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Maternal adaptation of the immune system aimed at limiting the immune response to fetal antigens is a necessary condition for a successful pregnancy. It involves various mechanisms (Th1/Th2 switching, Treg expansion, induction of anergy and apoptosis of T lymphocytes, development of T cell depletion) that are induced through the ligation of inhibitory receptors. Accordingly, the expression of inhibitory receptors on T cells, including PD-1, CTLA-4, and Tim-3 molecules, may reflect the effectiveness of immune adaptation and its impairment in pregnancy pathology. Preeclampsia (PE), the pathogenesis of which is associated with the impairments of immunological tolerance is a major complication of pregnancy. Accordingly, changes in the expression of inhibitory receptors on T cells may be biomarkers of abnormal gestation and potential therapeutic targets. The aim of this work was to study the expression of inhibitory molecules on peripheral blood T cells in women with PE. The study recruited 29 pregnant women with PE and 36 women with uncomplicated pregnancies in the second half of pregnancy. Pregnant women of the study groups were comparable in terms of gestational age, number of pregnancies and parity of childbirth. The control group consisted of 28 fertile women with children. Relative content of CD8+PD-1+, CD8+CTLA-4+, CD8+TIM-3+, CD8+PD-1+TIM-3+, CD4+PD-1+, CD4+CTLA-4+, CD4+TIM-3+, CD4+PD-1+TIM-3+T cells in blood were analyzed by flow cytometry. It has been shown that uncomplicated pregnancy is associated with increased expression of PD-1 and Tim-3 T cells, which is manifested by an increase in the relative content of CD4+Tim-3+, CD8+PD-1+ and PD-1+Tim-3+T lymphocytes . In PE, on the contrary, there is a reduction in the expression of PD-1 and Tim-3 by T cells, in particular, a decrease in the proportion of CD4+Tim-3+ and CD8+PD-1+ cells; the absence of elevated levels in PD-1+Tim-3+ cells (compared to uncomplicated gestation) and an increase in CTLA-4+ cells within CD4+ lymphocytes. Changes in the expression of inhibitory receptors are associated with the severity of PE. A decrease in CD4+Tim-3+ and CD8+PD-1+T cells is most typical for patients with moderate PE, and an increase in CD4+CTLA-4T cells for pregnant women with severe PE. The relationship between changes in the expression of inhibitory molecules and the onset of PE has also been demonstrated. A distinctive feature of early PE is a decrease in the proportion of CD8+CTLA-4+ cells and a more pronounced increase in CD4+CTLA-4+ cells, while late PE is associated with a decrease in CD4+PD-1+ cells and a more pronounced decrease in CD4+Tim-3+ cells. The results obtained indicate a changes in the expression of CTLA-4, PD-1 and Tim-3 molecules on circulating T cells in pregnant women with PE and the association of these changes with the severity and the onset of PE manifestation.

Innate immune cells, including myeloid cells — myeloid derived suppressor cells (MDSCs) — are supposed to play an important role in the pathogenesis of axial spondyloarthritis (AxSp). Myeloid derived suppressor cells represent a heterogeneous population of immature cells capable of suppressing innate and adaptive immune responses with the most pronounced suppressor activity against T cells. Biological disease-modifying antirheumatic drugs (bDMARDs) can reduce the clinical and laboratory disease activity, but their effectiveness varies widely in different patients with AxSp. The present study is aimed at studying MDSCs subpopulations and their suppressive function depending on the response to bDMARD therapy in AxSp. The study included AxSp patients with a disease duration of 16.5 years (median); HLA-B27 (+) status was detected in 79% of cases. All patients received bDMARDs at least the past 12 weeks, including TNF inhibitors (etanercept, certolizumab pegol, adalimumab, or golimumab) or IL-17 inhibitors (secukinumab, ixekizumab, or netakimab). Percentage of granulocytic MDSCs (G-MDSCs, Lin-HLA-DR-CD33+CD66b+), monocytic MDSCs (M-MDSCs, HLA-DRlow/-CD14+), MDSCs of early stage differentiation (E-MDSCs, Lin-HLA-DR- CD33+CD66b-), as well as intracellular expression of arginase-1 was assessed by flow cytometry. Frequency of circulating MDSC subpopulations of patients with a stable response to bDMARDs (responders) did not differ significantly compared to healthy donors. Patients not responding to bDMARDs therapy showed increased relative and absolute number of E-MDSCs compared to healthy donors (pU = 0.01 and pU = 0.02, respectively) and the responders (pU = 0.03 and pU = 0.07, respectively). Increased percentage of E-MDSCs was positively correlated to disease activity — ESR (Rs = 0.821; p = 0.023), CRP (Rs = 0.714; p = 0.07) and ASDASCRP (Rs = 0.829; p = 0.042) in the non-responder group. Responder patients exhibited no correlation between disease activity and circulating MDSCs. The suppressor potential of MDSCs was analyzed by the intracellular expression of arginase-1 molecule which is involved in the inhibition of T cell response. Patients with the stable response were characterized by increased expression of arginase-1 in E-MDSCs compared to donors (pU = 0.02). Non-responders did not demonstrate significant changes in Arg-1 expression, however, the percentage of arginase-1-expressing G-MDSCs was positively correlated to indexes ASDASESR (Rs = 0.857; p = 0.014) and BASDAI (Rs = 0.785; p = 0.036). Thus, E-MDSCs as well as arginase-1 expression in MDSCs may serve as biomarkers of effectiveness bDMARD therapy, and act as potential candidate predictors of response to therapy in AxSp.

