Hybridoma technology remains one of the most reliable and widely used platforms for generating highly specific monoclonal antibodies for use in diagnostics, fundamental research, and clinical practice. Moreover, the combination of unlimited proliferative capacity with the preservation of a key B lymphocyte function, antibody production, enables hybridoma cells to support a comprehensive functional evaluation of cellular responses. Thus, by integrating analysis of proliferation, viability, and productivity, hybridoma-based approaches enable the detection of differential modulatory effects and offer a nuanced assessment of compound bioactivity. This review provides a comprehensive analysis of the functional parameters of hybridoma cells: viability, proliferation, and productivity, as well as the methods used for their evaluation. The main stages of hybridoma cell generation, advances in hybridoma technology, and current applications of hybridoma cells are also reviewed. A key aspect of this review is the differential modulation of functional parameters of hybridoma cells. Modulation of culture conditions and bioactive compounds can differentially influence growth dynamics and specific antibody yield, often revealing an inverse relationship between proliferation and productivity.

Graphene oxide (GO) nanoparticles hold biomedical promise due to unique properties, but their immunomodulatory effects on phagocytes require evaluation, particularly regarding size- and coating-dependent interactions. Polyethylene glycol (PEG) coatings reduce cytotoxicity, yet long-term impacts of varied coatings remain critical. This study investigated PEGylated GO nanoparticles (P-GO) of two lateral sizes (≈100-300 nm and ≈1-1.5 μm) with linear or branched PEG coatings on THP-1 monocyte viability, apoptosis, metabolism, and wide-spectrum cytokine production. Only the larger branched PEG-coated GO (25 μg/mL) exhibited cytotoxicity after 72 h. Other variants showed no cytotoxicity but modulated THP-1 activity. Larger linear PEG-coated GO induced apoptosis within 24 h. All particles in a concentration of 25 μg/mL were internalized by/adhered to cells, suppressed ROS production, and altered cytokine profiles: TNF-α, MIP-1β, MIP-1α, and G-CSF increased, while HGF and SCGF-β decreased. Larger branched PEG-coated GO suppressed oxidative phosphorylation and glycolysis after 24 h. While a spectrum of effects of PEGylated graphene oxide on THP-1 cell functions was identified, predominantly observed at a dose of 25 μg/mL over a 24 to 72-h exposure period, no clear dependence of P-GO nanoparticle effects on THP-1 cells was observed with respect to PEG coating type (linear vs. branched) or particle size. At 5 μg/mL, P-GO caused minimal functional modulation. Thus, the study underscores the potential of low-concentration P-GO for therapeutic use while cautioning that even non-cytotoxic nanoparticles can profoundly alter immune cell behavior.

Water-soluble fullerene derivatives such as fullerenol C60(OH)24 are promising candidates for nanomedicine applications, yet their effects on innate immune cells remain poorly characterized. We investigated the interaction of fullerenol with human neutrophils isolated from healthy donors, exposed to concentrations of 0.25-200 μg/mL over 24-72 h. Using multi-parameter flow cytometry, we assessed viability, apoptosis, phagocytic activity, and intracellular reactive oxygen species (ROS) production, complemented by cell-free DPPH radical scavenging assays. Fullerenol was taken up by neutrophils in a concentration- and time-dependent manner. No significant cytotoxicity was observed up to 100 μg/mL, while viability declined at 200 μg/mL. Phagocytosis of opsonized E. coli was preserved at lower concentrations, though a statistically significant negative correlation with fullerenol concentration was detected at higher doses. In cell-free assays, fullerenol scavenged DPPH radicals with an EC50 of 48.90 ± 10.02 μg/mL, exhibiting slower kinetics than Trolox or ascorbic acid. Critically, fullerenol suppressed intracellular ROS production by >33% at 50 μg/mL following PMA stimulation of neutrophils. These findings demonstrate that fullerenol C60(OH)24 combines potent intracellular antioxidant activity with a favorable neutrophil safety profile, supporting its potential application in oxidative stress-related conditions.

