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

Abstract

Background: Immunotherapy 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.

Methods: Functional 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).

Results: Pembrolizumab 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.

Conclusions: Pembrolizumab 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.

Keywords: T-lymphocytes; head and neck neoplasms; immunotherapy; lymphocytes; programmed cell death 1 receptor; tumor-infiltrating.