Коваленко Елена Ивановна

Избранные публикации

  1. Streltsova M.A., Barsov E., Erokhina S.A., Kovalenko E.I. (2017). Retroviral gene transfer into primary human NK cells activated by IL-2 and K562 feeder cells expressing membrane-bound IL-21. J. Immunol. Methods 450, 90–94 [+]

    Natural killer (NK) cells are capable of rapidly recognizing and efficiently killing tumor cells. This makes them a potentially promising agent for cancer immunotherapy. Additional genetic modifications of NK cells may further improve their anti-tumor efficacy. Numerous technical challenges associated with gene delivery into NK cells have significantly tempered this approach. We achieved efficient retroviral vector transduction of primary human NK cells that were stimulated by a combination of IL-2 and engineered K562 cells expressing membrane-bound IL-21. The activated NK cells were in less differentiated state and expressed NK cell activation receptors NKG2D, NKp30, CD16, and were highly HLA-DR-positive. This NK cell population was highly susceptible to the transduction by both GFP- and NGFR-expressing retroviral vectors, with transduction efficiency exceeding 50%. More mature CD57(+) NK cell population was generally resistant to retroviral vector transduction because of poor response to the stimulation. Our findings may facilitate retroviral vector-mediated genetic engineering of human primary NK cells for future immunotherapies.

  2. Kovalenko E.I., Streltsova M.A., Kanevskiy L.M., Erokhina S.A., Telford W.G. (2017). Identification of Human Memory-Like NK Cells. Curr Protoc Cytom 79, 9.50.1–9.50.11 [+]

    Our understanding of NK biology is increased dramatically, a product of improved flow-cytometric techniques for analyzing these cells. NK cells undergo significant changes in repertoire during differentiation. A repeating stimulus, such as a cytomegalovirus infection, may result in accumulation of certain types of highly differentiated NK cells designated as memory-like, or adaptive NK cells. Adaptive NK cells are capable of rapid expansion and effective response to the recall stimulus. These cells differ significantly from conventional NK cells both functionally and phenotypically. Here we describe an approach for identification and analysis of adaptive NK cells in human peripheral blood. CD57-positive cells with high expression of activating-receptor NKG2C, increased expression of KIR receptors, lack of co-expression with inhibitory receptor NKG2A, and decreased expression of activating receptor NCR3 (NKp30) all characterize this cell type. The flow-cytometric method described below can identify this NK cell subset on a relatively simple flow cytometer. © 2017 by John Wiley & Sons, Inc.

  3. Streltsova M.A., Klinkova A.V., Kuchukova A.A., Kadin A.Y., Kanevskiy L.M., Kovalenko E.I. (2016). Ethanol-dependent expression of the NKG2D ligands MICA/B in human cell lines and leukocytes. Biochem. Cell Biol. , 1–9 [+]

    Alcohol consumption affects the human immune system, causing a variety of disorders. However, the mechanisms of development of these changes are not fully understood. We hypothesized that ethanol may influence the expression of MICA and MICB, stress-induced molecules capable of regulating the activity of cytotoxic lymphocytes through the interaction with receptor NKG2D, which substantially affects the functionality of cellular immunity. We analyzed the effects of ethanol on MICA/B expression in tumor cell lines and human leukocytes. In the cell line models, ethanol caused different changes in the surface expression of MICA/B; in particular, it induced the translocation of intracellular proteins MICA/B to the cell surface and shedding of MICA (in soluble and microparticle-associated forms) from the plasma membrane. The observed results are not linked with cell death in cultures, taking place only under higher doses of ethanol. Ethanol at physiologically relevant concentrations (and higher) stimulated expression of MICA/B genes in different cell types. The effect of ethanol was more pronounced in hepatocyte line HepG2 compared with hematopoietic cell lines K562, Jurkat, and THP-1. Among the tested leukocytes, the most sensitive to ethanol action were T cells activated ex vivo with IL-2, in which the increase of MICA/B mRNA expression was registered with the smallest dose of ethanol (0.125%). In human monocytes, ethanol may lead to elevations in surface MICA/B levels. Presumably, changes in MICA/B expression caused by ethanol can affect the functions of NKG2D-positive cytotoxic lymphocytes, modulating immune reactions at excessive alcohol consumption.

