Reduction of Ca2+ Entry by a Specific Block of KCa3.1 Channels Optimizes Cytotoxic Activity of NK Cells against T-ALL Jurkat Cells

Abstract

Degranulation mediated killing mechanism by NK cells is dependent on store-operated Ca²⁺ entry (SOCE) and has optimum at moderate intracellular Ca²⁺ elevations so that partial block of SOCE optimizes the killing process. In this study, we tested the effect of the selective blocker of KCa3.1 channel NS6180 on SOCE and the killing efficiency of NK cells from healthy donors and NK-92 cells against T-ALL cell line Jurkat. Patch-clamp analysis showed that only one-quarter of resting NK cells functionally express KCa3.1 current, which increases 3-fold after activation by interleukins 15 and 2. Nevertheless, blockage of KCa3.1 significantly reduced SOCE and intracellular Ca²⁺ rise induced by IL-15 or target cell recognition. NS6180 (1 μM) decreased NK degranulation at zero time of coculture with Jurkat cells but already after 1 h, the degranulation reached the same level as in the control. Monitoring of target cell death by flow cytometry and confocal microscopy demonstrated that NS6180 significantly improved the killing ability of NK cells after 1 h in coculture with Jurkat cells and increased the Jurkat cell fraction with apoptotic and necrotic markers. Our data evidence a strong dependence of SOCE on KCa3.1 activity in NK cells and that KCa3.1 specific block can improve NK cytotoxicity.

Keywords: Jurkat cells; KCa3.1 channel; NK cells; NK-92; NK-mediated killing; acute lymphoblastic leukemia; intracellular calcium; store-operated calcium entry.

Figure A1

Representative flow cytometry dot plots demonstrating the purity of the primary NK population. (A). Gate of the cell population, FSC(Size) vs. SSC (complexity). (B). The non-stained sample was used to determine autofluorescence levels (Q4). (C). Double-stained (FITC-conjugated anti-hCD56 antibody + PE-conjugated anti-hCD3 antibody) sample: 96% of the cell population are CD3⁻CD56⁺ NK cells (Q1).

Figure A2

Typical record of K⁺ (Kv and KCa3.1) currents from an activated NK-92 cell (C = 5.5 pF). From a holding potential of −50 mV, the voltage was stepped to −150 mV and then linearly changed to +50 mV, followed by a step to −100 mV (evoking tail current or Kv deactivation). The black trace is the control, green trace is after the application of 300 nM of specific KCa3.1 blocker NS6180. Note that instead of the expected 95% block at −150 mV, an apparent current reduction was by 83% only due to a significant background (leak) current (estimated seal resistance was 1.6 GΩ). The difficulty of obtaining of tight seals with the NK-92 cell line was a constant problem.

Figure A3

(A). Representative confocal micrograph of the heterogenous Fluo-4 staining of unstimulated NK-92 cells. (B). Frequency analysis of different Fluo-4 staining patterns for NK-92 cells. (C). Fluo 4 staining patterns of T (Jurkat, CCRF-CEM, and MOLT 4) and B (Reh)-ALL cells. Scale bar represents 10 µm for A and 50 µm for C.

Figure A4

Effect of NS6180 on antiCD107a surface expression in Jurkat cells. Jurkat cells were incubated with NS6180 (10 μM) in the presence of an antiCD107a antibody (green) and monitored for 2 h by confocal microscopy in order to trace the antiCD107a staining. The green fluorescence remained in the background. Scale bar represents for A 10 µm.

Figure A5

Effect of NS6180 on Jurkat cells viability. In total, 1 × 106 cells/mL were analyzed for each condition, and NS6180 was administered for 24 h prior to the cell count.

Figure 1

The experimental strategy is based on cooperative interactions between CRAC and KCa3.1 channel. CRAC mediates an increase of cytosolic Ca²⁺, which activates KCa3.1. K⁺ efflux via KCa3.1 provokes membrane hyperpolarization, hence relatively high sustained Ca²⁺ influx by CRAC. Disruption of this interaction by the selective block of KCa3.1 should reduce CRAC activity and global Ca²⁺ signal. See text for details.

