Subpopulation Heterogeneity of NK Cells during the Genetic Modification for Subsequent Use in Targeted Therapy.

Obtaining genetically engineered NK cells is a developing area of immunotherapy. In this work, we analyzed the subset heterogeneity of NK cells subjected to retroviral transduction, taking into account the content of adaptive NK cell progenitors. It was shown that subsets KIR2DL2/DL3+, as well as CD57-KIR2DL2/DL3+NKG2C+, can be modified with greater efficiency than the corresponding subsets that do not carry the KIR2DL2/DL3 and NKG2C markers. After genetic modification, the CD57-KIR2DL2/DL3+NKG2C+ cells began to express CD57 de novo, acquiring the adaptive NK cell phenotype.

A Novel Approach to Anticancer Therapy: Molecular Modules Based on the Barnase:Barstar Pair for Targeted Delivery of HSP70 to Tumor Cells.

One important distinction between many tumor cell types and normal cells consists in the translocation of a number of intracellular proteins, in particular the 70 kDa heat shock protein (HSP70), to the surface of the plasma membrane. It has been demonstrated that such surface localization of HSP70 on tumor cells is recognized by cytotoxic effectors of the immune system, which increases their cytolytic activity. The mechanisms behind this interaction are not fully clear; however, the phenomenon of surface localization of HSP70 on cancer cells can be used to develop new approaches to antitumor immunotherapy. At the same time, it is known that the presence of HSP70 on a cell's surface is not a universal feature of cancer cells. Many types of tumor tissues do not express membrane-associated HSP70, which limits the clinical potential of these approaches. In this context, targeted delivery of exogenous HSP70 to the surface of cancer cells with the aim of attracting and activating the cytotoxic effectors of the immune system can be considered a promising means of antitumor immunotherapy. Molecular constructs containing recombinant mini-antibodies specific to tumor-associated antigens (in particular, antibodies specific to HER2/neu-antigen and other markers highly expressed on the surface of a wide range of cancer cells) can be used to target the delivery of HSP70 to tumor tissues. In order to assess the feasibility and effectiveness of this approach, recombinant constructs containing a mini-antibody specific to the HER2/ neu-antigen in the first module and HSP70 molecule or a fragment of this protein in the second module were developed in this study. Strong selective interaction between the modules was ensured by a cohesive unit formed by the barnase:barstar pair, a heterodimer characterized by an unusually high constant of association. During testing of the developed constructs in in vitro models the constructs exhibited targeted binding to tumor cells expressing the HER2/neu antigen and the agents had a significant stimulating effect on the cytotoxic activity of NK cells against the respective cancer cells.

Current Approaches to Engineering of NK Cells for Cancer Immunotherapy.

Natural Killer (NK) cells belong to a unique subtype of lymphocytes with a great potential for cancer immunotherapy due to their ability to rapidly recognize and efficiently kill tumor cells. Their anti-cancer potential can be further increased by genetic and non-genetic modifications. However, the attempts of genetic improvements of NK cells over the past 20 years have been hampered by the difficulties of gene delivery into this cell type, thus preventing researchers from producing clinically relevant numbers of viable and biologically active NK cells. Currently, several successful approaches to genetic modification of NK cells have been described, and clinically applicable cell therapy products have been characterized. Now that we understand much better the ways of NK cell optimization to enhance their tumor regression-inducing capabilities, novel approaches to engineering NK surface receptors are being developed. In this review, we focus on the advantages and perspectives of various approaches to modification of NK cells. Positive results of several preclinical studies are described, demonstrating that genetically modified NK cells can be comparable to therapeutic T cells in their efficiency of recognizing and destroying tumor targets. Moreover, using allogenic NK cells to treat a number of cancer types might have even wider and eager clinical adoption than cytotoxic T cells due to a much decreased risk of graft versus host reaction inherent in NK cell-based immunotherapeutic products.

Effect of lipopeptide structure on gene delivery system properties: Evaluation in 2D and 3D in vitro models.