  4. Boyko A.A., Azhikina T.L., Streltsova M.A., Sapozhnikov A.M., Kovalenko E.I. (2016). HSP70 in human polymorphonuclear and mononuclear leukocytes: comparison of the protein content and transcriptional activity of HSPA genes. Cell Stress Chaperones , [+]

    Cell-type specific variations are typical for the expression of different members of the HSP70 family. In circulating immune cells, HSP70 proteins interact with units of signaling pathways involved in the immune responses and may promote cell survival in sites of inflammation. In this work, we compared basal HSP70 expression and stress-induced HSP70 response in polymorphonuclear and mononuclear human leukocytes. The intracellular content of inducible and constitutive forms of HSP70 was analyzed in relation to the transcriptional activity of HSPA genes. Hyperthermia was used as the stress model for induction of HSP70 synthesis in the cells. Our results demonstrated that granulocytes (mainly neutrophils) and mononuclear cells differ significantly by both basal HSP70 expression and levels of HSP70 induction under hyperthermia. The differences were observed at the levels of HSPA gene transcription and intracellular HSP70 content. The expression of constitutive Hsс70 protein was much higher in mononuclear cells consisting of monocytes and lymphocytes than in granulocytes. At the same time, intact neutrophils showed increased expression of inducible Hsp70 protein compared to mononuclear cells. Heat treatment induced additional expression of HSPA genes in leukocytes. The most pronounced increase in the expression was observed in polymorphonuclear and mononuclear leukocytes for HSPA1A/B. However, in granulocytes, the induction of the transcription of the HSPA8 gene encoding the Hsc70 protein was significantly higher than in mononuclear cells. These variations in transcriptional activity of HSPA genes and intracellular HSP70 content in different populations of leukocytes may reflect specified requirements for the chaperone activity in the cells with a distinct functional role in the immune system.

  5. Клинкова А.В., Кузьмина Е.Г., Абакушина Е.В., Каневский Л.М., Неприна Г.С., Павлов В.В., Коваленко Е.И. (2016). Циркулирующий белок MICА у больных злокачественными лимфомами. Медицинская иммунология 18 (2), 151–162 ID:2030
  6. Troyanova N.I., Shevchenko M.A., Boyko A.A., Mirzoyev R.R., Pertseva M.A., Kovalenko E.I., Sapozhnikov A.M. (2015). Modulating Effect of Extracellular HSP70 on Generation of Reactive Oxigen Species in Populations of Phagocytes. Russ. J. Bioorgan. Chem. 41 (3), 271–279 [+]

    Reactive oxygen species (ROS) produced by phagocytic cells of the innate immune system play an important role in the first line of defense protecting the host from pathogens. The NADPH oxidase multi-subunit complex is the main source of ROS in all types of the phagocytes. Formation of the membrane-associated enzyme complex and its activity are dependent on many different factors controlling both intensification and suppression of the ROS production rate. However, the evidences are emerging in recent years indicating existence of poorly studied mechanisms of restriction of ROS generation level in phagocytes directed at protection of host tissues in the sites of inflammation from destruction caused by the oxygen free radicals. Our previous data and results of other authors demonstrate that a mechanism of the limitation of ROS production by phagocytes may by connected with immunomodulating activity of extracellular pool. of HSP70. In the present work, we used inhibitors of NADPH oxidase and in vitro cultures of different phagocytes to study a possible relationship between down-regulating effect of exogenous HSP70 on ROS generation and the interaction of the protein with the enzyme subunits. Our results confirmed the literature data concerning the ability of extracellular HSP70 to modulate NADPH oxidase activity and demonstrated for the first time an inhibitory effect of the protein on intracellular ROS generation in phagocytes.

  7. Israel L.L., Kovalenko E.I., Boyko A.A., Sapozhnikov A.M., Rosenberger I., Kreuter J., Passoni L., Lellouche J.P. (2015). Towards hybrid biocompatible magnetic rHuman serum albumin-based nanoparticles: use of ultra-small (CeLn)3/4+ cation-doped maghemite nanoparticles as functional shell. Nanotechnology 26 (4), ID:1152
  8. Kovalenko E.I., Boyko A.A., Semenkov V.F., Lutsenko G.V., Grechikhina M.V., Kanevskiy L.M., Azhikina T.L., Telford W.G., Sapozhnikov A.M. (2014). ROS production, intracellular HSP70 levels and their relationship in human neutrophils: effects of age. Oncotarget 5 (23), 11800–12 [+]

    ROS production and intracellular HSP70 levels were measured in human neutrophils for three age groups: young (20-59 years), elders (60-89 years) and nonagenarians (90 years and older). Elders showed higher levels of spontaneous intracellular ROS content compared with young and nonagenarian groups, which had similar intracellular ROS levels. Zymosan-induced (non-spontaneous) extracellular ROS levels were also similar for young and nonagenarians but were lower in elders. However, spontaneous extracellular ROS production increased continuously with age. Correlation analysis revealed positive relationships between HSP70 levels and zymosan-stimulated ROS production in the elder group. This was consistent with a promoting role for HSP70 in ROS-associated neutrophils response to pathogens. No positive correlation between ROS production and intracellular HSP70 levels was found for groups of young people and nonagenarians. In contrast, significant negative correlations of some ROS and HSP70 characteriscics were found for neutrophils from young people and nonagenarians. The observed difference in ROS and HSP70 correlations in elders and nonagenarians might be associated with an increased risk of mortality in older individuals less than 90 years old.

