HLA-DR Expression in Natural Killer Cells Marks Distinct Functional States, Depending on Cell Differentiation Stage.

HLA-DR-positive NK cells, found in both healthy individuals and patients with different inflammatory diseases, are characterized as activated cells. However, data on their capacity for IFNγ production or cytotoxic response vary between studies. Thus, more precise investigation is needed of the mechanisms related to the induction of HLA-DR expression in NK cells, their associations with NK cell differentiation stage, and functional or metabolic state. In this work, HLA-DR-expressing NK cell subsets were investigated using transcriptomic analysis, metabolic activity assays, and analysis of intercellular signaling cascades. We demonstrated that HLA-DR+CD56bright NK cells were characterized by a proliferative phenotype, while HLA-DR+CD56dim NK cells exhibited features of adaptive cells and loss of inhibitory receptors with increased expression of MHC class II trans-activator CIITA. The activated state of HLA-DR-expressing NK cells was confirmed by higher levels of ATP and mitochondrial mass observed in this subset compared to HLA-DR- cells, both ex vivo and after stimulation in culture. We showed that HLA-DR expression in NK cells in vitro can be induced both through stimulation by exogenous IL-2 and IL-21, as well as through auto-stimulation by NK-cell-produced IFNγ. At the intracellular level, HLA-DR expression depended on the activation of STAT3- and ERK1/2-mediated pathways, with subsequent activation of isoform 3 of the transcription factor CIITA. The obtained results broaden the knowledge about HLA-DR-positive NK cell appearance, diversity, and functions, which might be useful in terms of understanding the role of this subset in innate immunity and assessing their possible implications in NK cell-based therapy.

Coordinated Loss and Acquisition of NK Cell Surface Markers Accompanied by Generalized Cytokine Dysregulation in COVID-19.

Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, is accompanied by a dysregulated immune response. In particular, NK cells, involved in the antiviral response, are affected by the infection. This study aimed to investigate circulating NK cells with a focus on their activation, depletion, changes in the surface expression of key receptors, and functional activity during COVID-19, among intensive care unit (ICU) patients, moderately ill patients, and convalescents (CCP). Our data confirmed that NK cell activation in patients with COVID-19 is accompanied by changes in circulating cytokines. The progression of COVID-19 was associated with a coordinated decrease in the proportion of NKG2D+ and CD16+ NK cells, and an increase in PD-1, which indicated their exhaustion. A higher content of NKG2D+ NK cells distinguished surviving patients from non-survivors in the ICU group. NK cell exhaustion in ICU patients was additionally confirmed by a strong negative correlation of PD-1 and natural cytotoxicity levels. In moderately ill patients and convalescents, correlations were found between the levels of CD57, NKG2C, and NKp30, which may indicate the formation of adaptive NK cells. A reduced NKp30 level was observed in patients with a lethal outcome. Altogether, the phenotypic changes in circulating NK cells of COVID-19 patients suggest that the intense activation of NK cells during SARS-CoV-2 infection, most likely induced by cytokines, is accompanied by NK cell exhaustion, the extent of which may be critical for the disease outcome.

Alterations in the CD56- and CD56+ T Cell Subsets during COVID-19.

The effectiveness of the antiviral immune response largely depends on the activation of cytotoxic T cells. The heterogeneous group of functionally active T cells expressing the CD56 molecule (NKT-like cells), that combines the properties of T lymphocytes and NK cells, is poorly studied in COVID-19. This work aimed to analyze the activation and differentiation of both circulating NKT-like cells and CD56- T cells during COVID-19 among intensive care unit (ICU) patients, moderate severity (MS) patients, and convalescents. A decreased proportion of CD56+ T cells was found in ICU patients with fatal outcome. Severe COVID-19 was accompanied by a decrease in the proportion of CD8+ T cells, mainly due to the CD56- cell death, and a redistribution of the NKT-like cell subset composition with a predominance of more differentiated cytotoxic CD8+ T cells. The differentiation process was accompanied by an increase in the proportions of KIR2DL2/3+ and NKp30+ cells in the CD56+ T cell subset of COVID-19 patients and convalescents. Decreased percentages of NKG2D+ and NKG2A+ cells and increased PD-1 and HLA-DR expression levels were found in both CD56- and CD56+ T cells, and can be considered as indicators of COVID-19 progression. In the CD56- T cell fraction, increased CD16 levels were observed in MS patients and in ICU patients with lethal outcome, suggesting a negative role for CD56-CD16+ T cells in COVID-19. Overall, our findings suggest an antiviral role of CD56+ T cells in COVID-19.