During pregnancy, the maternal immune system must maintain tolerance to paternal antigens, at the same time being able to eliminate pathogens, which is achieved by the weakening of adoptive immunity and the activation of innate immunity, in particular, monocytes. However, the question about the functional phenotype of monocytes, having not only pro-inflammatory, but also anti-inflammatory activity, remains open. In the given work, we have investigated the expression of M2-associated suppressive markers Arg1 and MerTK in monocyte subpopulations during uncomplicated pregnancy. Fifty-three pregnant women with uncomplicated gestation were recruited, including 14 pregnant in the 1st trimester, 20 – in the 2nd and 19 – in the third pregnancy trimester. The comparison group consisted of 15 fertile unpregnant women without aggravated somatic anamnesis, with a history of at least one childbirth. The findings showed that in the unpregnant group circulating Mo express Arg1 and MerTK, and the most relative number of Arg1+ and MerTK+ cells is concentrated in intermediate and nonclassic monocytes. During pregnancy the expression of researched molecules in monocytes reliably increases. An increase in MerTK expression is manifested by a simultaneous increase in the number of MerTK+ cells and the mean fluorescence intensity of this marker; it is observed in the 1st and 2nd trimesters and registered in all three monocyte subpopulations. At the same time, an increase in Arg1 expression is manifested either by an enhancement of Arg1+ cells, or an increase in receptor density; it is registered throughout pregnancy, including the 3rd trimester, and is maximally expressed in classic monocytes. There is a direct correlation between the number of Arg1+ and MerTK+ cells in intermediate Mo, which increases with the progression of pregnancy, and in the 3rd trimester is also detected in classical and non-classical Mo. In general, the revealed increase in the expression of Arg1 and MerTK by monocytes indicates an increase in the anti-inflammatory potential of monocytes during pregnancy, and the involvement of monocytes in the regulation of the inflammatory process at the system level. Moreover, the features of Arg1 and MerTK expression in various monocyte subpopulations during pregnancy suggest that monocytes expressing Arg1 and MerTK can mediate different mechanisms of immune adaptation during pregnancy.

In humans circulating monocytes include classical (CD14++CD16- ), intermediate (CD14++CD16+) and non-classical/alternative (CD14+CD16++) monocytes, which in turn can be activated via the classical or alternative pathway. Pregnancy is accompanied by significant changes in the monocyte compartment, which is manifested by an increase in the number of circulating monocytes, including the proportion of intermediate monocytes, and a change in their function. However, the functional properties of monocyte subsets during gestation remain largely unexplored. We hypothesized that circulating monocytes may be activated in an alternative pattern and acquire features of M2 polarization (anti-inflammatory / immunosuppressive properties). The aim of the investigation was to study M2-associated markers that characterize the anti-inflammatory and immunosuppressive potential of myeloid cells in subpopulations of circulating monocytes in fertile nonpregnant women and women with uncomplicated pregnancy in the 2nd trimester. It was shown that in fertile non-pregnant women intermediate and non-classical monocytes are characterized by a higher expression of M2-associated markers (CD206, Arginase 1, MerTK) compared to classical monocytes. In the 2nd trimester of pregnancy, the expression of these molecules on monocytes increases significantly, which is manifested by 1) an increase in the proportion of CD206+ cells in subpopulations of classical and intermediate monocytes, 2) an increase in the mean fluorescence intensity of Arginase 1 in all monocyte subsets, 3) an increase in the proportion of MerTK+ cells in subpopulations of classical and intermediate monocytes and mean fluorescence intensity across all monocyte subsets. The highest content of CD206+ and MerTK+ cells in pregnant women is detected in the subpopulation of intermediate monocytes, and the highest values of the mean fluorescence intensity of Arginase 1 and MerTK – in the subpopulations of intermediate and non-classical monocytes. The data obtained demonstrate that monocytes of pregnant women in the 2nd trimester of pregnancy are characterized by signs of M2 polarization. This is confirmed not only by an increase in the expression of the M2-associated mannose receptor CD206, but also by an increase in the expression of Arginase 1 and MerTK, which mediate the immunosuppressive activity of myeloid cells and, in particular, macrophages of the M2 phenotype. Further studies of M2-associated markers in monocyte subpopulations during gestation will allow a more detailed characterization of the regulatory role of circulating myeloid cells during pregnancy.

GM-CSF and M-CSF, the hematopoietic colony-stimulating factors, induce various phenotypic changes in macrophage lineage populations and promote cell differentiation, respectively, into M1- and M2-like macrophages. The pro- and anti-inflammatory properties of macrophages generated by these colony-stimulating factors are well described, but the contribution of differentiation and polarization signals to the fibromodulatory activity of macrophages remains unexplored. To clarify the differences in the fibrogenesis regulation mechanisms inherent in differently activated macrophages, we studied the effects of macrophage-conditioned media on proliferation and differentiation of dermal fibroblasts. In this study, the human macrophages generated from peripheral blood monocytes were investigated. They were induced for differentiation by M-CSF or GM-CSF, being further polarized in the M1 direction with lipopolysaccharide and, in the M2 direction, with IL-4 or dexamethasone. Proliferative response of the fibroblasts was determined radiometrically by [3H]-thymidine incorporation. Differentiation into myofibroblasts was determined with flow cytometry technique, as expression of a specific marker α-smooth muscle actin (α-SMA). The level of macrophage TGF-β1 production was assessed using an appropriate ELISA kit. The data obtained indicate that the macrophages differentiated under the influence of “homeostatic” M-CSF are characterized by a moderate stimulating effect upon fibroblast proliferation, and the effects of M2 (IL-4) and M2 (Dex) macrophages exceed that of M1 (LPS), but do not differ significantly from each other. The M-CSF-induced M1 (LPS) and M2 (IL-4) macrophages, but not M2 (Dex), enhance the fibroblast differentiation and show similar level of stimulation. In contrast to M-CSF, the macrophages induced by “pro-inflammatory” GM-CSF exhibit a pronounced stimulatory effect on fibroblast proliferation, and the effects of M2 macrophages exceed those of M1 cells, being most pronounced for M2 (Dex). At the same time, only GM-CSF-induced M2 (IL-4) macrophages enhance fibroblast differentiation. Dexamethasone-polarized macrophages do not significantly affect fibroblast differentiation regardless of the CSF used (M-CSF or GM-CSF). The content of TGF-β1 in the supernatants of differently activated macrophages does not correlate with the level of stimulating effect of macrophage-conditioned media upon fibroblast differentiation. In general, the data obtained suggest the involvement of differentiation and polarization signals into modulation of pro- and anti-fibrogenic properties of macrophages.