Regulatory T cells (Tregs) play a key role in immune tolerance and are promising targets for treating immune-mediated diseases. This study investigated the direct effects of PEGylated graphene oxide nanoparticles (LP-GO, BP-GO at 5–25 μg/mL) and fullerenol C60(OH)24 (25–200 μg/mL) on human Treg viability and differentiation in vitro. Tregs were induced from peripheral blood CD4+ T cells using IL-2, TGF-β, and CD2/CD3/CD28 activation beads for 72 h with nanoparticles. Assessments included viability, apoptosis (Zombie aqua/Annexin V), phenotype (CD45+CD4+CD25+CD127dim/−FOXP3+), nanoparticle sorption (intrinsic fluorescence), and IL-10 production. Neither PEGylated graphene oxide nor fullerenol C60(OH)24 affected T-helper (CD4+) viability (95.35–96.15%) nor early/late apoptosis levels. Despite this, we found a decrease in the percentage of CD4+ cells in cultures exposed to 50–200 μg/mL of fullerenol C60(OH)24. The percentage and absolute number of Treg cells decreased with 100–200 μg/mL of fullerenol, while IL-10 levels declined following treatment with 200 μg/mL of the same nanoparticles. Graphene oxide nanoparticles showed virtually no localization within or on cells. However, T helper and Treg cells demonstrated concentration-dependent sorption of fullerenol C60(OH)24 at concentrations of 100–200 μg/mL without a reduction in viability. These findings demonstrate good in vitro biocompatibility of the nanoparticles at pharmacological concentrations up to 25 μg/mL, alongside the inhibition of Treg differentiation with 100–200 μg/mL of fullerenol C60(OH)24.

Fullerenols are polyhydroxylated derivatives of fullerene (C60(OH)n) with antioxidant, antiviral, and antibacterial properties and potential biomedical applications due to their solubility and biocompatibility. However, comprehensive assessment of their cytotoxicity is required, particularly regarding their effects on immune system cells. This study investigated the effects of fullerenol C60(OH)24 (MST-Nano, St. Petersburg, Russia) on the viability, apoptosis, and metabolism of THP-1 human monocytic leukemia cells. Cells were treated with concentrations ranging from 0.25 to 1000 µg/mL and incubated for 24, 48, and 72 h. Viability, apoptosis, and nanoparticle association were assessed by flow cytometry; glycolysis and mitochondrial respiration were measured after 24 h on a Seahorse XFe96 analyzer (Agilent Technologies, Santa Clara, CA, USA). Results showed that the effects of fullerenol depend on concentration and exposure time. At 24 h, 750 µg/mL increased viability, while 1000 µg/mL induced apoptosis. After 48 and 72 h, apoptosis increased at concentrations ≥750 µg/mL, with reduced viability. Nanoparticle association correlated with concentration and inversely correlated with viability but was independent of incubation time. Metabolic analysis revealed decreased glycolysis at 750 µg/mL after 24 h, while mitochondrial respiration was unaffected. Thus, our study demonstrated that fullerenol nanoparticles were safe for the THP-1 monocytic cell line up to 500 µg/mL.

Fullerenols are water-soluble spherical carbon nanoparticles derived from fullerenes. Since the discovery of this class of nanomaterials in the late 20th century, a substantial body of literature has accumulated on their properties and potential applications in many areas including biomedicine. This review analyzes existing data on the size of fullerenol nanoparticles and their internalization by cells. Results of in vitro and in vivo studies assessing toxicity of fullerenol are discussed. A dedicated section explores the potential medical applications of fullerenol, considering its pronounced antioxidant properties, high stability, and ability to exert antiviral and antitumor effects. The review primarily focuses on fullerenol C60(OH)n, but is not strictly limited to it. Despite a significant number of publications, there are no known cases of fullerenol-based drugs successfully advancing to clinical trials. In this review, the authors critically evaluate published studies, aiming to identify aspects that require further investigation in the context of fullerenol's potential biomedical applications.

