Targeted Delivery of HSP70 to Tumor Cells via Supramolecular Complex Based on HER2-Specific DARPin9_29 and the Barnase:Barstar Pair.

(1) Background: We have previously shown that the use of an artificial supramolecular two-component system based on chimeric recombinant proteins 4D5scFv-barnase and barstar-heat shock protein 70 KDa (HSP70) allows targeted delivery of HSP70 to the surface of tumor cells bearing HER2/neu antigen. In this work, we studied the possibility to using DARPin9_29-barnase as the first targeting module recognizing HER2/neu-antigen in the HSP70 delivery system. (2) Methods: The effect of the developed systems for HSP70 delivery to human carcinomas SK-BR-3 and BT474 cells hyperexpressing HER2/neu on the activation of cytotoxic effectors of the immune cells was studied in vitro. (3) Results: The results obtained by confocal microscopy and cytofluorimetric analysis confirmed the binding of HSP70 or its fragment HSP70-16 on the surface of the treated cells. In response to the delivery of HSP70 to tumor cells, we observed an increase in the cytolytic activity of different cytotoxic effector immune cells from human peripheral blood. (4) Conclusions: Targeted modification of the tumor cell surface with molecular structures recognized by cytotoxic effectors of the immune system is among new promising approaches to antitumor immunotherapy.

Epithelial-Immune Cell Crosstalk Determines the Activation of Immune Cells In Vitro by the Human Cathelicidin LL-37 at Low Physiological Concentrations.

The only human cathelicidin, LL-37, is a host defense antimicrobial peptide with antimicrobial activities against protozoans, fungi, Gram(+) and Gram(-) bacteria, and enveloped viruses. It has been shown in experiments in vitro that LL-37 is able to induce the production of various inflammatory and anti-inflammatory cytokines and chemokines by different human cell types. However, it remains an open question whether such cytokine induction is physiologically relevant, as LL-37 exhibited its immunomodulatory properties at concentrations that are much higher (>20 µg/mL) than those observed in non-inflamed tissues (1-5 µg/mL). In the current study, we assessed the permeability of LL-37 across the Caco-2 polarized monolayer and showed that this peptide could pass through the Caco-2 monolayer with low efficiency, which predetermined its low absorption in the gut. We showed that LL-37 at low physiological concentrations (<5 µg/mL) was not able to directly activate monocytes. However, in the presence of polarized epithelial monolayers, LL-37 is able to activate monocytes through the MAPK/ERK signaling pathway and induce the production of cytokines, as assessed by a multiplex assay at the protein level. We have demonstrated that LL-37 is able to fulfill its immunomodulatory action in vivo in non-inflamed tissues at low physiological concentrations. In the present work, we revealed a key role of epithelial-immune cell crosstalk in the implementation of immunomodulatory functions of the human cathelicidin LL-37, which might shed light on its physiological action in vivo.

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.

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.

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.

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.

HLA-DR-expressing NK cells: Effective killers suspected for antigen presentation.

HLA-DR-expressing cells comprise an intriguing group of NK cells, which combine phenotypic characteristics of both NK cells and dendritic cells. These cells can be found in humans and mice; they are present in blood and tissues in healthy conditions and can expand in a spectrum of pathologies. HLA-DR+ NK cells are functionally active: they produce proinflammatory cytokines, degranulate, and easily proliferate in response to stimuli. Additionally, HLA-DR+ NK cells seem able to take in and then present certain antigens to CD4+ and CD8+ T cells, inducing their activation and proliferation, which puts them closer to professional antigen-presenting cells. It appears that these NK cells should be considerable players of the innate immune system, both due to their functional activity and regulation of the innate and adaptive immune responses. In this review, for the first time, we provide a detailed description and analysis of the available data characterizing phenotypic, developmental, and functional features of the HLA-DR+ NK cells in a healthy condition and a disease.

Extracellular heat shock proteins and cancer: New perspectives.