Expansion of myeloid-derived suppressor cells (MDSCs) due to impaired differentiation of myeloid progenitor cells under conditions of inflammation was described in a number of autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, and type 1 diabetes mellitus. Studying the role of MDSCs in ankylosing spondylitis is an important issue, given that increased concentration of proinflammatory mediators in this pathology can also cause myelopoiesis disorders. The aim of present work was to study the quantitative content of MDSC subpopulations in patients with different clinical phenotypes and activity of AS. 37 patients, including 10 patients without peripheral skeletal lesions (axial form) and 27 patients with simultaneous lesions of spine and peripheral joints (peripheral form) were recruited into the study. The control group consisted of 32 age/sex-related healthy donors. Evaluation of granulocytic (LinHLA-DRCD33+CD66b+; G-MDSC), monocytic (CD14+HLA-DRlow/-; M-MDSC) and early-stage MDSCs (LinHLA-DRCD33+CD66b- ; E-MDSC) was performed using corresponding antibodies (BD Biosciences, USA) in the population of peripheral blood mononuclear cells by flow cytometry. In general, the AS patients were characterized by an increased relative and absolute amount of M-MDSC (p = 0.00002 and p = 0.00003, respectively) and G-MDSC (p = 0.0002 and p = 0.0006, respectively). Patient gender, age, and HLA-B27 expression did not significantly affect the content of these cells in peripheral blood. An increase in the median values of M-MDSC was detected both in patients with axial (Ме 5.0 (3.2-6.3) versus 2.4 (1.7-3.5) %; p = 0.001) and peripheral form (Ме 5.0 (3.0-7.0) versus 2.4 (1.7-3.5) %; p = 0.0002) AS. At the same time, the G-MDSC expansion was observed only in patients with involvement of peripheral joints (Ме 0.16 (0.07-0.3) % versus 0.05 (0.04-0.09) %; p = 0.0001). The relative contents of E-MDSC, M-MDSC and G-MDSC in the axial form of AS was in direct correlation with the activity of the disease (R = 0.58, p = 0.02; R = 0.73, p = 0.08 and R = 0.65 p = 0.04, respectively). This relationship was not observed in peripheral form of AS. The data obtained suggest a potential involvement of MDSCs in pathogenesis and phenotypic heterogeneity of AS. Simultaneously, the revealed direct correlation between the MDSC contents and the disease activity suggests a decrease in suppressive activity and/or appearance of pro-inflammatory activity in MDSC, thus requiring further research in the field.

Early prediction for ischemic stroke (IS) outcome is a major challenge since it may help to optimize treatment program and to make it more personalized. Since T cells with regulatory activity are involved in different pathophysiological processes in brain stroke, including inflammation, immune suppression, brain damage and repair, the study of T cells as potential biomarkers has essential importance. The present work aimed to study the circulating T cell subsets with phenotype of type 1 T helper cells (Th1) and regulatory T cells (Treg), and their ratio during the acute phase of IS, depending on stroke severity, inflammatory response and 3-month outcome (according to modified Rankin scale, mRs). Patients and methods. The study included 61 patients with a newly diagnosed IS (severity according to NIHSS ≥ 5), in the first 24-48 h after stroke onset, and 20 age/sex-related healthy donors. Laboratory examination included assessment of leukocytosis, neutrophillymphocyte ratio (NLR) and CRP concentration. Mononuclear cells were isolated from peripheral blood to study T cell subsets. Th1 and Tregs were measured by FACS analysis as CD4+IFNγ+ and CD4+CD25hiT cells, respectively. During the first 24-48 h after stroke, the patients had elevated values of leukocyte counts, NLR and CRP. Higher levels of these parameters in severe stroke compared with mild stroke, as well as direct correlation of NIHSS with NLR and CRP evidenced that the stroke severity was associated with more pronounced inflammatory response. Patients were also characterized by a significant decrease in CD4+IFNγ+Th1 cells, an increase in CD4+CD25hiTreg, and a marked decrease in Th1/Treg ratio. Furthermore, in patients with NIHSS ≥ 8 (moderate and severe stroke), the percentage of CD4+IFNγ+T cells was in direct correlation, and the number of CD4+CD25hiT cells was inversely related to CRP and NLR values. The changes of T cell subsets were more pronounced in patients with a favorable 3-month outcome (mRs > 3). As a result, the patients with poor outcome (mRs ≤ 3) had higher CD4+IFNγ+T cell proportion, lower CD4+CD25hiT cell percentage and 4-fold higher CD4+IFNγ+/CD4+CD25hi ratio compared with opposing group. ROC analysis revealed a “good” quality of prognosis based on evaluation of the CD4+IFNγ+/CD4+CD25hi ratio as a monopredictor of adverse outcome (AUC = 0.75) and “very good” quality of prognosis when the indicated ratio was combined with NIHSS scale (AUC = 0.82). The data obtained suggest that a decrease of Th1/Тreg ratio, due to a decrease in CD4+IFNγ+ and increased CD4+CD25hiT cell counts during the acute phase of ischemic stroke is a compensatory reaction directed at inhibition of inflammatory response, and has a prognostic significance as early predictor of the outcome at 3 months.