Graphene oxide offers properties that make it a promising material for numerous biomedical applications, including various cancer treatment methods. Nanomaterials may overcome certain limitations of conventional chemotherapy. In cancer research, cell lines like Jurkat (T cell acute lymphoblastic leukemia cells) serve as models for investigating tumor treatment strategies. Previous studies have explored the effect of pegylated graphene oxide nanoparticles on some parameters of Jurkat cells, such as metabolism and apoptosis. This study aims to investigate how pegylated graphene oxide nanoparticles, varying in size, surface functionalization, and concentration, influence the functional activity of Jurkat cells, including cell viability, IL-2 production, and CD69 expression, both spontaneously, and following external activation. Jurkat cells were cultured with different types of graphene oxide nanoparticles (100-200 nm and 1-5 ìm; functionalized with linear and branched polyethylene glycol (PEG)) at concentrations of 5 ìg/ mL and 25 ìg/ mL for 24 hours. For assessment of cell parameters under stimulation with different types of particles, phytohemagglutinin (PHA, 50 ìg/mL) was used. Subsequently, the cells were stained with Zombie Aqua and CD69-APC antibody, followed by flow cytometry analysis (CytoFlex S) to determine the percentage of viable cells and CD69-expressing cells. The presence of IL-2 in cell culture supernatants was quantified using ELISA tests. It was observed that nanoparticles at low concentrations did not induce cytotoxic effects; cell viability improved after PHA stimulation. Small particles (100-200 nm) coated with linear PEG induced IL-2 production and CD69 expression. However, 1-5 ìm graphene oxide modified with branched PEG at a concentration of 25 ìg/mL led to a decrease in CD69 expression following PHA-stimulation. It was shown for the first time that pegylated graphene oxide nanoparticles affect the functional activity of Jurkat cells. The influence of particles is dependent on the size, concentration, surface functionalization of graphene oxide, and activation by PHA.

Objective. The range of peptide drugs is expanding, but no drug with immunosuppressive activity has yet been found. Considering the fact that the trophoblastic β1-glycoprotein (PSG) is a fetoplacental protein with immunosuppressive activity, short peptide fragments of this protein were studied in the formation of an immune response in a situation of allogeneic cell transplantation. To study the effect of PSG peptides (YECE, YQCE, YVCS, and YACS) on the levels of peripheral and local T-regulatory cells (Treg) during the formation of an immune response to the introduction of allogeneic bone marrow cells (BM) in a dynamic experiment on Wistar rats. Materials and methods. We used an original host-versus-graft model in male Wistar rats without preconditioning of recipients. Animals were injected with PSG peptide fragment composition against a background of allogeneic intraperitoneal transplantation of BM cells in a dynamic experiment, in which the following parameters were evaluated: the level of peripheral "true" Tregs (CD4+CD25+FOXP3+), CD4+CD25-FOXP3+ cells, and FOXP3 expression in mesenteric lymph nodes. Material was collected on days 3 and 21 of the experiment. Results. PSG peptide administration against a background of allogeneic BM cells was found to reduce the absolute and relative amount of Treg in the peripheral blood of rats on days 3 and 21 of the experiment. PSG peptides against the background of the introduction of allogeneic BM cells reduced the absolute and relative amounts of CD4+CD25-FOXP3+ cells on the day 3 of the experiment. The introduction of PSG peptides against the background of the introduction of BM cells resulted in a relative decrease in FOXP3 expression in the T zone of mesenteric lymph nodes on the day 21 of the experiment. Conclusions. Thus, the PSG peptides did not have the expected effect on the level of peripheral and local Treg cells; moreover, the presence of the peptides led to a decrease in the number of these cells.

Amniotic variant of glycodelin (GdA) has pronounced immunomodulatory properties, participating in the formation of immune tolerance during pregnancy. We investigated the effect of glycodelin on the level of T regulatory lymphocytes (Treg) and the level of acute phase proteins (α-2-macroglobulin (α-2M), orosomucoid, C-reactive protein (CRP)) upon administration of allogeneic red bone marrow (BM) cells to Wistar rats in a dynamic experiment in vivo. It was found that the introduction of GdA in animal whith allogeneic BM led to an increase in the proportion of peripheral Treg among CD4+ lymphocytes at the end of the experiment (on the 21st day) in comparison with the group that was injected with BM. It was shown that glycodelin reduced the level of CRP and α-2M, but increased the level of orosomucoid in the serum of experimental animals at the beginning of the experiment (day 3), however, by the end of the experiment (day 21), normalization of protein values was observed in all groups of experimental animals acute phase to the level of intact animals. Thus, glycodelin is able to realize an immunosuppressive effect on allogeneic cells through an increase in the level of Treg and orosomucoid, as well as a decrease in the concentration of CRP and α-2M.