Development of efficient biodegradable, environmentally responsive, biocompatible and non-toxic delivery system is needed for efficient gene delivery. As well known, properties of the vehicle are determined by the structure of carrier components. The aim of the current study was to estimate in vitro transfection efficacy of aliphatic di-, tri- and tetrapeptide-based cationic lipoplexes loaded with siRNA in function of a number of cationic groups using 2D (monolayer culture) and 3D (multicellular tumor spheroids) in vitro models. Physicochemical properties and cytotoxicity of the liposomes were found to be dependent upon a number of amino acid derivatives in an amphiphilic polar head. Uptake of liposomes loaded with nucleic acid (lipoplexes) and their localization in HEK293T cells was studied by confocal microscopy. The liposomes based on lipotripeptides had the highest transfection efficiency which was 20-fold higher than those fabricated from lipotetrapeptides.

Study of Immunomodulatory Effects of Extracellular HSP70 in a Mouse Model of Allergic Airway Inflammation.

Immunostimulatory properties of extracellular heat shock proteins 70 kDa (HSP70) became interesting for investigators a long time ago. However, in recent years a series of works showing a significant relation of the immunostimulating effects of recombinant HSP70 to contamination of the protein samples with bacterial endotoxins (lipopolysaccharide, LPS) has been published. The authors showed that intensive elimination of LPS from the protein samples resulted in inversion of immunostimulating effects of HSP70 to immunosuppressive activity of the protein. Nevertheless, at present the conception of immunostimulating, proinflammatory action of extracellular HSP70 is the most common. In this work, we studied immunomodulatory effects of exogenous HSP70 in a mouse model of allergic inflammation of airways. We also analyzed the dynamics of the level of the extracellular pool of HSP70 in the site of inflammation. The results demonstrated a considerable content of extracellular HSP70 in bronchoalveolar lavages with dynamics reflecting the stages of development of the induced inflammation. Oropharyngeal injection of exogenous HSP70 in the acute phase of allergic inflammation of airways resulted in significant suppression of the inflammatory process, which conforms to published data demonstrating an immunosuppressive activity of the extracellular pool of HSP70.

[Modulating Effect of Extracellular HSP70 on Generation of Reactive Oxigen Species in Populations of Phagocytes].

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.

Bacterial lipopolysaccharide activates CD57-negative human NK cells.

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.

[Alterations in heat shock protein 70 kDa levels in human neutrophils under the heat shock conditions].

Intracellular content of heat shock proteins of 70 kDa family (HSP70) possessing chaperone and cytoprotective functions depends on specialization and functional activity of the cells. The aim of this study was to analyze the dynamics of constitutive and inducible HSP70 levels evoked by heat shock in human neutrophils, the short-lived fraction of white blood cells providing non-specific defense against bacterial pathogens. Biphasic dynamics of the intracellular HSP70 level with an increase and following decrease in 15-30 min after the heat shock was revealed by flow cytometry. This dynamics was similar for constitutive and inducible forms of HSP70. Pre-incubation of neutrophils with cycloheximide, the inhibitor of protein synthesis, did not change the intracellular HSP70 dynamics registered by flow cytometry indicating that the increased HSP70 level detected immediately after the heat shock was not mediated by de novo protein synthesis. It was confirmed by Western blotting analysis of HSP70 intracellular content. It was suggested that the elevated HSP70 level was related to conformational HSP70 molecule changes and to increased availability of HSP70 epitopes for antibody binding. Using a panel of antibodies specific to the N-terminal ATP-binding or C-terminal substrate-binding domains of HSP70 it has been demonstrated by cell immunofluorescence and flow cytometry methods that the heat shock-associated increase of the intracellular HSP70 level was mediated by HSP70 interaction with antibodies recognizing HSP70 substrate-binding domain. It was demonstrated that the decrease of intracellular HSP70 level after heat treatment could be connected with a release of both inducible and constitutive HSP70 into extracellular space. Our data suggest that stress-induced release of HSP70 from neutrophils is regulated by ABC-transporters.

Relationship between apoptosis and expression of heat shock proteins in peripheral blood lymphocytes of patients with myocardial infarction.

We studied activity and dynamics of apoptosis of peripheral blood lymphocytes in patients with myocardial infarction and analyzed the relationship of these processes with expression of heat shock proteins with a molecular weight of 70 kDa playing an essential role in preventing cell death. Thus, we first demonstrated activation of apoptosis in peripheral blood cells of patients with myocardial infarction compared to the control (healthy individuals) and revealed the expected negative correlation between the expression of heat shock proteins with a molecular weight of 70 kDa by lymphocytes and intensity of their death. The observed dynamics of mononuclear cell apoptosis in the peripheral blood of patients with myocardial infarction can reflect activity of programmed cardiomyocyte death in the focus of ischemic injury.

