remaining at resection; responders: ≤80% viable tumor; non-responders: >80% viable tumor).ResultsPembrolizumab increased K+ channel activity and Ca2+ fluxes in TILs independently of treatment response. However, in PBTs from responder patients there was an increased KCa3.1 activity immediately after pembrolizumab treatment that was accompanied by a characteristic increase in Kv1.3 and Ca2+ fluxes as compared with PBTs from non-responder patients. The effects on Kv1.3 and Ca2+ were prolonged and persisted after tumor resection. Chemotaxis was also improved in responder patients’ PBTs. Unlike non-responders’ PBTs, pembrolizumab increased their ability to chemotax in a tumor-like, adenosine-rich microenvironment immediately after treatment, and additionally they maintained an efficient chemotaxis after tumor resection.ConclusionsPembrolizumab enhanced K+ channel activity, Ca2+ fluxes and chemotaxis of CD8+ T cells in patients with HNSCC, with a unique pattern of response in responder patients that is conducive to the heightened functionality of their cytotoxic T cells.

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

“…PD-1 Blockade improves KCa3.1 and Kv1.3 Activity in CD8 + PBTs of HNSCC Patients Ion channels are fundamental regulators of T cell Ca 2+ signaling and effector functions (Feske et al, 2015) and we have shown that treatment of HNSCC patients with pembrolizumab increases KCa3.1 and Kv1.3 activity in CD8 + PBTs (Newton et al, 2020). Herein, we conducted in vitro experiments to study in detail the effect of pembrolizumab on ion channels in HNSCC T cells.…”

“…The G of Kv1.3 channels was determined from the same recordings by measuring the peak currents at +50 mV after subtraction of KCa3.1 currents extrapolated by linear regression. This protocol accurately record and separate KCa3.1 and Kv1.3 currents (Newton et al, 2020). LY294002 was delivered extracellular via a manual perfusion system.…”

“…Cells were then stained for rabbit anti-human STIM1 (Proteintech) primary antibodies followed by secondary antibodies (Alexa Fluor 594 goat antirabbit IgG/Thermo Fisher). Specificity of these antibodies was previously reported by our laboratory (Chimote et al, 2018;Newton et al, 2020). All flow cytometry data were collected on LSR Fortessa or LSR II flow cytometer (BD Biosciences), using the FACS Diva software v.6.0.…”

“…Furthermore, TILs of melanoma patients with high expression of PD-1 have decreased Ca 2+ responses to TCR stimulation (Chapon et al, 2011). We have recently shown that treatment of HNSCC patients with pembrolizumab, a monoclonal blocking antibody against PD1 (αPD-1), increases KCa3.1 and Kv1.3 activities in CD8 + TILs and PBTs, along with their Ca 2+ fluxing and migratory abilities (Newton et al, 2020). Furthermore, a specific pattern of K + channel reinvigoration was associated with a pathological response to therapy (Newton et al, 2020).…”

“…We have recently shown that treatment of HNSCC patients with pembrolizumab, a monoclonal blocking antibody against PD1 (αPD-1), increases KCa3.1 and Kv1.3 activities in CD8 + TILs and PBTs, along with their Ca 2+ fluxing and migratory abilities (Newton et al, 2020). Furthermore, a specific pattern of K + channel reinvigoration was associated with a pathological response to therapy (Newton et al, 2020). However, these studies did not allow us to dissect the mechanisms by which PD-1 signaling affects ion channel activity.…”