Julia D. Vavilova

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.

Targeting of adoptive immune cells is among modern approaches to therapy of solid tumors. Expression of an epidermal growth factor receptor HER2 is a common finding (20% of breast tumors) and is associated with a negative outcome. In this regard, obtaining HER2-specific effector cells carrying a chimeric CAR receptor is a relevant task. NK cells have a wide spectrum of activating receptors capable of recognizing tumor-associated markers and do not initiate a graft-versus-host reaction. The goal of this study was to obtain effector CAR-NK cells capable of eliminating the HER2-positive tumor targets. The NK cells were obtained by negative magnetic separation from peripheral mononuclear cells isolated from volunteers’ blood using density gradient centrifugation. Activated NK cells were modified by retroviral transduction. Preliminarily transfected Phoenix Ampho cells were used to accumulate retroviral particles carrying HER2- CAR construct. The DARPin 9-29-HER2 molecule with affinity for the HER2 distal domain I was used as the antigen-recognizing domain. The proportion of transduced NK cells was measured by means of GFP reporter protein expression. The surface emergence of HER2-CAR receptors was detected by expression of the c-Myc extracellular domain. All modified GFP+NK cells were capable of expressing HER2-CAR receptors on the cell membrane. Functional activity of HER2-CAR NK cells was measured using flow cytometry, by degranulation intensity and IFNγ production in the presence of HER2-positive target cells BT-474. To assess lytic activity of HER2-CAR NK cells, the cultures of HER2-CAR-expressing GFP+NK cells and unmodified GFP-NK cells were obtained by cell sorting. Lysis of BT-474 targets was measured by calcein release upon incubation with HER2-CAR and GFP- effectors. HER2-CAR NK cells were characterized by higher levels of degranulation and IFNγ production compared to GFP-NK cells. Moreover, HER2-CAR-NK cells had higher lytic activity towards BT-474. Thus, by means of genetic modification based on primary NK cells, we obtained highly effective HER2-CAR-NK agents capable of HER2-positive target recognition and possessing cytotoxic and cytokine-producing potential in presence of tumor cells. Expanding the variety of cellular effectors aimed at treating HER2-positive breast tumors will increase the potential of personalized tumor therapy in the future.

Natural killer (NK) cells are vital in the antiviral response regulated by inhibitory and activating receptors, including NKG2 and KIR families, which bind HLA‐I. While the adaptive features of NK cells in response to human cytomegalovirus (hCMV) have been well described, their behavior during Epstein‐Barr virus (EBV) infection and the influence of KIR‐HLA combinations in healthy carriers of these viruses remains unclear. We performed high‐resolution HLA genotyping, phenotypic profiling of NK cell subsets, and serological testing for hCMV and EBV‐specific IgG in 85 healthy adult donors. hCMV‐seropositive individuals exhibited significant expansions of NKG2C ⁺ and HLA‐DR ⁺ NK cell subsets, with the proportion of NKG2C ⁺ cells strongly correlating with hCMV‐IgG titers. In contrast, EBV infection was associated with increased frequencies of terminally differentiated CD56 ^dim , NKG2A ^– , CD57+ NK cells and elevated expression of inhibitory KIRs, but not NKG2C or HLA‐DR. EBV‐IgG titers correlated with CD57 and KIR2DS4 levels. Among KIR2DS4‐expressing donors, carriage of at least one HLA‐C2 allele was associated with elevated EBV‐IgGs. The precise analysis of KIR2DL2/DL3, KIR2DS4, and KIR2DL1 revealed dependencies on EBV‐IgG titers, with no associations with hCMV. These findings highlight the differential impacts of hCMV and EBV on NK cells and underscore the relevance of HLA‐KIR landscapes in shaping antiviral immunity.

The ability of NK cells to establish antigen-specific responses has been demonstrated in various infections. NK cell receptors of the diverse family of Killer-cell Immunoglobulin-like Receptors (KIR) interact with HLA class I molecules, and this interaction is peptide-dependent. The activating receptor KIR2DS4 enables NK cell degranulation following interaction with specific peptides presented within HLA-C*05. However, the mechanism underlying the differential NK cell response depending on a peptide remains poorly understood and lacks explanation based on the structure of ligand-receptor interaction. Using AlphaFold 3, we generated models of KIR2DS4-peptide-HLA-C*05 complexes to analyze the contact interfaces. We confirmed the substantial role of the aromatic ring in the 8th amino acid residue of peptide sequences in mediating interactions with KIR2DS4. Even with the same amino acid residue at position 8, different peptides exhibited variability in polar contacts with KIR2DS4. Our results may contribute to the prediction of KIR-HLA interactions and facilitate the identification of specific peptides capable of activating NK cells.