Figure 2

KCa3.1 and Kv1.3 currents in NK cells of healthy donors. (A–C) Examples of whole-cell current recordings in response to voltage ramps from −150 to +50 mV. A. Activated NK cell, mainly expressing NS6180-sensitive KCa3.1 current (KCa type). (B). Resting NK cell expressing only margatoxin-sensitive Kv1.3 current (Kv type). (C). Activated NK cells, expressing both KCa.1 and Kv1.3 currents (KCa + Kv). Dashed lines mark zero current level. (D). KCa3.1 single channel recording in the whole-cell mode. Steady state record at −100 mV (top). Unitary current–voltage relation (bottom), leak current was subtracted from recordings, displaying 1 or 2 open KCa3.1 channels. Slope conductance was calculated from linear regression (dashed line).

Figure 3

Changes in cell morphology and K⁺ current density upon activation of NK cells. (A). Representative micrographs of unstimulated primary NK cells (resting; upper micrograph) and activated primary NK cells (IL-2 and IL-15, 500UI/15 ng/mL respectively; 2 h; lower micrograph). Scale bar, 10 µm. (B,C). Time course of the change in cell size (B) and circularity (C) upon primary NK cell activation (n = 500 individual cells for each time point). One-way ANOVA was performed, followed by a Tukey’s Multiple comparison test to determine statistical significance between groups (****: <0.0001). (D). Relative distribution of KCa, Kv, KCa + Kv, and KCa/Kv −/− whole-cell current patterns in resting and activated NK cells. A significant Kv1.3 or KCa3.1 current presence was considered when the respective current density exceeded 20 pA/pF at −150 mV for KCa3.1 and +50 mV for Kv1.3. NK cells from 4 donors were analyzed. (E,F). Kv1.3 (olive) or KCa3.1 (blue) current density in resting and activated primary NK cells (E) and NK-92 cell lines (F). Open and filled circles are for NK cells from 2 different healthy donors. Independent t-test was employed to determine statistical significance (n = 51 for resting and n = 21 for activated primary NK cells; n = 23 for NK-92 either resting or activated. ns: p > 0.05, *: p < 0.05, **: p < 0.01, ****: p < 0.0001).

Figure 4

Effect of NS6180 on the basal [Ca²⁺]_i level and NK activation. (A,B). Effect of NS6180 on the basal [Ca²⁺]_i levels in NK-92 cells (A, flow cytometry; n = 3; 10,000 events per sample) and primary NK cells (B; fluorescent plate reader; n > 12; 1 × 10⁶ cells/mL/sample). (C,D). Effect of NS6180 on SOCE in primary NK (n = 4; 1 × 10⁶ cells/mL/sample by spectrofluorometry) and NK-92 cell line (n = 3; 1 × 10⁶ cells/mL/sample by fluorescent plate reader). In C, primary NK cells were preloaded with ratiometric Ca²⁺ indicator Fura-2 and fluorescence Ex.340/380 ratio was monitored (Em. 510 nm). In D, NK-92 was preloaded with Ca²⁺ indicator Fluo-4 and fluorescence intensity was measured at Ex: 475 nm, Em: 500–550 nm. (E). [Ca²⁺]_i oscillations of a primary NK cell after target cell recognition. (F) Recognition of target cells (Jurkat, stained with Deep Red) by an NK cell (stained with Fluo-4). (G–I). NS6180 effects on [Ca²⁺]_i of primary NK cells (G) or NK-92 cells (H) in response to the addition of Jurkat cells or IL-15 (I) (n = 4). Statistical analysis: (A–C). One-way ANOVA was performed, followed by a Tukey’s Multiple comparison test to determine statistical significance between groups (***: p < 0.001, ****: p < 0.0001). (G–I). Independent t-test was employed to determine statistical significance (*: p < 0.05, **: p < 0.01).