Pregnancy-specific β1-glycoprotein (PSG), one of the most important proteins of pregnancy, has a pronounced immunosuppressive effect. Short peptides of PSG, the so-called SLiMs (short linear motifs), are promising molecules for mild immunosuppression. We studied in vitro effect of short PSG peptides (YACS, YQCE, YVCS, and YECE) on differentiation and cytokine profile of human T-regulatory lymphocytes (Treg). T helpers isolated from the peripheral blood and polarized into the Treg phenotype with a T-cell activator (anti-CD2/3/28) and the cytokines IL-2 and transforming grown factor β (TGFβ) were used. PSG peptides were shown to have no direct modulatory effect on Treg differentiation in a culture of CD4+ cells polarized to the Treg phenotype. At the same time, PSG peptides had no effect on the viability and number of CD4+ cells in the in vitro culture. PSG peptides also had no effect on the levels of TNFα, IL-8, IL-2, macrophage inflammatory protein 1β, IL-17, IL-10, IL-6, granulocyte-macrophage CSF, monocyte chemoattractant protein 1, IL-13, IL-5, IL-7, IL-12(p70), IL-1β, granulocyte CSF, IL-4, but decreased IFNγ levels. The observed ability of the YQCE peptide to reduce the production of this proinflammatory Th1 cytokine by T helper cells can be interpreted as a positive effect. Our findings can be used for further development of safe peptide drugs based on SLiMs sequences.

Glycodelin (Gd) has pronounced immunomodulatory properties and participates in the development of immune tolerance during pregnancy. The role of recombinant Gd in physiological (0.2 and 2 μg/mL) and superphysiological (10 μg/mL) concentrations in the regulation of differentiation and functional activity of human myeloid-derived suppressor cells (MDSCs) was investigated in vitro. MDSCs were obtained from peripheral blood CD11b+ cells of healthy donors by two-step induction (IL-1β + granulocyte–monocyte colony-stimulating factor (GM-CSF) and lipopolysaccharide). The effect of Gd on the level of polymorphonuclear MDSCs (PMN-MDSCs) and monocyte MDSCs (M-MDSCs) was assessed. The intracellular level of indoleamine 2,3-dioxygenase (IDO) and arginase 1 (Arg1), as well as the cytokine profile in cultures of these cells, was measured. In general, the conversion of CD11b+ cells into MDSCs has the following features: as a result of cytokine induction, predominantly M-MDSCs are generated, but not PMN-MDSCs, and the level of Arg1 is practically not detected. It was found that Gd increased the number of M-MDSCs at concentrations of 2 and 10 μg/mL. It was shown that Gd did not affect the content of Arg1, but increased the number of MDSCs expressing IDO (10 μg/mL). Gd also modulated the cytokine profile of CD11b+ cells (at a physiological concentration of 2 μg/mL), suppressing IL-19, IL-26, and TWEAK/TNFsF12 production and, at a supraphysiological concentration, the production of IFN-α2 and IL-26.

Scientific and technological progress contributes to the discovery and production of innovative materials. The emergence of graphene is a clear example of this. Graphene is considered a promising material for use in nanobiomedicine and nanobiotechnology. It is therefore important to understand how it affects human immune cells. In a study, the effects of 5 and 25 μg/mL graphene oxide nanoparticles with lateral sizes of 100-200 nm and 1-5 μm, modified with linear and branched polyethylene glycol, on human neutrophils were investigated. The formation of reactive oxygen species was evaluated with a lucigenin as a chemiluminescence activator.Inaddition, we investigated theeffect of a 60-minute incubation of neutrophils with pegylated graphene oxide nanoparticles on the viability of these cells by staining with trypan blue and a 30-minute incubation on the uptake of fluorescein isocyanate-labelled E. coli. The percentage of neutrophils which engulfed E. coli and the uptake index were determined. Samples without added nanoparticles served as controls.A decrease in lucigenin-enhanced chemiluminescence of neutrophils was observed under the influence of two types of graphene oxide nanoparticles: 1-5 μm in size coated with linear polyethylene glycol, and 100-200 nm in size coated with branched polyethylene glycol, at a concentration of 25 μg/mL in the zymosan-stimulated version of the assay. No dependence of the effect on the particle size and the type of polyethylene glycol was observed. The indicators for spontaneous chemiluminescence of neutrophils did not change with the addition of PEGylated graphene oxide nanoparticles.A thirty-minute incubation of human neutrophils at 37 °C with PEGylated graphene oxide nanoparticles with lateral dimensions of 100-200 nm and 1-5 μm had no effect on the viability of these cells and on the percentage of neutrophils that engulfed E. coli. However, 1-5 μm graphene oxide modified with linear polyethylene glycol at a concentration of 25 μg/mL increased the amount of E. coli engulfed by neutrophils per cell.Thus, in the absence of cytotoxicity, PEGylated graphene oxide particles have multidirectional immunomodulatory effects on neutrophils. In this case, their concentration is decisive and not the size of the graphene oxide particles and the type of polyethylene glycol.