Background/Objectives: Airborne exogenous antigen inhalation can induce neutrophil infiltration of the airways, while eosinophils migrate to the airways in allergic airway inflammation. During a bacterial infection, Th2-associated cytokine IL-4, by binding to the IL-4 receptor (IL-4R), can suppress neutrophil recruitment to the site of inflammation. In the present study, we estimated whether the IL-4-dependent suppression of neutrophil recruitment contributed to the development of an immune response in asthma. Methods: Using a mouse model of Aspergillus fumigatus extract-induced allergic airway inflammation, we investigated the proportions of eosinophils and neutrophils in blood, lungs, and bone marrow over time. Bronchoalveolar lavage (BAL) fluid cytokine (including IL-4) levels and the proportions of bone marrow IL-4Rα (CD124)-expressing neutrophils were estimated. Results: We identified skewing from the neutrophil- to eosinophil-mediated immune response in the blood after five extract applications. At this point, the BAL fluid IL-4 level was not elevated, while IL-12p40 and CXCL1 levels were considerably increased. At the early stage of allergic airway inflammation, the proportions of neutrophils expressing CD124 and circulating neutrophils expressing CXCR2 (CD182) were significantly increased. Upon inflammation progression, the former remained elevated, but the latter significantly decreased. Conclusions: Thus, in allergic airway inflammation, bone marrow neutrophils become insensible to the attractive chemokine CXCL1 signals and susceptible to IL-4 effects.

Natural killer (NK) cells play a pivotal role in the immune response against viral infections, including SARS‐CoV‐2. However, our understanding of memory NK cell responses in the context of SARS‐CoV‐2 remains limited. To address this, we investigated the memory‐like response of NK cells to SARS‐CoV‐2 peptides, presented by autologous cells. Blood samples from 45 donors underwent analysis for SARS‐CoV‐2 IgG antibodies, categorizing them into four groups based on the antibody kind and level. NK cells from SARS‐CoV‐2‐experienced donors demonstrated enhanced degranulation and activation levels, IFNγ production and proliferative potential in response to SARS‐CoV‐2 peptides. Investigation of highly proliferating NK cells demonstrated the formation of distinct clusters depending on the SARS‐CoV‐2 peptide supplementation and the donor group. RNA sequencing revealed differential gene expression patterns, highlighting metabolism, protein transport, and immune response genes. Notably, KIR2DS4 expression correlated with enhanced IFNγ production, degranulation and proliferation levels, suggesting a role in SARS‐CoV‐2 recognition. Collectively, these findings provide detailed insights into antigen‐specific NK cell responses to SARS‐CoV‐2 peptides, indicating potential mechanisms underlying NK cell activation in antiviral immunity.

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.

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.

Human cytomegalovirus (hCMV) is a prevalent virus that affects a large proportion of the population worldwide. Natural Killer (NK) cells are essential immune cells that play a crucial role in controlling hCMV infection. Despite the wide spread of hCMV infection, there is still not enough data related to the association between innate and adaptive immunity. This study investigated the coordination between some of the NK cell markers expression and humoral immune response during hCMV infection. Thirty-three samples obtained from different healthy donors were investigated. The anti-hCMV IgG antibody titer was measured in serum samples, and expression of NKG2C, HLA-DR, CD57, KIR2DL2/DL3, and KIR2DL1 were analyzed in CD56⁺CD3^- cells in PBMC samples by flow cytometry. To evaluate the dependence of proportions of different NK cell subsets on IgG titers, cluster analysis was first performed on all the obtained data, resulting in the identification of four main clusters. The identified clusters demonstrated a dependence on the levels of hCMV antibodies, according to which clusters corresponding to seronegative and low-positive were grouped. The results confirmed that hCMV infection leads to an expansion of NK cell populations expressing the NKG2C marker, which correlates with higher levels of IgG response to hCMV. Besides, we identified increased HLA-DR⁺ and decreased of KIR2DL1⁺ NK cells proportions in the middle anti-CMV-IgG level group compared to samples obtained from seronegative and low-positive donors. Moreover, the statistically significant negative correlation was found between KIR2DL1⁺NK cell percentage and anti-CMV IgG antibody titer, while the positive correlation between HLA-DR⁺NK cell proportion and the IgG level was noticed only without the cluster corresponded to high level of anti-hCMV IgG. In this cohort, we did not find any association between KIR2DL3 and CD57 expression in NK cells and levels of IgG response to hCMV. This may indicate that different subsets of NK cells may have distinct roles in regulating humoral immunity to hCMV. Overall, the results of the study provide valuable insights into the coordination of NK cell marker expression and IgG response in hCMV infection.