Peripheral blood monocytes are heterogeneous CD14+ cell population, some of which also express CD16 molecule. Differences in phenotype between monocyte subpopulations can affect their functional activity, as well as the ability to further differentiate into dendritic cells (DCs). DCs are professional antigen-presenting cells which induce the immune response or, conversely, maintain the immunological tolerance. The aim of the present study was to analyze the relationship between monocyte subpopulations and the functional activity of monocyte-derived DCs, as well as DC sensitivity to the tolerogenic effect of dexamethasone. DCs were generated by cultivating enriched fractions of CD14+ monocytes with or without CD16+ cell depletion (CD16-Mo-DCs or CD16+Mo-DCs, respectively) in the presence of IFNα and GM-CSF. Monocyte subpopulations were obtained by immunomagnetic negative selection. CD16+Mo-DCs were characterized by lower ability to take up FITC-dextran and higher allostimulatory activity compared to CD16-Mo-DCs. In addition, CD16+Mo-DCs showed higher apoptosis-inducing activity against autologous CD4+T lymphocytes and allogeneic CD8+T lymphocytes, but were similar to CD16-Mo-DCs in their ability to induce apoptosis in allogeneic CD4+T lymphocytes. TNFa production level, similar for both types of DCs, was negatively correlated with CD16-Mo-DC allostimulatory activity and directly correlated with apoptosis-inducing activity of CD16+Mo-DCs towards allogeneic CD4+T cells. CD16-Mo-DCs and CD16+Mo-DCs were similar by their IL-10 production, which was inversely related to allostimulatory activity of both types of DCs. Dexamethasone increased endocytic activity, decreased the ability to stimulate autologous and allogeneic T cells, inhibited TNFα production of CD16-Mo-DCs and CD16+Mo-DCs. However, CD16+Mo-DCs demonstrated a more pronounced increase in endocytic activity and more dramatic decrease in their ability to stimulate the proliferation of CD4+T cells in auto-MLR. Also, addition of dexamethasone into CD16+Mo-DCs cultures led to the increase in DC pro-apoptogenic activity against autologous CD8+T lymphocytes. Thus, the presence of CD16+ cells among monocyte population affects the properties of IFNα-induced monocyte-derived DCs and DC sensitivity to the immunomodulatory effects of dexamethasone.

Dendritic cells (DCs) play a key role in maintaining the peripheral tolerance of lymphocytes to autoantigens. Recovery of immunological tolerance in autoimmune diseases, particularly, in rheumatoid arthritis (RA) is considered a new therapeutic strategy. The aim of this work was to study the effect of dexamethasone-modified DCs generated from monocytes of RA patients in the presence of IFNα (DCsDex), upon autologous T lymphocytes in mixed leukocyte culture (auto-MLC), and to investigate possible mechanisms of the DCsdex tolerogenic effect upon autoreactive T cells. We have shown, that DCsDex from RA patients induce T cell hyporeactivity in auto-MLC. Hyporeactivity of T cells is associated with cell cycle blockage in CD4+T lymphocytes and decreased IFNγ, IL-17, IL-4 and IL-13 production, which indicates the induction of CD4+T cell anergy. In this case, inhibition of Th1/Th17 has been more pronounced than the suppression of Th2 cells producing IL-4 and IL-13. Along with T cell anergy, the decrease of proliferative response in auto-MLC is associated with increased CD3+T lymphocyte apoptosis. In addition, the DCsDex of RA patients suppresses the proliferation of autologous T cells stimulated by unmodified DCs. This effect is associated with enhancement of IL-10-producing CD4+T cells in the auto-MLC, thus being indicative for an ability of DCsDex to induce conversion of CD4+T lymphocytes into regulatory T cells (Tr1). The data obtained characterize a new type of tolerogenic DCs, generated from blood monocytes of RA patients in the presence of IFNα and modified by dexamethasone, thus revealing a mechanism for tolerogenic effect of DCsDex upon T cells that recognize self-antigens in auto-MLC.

The immunomodulatory activity of vascular endothelial growth factors (VEGFs) reveals a new role of neoangiogenesis in tumor development. Most of VEGF effects on T cells are mediated through the VEGF-R2 receptors. Placental growth factor (PlGF) belongs to the VEGFs family and is a selective ligand for VEGF-R1. In order to study the role of VEGF-R1-signaling in the regulation of T-cell functions, the effect of PlGF on the proliferation of donor T cell has been investigated. PlGF has been shown to inhibit the proliferation of T-lymphocytes in cultures of anti-CD3-stimulated mononuclear cells in a wide dose range, suppressing the proliferative response of both CD4 + and CD8 + T cells. The suppressive effect of PlGF was mediated through the direct interaction with VEGFR-1 on T-cells that was evidenced by the expression of VEGFR-1 by T-lymphocytes (especially after their activation) and by blocking the suppressive effect of PlGF with neutralizing anti-VEGFR-1 antibodies. Given the increased levels of PlGF in many tumors, this factor may play an important role in immunomodulation during tumor growth, mediating its effect through the VEGFR-1 signaling pathway.

The aim of the study was to evaluate FoxP3+ regulatory T cell (Тreg) subsets in the follicular fluid (FF) of the women undergoing in vitro fertilization (IVF), and their relationships with features of folliculogenesis and oogenesis, as well as quality of embryos and retrospective assessment of IVF outcomes. The study included 53 women at the age of 25 to 46 years stimulated by superovulation. The count of Tregs in the FF samples was determined by flow cytometry using appropriate monoclonal antibodies. The FF examination revealed of FoxP3+ T cells from both CD4+ (CD4+FoxP3+) and CD4(CD4-FoxP3+) T lymphocyte subsets. Moreover, the FoxP3+ cells were registered in CD4+CD25+ and CD4+CD25T lymphocyte subsets. Follicular fluid of women with a relatively small number of follicles contained higher numbers of CD4+CD25-FoxP3+ T cells, whereas a reduced number of oocytes was associated with the highest count of CD4-FoxP3+ T cells in FF. Retrospective analysis showed the a relationship between percentage of Tregs, and quality of oocytes and embryos. High fertilization index (0.75-1.0), reflecting maturity of the oocytes was associated with higher count of CD4-FoxP3+ T cells in the FF. Better quality of blastocysts was associated with a higher count of both CD4-FoxP3+ and CD4+FoxP3+ T cells. Onset and progression of pregnancies was also registered in women with relatively high counts of CD4-FoxP3+ T cells. The obtained data showed presence of different FoxP3+ T cell subtypes in follicular fluid, and its possible role in controlling early stages of reproductive process. CD4-FoxP3+ T cells seem to be the most important subpopulation; their counts are associated with efficacy of oogenesis, blastulation and pregnancy occurence.

