Telomerase reverse transcriptase (TERT), a core part of telomerase, has been known for a long time only for its telomere lengthening function by reverse transcription of RNA template. Currently, TERT is considered as an intriguing link between multiple signaling pathways. The diverse intracellular localization of TERT corresponds to a wide range of functional activities. In addition to the canonical function of protecting chromosome ends, TERT by itself or as a part of the telomerase complex participates in cell stress responses, gene regulation and mitochondria functioning. Upregulation of TERT expression and increased telomerase activity in cancer and somatic cells relate to improved survival and persistence of such cells. In this review, we summarize the data for a comprehensive understanding of the role of TERT in cell death regulation, with a focus on the interaction of TERT with signaling pathways involved in cell survival and stress response.

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.

The NK cell exhaustion state evolving during extensive and prolonged cultivation is still one of the limitations of NK cell approaches. In this research, we transduced NK cells with the hTERT and iCasp9 genes. hTERT overexpression can prevent the functional exhaustion of NK cells during long-term cultivation, but, still, the therapeutic use of such cells is unsafe without irradiation. To overcome this obstacle, we additionally transduced NK cells with the iCasp9 transgene that enables the rapid elimination of modified cells. We compared the proliferative and functional activities of the hTERT- and/or iCasp9-modified NK cells, determined their exhaustion state and monitored the levels of EOMES and T-BET, the main NK cell transcription factors. The hTERT and iCasp9 genes were shown to affect the EOMES and T-BET levels differently in the NK cells. The EOMES⁺T-BET⁺ phenotype characterized the functionally active NK cells during two months of culture upon stimulation with IL2 and K562-mbIL21 feeder cells, which induced the greatest expansion rates of the NK cells, independently of the transgene type. On the other hand, under cytokine stimulation, the hTERT-iCasp9-NK cells displayed improved proliferation over NK cells modified with iCasp9 alone and showed an increased proliferation rate compared to the untransduced NK cells under stimulation with IL2 and IL15, which was accompanied by reduced immune checkpoint molecule expression. The individual changes in the EOMES and T-BET levels strictly corresponded to the NK cell functional activity, the surface levels of activating and inhibitory receptors along with the expansion rate and expression levels of pro-survival and pro-apoptotic genes.

Currently, a large number of studies on genetic modification of cord blood NK cells (UCB-NK) are carried out at both clinical and preclinical levels. Immunotherapy based on UCB-NK cells has great potential for antitumor therapy. However, despite having known several advantages over peripheral blood NK cells (PB- NK), including a high concentration in cord blood and low virulence rate, UCB-NK cells are predominantly characterized in the scientific literature as immature and low-functioning NK cells. In this work, we studied the phenotypic characteristics of UCB-NK cells and the possibility of stimulatory compensation of the decreased functional activity of UCB-NK cells. Our studies revealed UCB-NK cells can be characterized as poorly differentiated and weakly activated cells with high level of inhibitory receptor NKG2A and low level of activating receptor NKG2C and HLA-DR, accordingly with the literature data. Two types of stimuli were chosen to stimulate freshly isolated UCB-NK cells: 1) 100 units of IL-2; 2) combinations of 100 units IL-2 and K-562 feeder cells expressing membrane-bound IL-21 (K562-mbIL21). It was shown the degranulation (LAMP-1) and proliferative activity was higher than for parallel cultured ex vivo PB-NK cells under the same conditions for UCB-NK cells stimulated for 7 days with IL-2 + K562-mbIL21. Moreover, stimulation in the way of IL-2 + K562-mbIL21 seemed to be a more perspective way to obtain a large number of proliferatively active UCB-NK cells compared to stimulation with IL-2 only. Since genetic modification of NK cells is a promising way to improve the antitumor properties of NK cells, retroviral transduction procedure was performed to study of the stimulated UCB-NK cells. UCB-NK cells stimulated with IL-2 + K562-mbIL21 were transduced on day 8 of cultivation. In this study, we used targeted overexpression of the adaptor molecule DAP12, which is involved in the signaling of activating NK cell receptors. PB-NK cells and UCB-NK cells were transduced under the equal experimental conditions in same volume of viral particles. As a result, the transduction efficiency was found to be more than 4-fold higher for UCB-NK cells compared to PB-NK cells. Thus, UCB-NK cells appear to be a promising tool for further research in cancer immunotherapy.

Cancer progression is often accompanied with the acquisition of functional anergy by natural killer (NK) cells and their aging. As a result, persistent herpesviruses, such as hCMV and EBV, reactivate. The catalytic subunit of telomerase encoded by hTERT gene may enhance functional and proliferative activity. We aimed to elucidate if NK cells modified for sustained hTERT expression acquire these beneficial characteristics. We examined the proliferative and functional activities of hTERT-NK cells and combined observations with RNA-seq results. Thus, hTERT-NK cells were characterized with an increase in the expression levels of cell cycle genes and better proliferative activity in the third month after isolation. Increased degranulation upon K562 target cell recognition simultaneously with a higher expression level of granzyme B was observed for hTERT-NK cells. An increased level of IFNγ was also noted in hTERT-NK cells. These results reveal that hTERT-NK cells obtain additional advantages due to the stable hTERT expression. hTERT-NK cells are likely to perform high levels of functional activity over long time periods that are commonly needed during cancer treatment to perform immune surveillance and minimize relapse rates.