Effect of delta-sleep-inducing peptide on expression of heat shock protein 70 kDa in K562 cells.

For evaluation of the stress-protective influence of delta-sleep inducing peptide we studied its effects on the system of heat-shock proteins in immune cells using the method of flow cytometry. The peptide affected the expression of heat-shock protein 70 kDa in cultured human myeloleukemia K562 cells. Delta-sleep-inducing peptide reduces accumulation of intracellular heat shock proteins 70 kDa in cells cultured under conditions of high density.

[Dynamics of apoptosis and proliferation in the rat thymus and spleen during perinatal development].

The levels of spontaneous apoptosis and proliferation of the rat thymic and spleen cells, as well as their regulation by the hypothalamo-hypophysial system were studied during perinatal development. The apoptotic and proliferating cells in the thymus and spleen were assayed using flow cytometry with the DNA-specific dye propidium iodide. The level of apoptosis in the thymus reached 25% on day 18 of embryogenesis (E 18) and decreased to 5% thereafter. In the spleen, the level of apoptosis gradually increased from 15 to 37% during the period of E18 to day 30 of postnatal development (P30). The level of dividing cells in the thymus was 20-25% at all developmental stages studied. In the spleen, it was at a maximum on E18 (32%) and decreased almost twice on E21 (17%). On P7, the amount of proliferating cells again increased to 22% and then gradually decreased to 7% by P30. The surgical ablation of hypothalamus in utero on E18 did not affect cell apoptosis or proliferation in the thymus and spleen. The surgical ablation of both hypothalamus and pituitary led a twofold decrease of the level of apoptosis in the spleen and insignificant increase of the level of proliferation in the thymus. Thus, the numbers of cells in the embryonic thymus is regulated not only by the thymus itself, but also by the hypothalamo-hypophysial system. The programmed cell death in the embryonic spleen appears to be regulated by the hypothalamo-hypophysial system as well.

[Modification of the target cell surface with lipophilic glycoconjugates and interaction of the modified cells with natural killer cells]. / Modifikatsiia poverkhnosti kletok s pomoshch'iu lipofil'nykh glikokon''iugatov i vzaimodeistvie modifitsirovannykh kletok s natural'nymi killerami.

An experimental model system involving the modification of carbohydrate composition of the target cell surface with neoglycolipids was developed for studying the role of surface carbohydrates of target cells in the NK-cell-mediated cytotoxicity. The polymeric glycoconjugates of the Glyc-PAA-PEA and Glyc-PAA(Flu)-PEA types (where Glyc was an oligosaccharide residue, PAA poly(acrylamide) polymer, and PEA the phosphatidylethanolamine residue, and Flu fluorescein residue) capable of incorporation into the cell membrane were synthesized. The optimum structures of neoglycoconjugates and conditions for their incorporation into K562 and Raji cell lines, which differ in their sensitivity to the NK-cell-mediated lysis were selected. The mechanism of association of glycoconjugates with the plasma cell membrane and the kinetics of their elimination from the cell surface were investigated using the fluorescent-labeled Glyc-PAA(Flu)-PEA derivatives. The spatial accessibility of the carbohydrate ligands for the interaction with human NK cells was demonstrated. The target cells modified with the Le(x) trisaccharide were shown to be more sensitive to the cytotoxic effect of human NK cells than the intact cells. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

Splenic cytotoxic cells recognize surface HSP70 on culture-adapted EL-4 mouse lymphoma cells.

