CD8⁺ T cells play a role in the suppression of tumor growth and immunotherapy. Ion channels control the Ca²⁺-dependent function of CD8⁺ lymphocytes such as cytokine/granzyme production and tumor killing. Kv1.3 and KCa3.1 K⁺ channels stabilize the negative membrane potential of T cells to maintain Ca²⁺ influx through CRAC channels. We assessed the expression of Kv1.3, KCa3.1 and CRAC in CD8⁺ cells from ovarian cancer (OC) patients (n = 7). We found that the expression level of Kv1.3 was higher in patients with malignant tumors than in control or benign tumor groups while the KCa3.1 activity was lower in the malignant tumor group as compared to the others. We demonstrated that the Ca²⁺ response in malignant tumor patients is higher compared to control groups. We propose that altered Kv1.3 and KCa3.1 expression in CD8⁺ cells in OC could be a reporter and may serve as a biomarker in diagnostics and that increased Ca²⁺ response through CRAC may contribute to the impaired CD8⁺ function.

Ion channels of T cells (Kv1.3, KCa3.1, and CRAC) participate in the regulation of activation and effector functions via modulation of the Ca²⁺-dependent pathway. T cells expressing chimeric antigen receptors (CAR T cells) showed a remarkable role in anti-tumor therapy, especially in the treatment of chemotherapy-resistant liquid cancers. Nevertheless, many challenges remain to be overcome to improve the treatment for solid tumors. In this study, we assessed the expression and role of ion channels in CAR T cells. We found that HER2-specific CAR T cells had higher KCa3.1 conductance compared to the non-transduced (NT, control) cells, which was more prominent in the CD8⁺ population (CD4⁺ cell also showed elevation). Conversely, the Kv1.3 expression level was the same for all cell types (CD4⁺, CD8⁺, CAR, and NT). Single-cell Ca²⁺ imaging revealed that thapsigargin-induced SOCE via CRAC is suppressed in CD8⁺ CAR T cells, unlike for CD4⁺ and CD8⁺ NT cells. To dissect the functional role of Kv1.3 and KCa3.1, we used specific antagonists (Kv1.3: Vm24; KCa3.1: TRAM-34): the target cell elimination capacity of the CD8⁺ CAR T cells was improved either by blocking KCa3.1 or Kv1.3. These results imply that ion channels could be a target in CAR T cell immunotherapy elaboration.

Genetic modification of T cells to express chimeric antigen receptors (CAR, CAR-T cells) enable them to recognize the specific antigen on tumor surface and then eliminate the tumor. T lymphocyte ion channels such as Kv1.3, KCa3.1 and CRAC influence T cell activation and proliferation by regulating Ca2+ signaling, as well as other effector functions such as cytokine release, migration and even target cell killing. Here we established two CAR cell lines (using CEM T cell line and primary T cells) recognizing CD19 antigen on surface of Raji B and human breast cancer MCF-7 expressing CD19 cell lines. First, we exposed that KCa3.1 and Kv1.3 functional expressions of CEM cells were comparable to those in T cells, which demonstrated their suitability for primary T cell mimics. Next, we studied the tumor cell killing efficiency of CAR-T and CEM-CAR cells in monolayer and 3D spheroid tumor models. We could show that CAR expressing cells specifically eliminate tumor cells regardless of tumor models. Furthermore, the application of Kv1.3 (Vm24) and KCa3.1 (TRAM34) inhibitors significantly improved the tumor eradication efficiency for both CEM-CAR and CAR-T cells in spheroids, however, the infiltration rate was not influenced upon addition of antagonists. We could conclude that modification of Kv1.3 and KCa3.1 ion channels could contribute to a more effective immunotherapy of solid tumor.

Genetically modified chimeric antigen receptor (CAR) T cells eliminate tumors by recognizing specific antigens on the cell surface. T cell ion channels (e.g. Kv1.3, KCa3.1) influence activation, proliferation, and effector functions such as target cell killing, through the regulation of Ca2+ signaling. We showed that CAR-expressing cells (Jurkat) specifically eliminate tumor cells (Raji B cells) in monolayer culture and inhibition of KCa3.1 by TRAM34 increased the tumor cell–killing ability of KCa3.1+ Jurkat CARs. Blockage of KCa3.1 facilitated the migration of KCa3.1+ Jurkat CARs (mean speed, displacement and distance). The application of TRAM34 lowered the baseline Ca2+ level in mCherry-KCa3.1+ Jurkat CARs. Finally, TRAM34 significantly reduced the time needed to eliminate tumor cells. We concluded that expression and modification of KCa3.1 ion channels shifts the intracellular Ca2+ concentration into the range where cytotoxicity dominates. Hence, modification of KCa3.1 channels could contribute to a more effective anticancer immunotherapy.