Figure 5

Effect of NS6180 on the degranulation of NK cells. (A). Effect of IL-15 (0–10 ng/mL) on NK-92 cell degranulation. Left: Fluorescent images by confocal microscopy show the CD107a surface expression in resting NK-92 cells and after stimulation with IL-15 (0.3 ng/mL, 24 h). Scale bar, 20 µm. Right: representative data of the effect of IL-15 concentration on NK-92 cells degranulation by flow cytometry (10,000 events/sample). (B). Example of NK-92 cell degranulation upon Jurkat cell recognition (1 h in coculture, E/T = 1:1). NK-92 cells were stained with Deep Red. Red arrows indicate morphological changes (cells increase their surface, and their shape becomes irregular towards the target cell) induced by the degranulation process. The blue arrow points to a single cytotoxic granule. The pink arrow points to the apoptotic body induced in the Jurkat cell. (C). Time course of primary NK cell degranulation upon target cell recognition at control conditions and in the presence of 1 and 10 µM of NS6180. Two-way ANOVA was performed to determine statistical significance (* is used to represent differences within a condition while # is employed to represent differences between conditions; *: p < 0.05, **: p < 0.01, ****: <0.0001). (D). Representative images of CD107a acquisition by target Jurkat cells in contact with primary NK cells. E stays for effector (NK) and T for target (Jurkat) cells. White arrows indicate NK granule delivery to target cells upon IS formation (2 h coculture, scale bar 10 µm). (E). CD107a is not expressed at the cell surface of Jurkat cells monoculture (see also Appendix E at larger magnification). Scale bar, 10 µm. (F). Flow cytometry analysis of the effect of NS6180 on the CD107a acquisition by Jurkat cells upon 1 h and 4 h in 1:1 coculture with primary NK cells. (n:4, One-way ANOVA was performed followed by a Tukey’s Multiple comparison test to determine statistical significance between groups; *: p < 0.05, **: p < 0.01).

Figure 6

NS6180 improves the NK-mediated killing of Jurkat cells. (A). The strategy employed to evaluate NK-induced death of target cells. (B). Estimation of Deep Red release by Jurkat cells exposed for 4 h to primary NK cells; data was collected by fluorescent plate reader. (C). Representative confocal microscopy images of Jurkat cells cocultured with primary NK cells (1:1) in the absence and presence of 1 μM of NS6180. Blue fluorescence: primary NK cells stained with Hoechst; red: propidium iodide (necrosis); green: annexin-V A488 (apoptosis), scale bar 10 µm. (D,E). Effect of NS6180 on the time course of Jurkat cells death, induced by primary NK cells in 1:1 and 3:1 T/E ratio. n > 2500 cells were analyzed by confocal microscopy for each time point. (F). Analysis of Jurkat cell death type induced by primary NK cells at 6 h. Open and filled circles are for the control coculture and NS6180-treated one, respectively (>1000 cells per condition). (G). left: Representative dot plot of the characteristics of NK and Jurkat cell populations by flow cytometry. Blue dot plots represent a cell death analysis (x-axis: apoptosis, y-axis: necrosis) of the target cells gate prior to and after coculture (left and middle, respectively) and in the coculture with NS6180, 1 µM (right). (H). Estimation of the NK-mediated Jurkat cells killing by flow cytometry (n = 3, 10,000 events/sample). Statistical analysis: (B,D,E,H). One-way ANOVA was performed, followed by a Tukey’s Multiple comparison test to determine statistical significance between groups; * was used to indicate a difference from the control, whereas # indicates a difference between conditions; *: p < 0.05, **: p < 0.01, ***: <0.001, ****: <0.0001). (F). t-test was performed (*: p < 0.05).

Figure 7

(A,B) Disruption of the positive feedback between CRAC and KCa3.1 channels by KCa3.1 specific block optimized degranulation and enhanced the killing ability of NK against Jurkat cells. Recognition of a target cell results in the assembly of CRAC/activation of SOCE. This resulted in an increase of [Ca²⁺]_i in the effector (NK) cell triggers degranulation, an excessive one in control, and a moderate one in the presence of the KCa3.1 blocker. Ca²⁺-activated KCa3.1 channels mediate K⁺ efflux and hyperpolarization, which in turn potentiates CRAC/ SOCE, thus sustaining high [Ca²⁺]_i and high activity of CRAC and KCa3.1 channels. Elimination of this positive feedback loop reduces target-induced [Ca²⁺]_i signal and makes the reserve of lytic granules available for longer times, allowing the killing of a greater number of target cells.