Heat shock proteins (HSPs) are a large family of molecular chaperones aberrantly expressed in cancer. The expression of HSPs in tumor cells has been shown to be implicated in the regulation of apoptosis, immune responses, angiogenesis and metastasis. Given that extracellular vesicles (EVs) can serve as potential source for the discovery of clinically useful biomarkers and therapeutic targets, it is of particular interest to study proteomic profiling of HSPs in EVs derived from various biological fluids of cancer patients. Furthermore, a divergent expression of circulating microRNAs (miRNAs) in patient samples has opened new opportunities in exploiting miRNAs as diagnostic tools. Herein, we address the current literature on the expression of extracellular HSPs with particular interest in HSPs in EVs derived from various biological fluids of cancer patients and different types of immune cells as promising targets for identification of clinical biomarkers of cancer. We also discuss the emerging role of miRNAs in HSP regulation for the discovery of blood-based biomarkers of cancer. We outline the importance of understanding relationships between various HSP networks and co-chaperones and propose the model for identification of HSP signatures in cancer. Elucidating the role of HSPs in EVs from the proteomic and miRNAs perspectives may provide new opportunities for the discovery of novel biomarkers of cancer.

CD56dim CD57- NKG2C+ NK cells retaining proliferative potential are possible precursors of CD57+ NKG2C+ memory-like NK cells.

Formation of the adaptive-like NK cell subset in response to HCMV infection is associated with epigenetic rearrangements, accompanied by multiple changes in the protein expression. This includes a decrease in the expression level of the adapter chain FcεRIγ, NKp30, and NKG2A receptors and an increase in the expression of NKG2C receptor, some KIR family receptors, and co-stimulating molecule CD2. Besides, adaptive-like NK cells are characterized by surface expression of CD57, a marker of highly differentiated cells. Here, it is shown that CD57-negative CD56dim NKG2C+ NK cells may undergo the same changes, as established by the similarity of the phenotypic expression pattern with that of the adaptive-like CD57+ NKG2C+ NK cells. Regardless of their differentiation stage, NKG2C-positive NK cells had increased HLA-DR expression indicating an activated state, both ex vivo and after cultivation in stimulating conditions. Additionally, CD57- NKG2C+ NK cells exhibited better proliferative activity compared to CD57+ NKG2C+ and NKG2C- NK cells, while retaining high level of natural cytotoxicity. Thus, CD57- NKG2C+ NK cells may represent a less differentiated, but readily expanding stage of the adaptive-like CD57+ NKG2C+ NK cells. Moreover, it is shown that NK cells have certain phenotypic plasticity and may both lose NKG2C expression and acquire it de novo during proliferation, induced by IL-2 and K562-mbIL21 feeder cells.

Murine Intraepithelial Dendritic Cells Interact With Phagocytic Cells During Aspergillus fumigatus-Induced Inflammation.

People are constantly exposed to airborne fungal spores, including Aspergillus fumigatus conidia that can cause life-threatening conditions in immunocompromised patients or acute exacerbations in allergics. However, immunocompetent hosts do not exhibit mycoses or systemic inflammation, due to the sufficient but not excessive antifungal immune response that prevent fungal invasion. Intraepithelial dendritic cells (IE-DCs) of the conducting airway mucosa are located in the primary site of the inhalant pathogen entry; these cells can sense A. fumigatus conidia and maintain homeostasis. The mechanisms by which IE-DCs contribute to regulating the antifungal immune response and controlling conidia dissemination are not understood. To clarify the role of IE-DCs in the balance between pathogen sensing and immune tolerance we investigated the A. fumigatus conidia distribution in optically cleared mouse lungs and estimated the kinetics of the local phagocytic response during the course of inflammation. MHCII+ antigen-presenting cells, including IE-DCs, and CD11b+ phagocytes were identified by immunohistochemistry and three-dimensional fluorescence confocal laser-scanning microscopy of conducting airway whole-mounts. Application of A. fumigatus conidia increased the number of CD11b+ phagocytes in the conducting airway mucosa and induced the trafficking of these cells through the conducting airway wall to the luminal side of the epithelium. Some CD11b+ phagocytes internalized conidia in the conducting airway lumen. During the migration through the airway wall, CD11b+ phagocytes formed clusters. Permanently located in the airway wall IE-DCs contacted both single CD11b+ phagocytes and clusters. Based on the spatiotemporal characteristics of the interactions between IE-DCs and CD11b+ phagocytes, we provide a novel anatomical rationale for the contribution of IE-DCs to controlling the excessive phagocyte-mediated immune response rather than participating in pathogen uptake.