  9. Kanevskiy L.M., Erokhina S.A., Streltsova M.A., Telford W.G., Sapozhnikov A.M., Kovalenko E.I. (2014). Bacterial lipopolysaccharide activates CD57-negative human NK cells. Biochemistry Mosc. 79 (12), 1339–48 [+]

    NK cells play an important regulatory role in sepsis by induction and augmentation of proinflammatory reactions in early stages of the septic process and by suppression of immune response in later stages of inflammation. The present work was aimed at the effect of bacterial lipopolysaccharide (LPS), the main pathogenic factor of sepsis development, on human NK cells ex vivo. We show that LPS activates immature CD57-negative NK cells, which typically constitute less than half of the normal NK cell population in human peripheral blood. Under conditions of NK cell stimulation with IL-2, addition of LPS provokes an increase in IFN-γ production. However, LPS both increased and inhibited NK cell cytotoxic activity. It is important to note that the activation of NK cells on LPS addition was observed in the absence of TLR4 on the NK cell surface. These results confirm our previous data arguing for a direct interaction of LPS with NK cells and evidence an atypical mechanism of LPS-induced NK cell activation without the involvement of surface TLR4.

  10. Balabashin D., Kovalenko E., Toporova V., Aliev T., Panina A., Svirshchevskaya E., Dolgikh D., Kirpichnikov M. (2014). Production of anti TNF-α antibodies in eukaryotic cells using different combinations of vectors carrying heavy and light chains. Cytotechnology , ID:1153
  11. Бойко А.А., Ветчинин С.С., Сапожников А.М., Коваленко Е.И. (2014). Изменение уровня белков теплового шока семейства 70 кДа в нейтрофилах человека под действием теплового шока. Биоорг. хим. 40 (5), 528–540 ID:1156
  12. Kanevskiy L.M., Telford W.G., Sapozhnikov A.M., Kovalenko E.I. (2013). Lipopolysaccharide induces IFN-γ production in human NK cells. Front Immunol 4, 11 [+]

    Natural killer (NK) cells have been shown to play a regulatory role in sepsis. According to the current view, NK cells become activated via macrophages or dendritic cells primed by lipopolysaccharide (LPS). Recently, TLR4 gene expression was detected in human NK cells suggesting the possibility of a direct action of LPS on NK cells. In this study, effects of LPS on NK cell cytokine production and cytotoxicity were studied using highly purified human NK cells. LPS was shown to induce IFN-γ production in the presence of IL-2 in NK cell populations containing>98% CD56(+) cells. Surprisingly, in the same experiments LPS decreased NK cell degranulation. No significant expression of markers related to blood dendritic cells, monocytes or T or B lymphocytes in the NK cell preparations was observed; the portions of HLA-DR(-bright), CD14(+), CD3(+), and CD20(+) cells amounted to less than 0.1% within the cell populations. No more than 0.2% of NK cells were shown to be slightly positive for surface TLR4 in our experimental system, although intracellular staining revealed moderate amounts of TLR4 inside the NK cell population. These cells were negative for surface CD14, the receptor participating in LPS recognition by TLR4. Incubation of NK cells with IL-2 or/and LPS did not lead to an increase in TLR4 surface expression. TLR4(-)CD56(+) NK cells isolated by cell sorting secreted IFN-γ in response to LPS. Antibody to TLR4 did not block the LPS-induced increase in IFN-γ production. We have also shown that R(e)-form of LPS lacking outer core oligosaccharide and O-antigen induces less cytokine production in NK cells than full-length LPS. We speculate that the polysaccharide fragments of LPS molecule may take part in LPS-induced IFN-γ production by NK cells. Collectively our data suggest the existence of a mechanism of LPS direct action on NK cells distinct from established TLR4-mediated signaling.