Heat shock proteins (HSPs) are intracellular proteins which function as molecular chaperones. At the same time, translocation of HSPs to the cell surface has been observed in stressed, infected and transformed cells. It seems plausible that surface HSPs may represent molecular targets for recognition and elimination of 'altered' cells by cytotoxic lymphocytes. Previously we demonstrated that EL-4 mouse lymphoma cells growing in vitro express HSPs on their plasma membrane. In this study, we tested the hypothesis that surface HSPs present on EL-4 cells may mediate their recognition and killing by cytotoxic lymphocytes. We have found that susceptibility of culture-adapted EL-4 cells to in vitro lysis by syngeneic and allogeneic splenocytes correlated with the expression of HSP70 on EL-4 cells. Moreover, cytotoxicity was blocked by pretreatment of EL-4 target cells with anti-HSP70 antibody, whereas antibodies to MHC class I molecules and Thy1 did not have such effect. Cytotoxicity against EL-4 lymphoma was not MHC class I-restricted, and was not decreased after depletion of CD8(+) cells from the effector cell population. We conclude that in vitro killing of EL-4 cells is mediated, at least in part, by NK cells via recognition of HSPs present on the surface of tumor cells. Thus, cytotoxic response against EL-4 lymphoma should serve as a good model to study the role of HSPs in anti-tumor immunity.

Gangliosides induce cell apoptosis in the cytotoxic line CTLL-2, but not in the promyelocyte leukemia cell line HL-60.

Gangliosides induce apoptosis in the cells of the IL-2-dependent cytotoxic mouse line CTLL-2. Upon incubation with gangliosides for 24 h, their effect resulting in appearance of apoptotic cells, falls in a series GM2 > GM3 > GM1 > GD1a > GD1b > GT1b. In the presence of rIL-2, apoptosis induced by GM1 is suppressed, whereas that induced by GM2 is enhanced (the effect of intracellular agent C2-Cer is independent of this cytokine). The GM1-induced apoptosis is cancelled by the caspase I inhibitor. The gangliosides under study are not able to induce apoptosis in the promyelocyte leukemia cell line HL-60. Physiological aspects of the phenomenon found are discussed.

Immunosuppressive activity of glycosphingolipids. Influence of serum factors on ganglioside inhibition of IL-4-dependent cell proliferation.

Gangliosides have been shown to inhibit proliferation of the interleukin-4 (IL-4) responsive cell line CT.4R. Kinetic analysis has revealed that ganglioside GT1b is a competitive inhibitor of proliferation, while GM and GM3 show a mixed pattern of inhibition, i.e., exhibit more than one inhibition type. Contribution of the competitive cell inhibition for GM1 and GM3 depends on serum factors added: the higher is the percentage of FCS, the larger is the contribution of competitive inhibition. The pattern of proliferation inhibition shown for GT1b does not depend on the FCS content. We have also studied the interaction of the recombinant IL-4 with fluorescent (anthrylvinyl-labelled) gangliosides GM1 and GM3 and lactosylceramide incorporated into liposomes. Dissociation constants of the IL-4-ganglioside complexes have been determined; lactosylceramide does not interact with rIL-4. The K(d) values for the lymphokine complexes with gangliosides support the conclusion based on the kinetic analysis that IL-4 has a higher affinity for GM3 (K(d) = 5 nM) than for GM1 (K(d) = 0.28 microM).

Spontaneous apoptosis and expression of cell surface heat-shock proteins in cultured EL-4 lymphoma cells.

The expression of heat-shock proteins (HSPs) is enhanced in stressed cells and can protect cells from stress-induced injury. However, existing data about the relationship between apoptosis and HSP expression is contradictory. In this paper, a mouse lymphoma cell death model system is used to detect simultaneously both the process of apoptosis and the level of HSP expression. The model was established after discovering that spontaneous apoptosis and spontaneous cell surface HSP expression occurs in EL-4 mouse lymphoma cells during normal optimal culture conditions. The data show that apoptotic EL-4 cells had higher levels of hsp25, hsp60, hsp70 and hsp90 exposed on the plasma membrane surface than viable cells. The level of surface HSPs was found to increase through several stages of early and late apoptotic death as measured by flow cytometry, with the highest levels observed during the loss of cell membrane phospholipid asymmetry. Heat shock and actinomycin D significantly increased the proportion of apoptotic cells in culture. However, hyperthermia only stimulated a weak and temporary increase in surface HSP expression, whereas actinomycin D strongly elevated the level of surface and intracellular HSPs, particularly in live cells. These results show an associative relationship between apoptosis and HSP expression. The relationship between the progression of cell death and HSP expression suggests a role for membrane HSP expression in programmed cell death.