HSP70 Multi-Functionality in Cancer.

The 70-kDa heat shock proteins (HSP70s) are abundantly present in cancer, providing malignant cells selective advantage by suppressing multiple apoptotic pathways, regulating necrosis, bypassing cellular senescence program, interfering with tumor immunity, promoting angiogenesis and supporting metastasis. This direct involvement of HSP70 in most of the cancer hallmarks explains the phenomenon of cancer "addiction" to HSP70, tightly linking tumor survival and growth to the HSP70 expression. HSP70 operates in different states through its catalytic cycle, suggesting that it can multi-function in malignant cells in any of these states. Clinically, tumor cells intensively release HSP70 in extracellular microenvironment, resulting in diverse outcomes for patient survival. Given its clinical significance, small molecule inhibitors were developed to target different sites of the HSP70 machinery. Furthermore, several HSP70-based immunotherapy approaches were assessed in clinical trials. This review will explore different roles of HSP70 on cancer progression and emphasize the importance of understanding the flexibility of HSP70 nature for future development of anti-cancer therapies.

Surface NKG2C Identifies Differentiated αßT-Cell Clones Expanded in Peripheral Blood.

T cells that express CD56 in peripheral blood of healthy humans represent a heterogeneous and poorly studied subset. In this work, we analyzed this subset for NKG2C expression. In both CD56+ and CD56- subsets most of the NKG2C+ T cells had a phenotype of highly differentiated CD8+ TEMRA cells. The CD56+NKG2C+ T cells also expressed a number of NK cell receptors, such as NKG2D, CD16, KIR2DL2/DL3, and maturation marker CD57 more often than the CD56-NKG2C+CD3+ cells. TCR ß-chain repertoire of the CD3+CD56+NKG2C+ cell fraction was limited by the prevalence of one or several clonotypes which can be found within the most abundant clonotypes in total or CD8+ T cell fraction TCRß repertoire. Thus, NKG2C expression in highly differentiated CD56+ T cells was associated with the most expanded αß T cell clones. NKG2C+ T cells produced almost no IFN-γ in response to stimulation with HCMV pp65-derived peptides. This may be partially due to the high content of CD45RA+CD57+ cells in the fraction. CD3+NKG2C+ cells showed signs of activation, and the frequency of this T-cell subset in HCMV-positive individuals was positively correlated with the frequency of NKG2C+ NK cells that may imply a coordinated in a certain extent development of the NKG2C+ T and NK cell subsets under HCMV infection.

The Role of O-Antigen in LPS-Induced Activation of Human NK Cells.

NK cells can be stimulated by bacterial lipopolysaccharides (LPS). Unlike macrophages, human NK cells do not express or express very low level of surface TLR4 receptor normally required for the LPS stimulation. This has led to the assumption that the mechanisms of stimulating action of LPS on macrophages and NK cells differs. In this work, we investigated the effects of different forms of E. coli LPS, including mutants lacking O-antigen structures, and deacylated LPS on IFNγ production by purified human NK cells. The main findings were the following: (1) NK cells were more sensitive to the S-forms of LPS than the R-forms (LPS lacking O-antigen); (2) LPS triggered a significant increase in IFNγ production by NK cells in concentrations about 1000 times higher than those that can induce cytokine production by macrophages; (3) the composition and structure of saccharide part of LPS have a strong influence on its observed effects on NK cells; and (4) LPS fully retained the ability to trigger cytokine production in NK cells in serum-free media. The acquired data demonstrated that the presence and structure of O-antigen affects the LPS-induced activation of human NK cells.