  13. Kovalenko E., Kanevskiy L., Klinkova A., Kuchukova A., Streltsova M., Telford W., Sapozhnikov A. (2012). Stress-Induced Molecules in Regulation of NK Cell Activity. Chapter in a book “Cell Interaction” , 93–120 ID:1168
  14. Абакушина Е.В., Кузьмина Е.Г., Коваленко Е.И. (2012). Основные свойства и функции NK-клеток человека. Иммунология 33 (4), 220–225 ID:1174
  15. Kovalenko E.I., Ranjbar S., Jasenosky L.D., Goldfeld A.E., Vorobjev I.A., Barteneva N.S. (2011). The use of HaloTag-based technology in flow and laser scanning cytometry analysis of live and fixed cells. BMC Res Notes 4, 340 [+]

    Combining the technologies of protein tag labeling and optical microscopy allows sensitive analysis of protein function in cells.

  16. Mashkina A.P., Tyulina O.V., Solovyova T.I., Kovalenko E.I., Kanevski L.M., Johnson P., Boldyrev A.A. (2009). The excitotoxic effect of NMDA on human lymphocyte immune function. Neurochem. Int. 51 (6-7), 356–60 [+]

    N-Methyl-d-aspartate (NMDA)-activated glutamate receptors are expressed in lymphocytes, but their roles have not yet been defined. We show that incubation of human peripheral blood lymphocytes with NMDA resulted in increased intracellular calcium and reactive oxygen species (ROS) levels through effects on NMDA-activated glutamate receptors. In terms of ROS production, T cells were most affected, followed by NK cells, whereas B cell ROS levels were not increased. In unstimulated T and NK cells, interferon-gamma (IFN-gamma) production was unaffected by NMDA, whereas interleukin-2 stimulation of IFN-gamma production was significantly suppressed by NMDA. Simultaneous incubation of the cells with NMDA and IL-2 resulted in a dramatic increase in the amount of cells expressing the NR1 subunit of the NMDA-activated receptors. We conclude that NMDA-activated glutamate receptor activation, accompanied by the changes in intracellular calcium and ROS levels, may be involved in the modification of immune functions of human T and NK cells.

  17. Синцов А.В., Коваленко Е.И., Ханин М.А. (2008). Апоптоз, индуцированный гранзимом B. Биоорг. хим. 34 (6), 725–733 ID:1194
  18. Kovalenko E.I., Abakushina E., Telford W., Kapoor V., Korchagina E., Khaidukov S., Molotkovskaya I., Sapozhnikov A., Vlaskin P., Bovin N. (2007). Clustered carbohydrates as a target for natural killer cells: a model system. Histochem. Cell Biol. 127 (3), 313–26 [+]

    Membrane-associated oligosaccharides are known to take part in interactions between natural killer (NK) cells and their targets and modulate NK cell activity. A model system was therefore developed using synthetic glycoconjugates as tools to modify the carbohydrate pattern on NK target cell surfaces. NK cells were then assessed for function in response to synthetic glycoconjugates, using both cytolysis-associated caspase 6 activation measured by flow cytometry and IFN-gamma production. Lipophilic neoglycoconjugates were synthesized to provide their easy incorporation into the target cell membranes and to make carbohydrate residues available for cell-cell interactions. While incorporation was successful based on fluorescence monitoring, glycoconjugate incorporation did not evoke artifactual changes in surface antigen expression, and had no negative effect on cell viability. Glycoconjugates contained Le(x), sulfated Le(x), and Le(y) sharing the common structure motif trisaccharide Le(x) were revealed to enhance cytotoxicity mediated specifically by CD16 +CD56+NK cells. The glycoconjugate effects were dependent on saccharide presentation in a polymeric form. Only polymeric, or clustered, but not monomeric glycoconjugates resulted in alteration of cytotoxicity in our system, suggesting that appropriate presentation is critical for carbohydrate recognition and subsequent biological effects.

  19. Власкин П.А., Каневский Л.М., Стрельникова Ю.И., Сапожников А.М., Коваленко Е.И. (2007). Активирующее действие белка теплового шока 70 кДа на НК-клетки человека. Иммунология 28 (2), 74–79 ID:1198
  20. Коваленко Е.И., Власкин П.А., Каневский Л.М., Стрельникова Ю.И., Сапожников А.М. (2006). Влияние белков теплового шока 70 кДа на продукцию γ-интерферона натуральными киллерами человека. ДАН 406 (1), 1–4 ID:1202
  21. Коваленко Е.И., Хирова Е.В., Молотковская И.М., Овчинникова Т.В., Саблина М.А., Сапожников А.М., Хайдуков С.В., Бовин Н.В. (2004). . Модификация поверхности клеток с помощью липофильных гликоконъюгатов и взаимодействие модифицированных клеток с натуральными киллерами. Биоорг. хим. 30 (3), 281–292 ID:1216