We investigated the direct effect of PEGylated graphene oxide (P-GO) nanoparticles on the differentiation, viability, and cytokine profile of activated T helper type 17 (Th17) in vitro. The subject of the study were cultures of “naive” T-helpers (CD4+) isolated by immunomagnetic separation and polarized into the Th17 phenotype with a TCR activator and cytokines. It was found that P-GO at low concentrations (5 µg/mL) had no effect on the parameters studied. The presence of high concentrations of P-GO in T-helper cultures (25 μg/mL) did not affect the number and viability of these cells. However, the percentage of proliferating T-helpers in these cultures was reduced. GO nanoparticles modified with linear polyethylene glycol (PEG) significantly increased the percentage of Th17/22 cells in cultures of Th17-polarized T helpers and the production of IFN-γ, whereas those modified with branched PEG suppressed the synthesis of IL-17. Thus, a low concentration of PEGylated GO nanoparticles (5 μg/mL), in contrast to a concentration of 25 μg/mL, has no effect on the Th17-polarization of T helpers, allowing their further use for in-depth studies of the functions of T lymphocytes and other immune cells. Overall, we have studied for the first time the direct effect of P-GO nanoparticles on the conversion of T helper cells to the Th17 phenotype.

Glycodelins, the glycosylated proteins of reproductive tract are characterized by immunomodulatory functions, are of interest because of their role in the development of immune tolerance. Interleukin-17-producing T helpers (Th17) bearing the surface marker CCR6, are a heterogeneous cell population with increased plasticity and functional dichotomy. On the one hand, these cells support antimicrobial and antifungal immunity and microbiota composition; on the other hand, they are involved in the pathogenesis of autoimmune diseases, graft rejection, and pregnancy complications. Despite the scientific interest in glycodelin as an immunomodulator, its direct effects on pro-inflammatory Th17 have not been studied. Therefore, the aim of our work was to investigate the effect of recombinant human glycodelin on Th17 polarization of naïve human T helper cells cells by assessing surface expression of CCR6, CCR4, and CXCR3 molecules. Naïve T helper cells were polarized for 7 days in vitro to Th17 cells with a TCR activator and cytokines for 7 days, supplemented with glycodelin at concentrations appropriate for the 1st and 2nd trimesters of pregnancy. The percentages of CD4+CCR6+ cell population (Th17 cells), and their CCR4+CXCR3-(Th17/Th22) and CCR4-CXC3+ subpopulations (Th17.1) was then determined. Moreover, the levels of IL-17, IL-2, and other cytokines/chemokines were determined in the culture supernatants of Th17-polarized T helper cells. Treatment with recombinant glycodelin at concentrations equivalent to those in pregnancy (0.2, 2, and 10 μg/mL) did not alter the percentage of CD4+CCR6+ cells in culture, or their IL-17 production. However, at a concentration of 10 μg/mL, it caused a decrease in Th17.1 (CCR6+CCR4-CXCR3+) percentage in the T helper culture, and increased the production of IL-2. In addition, glycodelin was found to have selective pro-apoptotic activity against Th17.1 if applied at 2 μg/mL. Given the known involvement of these cells in pathological processes, the observed effect of glycodelin could be of interest from a biopharmaceutical perspective. However, the mechanism of the revealed selective effects of this pregnancy protein needs further investigation.

The interaction of graphene oxide nanoparticles with human peripheral blood mononuclear cells was studied using the Cell-IQ continuous monitoring system for living cells. We used graphene oxide nanoparticles of various sizes coated with linear or branched polyethylene glycol (PEG) in concentrations of 5 and 25 μg/ml. After 24-h incubation with graphene oxide nanoparticles, the increase in the number of peripheral blood mononuclear cells at visualization points decreased; nanoparticles coated with branched PEG more markedly suppressed cell growth in culture. In the presence of graphene oxide nanoparticles, peripheral blood mononuclear cells retained high viability in culture after daily monitoring in the Cell-IQ system. The studied nanoparticles were engulfed by monocytes and the type of PEGylation had no effect on this process. Thus, graphene oxide nanoparticles reduced the increase in peripheral blood mononuclear cell mass during dynamic observation in the Cell-IQ system without reducing their viability.

