Activated NK cells are potent effectors against glioblastoma cells due to activating KIR2DS2 and KIR2DS4 - HLA ligand interactions - In vitro study
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Background: Glioblastoma (GBM) is the most malignant brain tumor, where patients' median survival is only 14,6 months after diagnosis, thus novel, effective adjuvant therapies are required. Natural Killer (NK) cells are potent effectors for adoptive immunotherapy against GBM due to their selective cytotoxicity against tumor cells. We investigated whether purified NK cells are more effective than Lymphokine Activated Killer (LAKs) cells against GBM and the role of Killer Immunoglobulin-like Receptor - HLA-ligand mismatch in the killing potency of NK cells. Methods: We used flow cytometry based in vitro cytotoxicity assays comparing killing potential of purified, resting or activated NK cells with LAK cells derived from the same healthy donors (n=8), in different effector: target ratios against patient-derived GBM cell lines (n=3) maintained in stem cell medium. The GBM cells were phenotyped for the expression of stress ligands MICA/B, ULPB -1,-2,-3, as well as HLA-A,-B-,C and HLA-G and HLA-E. Multicolor flow cytometry was used to characterize the effector populations involved in each cytotoxicity and their expression of activating and inhibitory KIR receptors. KIR genotyping of donors was performed by ssPCR and HLA-genotyping of the GBM cells was performed by SSO to determine the degree of KIR receptor-HLA ligand mismatch. K562 cell line was used as a positive control. Results: Resting NK cells major subpopulation was CD56dim/CD16+ in contrast to CD56brightCD16+ in activated NK cells. LAK cells major subpopulation was T cells (72±12,5%, 47,44±11,9% of CTL and 28±13% of Th) in addition to a minor NK cell population (18,1±6,4%), predominantly CD56bright/CD16dim. After culture, NK cells upregulated activating receptors NKp46 and NKG2D that were not abundantly expressed by the resting NK cells. Cytotoxicity assays demonstrated donor and dose dependent efficacy of NK cells against GBM cells. Activated NK cells were more cytotoxic than resting NK cells and LAK cells against the same GBM, and at similar effector: target ratios. KIR-HLA ligand mismatch determined the greater cytotoxic potential of resting NK cells while KIR2DS2 and KIR2DS4 activating receptors demonstrated an important role in the cytotoxicity efficiency of the activated NK cell. Conclusions: Activated NK cells are more potent therapeutic effectors against GBM compared to LAK cells. NK cells exhibit donor-dependent efficacy against different patient-derived GBMs due to the distinct KIR -HLA ligand mismatch between resting NK cells and GBM cells. However, for activated NK cells the activating KIR2DS2 and KIR2DS4 receptor- ligand interaction display a more important in the cytolytic capability.