Increased Susceptibility of the CD57- NK Cells Expressing KIR2DL2/3 and NKG2C to iCasp9 Gene Retroviral Transduction and the Relationships with Proliferative Potential, Activation Degree, and Death Induction 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.

Autologous T-Cell-Free Antigen Presentation System Unveils hCMV-Specific NK Cell Response.

NK cells play a decisive role in controlling hCMV infection by combining innate and adaptive-like immune reactions. The hCMV-derived VMAPRTLFL (LFL) peptide is a potent activator of NKG2C+ NK cells. Proposed here is an autologous system of LFL stimulation without T lymphocytes and exogenous cytokines that allows us to evaluate NK-cell hCMV-specific responses in more native settings. In this model, we evaluated LFL-induced IFNγ production, focusing on signaling pathways and the degranulation and proliferation of NK cells orchestrated by microenvironment cytokine production and analyzed the transcriptome of expanded NK cells. NK cells of individuals having high anti-hCMV-IgG levels, in contrast to NK cells of hCMV-seronegative and low-positive donors, displayed increased IFNγ production and degranulation and activation levels and enhanced proliferation upon LFL stimulation. Cytokine profiles of these LFL-stimulated cultures demonstrated a proinflammatory shift. LFL-induced NK-cell IFNγ production was dependent on the PI3K and Ras/Raf/Mek signaling pathways, independently of cytokines. In hCMV-seropositive individuals, this model allowed obtaining NK-cell antigen-specific populations proliferating in response to LFL. The transcriptomic profile of these expanded NK cells showed increased adaptive gene expression and metabolic activation. The results complement the existing knowledge about hCMV-specific NK-cell response. This model may be further exploited for the identification and characterization of antigen-specific NK cells.

Obtaining Gene-Modified HLA-E-Expressing Feeder Cells for Stimulation of Natural Killer Cells.

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 CD56brightHLA-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.

Phenotypic Changes in T and NK Cells Induced by Sputnik V Vaccination.

A highly effective humoral immune response induced by the Sputnik V vaccine was demonstrated in independent studies, as well as in large-scale post-vaccination follow-up studies. However, the shifts in the cell-mediated immunity induced by Sputnik V vaccination are still under investigation. This study was aimed at estimating the impact of Sputnik V on activating and inhibitory receptors, activation and proliferative senescence markers in NK and T lymphocytes. The effects of Sputnik V were evaluated by the comparison of PBMC samples prior to vaccination, and then three days and three weeks following the second (boost) dose. The prime-boost format of Sputnik V vaccination induced a contraction in the T cell fraction of senescent CD57+ cells and a decrease in HLA-DR-expressing T cells. The proportion of NKG2A+ T cells was down-regulated after vaccination, whereas the PD-1 level was not affected significantly. A temporal increase in activation levels of NK cells and NKT-like cells was recorded, dependent on whether the individuals had COVID-19 prior to vaccination. A short-term elevation of the activating NKG2D and CD16 was observed in NK cells. Overall, the findings of the study are in favor of the Sputnik V vaccine not provoking a dramatic phenotypic rearrangement in T and NK cells, although it induces their slight temporal non-specific activation.

Telomerase Reverse Transcriptase Increases Proliferation and Lifespan of Human NK Cells without Immortalization.

NK cells are the first line of defense against viruses and malignant cells, and their natural functionality makes these cells a promising candidate for cancer cell therapy. The genetic modifications of NK cells, allowing them to overcome some of their inherent limitations, such as low proliferative potential, can enable their use as a therapeutic product. We demonstrate that hTERT-engineered NK cell cultures maintain a high percentage of cells in the S/G2 phase for an extended time after transduction, while the life span of NK cells is measurably extended. Bulk and clonal NK cell cultures pre-activated in vitro with IL-2 and K562-mbIL21 feeder cells can be transduced with hTERT more efficiently compared with the cells activated with IL-2 alone. Overexpressed hTERT was functionally active in transduced NK cells, which displayed upregulated expression of the activation marker HLA-DR, and decreased expression of the maturation marker CD57 and activating receptor NKp46. Larger numbers of KIR2DL2/3+ cells in hTERT-engineered populations may indicate that NK cells with this phenotype are more susceptible to transduction. The hTERT-modified NK cells demonstrated a high natural cytotoxic response towards K562 cells and stably expressed Ki67, a proliferation marker. Overall, our data show that ectopic hTERT expression in NK cells enhances their activation and proliferation, extends in vitro life span, and can be a useful tool in developing NK-based cancer cell therapies.