A key role of T cells in viral elimination and absence of strong T cell responses in patients with chronic hepatitis C virus (HCV) infection presumes that activation of antigen-specific T cells may be a promising approach to enhance treatment efficacy. Given the central role of dendritic cells (DCs) in the induction of T cell response, the aim of our study was to evaluate effects of DC immunotherapy upon immunological parameters in chronic HCV infection. Ten patients with chronic hepatitis C (genotype 1) were vaccinated with monocytederived DCs, generated in presence of IFNα (IFN-DCs) and pulsed with recombinant HCV Core (1–120) and NS3 (1192–1457) proteins. The vaccination protocol included as initiating procedure (one injection per week, ns = 4) and maintaining treatment (one monthly injection, ns = 6), with subsequent follow up for 6 months. The immunotherapy was not associated with serious adverse events, significant post-vaccination reactions, or increased hepatitis C activity, according to biochemical tests. Ex vivo studies have shown that immunotherapy elicited strong and stable immune response to Core and moderate response to NS3 protein, which manifested as a significant increase of MNC proliferation and IFNγ production in response to Core and enhancement of IFNγ production (without higher proliferation rates), in response to NS3. DC immunotherapy also led to increase of ConA-induced MNC proliferation up to normal levels indicating a recovery of mitogenic T cell reactivity. Meanwhile, T cell activation did not elicit antigen-specific Th2 response and expansion of CD4+CD25+CD127 regulatory T cells. Despite induced immune response, the immunotherapy with DCs was not accompanied by decreased viremia levels. Nevertheless, a transitory decrease of viral load in four patients and stable decrease of viremia in two patients as well as an inverse correlation between NS3-specific proliferation and viremia (Rss = 0.62; p < 0.05) by the end of 6-month follow-up indicated that the antigenspecific T cells may have a potential to control viral replication.

Granule-mediated cytotoxicity of effector cells is a universal mechanism of tumor growth inhibition and induction of tumor cell death. The aim of present study was to evaluate expression of lytic molecules in DCs generated in presence of IFNα (IFN-DCs), and to analyze the role of granule-mediated mechanism for IFN-DC cytotoxic activity against tumor cell lines. IFN-DCs were generated by culturing of plastic-adherent peripheral blood mononuclear cells in presence of GM-CSF and IFNα for 4 d followed by LPS addition for 24 h. The tumor cell lines were obtained from malignant tissues from patients with glioblastoma multiforme. Maturation of IFN-DCs in presence of LPS was accompanied by accumulation of intracellular perforin and granzyme B molecules. Perforin expression showed a direct correlation with intracellular lysosome-associated membrane protein-1 (LAMP-1/CD107a) expression in LPS-stimulated IFN-DCs. However, CD107a expression did not increase under LPS stimulation. At the same time, LPS caused upregulated degranulation in IFN-DCs, as shown by an increase of surface CD107a expression on IFN-DCs. LPS activation of DCs generated from the same donors in the presence of GM-CSF and IL-4 (IL-4-DCs) did not influence perforin and granzyme B expression in IL-4-DCs which was significantly lower than in IFN-DCs. Intracellular pool of CD107a molecules was increased in response to LPS stimulation of IL-4-DCs, but surface CD107a expression did not change on IL-4-DCs. Studies of cytotoxic activity of LPS-stimulated IFN-DCs revealed that concanamicyn A (CMA), an inhibitor of vacuolar H+-ATPase and of perforin/granzyme B-mediated signaling pathway, caused reduced cytotoxicity of donor DCs towards glioblastoma cell lines. Involvement of perforin/granzyme B-signaling pathway into the DCs cytotoxicity was confirmed with glioblastoma cell lines, since blockage of this mechanism with vacuolar H+ ATPase blocker (CMA) caused inhibition of the IFN-DC cytotoxicity. Differently reduced DC cytotoxic activity by CMA may suggest that the glioblastoma cell lysis can be mediated via perforin/granzyme B-independent mechanisms.

Dendritic cells (DCs) play an important role in pathogenesis of rheumatoid arthritis (RA) and are considered a novel target for immune therapy. Under inflammatory conditions, local dendritic cells of non-lymphoid organs are thought to be differentiated from monocytes. Moreover, DCs differentiation and activation in RA may be largely controlled by interferon-alpha. The aim of the present study was to investigate phenotypic and functional properties of monocyte-derived DCs generated in the presence of interferonalpha (IFN-DCs) in RA patients, and to specify, whether IFN-DCs are sensitive to a tolerogenic effect of dexamethasone. Fourteen RA patients with moderate-to-high disease activity treated with disease-modifying drugs have been included into the study. Twenty sex- and age-related healthy donors were used as a control. IFN-DCs were generated from monocytes by culturing adherent fraction of mononuclear cells for 5 days with GM-CSF and IFNα in the absence or presence of dexamethasone (10-6 M). IFN-DCs from RA patients were characterized by increased numbers of CD14+CD83- and lower amounts of CD14- CD83+ cells, thus presuming a delayed maturation. Furthermore, IFN-DCs from patients were characterized by higher expression of B7-H1 and TLR2. The phenotypic changes did not significantly influence specific functional activities of DCs, in particular, the capacity of DCs to produce TNFα, IL-10, IL-6, to stimulate proliferation of allogeneic T-cells and to activate T-cells for Th1 and Th2 cytokine production. Generation of patients’ DCs in presence of dexamethasone caused a decrease in CD83 and CD86 expression, reduced TNFα production, and suppressed allostimulatory activity of the DCs. Moreover, dexamethasone inhibited the ability of DC to stimulate Th1 response, along with shifting the balance towards Th2-stimulating activity. The data obtained provide an evidence that IFN-DCs from RA patients remain sensitive to the tolerogenic effects of dexamethasone. Furthermore, the revealed variations in sensitivity of patient’s DCs to dexamethasone-mediated inhibitory effect may be of interest for prediction of therapeutic response to glucocorticoid therapy. Our results also provide an evidence for possible implementation of IFN-DCs as a new cell platform for obtaining tolerogenic DCs.