We studied the role of alpha-fetoprotein (AFP) in regulation of differentiation and functional activity of human myeloid-derived suppressor cells (MDSC) in vitro . To obtain MDSC, CD11b + cells were isolated from the peripheral blood of healthy donors followed by cytokine induction (IL-1β+GM-CSF) into the MDSC phenotype. The cell functions were assessed by the expression of indoleamine 2,3-dioxygenase (IDO) and arginase-1 (Arg1) and cytokine profile of the cell cultures. Native AFP did not affect the total number of MDSC and the percentage of polymorphonuclear MDSC (PMN-MDSC), but increased the number of monocytic MDSC (M-MDSC). AFP did not change the expression of Arg1, but in low concentrations (10 and 50 U/ml) increased the number of IDO-containing cells. AFP modulated the cytokine profile of CD11b + cells: it reliably decreased the level of IL-19 (50 and100 U/ml) and showed a tendency to decrease the levels of IL-34, MMP-2, sCD163, CHI3L1, OPN and to increase the levels of IL-29, IL-32, APRIL, PTX3, and sTNF-R1. Thus, we have demonstrated a regulatory effect of native AFP at the level of MDSC generated from CD11b + cells under conditions of cytokine induction in vitro .

Objective. To study the effect of recombinant glycodelin (Mybiosource, Germany) on the morphofunctional state of the spleen in case of transplantation of the allogeneic red bone marrow cells to Wistar rats in dynamics of in vivo experiment. From the point of view of immunology, pregnancy is a physiologically conditioned state of the tolerance of mothers immune system to genetically foreign embryo. Glycodelin is a protein associated with pregnancy; it has an immunosuppressive effect and is perspective for medicine. Materials and methods. The morphological picture of the organ was assessed; the following immunohistochemical indicators were studied: monoclonal antibodies to 1) CD68 for identification of macrophages, membrane staining; 2) Ki-67 for cells divided with mitosis and being in different phases of cellular cycle; 3) determination of macrophage colony-stimulating factor (M-CSF). Results. When studying the histological slices of the spleen, it was shown that glycodelin against the background of allogeneic transplantation of the bone marrow contributes to the activation of immune system cells in the spleen, stimulates the proliferation of immune cells (Ki-67) and their differentiation that was manifested by an increase in the number of plasmacytes. By the end of the study, macrophage content is essentially reduced; eosinophil infiltration is verified that is an indirect positive sign of reaction to the transplant. Against the background of the bone marrow cells allotransplantation, there was observed an increase in M-CSF level in animals on the day 21st from the onset the experiment compared with the group of intact animals. Introduction of glycodelin against the background of BM cells allotransplantation caused the cancellation of this effect. Conclusions. Thus, the action of glycodelin qualitatively determined the function of the spleen in direction of the development of a tolerant immune response to allogenate and excluded the development of severe post-transplantation complications.

