Journal: Frontiers in Pharmacology

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

doi: 10.3389/fphar.2021.742862

Figure Lengend Snippet: αPD-1 treatment increases K + channel activity in HNSCC T cells. (A) Representative current traces of KCa3.1 and Kv1.3 channels recorded in whole-cell mode of voltage-clamp configuration in activated CD8 + PBTs cells from a HNSCC patient in absence or presence of αPD-1 (10 μg/ml, for 6 h). Data are normalized to maximum current at +50 mV recorded using a ramp pulse protocol from −120 mV to +50 mV for 200 ms every 15 s. The holding potential used was −70 mV. (B,C) KCa3.1 (B) and Kv1.3 (C) conductance (G) measured in the absence or presence of αPD-1 (10 μg/ml, 6 h incubation) in CD8 + PBTs of HNSCC patients ( n = 68 cells without pembrolizumab and n = 55 cells with pembrolizumab from 14 patients). (D) Representative current traces of divalent free current (DVF) through CRAC channels recorded in whole-cell mode of voltage-clamp configuration in activated CD8 + PBTs from a HNSCC patient. Data were recorded using a ramp pulse protocol from −100 to +100 mV with at holding potential of +30 mV every 1.5 s. Cells were perfused with 0 mM Ca 2+ solution (1 min) followed by 20 mM Ca 2+ (1 min) and DVF solutions (2 min, see methods) to amplify currents during recordings. (E) Peak DVF current values measured in absence and presence of αPD-1 (10 μg/ml, 6 h incubation) in CD8 + PBTs of HNSCC patients ( n = 34 cells without αPD-1 and n = 31 cells with αPD-1 from 8 patients). The values in panels (B,C) and (E) are represented as box plots: the horizontal line indicates the median; the lower box is the 25 th percentile; the upper box is the 75 th percentile; and the whiskers represent the 10 th and 90 th percentiles. (F) Ion channel expression (KCa3.1, Kv1.3, Orai1 and STIM1) in HNSCC patient T cells after treatment with αPD-1 (10 μg/ml for 6 h). Effect of αPD-1 treatment is shown as ratio of mean fluorescence intensity (MFI, fold change) values of treatment versus control group. Data are represented as scatter plot where each symbol represents an individual patient ( n = 4–5). Horizontal line represents mean values for each group. Data in panels (B,C,E) were analyzed by Mann-Whitney rank sum test.

Article Snippet: The cells were then fixed with 1% paraformaldehyde (ThermoFisher), washed with 1x PBS and stained overnight with mouse anti-human anti-KCa3.1 biotinylated antibodies (clone 6C1, Alomone).

Techniques: Activity Assay, Incubation, Expressing, Fluorescence, MANN-WHITNEY

Journal: Frontiers in Pharmacology

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

doi: 10.3389/fphar.2021.742862

Figure Lengend Snippet: αPD-L1 treatment increases K + channel activity in HNSCC patients. (A) KCa3.1 and (B) Kv1.3 conductance values (G) measured with and without the αPD-L1 antibody, atezolizumab (1 and 10 μg/ml for 6 h) in activated CD8 + PBTs of HNSCC patients. (C) KCa3.1 and (D) Kv1.3 G measured in presence of PD-L1 and αPD-1 antibody pembrolizumab in activated CD8 + PBTs of HDs. Activated cells were treated with plate-bound PD-L1 (PD-L1-Fc 10 μg/ml) +/- αPD-1 (untreated cells were used as a control) and activated for 72 h using PMA/Ionomycin. αPD-1 was added to treatment group for 6 h. Data in the lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal lines. The lower and upper error bars represents 10 th and 90 th percentile respectively, n = 8–23 cells from 3 HNSCC patients, n = 30 cells from 6 HDs (control and PD-L1) and n = 15 cells from 3 HDs (PD-L1 + αPD-1). Five cells were recorded for each individual donor. Data in (A,C,D) were analyzed by ANOVA on ranks test ( p < 0.001) followed by Dunn’s post hoc analysis. Data in (B) were analyzed by One way ANOVA ( p < 0.001) followed by Holm-Sidak test.

Article Snippet: The cells were then fixed with 1% paraformaldehyde (ThermoFisher), washed with 1x PBS and stained overnight with mouse anti-human anti-KCa3.1 biotinylated antibodies (clone 6C1, Alomone).