Type I Interferons are potent inducers of monocyte’s differentiation into dendritic cells (DCs). However, sensitivity of these DCs to tolerogenic effect of glucocorticoids has not been previously investigated. The aim of this study was to investigate the effect of dexamethasone upon maturation and functions of interferonalpha-induced DCs (IFN-DC) derived from healthy donors. DCs were generated from blood monocytes cultured for 5 days with GM-CSF and IFNα, in absence or with addition of dexamethasone (10-6 M), applied on the 3rd day. Addition of dexamethasone inhibited IFN-DC maturation, which manifested with increasing numbers of CD14+ cells and decreased percentage of CD83+ DCs. Dexamethasone did not significantly influence HLA-DR, CD86 and B7H1 expression. However, it caused a 2-fold increase of tolerogenic TLR-2 molecule expression. Along with suppression of IFN-DC maturation, dexamethasone inhibited production of proinflammatory/Th1 cytokines (TNFα, IL-1, IL-2, IFNγ, IL-12), and some chemokines (MIP-1α, RANTES). Dexamethasone-treated IFN-DCs exhibited a 2-fold lower allostimulatory activity in mixed lymphocyte culture (MLC). Worth of note, the capacity of IFN-DCs to stimulate T cell proliferative response in allo-MLC showed direct correlation with CD83 expression on DCs, and an inverse correlation with CD14 and TLR-2. Evaluation of Th1/Th2-polarizing activity of IFN-DCs showed that dexamethasone exerted a pronounced inhibitory effect upon ability of DCs to stimulate T cells for IFNγ production, along with lowgrade suppressive effect upon ability of DCs to induce IL-6 production, thus being indicative for a dominance of Th2-polarizing activity of IFN-DCs under the influence of dexamethasone. In general, the data obtained show that IFN-DCs are sensitive to tolerogenic action of dexamethasone, and, hence, the IFN-DCs may mediate the immunomodulatory effect of glucocorticosteroids and represent novel candidate cells for the development of therapeutic tolerogenic DC-based vaccines applicable for management of autoimmune disorders.

The aim of present work was to evaluate the effect of mesenchymal stromal cells (MSCs) from lymphoma patients on the in vitro and in vivo homeostatic proliferation of lymphocytes. Our data have demonstratedfor the first time, that MSCs from lymphoma patient are able to enhance the in vitro proliferation of donor and patient mononuclear cells (MNCs) in response to IL-2 or IL-7. This effect was observed within a wide rangeof MSC-to-MNC ratios (1:50-1:2). Lymphocyte reactivity to IL-2 or IL-7 was found to be an important factor determining the stimulatory effect of MSCs. Ex vivo studies in the patients undergoing autologous hematopoietic stem cell transplantation (AHSCT) have shown that the patients co-transplanted with MSCs exhibited higher proportions of proliferating CD8+T cells (especially, memory cells) at the day of engraftment, than before AHSCT, as compared to the patients subjected to standard AHSCT protocols. Moreover, there was no an increase in apoptosis of naive CD4+T cells that was typical for standard AHSCT. These data indicate that the stimulatory effect of MSCs upon T lymphocyte proliferation following chemotherapy-induced lymphopeniaconcerns, mainly, the memory CD8+T cell population, whereas more efficient recovery of naive CD4+T cells is due to anti-apoptotic effects of MSCs.

Abstract. Some functional properties of IFNα-induced dendritic cells (DCs) from the patients with pulmonary tuberculosis (PT) were under study. It was revealed that production of IFNγ and IFNα by DCs of the patients was decreased, but TNFα production was preserved at normal level. NO production by DCs from the patients was also reduced. In addition, it has been shown, that DCs of PT patients were characterized by low allostimulatory activity in mixed lymphocyte reaction (MLR), and this defect was more marked in the patients with PPD-anergy. As compared to healthy donors, DCs from the patients with PT (in particular, with PPD-anergy) exhibited a decreased capacity for induction of intracellular expression and production of IFNγ by T-cells. However, DCs from the patients with PPD-anergy were able to stimulate intracellular expression of IL-4 in CD3+T-cell subset. Hence, the results suggest that functional activity of DCs in the patients with PT is altered, and some of these changes are distinctly associated with decreased antigen-specific response. (Med. Immunol., 2008, vol. 10, N 2-3, pp 151-158).

We have performed a comparative analysis of allostimulatory, Th1/Th2-stimulatory andtolerogenic activities of dendritic cells (DCs) derived from healthy donors (n = 52). The DCs were generatedin vitro with IFN-alpha (IFN-DCs) or IL-4 (IL-4-DCs). It was shown that IL-4-DCs and IFN-DCs didnot differ in their key functional characteristics, i.e., in their ability to stimulate proliferation of T-cells inresponse to alloantigens, and to induce generation of T-regulatory cells in mixed leukocyte culture. IFN- andIL-4-induced DCs possess similar ability of boosting T cells to produce Th1/proinflammatory (IFNγ, IL-2,IL-1β, TNFα, IL-12, IL-17) and Th2/antiinflammatory cytokines (IL-4, IL-6, IL-10, IL-13), growth factors(G-CSF, GM-CSF, IL-7) and chemokines (IL-8, MIP-1β). Nevertheless, IFN-DCs have more pronouncedstimulatory effect upon Th1 and Th2 cells, thus manifesting as a significantly higher IFNγ, IL-5 and MIP-1βproduction. IFN-DCs were characterized by more prominent ability to activate Th1-cells, and by moderateTh2-stimulatory activity, which is absent in IL-4-DCs. These data strongly suggest that IFN-DCs may presenta quite promising cellular tool for development of therapeutic vaccines aiming to induce/enhance the immuneresponse.

