Methodological Approaches for Increasing the Retroviral Transduction Efficiency of Primary NK Cells.

BACKGROUND: The growing attention to NK cells for cancer cell therapy is associated with the need to establish highly efficient protocols for their genetic modification, particularly by retroviral transduction. OBJECTIVE: In this work, we have optimized several stages of the retroviral-based modification process, and determined the distribution of the amino acid transporter ASCT2 between NK cell subsets. METHODS: Retroviral particles were produced using the Phoenix Ampho cell line transfected with the calcium phosphate method . We used RD114-based retroviral transduction for lymphocyte cell lines and primary NK cells. RESULTS: We have determined the optimal time to collect the RD114-pseudotyped viral supernatants resulting in the titer of viral particles required for efficient NK cell modification to be between 48 and 72 hours. Retroviral modification by retronectin-based method did not alter NK cell functional activity and cell survival. We identified differences in the Multiplicity of Infection (MOI) among cell lines that were partially associated with the ASCT2 surface expression. Cells with higher ASCT2 levels were more susceptible to transduction with RD114-pseudotyped viral particles. Higher ASCT2 expression levels were revealed in activated CD57+ and KIR2DL2DL3+ NK cells compared to their negative counterparts. CONCLUSION: Our findings provide a more nuanced understanding of NK cell transduction, offering valuable insights for improving therapeutic applications involving NK cell modification.

Antigenic Cartography of SARS-CoV-2.

Antigenic cartography is a tool for interpreting and visualizing antigenic differences between virus variants based on virus neutralization data. This approach has been successfully used in the selection of influenza vaccine seed strains. With the emergence of SARS-CoV-2 variants escaping vaccine-induced antibody response, adjusting COVID-19 vaccines has become essential. This review provides information on the antigenic differences between SARS-CoV-2 variants revealed by antigenic cartography and explores a potential of antigenic cartography-based methods (e.g., building antibody landscapes and neutralization breadth gain plots) for the quantitative assessment of the breadth of the antibody response. Understanding the antigenic differences of SARS-CoV-2 and the possibilities of the formed humoral immunity aids in the prompt modification of preventative vaccines against COVID-19.

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.

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.

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.

Short-Term Effect of SARS-CoV-2 Spike Protein Receptor-Binding Domain-Specific Antibody Induction on Neutrophil-Mediated Immune Response in Mice.

Vaccination protects against COVID-19 via the spike protein receptor-binding domain (RBD)-specific antibody formation, but it also affects the innate immunity. The effects of specific antibody induction on neutrophils that can cause severe respiratory inflammation are important, though not completely investigated. In the present study, using a mouse model mimicking SARS-CoV-2 virus particle inhalation, we investigated neutrophil phenotype and activity alterations in the presence of RBD-specific antibodies. Mice were immunized with RBD and a week after a strong antibody response establishment received 100 nm particles in the RBD solution. Control mice received injections of a phosphate buffer instead of RBD. We show that the application of 100 nm particles in the RBD solution elevates neutrophil recruitment to the blood and the airways of RBD-immunized mice rather than in control mice. Analysis of bone marrow cells of mice with induced RBD-specific antibodies revealed the increased population of CXCR2+CD101+ neutrophils. These neutrophils did not demonstrate an enhanced ability of neutrophil extracellular traps (NETs) formation compared to the neutrophils from control mice. Thus, the induction of RBD-specific antibodies stimulates the activation of mature neutrophils that react to RBD-coated particles without triggering excessive inflammation.

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.

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.