Background/objectives: The functional activity of a certain tumor determines the effectiveness of primary NK cells and NK-92 cell line-based cancer therapy; their therapeutic effectiveness against different tumors can vary. This work provides a direct simultaneous comparison of the cytotoxic effects of in vitro-activated peripheral NK (pNK) cells and NK-92 cells in spheroid models of BT-474, MCF7 and SKOV-3 carcinomas and uncovers the reasons for the differential effectiveness of NK cells against tumors. Methods: Tumor spheroids of similar size and shape, obtained from agarose molds, were incubated with NK-92 or pNK cells for 24 h. Tumor cell death was detected using flow cytometry or confocal microscopy. Cytokine production, granzyme B levels and NK cell degranulation analyses were performed, along with pNK and target-cell phenotypic characterization. Results: While NK-92 and pNK cells lysed BT-474 spheroids with comparably low efficiency, pNK cells were more capable of eliminating MCF7 and SKOV-3 spheroids than NK-92 cells were. The results of the functional and phenotypic analyses strongly support the participation of the NKG2D-NKG2DL pathway in pNK cell activation induced by the most sensitive cytotoxic attack on SKOV-3 spheroids, whereas the CX3CR1-CX3CL1 axis appears to be involved in the pNK reaction against MCF-7 spheroids. Conclusions: We provide a new approach for the preliminary identification of the most promising NK cell receptors that can alter the effectiveness of cancer therapy depending on the specific tumor type. Using this approach, NK-92 cells or pNK subsets can be selected for further accumulation and/or genetic modification to improve specificity and reactivity.

Nowadays, the use of genetically modified NK cells is a promising strategy for cancer immunotherapy. The additional insertion of genes capable of inducing cell suicide allows for the timely elimination of the modified NK cells. Different subsets of the heterogenic NK cell population may differ in proliferative potential, in susceptibility to genetic viral transduction, and to the subsequent induction of cell death. The CD57⁻NKG2C⁺ NK cells are of special interest as potential candidates for therapeutic usage due to their high proliferative potential and certain features of adaptive NK cells. In this study, CD57⁻ NK cell subsets differing in KIR2DL2/3 and NKG2C expression were transduced with the iCasp9 suicide gene. The highest transduction efficacy was observed in the KIR2DL2/3⁺NKG2C⁺ NK cell subset, which demonstrated an increased proliferative potential with prolonged cultivation. The increased transduction efficiency of the cell cultures was associated with the higher expression level of the HLA-DR activation marker. Among the iCasp9-transduced subsets, KIR2DL2/3⁺ cells had the weakest response to the apoptosis induction by the chemical inductor of dimerization (CID). Thus, KIR2DL2/3⁺NKG2C⁺ NK cells showed an increased susceptibility to the iCasp9 retroviral transduction, which was associated with higher proliferative potential and activation status. However, the complete elimination of these cells with CID is impeded.

Human cytomegalovirus (HCMV)-specific adaptive NK cells are capable of recognizing viral peptides presented by HLA-E on infected cells via the NKG2C receptor. Using retroviral transduction, we have generated a K562-cell-based line expressing HLA-E in the presence of the HLA-E-stabilizing peptide, which has previously shown the capacity to enhance adaptive NK cell response. The obtained K562-21E cell line was employed to investigate proliferative responses of the CD57⁻ NK cell subset of HCMV-seropositive and seronegative donors. Stimulation of CD57⁻ NK cells with K562-21E/peptide resulted in an increased cell expansion during the 12-day culturing period, regardless of the serological HCMV status of the donor. The enhanced proliferation in response to the peptide was associated with a greater proportion of CD56^brightHLA-DR⁺ NK cells. In later stages of cultivation, the greatest proliferative response to K562-21E/peptide was shown for a highly HCMV-seropositive donor. These expanded NK cells were characterized by the accumulation of CD57⁻KIR2DL2/3⁺NKG2C⁺NKG2A⁻ cells, which are hypothesized to represent adaptive NK cell progenitors. The K562-21E feeder cells can be applied both for the accumulation of NK cells as therapeutic effectors, and for the study of NK cell maturation into the adaptive state after the HLA-E peptide presentation.

NK cells co-expressing NKG2C and inhibitory KIRs represent promising effector candidates when directed against HLA-E⁺ tumors. These cells may propagate in vitro upon HCMV peptide (LFL) presentation within HLA-E by interacting cells due to KIR⁺NKG2C⁺ cell expansion or via NKG2C or KIR de novo expression. This study aimed to clarify whether these accumulation mechanisms are connected to the healthy individual’s ex vivo NK cell phenotype. The architecture of the phenotype was characterized by the distribution of markers known to be associated with NK cell differentiation and the HCMV adaptive response, including NKG2C, KIR2DL2/3, and HLA-DR. We also analyzed NK cells that differed in marker expression cultured with or without LFL in vitro. The most pronounced NKG2C expression de novo was observed in KIR⁺ NK cells, associated with a higher HLA-DR⁺ ex vivo proportion. Enhanced LFL-dependent KIR⁺NKG2C⁺ cell expansion of the CD57⁻ fraction was associated with a high content of adaptive NK cells ex vivo, while the CD57⁺KIR⁺NKG2C⁺ expansion rate correlated with a high level of NKG2A ex vivo. Moreover, LFL-activated cultures more effectively eliminated HLA-E⁺ tumor spheroids. These data are of interest both for KIR⁺NKG2C⁺ NK cell therapeutic application and for widening understanding of virus-induced NK cell repertoire modulation.