In present work, a comparative analysis of cytostatic and cytotoxic activity of IL-4 and IFNα-induced dendritic cells (IL-4- and IFN-DCs) has been performed in healthy donors and patients with brain tumors. Cytostatic activity was evaluated, as a capacity of DCs to inhibit growth of NK-resistant HEp-2 and A-549 tumor cell lines, whereas cytotoxic activity was measured by assessment of HEp-2 lysis. As compared with IL4-DC, IFN-DCs from healthy donors displayed a more pronounced cytostatic effect and similar cytotoxic activity against HEp-2 cells. In the patients with brain tumors, IFN-DCs were characterized by decreased cytotoxic activity. Meanwhile, DCs from the patients with malignant gliomas virtually did not exhibit a cytotoxic potential, whereas IFN-DCs from the patients with benign brain tumors retained their cytotoxic properties. A comparative study has shown that, in contrast to healthy donors, IFN-DCs from the patients with either malignant or benign brain tumors did not inhibit HEp-2 proliferation. The loss of DC-mediated cytostatic activity was not associated with decrease of CD123+ DCs (known as cells with maximal tumor-inhibitory potential), and, most likely, it resulted from functional changes in IFN-DC populations.

Abstract. The study deals with evaluation of dehydroepiandrosterone sulfate (DHEAS) effects upon phenotype and functions of dendritic cells (DCs), generated from blood monocytes of pregnant women by means of GM-CSF and IFN-alpha. It was shown, that initial supplementation with DHEAS (10-6 M, day 1 of culture) did not influence the pattern of DC subsets. Meanwhile, addition of DHEAS at the stage of DC maturation (last day of culture) is accompanied by a significant increase in mature CD83+ cells and activated DCs (CD25+), like as their precursors (CD1a+). Furthermore, the DCs generated in presence of DHEAS were characterized by marked allostimulating activity and decreased ability to downregulate the numbers of CD56+CD16+ NK cells. Hence, DHEAS promotes generation, maturation, and allostimulating activity of DCs, along with decreased negative regulation towards CD56+CD16+NK cell amounts. DHEAS-mediated changes in DCs’ phenotype and functioning are discussed as a possible mechanism of disturbed immunological tolerance to fetal antigens in pregnant women with suprarenal hyperandrogenia.

The relationship between various subpopulations of regulatory T-cells (Treg) and serum level of CA-125 and dynamics of Treg following surgery in ovarian cancer (OC) patients have been investigated. The typical for OC peripheral blood Treg changes (the enchantment of CD4+FoxP3+ и CD8+FoxP3+ T-cells and decrease of CD4+CD25+/CD4+CD25high Т-cells due to their migration to ascites) have been revealed only in patients with elevated CA-125 level and were not registered in patient's group with normal level of this antigen. CA-125 serum level was directly correlated to absolute count of CD4+CD25high Т-cells and was in reverse relation to CD8+FoxP3+ T-cells. Surgical removal of a tumor was accompanied by remarkable reduction of CA-125 level in all cases. However Treg changes were observed only in patients with optimal cytoreductive surgery and were evidenced by CD4+CD25+/CD4+CD25high Т-cell normalization, whereas the number of these cells in peripheral blood of patients with residual tumors after surgery remained decreased. At that percentage of CD4+FoxP3+ и CD8+FoxP3+ Т-cells 2 weeks after surgery was unchanged and their increased number was associated with persisted decrease of T-cell proliferation in response to anti-CD3 stimulation.

Abstract. Phenotypic and functional features of IFNα-induced dendritic cells (DCs) were studied in patients with chronic viral hepatitis B and C (HBV and HCV), and in cases with hepatitis-related liver cirrhosis (LC). It was shown that DCs are characterized by delayed differentiation/maturation which was more pronounced in HCV patients, as well as in all patients with LC, regardless of virus type. DCs from HBV patients were characterized by increased IFNγ secretion. Transformation of HBV-infection to LC is accompanied by a moderate decrease in IFNγ production, combined with a significantly increased IL-10 secretion. Irrespectively of fibrosis severity, the IFNα-induced DCs of HCV patients displayed active IL-10 synthesis. Moreover, ability of DCs to secrete IFNγ was significantly decreased only in cases of fibrosis-complicated HCV-infection. With respect to TNFα and IL-4 production levels, DCs of the patients were compatibe to normal donor cells, independently on the type of virus, or fibrosis severity. DCs from HBV- and HCV-patients were characterized by intact allostimulatory and Th1/Th2-stimulatory activities in MLC. At the same time, IFNα-induced DCs exhibited suppression of allostimulatory and increase in Th2-polarizing activity upon LC development, both in HBV and HCV patients.

