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.

The Biological Role and Therapeutic Potential of NK Cells in Hematological and Solid Tumors.

NK cells are an attractive target for cancer immunotherapy due to their potent antitumor activity. The main advantage of using NK cells as cytotoxic effectors over T cells is a reduced risk of graft versus host disease. At present, several variants of NK-cell-based therapies are undergoing clinical trials and show considerable effectiveness for hematological tumors. In these types of cancers, the immune cells themselves often undergo malignant transformation, which determines the features of the disease. In contrast, the current use of NK cells as therapeutic agents for the treatment of solid tumors is much less promising. Most studies are at the stage of preclinical investigation, but few progress to clinical trials. Low efficiency of NK cell migration and functional activity in the tumor environment are currently considered the major barriers to NK cell anti-tumor therapies. Various therapeutic combinations, genetic engineering methods, alternative sources for obtaining NK cells, and other techniques are aiming at the development of promising NK cell anticancer therapies, regardless of tumorigenesis. In this review, we compare the role of NK cells in the pathogenesis of hematological and solid tumors and discuss current prospects of NK-cell-based therapy for hematological and solid tumors.

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.

Reduced Immunosenescence of Peripheral Blood T Cells in Parkinson's Disease with CMV Infection Background.

Immunosenescence is a process of remodeling the immune system under the influence of chronic inflammation during aging. Parkinson's disease (PD) is a common age-associated neurodegenerative disorder and is frequently accompanied by neuroinflammation. On the other hand, cytomegalovirus (CMV), one of the most spread infections in humans, may induce chronic inflammation which contributes to immunosenescence, differentiation and the inflation of T cells and NK cells. Currently, there is no clear understanding of immunosenescence severity in PD patients infected with CMV. In this study, we analyzed differentiation stages and immunosenescence characteristics of T cells and NK cells in 31 patients with mild and moderate PD severity, 33 age-matched and 30 young healthy donors. The PD patients were 100% CMV-seropositive compared to 76% age-matched and 73% young CMV-infected healthy donors. The proportion of effector memory T cells re-expressing CD45RA, CD57+CD56- T cells and CD57+CD56+ T cells was significantly reduced in PD patients compared with CMV-seropositive age-matched healthy individuals. The CD57+CD56- T cell proportion in PD patients was similar to that of CMV-seropositive young healthy donors. Thus, PD is characterized by reduced peripheral blood T cell immunosenescence, even against the background of CMV infection.

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.

Alterations in Proteostasis System Components in Peripheral Blood Mononuclear Cells in Parkinson Disease: Focusing on the HSP70 and p62 Levels.

Parkinson disease (PD) is attributed to a proteostasis disorder mediated by α-synuclein accumulating in a specific brain region. PD manifestation is often related to extraneuronal alterations, some of which could be used as diagnostic or prognostic PD biomarkers. In this work, we studied the shifts in the expression of proteostasis-associated chaperones of the HSP70 family and autophagy-dependent p62 protein values in the peripheral blood mononuclear cells (PBMC) of mild to moderate PD patients. Although we did not detect any changes in the intracellular HSP70 protein pool in PD patients compared to non-PD controls, an increase in the transcriptional activity of the stress-associated HSPA1A/B and HSPA6 genes was observed in these cells. Basal p62 content was found to be increased in PD patients' PBMC, similarly to the p62 level in substantia nigra neural cells in PD. Moreover, the spontaneous apoptosis level was increased among PBMC and positively correlated with the p62 intracellular level in the PD group. A combined HSPA6- and p62-based analysis among 26 PD patients and 36 age-matched non-PD controls pointed out the diagnostic significance of these markers, with intermediate sensitivity and high specificity of this combination when observing patients diagnosed with PD.

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.

HLA-DR-Positive NK Cells Expand in Response to Mycobacterium Tuberculosis Antigens and Mediate Mycobacteria-Induced T Cell Activation.

NK cells play an important role in the control of tuberculosis infection: they are not only able to kill the infected cells, but also control the activity of macrophages and development of the adaptive immune response. Still, there is little information on the role of specific NK cell subsets in this network. In this study, we focused on the mycobacteria-driven responses of the NK cells expressing HLA-DR - a type of MHC class II. We have revealed that this subset is increased in the peripheral blood of patients with primary diagnosed tuberculosis, and expands in response to in vitro stimulation with ultrasonically destroyed Mycobacterium tuberculosis cells (sonicate). The expanded HLA-DR+ NK cells had less differentiated phenotype, higher proliferative activity and increased expression of NKp30 and NKp46 receptors. HLA-DR+CD56dim NK cells showed higher IFNγ production and degranulation level than the respective HLA-DR- NK cells in response to both 24 h and 7 day stimulation with sonicate, while HLA-DR+CD56bright NK cells mostly demonstarted similar high responsiveness to the same stimulating conditions as their HLA-DR-CD56bright counterparts. After preliminary incubation with destroyed mycobacteria, cytokine-activated HLA-DR-expressing NK cells were able to mediate mycobacteria-induced and HLA-DR-dependent cytokine production in autologous CD4+ T cells. Thus, functionally active HLA-DR+ cells seem to be one of the NK cell subsets providing an important link to the adaptive immunity.