Reduction of Ca2+ Entry by a Specific Block of KCa3.1 Channels Optimizes Cytotoxic Activity of NK Cells against T-ALL Jurkat Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents
2.2. Cell Lines and Culture Conditions
2.3. Isolation and Purification of Primary NK Cells
2.4. Electrophysiology
2.5. Activation of Primary NK and NK-92 Cells
2.6. Morphological Analysis of Activated Primary NK Cells
2.7. Intracellular Ca2+ Measurements
2.8. Degranulation Assay
2.9. Cytotoxic Activity of NK Cells
2.10. NK Mediated Cell Death
3. Results
3.1. Patch-Clamp Analysis of K+ Currents in Resting and Activated NK Cells and NK-92 Cell Line
3.1.1. Primary Human Donor NK Cells and the NK-92 Cell Line Functionally Express Kv1.3 and KCa3.1 Channels
3.1.2. Activation of NK Cells Changes Cell Morphology and Strongly Increases the Percentage of Cells Expressing Large KCa3.1 Current
3.2. Effect of KCa3.1 Block on Intracellular Ca2+ Dynamics in NK Cells and NK-92 Cell Line
3.2.1. KCa 3.1 Controls Basal [Ca2+]i Level in Resting NK Cells
3.2.2. KCa 3.1 Assists SOCE in NK Cells
3.2.3. KCa 3.1 Regulates [Ca2+]i Response of NK Cells to Target Cells
3.3. KCa 3.1 Blockade Optimizes NK Degranulation after Target Cell Recognition
3.4. KCa 3.1 Blockade Improves NK-Mediated Target Cell Killing
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
Product | Supplier | Catalog/Specification Number | Stock Concentration | Solvent |
---|---|---|---|---|
Culture media components | ||||
RPMI advanced | Gibco | 12633-012 | 1X | NS |
HEPES | Gibco | 15630-080 | 1M | H2O |
GLUTAMAX | Gibco | 35050-061 | 100X | NS |
FBS | Gibco | 0622 | NS | NS |
Antibiotic-antimycotic | Gibco | 15240-062 | 100X 103 UI/mL penicillin, 103 μg/mL streptomycin, 25 μg/mL B-amphotericin | NS |
αMEM | Gibco | 12561-056 | 1X | NS |
Horse serum | Equitech-Bio, Inc. | SE30 | NS | NS |
Myo-Inositol | Sigma | I7508-59G | 100 mM | H2O |
2-Mercaptoethanol | Sigma | M3148 | 14.3 M | NS |
Folic acid | Sigma | F8758 | 50 mg/mL | 1M NaOH in H2O |
IL2 | Stemcell | 78036.2 | 1 μg/mL | Sterile water |
IL15 | BioLegend | 570314 | 20 ng/mL | Sterile water |
Reagents for NK isolation | ||||
Ficollpaque plus o histopaque | Cytiva | 17144003 | NA | NS |
NK isolation kit | Miltenyi Biotec | 130-092-657 | NA | NA |
Cell death kit | ||||
Dead Cell Apoptosis Kit | Invitrogen by Thermo Fisher Scientific | V13245 | NA | NA |
Annexin binding buffer | Invitrogen by Thermo Fisher Scientific | V13246 | 1x | PBS |
Fluorescent dyes and antibodies | ||||
Anti-CD3-PE | Biolegend | 300308 | NS | NS |
Anti-CD56-FITC | Biolegend | 362546 | NS | NS |
Anti-CD107a-FITC | Biolegend | 326810 | NS | NS |
Fluo-4 AM | Invitrogen by Thermo Fisher Scientific | F1221 | 2 mM | DMSO |
Fura-2 AM | Invitrogen by Thermo Fisher Scientific | F14201 | 2 mM | DMSO |
CellTracker Deep Red | Invitrogen by Thermo Fisher Scientific | C34565 | 2 mM | DMSO |
Hoechst 33342 | Thermo Fisher Scientific | 62249 | 20 mM | H2O |
Blockers, salts, buffers | ||||
Monensin | Sigma-Aldrich | 22373-78-0 | 2 mM | Methanol |
NS6180 | Tocris | 4864 | 10 mM | DMSO |
MgTx | Tocris | 3563 | 10 mM | H2O |
PBS | Gibco | 70013-032 | 10X | H2O |
CaCl2 | Sigma | C-7902 | 1 M | H2O |
MgCl2 | Sigma | M-2670-100G | 1 M | H2O |
KCl | Sigma | P-5405 | NA | H2O |
EGTA | Sigma | E-4378 | 100 mM | H2O |
HEPES | Sigma | H-7523 | NA | H2O |
KOH | Sigma | P-5958 | 1 M | H2O |
NaOH | Sigma | S-0899 | 1 M | H2O |
Appendix B
Appendix C
Appendix D
Appendix E
Appendix F
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Olivas-Aguirre, M.; Cruz-Aguilar, L.H.; Pottosin, I.; Dobrovinskaya, O. Reduction of Ca2+ Entry by a Specific Block of KCa3.1 Channels Optimizes Cytotoxic Activity of NK Cells against T-ALL Jurkat Cells. Cells 2023, 12, 2065. https://doi.org/10.3390/cells12162065
Olivas-Aguirre M, Cruz-Aguilar LH, Pottosin I, Dobrovinskaya O. Reduction of Ca2+ Entry by a Specific Block of KCa3.1 Channels Optimizes Cytotoxic Activity of NK Cells against T-ALL Jurkat Cells. Cells. 2023; 12(16):2065. https://doi.org/10.3390/cells12162065
Chicago/Turabian StyleOlivas-Aguirre, Miguel, Laura Hadit Cruz-Aguilar, Igor Pottosin, and Oxana Dobrovinskaya. 2023. "Reduction of Ca2+ Entry by a Specific Block of KCa3.1 Channels Optimizes Cytotoxic Activity of NK Cells against T-ALL Jurkat Cells" Cells 12, no. 16: 2065. https://doi.org/10.3390/cells12162065
APA StyleOlivas-Aguirre, M., Cruz-Aguilar, L. H., Pottosin, I., & Dobrovinskaya, O. (2023). Reduction of Ca2+ Entry by a Specific Block of KCa3.1 Channels Optimizes Cytotoxic Activity of NK Cells against T-ALL Jurkat Cells. Cells, 12(16), 2065. https://doi.org/10.3390/cells12162065