Heat Shock Protein 70 kDa as a Target for Diagnostics and Therapy of Cardiovascular and Cerebrovascular Diseases.

Acute focal ischemia is a main factor of pathogenesis of a number of widespread cardiovascular and cerebrovascular diseases, in particular, myocardial infarction and ischemic stroke. It is known that under the conditions of ischemia expression of intracellular heat shock proteins (HSPs), especially HSP70, grows greatly irrespective of the cell type. This stress-induced cell response is connected with cytoprotective properties of HSP70. The protective functions of HSP70 contribute to the cell survival under adverse conditions and inhibit development of programmed cell death. It was shown, that the level of HSP70 increases in cardiomyocytes and brain cells in response to ischemia, that was connected with cardioprotective and neuroprotective effects. Besides, in recent years, clinical studies of HSP70 have demonstrated elevated level of HSP70 in peripheral blood lymphocytes in groups of patients with ischemic stroke and myocardial infarction. This review indicates that HSP70 can serve as a target for developing new approaches to diagnostics and therapy of cardiovascular and cerebrovascular diseases.

Recurrent Stimulation of Natural Killer Cell Clones with K562 Expressing Membrane-Bound Interleukin-21 Affects Their Phenotype, Interferon-γ Production, and Lifespan.

A pattern of natural killer cell (NK cell) heterogeneity determines proliferative and functional responses to activating stimuli in individuals. Obtaining the progeny of a single cell by cloning the original population is one of the ways to study NK cell heterogeneity. In this work, we sorted single cells into a plate and stimulated them via interleukin (IL)-2 and gene-modified K562 feeder cells that expressed membrane-bound IL-21 (K562-mbIL21), which led to a generation of phenotypically confirmed and functionally active NK cell clones. Next, we applied two models of clone cultivation, which differently affected their phenotype, lifespan, and functional activity. The first model, which included weekly restimulation of clones with K562-mbIL21 and IL-2, resulted in the generation of relatively short-lived (5⁻7 weeks) clones of highly activated NK cells. Levels of human leukocyte antigen class II molecule-DR isotype (HLA-DR) expression in the expanded NK cells correlated strongly with interferon-γ (IFN-γ) production. The second model, in which NK cells were restimulated weekly with IL-2 alone and once on the sixth week with K562-mbIL21 and IL-2, produced long-lived clones (8⁻14 weeks) that expanded up to 107 cells with a lower ability to produce IFN-γ. Our method is applicable for studying variability in phenotype, proliferative, and functional activity of certain NK cell progeny in response to the stimulation, which may help in selecting NK cells best suited for clinical use.

Analysis of NK cell clones obtained using interleukin-2 and gene-modified K562 cells revealed the ability of "senescent" NK cells to lose CD57 expression and start expressing NKG2A.

In this work, we analyzed the phenotype and growth of human NK cell clones obtained by the stimulation of individual NK cells with IL-2 and gene-modified K562 feeder cells expressing membrane-bound IL-21 (K562-mbIL21). We generated clones from NK cells at distinct differentiation and activation stages, determined by CD56, CD57 and HLA-DR expression levels. Less differentiated CD56bright NK cell subsets showed higher cloning efficiency compared with more differentiated CD56dim subsets, especially with the CD57bright subset. However, clones from the CD56dimCD57- subset lived longer on average than other subsets. Moreover, several clones with the highest cell numbers were derived from CD56dimCD57-HLA-DR-cells. Most of the clones including those derived from more differentiated CD56dimCD57+/-NKG2A- NK cells showed a less-differentiated NKG2A+ phenotype. Also, CD57- cells were frequently observed in clones derived from CD57+ NK cells suggesting the loss of CD57 during the cloning process. On the other hand, KIR surface expression once detected for a clone never disappeared entirely, confirming irreversibility of the KIR expression. In summary, we have demonstrated that in specific conditions terminally differentiated CD57+ human NK cells are able to acquire the CD57- phenotype that was previously not observed and, thus, was considered impossible.