Glycodelin, a protein associated with pregnancy, has an immunosuppressive effect and is promising for use in biomedicine. The aim of this work was to investigate the effect of recombinant glycodelin (MyBioSource, Germany) on the morphofunctional state of the spleen in case of allogeneic transplantation of red bone marrow cells in Wistar rats in a dynamic experiment in vivo. In the intact group, animals received a suspension of bone marrow cells treated once with camptothecin; in the experimental group, in addition to the introduction of the same bone marrow cells, the animals received 4 injections of glycodelin. Glycodelin was shown to promote the activation of immune system cells in the spleen against the background of allogeneic bone marrow transplantation and to stimulate the proliferation (Ki-67) and differentiation of immune cells. However, the number of macrophages (CD68) is reduced under the influence of glycodelin. As a result, glycodelin may affect the development of adaptive responses in bone marrow transplantation. Apparently, glycodelin is able to participate in the development of an adaptive mechanism in the spleen.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell population that primarily suppress T lymphocytes in healthy pregnancies and pathologies. MDSCs are one of the key regulators of immune responses. Finding ways to control them is important for the treatment of cancer, autoimmune diseases, miscarriage, and post-transplant complications. The mechanisms of immune suppression by MDSC are: expression of CD73, ADAM17, PD -L1, production of Arg 1, iNOS, IDO, IL -10 and TGF-b1. Pregnancy-specific b1-glycoprotein (PSG) has modulatory effects on dendritic cells and macrophages that mediate the shift of T cell phenotypes toward Th2 and Treg. We have previously shown that native PSG suppresses Th17 differentiation and cytokine production, stimulates the production of IDO by monocytes and the differentiation of Tregs. Considering the immunomodulatory properties of PSG and the key role of MDSCs in pathologies, the aim of our work was to investigate the effect of native and recombinant PSG on the differentiation of MDSCs in vitro . MDSCs were differentiated from CD11b + peripheral blood cells. Cells were cultured for 7 days and received stepwise GM-CSF, IL-1b, and LPS. Native (n) (1; 10 and 100 mg/mL) and recombinant (r) (1 and 10 mg/mL) PSG were introduced into the cultures three days before the end of incubation. Flow cytometry was used to determine the percentage of MDSC among the cells in culture and the percentage of M-, PMN-, and e-MDSC among the total number of MDSCs. It was found that rPSG (1 mg/mL) increased the percentage of MDSCs in culture. Both nPSG (1 and 10 mg/mL) and rPSG (10 mg/mL) increased the proportion of M-MDSC, whereas rPSG (10 mg/mL) decreased the number of PMN-MDSC. Thus, the cytokine background in CD11b + cell cultures favored the differentiation of predominantly M-MDSC, similar to the tumor microenvironment, whereas native and recombinant PSG enhanced this effect. Thus, nPSG and rPSG are able to modulate the differentiation of MDSCs by increasing their number, mainly due to the monocytic subpopulation. This fact opens perspectives for new research on targeted manipulation of MDSCs.

Myeloid-derived suppressor cells (MDSCs) are of interest as key regulators of the immune response for the development and improvement of cellular technologies in biomedicine. Enhancing the suppressive activity of these cells is important for developing therapies for autoimmune diseases and miscarriages, and their suppression may be useful in the treatment of cancer, since MDSCs are known to suppress antitumor immunity. However, there is a problem that prevents the active study of MDSCs, i.e., the difficulty in obtaining sufficient numbers of this cell population. Isolation of MDSCs in cancer patients poses an ethical challenge. Moreover, these MDSC may differ in subpopulation composition and suppressive activity due to individual factors. Researchers who generate human MDSC from bone marrow cells may also face similar problems. Therefore, finding a reliable and affordable source of these cells to facilitate the study of their functions is extremely important. Attempts to obtain human MDSCs in vitro have been ongoing for a long time. GM- CSF, IL-6, IL- 1β, IL-4, PGE2, LPS, M-CSF, IFNγ are described as factors that induce the ex vivo MDSC differentiation. However, despite multiple factors used, not all protocols are clearly reproducible, leading to generation of a sufficient number of cells in the target population. Previously, we had also developed a scheme for MDSC differentiation from CD11b+ cells derived from human peripheral blood, which made it possible to obtain a tangible but still insufficient percentage of cells to study functional activity. To increase the number of MDSCs in cultures, we developed a protocol aimed for differentiation of these cells from peripheral blood monocytes (CD14+ cells) previously transformed into PCMO (programmed cells of monocytic origin). The monocytes isolated by immunomagnetic separation were cultured in a de-differentiating medium (complete culture medium supplemented with M-CSF, IL-3 and β-mercaptoethanol) for one week. Later on, the medium was replaced by the addition of GM-CSF, being cultured for three days, followed by addition of LPS and IL-1β in order to induce suppressive activity. We have found that culturing CD14+ cells on a two-week schedule with prior creation of dedifferentiation conditions resulted in a slightly decreased percentage of viable cells in culture. However, there was a trend towards an increased ratio of MDSCs in culture (from an average of 34 to 40%) and an increase in their suppressive activity (arginase and IDO expression). The percentage of Arg+ cells increased by average of 10%, and IDO+ cells, by 16%. Moreover, the percentage of mature M-MDSCs was significantly (several-fold) higher when compared with differentiation protocol using CD11b+ cells. Hence, this method of MDSCs production enables us to increase the number of cells belonging to the conditionally “mature” monocyte subpopulation of MDSCs, as well as the percentage of functional suppressor cells in the population. The described scheme may be used to improve the quality of studies aimed at modulating MDSC functions in order to develop new therapeutic approaches.