Techniques: Activity Assay

Journal: Frontiers in Pharmacology

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

doi: 10.3389/fphar.2021.742862

Figure Lengend Snippet: Short-time treatment with PD-L1 decreases KCa3.1 activity in a calmodulin-independent manner. (A) Flow cytometry histogram and geometric mean fluorescence intensity (gMFI) values (B) for CaM expression in activated CD8 + PBTs from HD donors ( n = 3) in the absence and presence of PD-L1. (C) Representative recordings of KCa3.1 currents in activated CD8 + PBTs from HDs showing the effect of PD-L1 (PD-L1-Fc, 10 μg/ml) and CaM (50 µM). (D) Average normalized KCa3.1 conductance ( G , nS) measured in the absence and presence of PD-L1, with and without CaM. All conductance values are normalized to average conductance value obtained from control recordings. Cells were pre-incubated with plate-bound PD-L1 (PD-L1-Fc,10 μg/ml, for 72 h) activated using anti-CD3/CD28 antibodies and treated with or without CaM (50 µM), that was delivered intracellularly via patch pipette during recordings ( n = 15–18 cells per group from 3 HDs). The values in panel (B) are represented as bar graphs. Each symbol represent an individual HD. The values are represented as mean ± SEM. The values in panel (D) are represented as box and whisker plots. The lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal line. The lower and upper error bars represents 10 th and 90 th percentile respectively. Data in panel (B) were analyzed by t -test and data in panel (D) were analyzed by ANOVA on ranks ( p = 0.008) with Dunn’s post hoc analysis.

Article Snippet: The cells were then fixed with 1% paraformaldehyde (ThermoFisher), washed with 1x PBS and stained overnight with mouse anti-human anti-KCa3.1 biotinylated antibodies (clone 6C1, Alomone).

Techniques: Activity Assay, Flow Cytometry, Fluorescence, Expressing, Incubation, Transferring, Whisker Assay

Journal: Frontiers in Pharmacology

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

doi: 10.3389/fphar.2021.742862

Figure Lengend Snippet: Differential time-dependent involvement of PI3K and calmodulin on PD-L1 mediated inhibition of KCa3.1 channels. (A,B) Representative recordings of KCa3.1 channels in activated CD8 + PBTs from HDs showing the effect of the PI3K inhibitor LY294002 (10 µM) +/− phosphatidylinositol-3 phosphatase (PI3P) (100 nM) (A) and PD-L1 (PD-L1-Fc, 10 μg/ml) +/− PI3P (100 nM) (B). (C) Summary of the pharmacological modulation of KCa3.1 channels byLY294002 and PI3P in the absence and presence of plate bound PD-L1 in activated CD8 + PBTs of HDs. Cells were activated using anti-CD3/CD28 antibodies for 72 h. Cells were perfused with LY294002 for 15 min followed by patch clamp recordings with and without PI3P, delivered intracellularly via patch pipette ( n = eight to nine cells per group from 3 HDs). All KCa3.1 conductance (G) values are normalized to the average G of the control group (drug-free). (D) KCa3.1 G measured in absence or presence of PD-L1 in activated CD8 + PBTs of HDs. Cells were treated with plate-bound PD-L1 (PD-L1-Fc, 10 μg/ml) and activated using anti-CD3/CD28 antibodies for 120 h PI3P was delivered intracellularly via the patch pipette during the electrophysiological experiments (drug-free control). Cells were held at −70 mV, n = four to five cells per group from one HD. The values in panel (C,D) are represented as box and whiskers plot. The lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal line. The lower and upper error bars represents 10 th and 90 th percentile respectively. (E) Percentage change in mean fluorescence intensity (MFI) of ion channels (Kv1.3, KCa3.1, Orai1, Stim1) and Calmodulin (CaM) measured using flow cytometry. Each dot represents an individual HD and the horizontal black line represents the mean value. Data in panel (C) were analyzed by One Way ANOVA ( p < 0.001) followed by Holm-Sidak’s post hoc analysis. Data in (D) were analyzed by One way ANOVA followed by Holm-Sidak’s post hoc analysis.

Article Snippet: The cells were then fixed with 1% paraformaldehyde (ThermoFisher), washed with 1x PBS and stained overnight with mouse anti-human anti-KCa3.1 biotinylated antibodies (clone 6C1, Alomone).

Techniques: Inhibition, Patch Clamp, Transferring, Fluorescence, Flow Cytometry