Abstract. The phenotype and functional properties of antigen-presenting cells (APCs: blood monocytes and in vitro generated macrophages/dendritic cells) were investigated in patients with pulmonary tuberculosis (TB, n = 192) with different levels of proliferative response to M. tuberculosis antigens (PPD-responsive vs PPD - anergic patients, n = 118 and 74, respectively). A functional deficiency of all 3 types of APCs was revealed in patients with TB. I.e., a monocyte disfunction was displayed by low CD86 and HLA-DR expression, 2-fold increase of CD14+CD16+ subset, high level of FasL+ and IL-10+ cells, and enhanced IL-10 and IL-6 production upon LPS-stimulation. The in vitro generated macrophages from blood monocytes challenged with GM-CSF, were characterized by shifted Th1/Th2 balance (down-regulated production of IFNγ and IL-18 combined with up-regulation of IL-6 and IL-10), and reduced allostimulatory activity in mixed lymphocyte culture. The dendritic cells were characterized by decrease of mature, activated CD25+ cells, low level of IFNγ production in conjunction with enhanced capacity to produce IL - 10 and IL-6, and profound reduction of functional (allostimulatory) activity. The APC disfunction of were most prominent in PPD-anergic patients. A possible role of APC disfunctions in disturbed antigen-specific T-cell response to M. tuberculosis is discussed.

Abstract. Cytotoxic/pro-apoptogenic effects of IFNα-induced dendritic cells (IFN-DCs) directed against Т-lymphocytes and NK cells were investigated in healthy donors. Using an allogenic MLC system, it was revealed that IFN-DCs induce apoptosis of both activated CD4+ and CD8+ T-lymphocytes, and NK cells. Apoptosis of CD4+ and CD8+ T-lymphocytes induced by their interaction with IFN-DCs was mediated by various signaling pathways. In particular, activated CD4+Т-lymphocytes were most sensitive to TRAIL- и Fas/ FasL-transduction pathways, whereas activated CD8+ T-lymphocytes were induced to apoptosis via TNFα-mediated pathway. PD-1/B7-H1-signaling pathway also played a distinct role in cytotoxic activity of IFNDCs towards both types of T lymphocytes and activated NK cells. The pro-apoptogenic/cytotoxic activity of IFN-DC against activated lymphocytes may be regarded as a mechanism of a feedback regulation aimed at restriction of immune response and maintenance of immune homeostasis. Moreover, upregulation of proapoptogenic molecules on DCs under pathological conditions may lead to suppression of antigen-specific response, thus contributing to the disease progression.

Abstract. The present work was aimed to evaluation of phenotype and functional properties of IFNα-induced dendritic cells (DCs) in normal pregnancy, and in cases complicated with suprarenal hyperandrogenia (HA), as well as in vitro assessment of dehydroepiandrosterone sulfate (DHEAS) effects upon DC functions. As compared with non-pregnant women, DCs from healthy pregnant women are notable for impaired maturation/activation, whereas fractions of mature (CD83+) and activated (CD25+) cells DC were similar in normal pregnancy and in women with HA. Blood sera from healthy pregnant women inhibited generation of mature DCs, whereas sera of women with HA and direct supplementation with DHEAS enhanced maturation and activation of DCs. Functional analysis of DC capacity to stimulate Th1 (IFNγ) and Th2 (IL-4) cytokine expression in MLC has shown that, in contrast to non-pregnant women with T1-cell activation by DCs, the DCs from healthy pregnant women caused predominant activation of CD3+IL-4+Т-cells. In HA-complicated pregnancy, DCs were capable to stimulate both Th1- and Th2-cytokine production in T-cell populations. Meanwhile, addition of DHEAS to DCs cultures were accompanied by enhancement of their T1- polarizing activity. DCs from women with normal pregnancy exerted inhibitory effect upon activated NK-cells that was evidenced by a relative decrease in CD56+CD16+ cells counts and increased apoptosis levels. This function of DCs was partially abrogated in presence of DHEAS. The results obtained reveal new mechanisms of immune/ endocrine nteractions in normal and complicated pregnancy.

Abstract. The aim of present study was to investigate PD-1/B7-H1-mediated induction of T-cell apoptosis/ anergy, a suggested mechanism of reduced antigen-specific immune response against M. tuberculosis. We examined 76 patients with pulmonary tuberculosis (PT) who differed in levels of proliferative response to specific antigen (purified protein derivative, PPD). It was revealed that in vitro generated dendritic cells (DCs) from the patient’s blood monocytes with GMCSF+IFNα, were characterized by increased B7-H1 expression, upregulation of IL-10 production, and reduced allostimulatory activity in mixed lymphocyte culture (MLC). Moreover, DCs of PT patients were able to enhance T-cell apoptosis, and to block T-cell division in MLC. Thus, the patients’ DCs exhibited the higher tolerogenic potential since these DCs could induce apoptosis/anergy in responsive T-cells via PD-1/B7-H1-mediated pathway combined with IL-10 effects. It was shown that neutralizing anti-PD1-antibodies partially abolished the pro-apoptogenic/tolerogenic effect of DCs. The revealed phenomenon of PD-1/B7-H1-mediated pro-apoptogenic activity should be of obvious clinical significance, since the cytotoxic/tolerogenic potential of DCs was especially pronounced in the patients with low antigen-specific response to PPD.

Abstract. Present work aims to investigate functioning of the regulatory T-cells with suppressor activity in surgical sepsis. It has been shown that the septic patients with decreased T cell proliferation are characterized by enhanced relative contents of СD4+СD25+ lymphocytes, including СD4-CD25high. Moreover, a reverse correlation has been revealed between СD4+CD25+high and anti-CD3-induced proliferative response of mononuclear cells (MNC).Increased ratio of СD4+СD25+ cells is associated with arising suppressive activity of MNC, which is partially abrogated by addition of rIL-2. It should be noted that the depletion of CD25+ subpopulation is accompanied by increased T cell proliferation. Hence, a decrease in T cell proliferative activity in surgical sepsis is coupled with naturally occurring СD4+СD25+ regulatory T-cells (Trn).Furthermore, it has been demonstrated that the suppressive activity of MNCs in septic patients is mediated by soluble factors, in particular, IL-10. Indeed, septic patients are characterized by increased level of IL-10 production and enhanced number of CD4+IL-10+ T-cells. Addition of neutralizing anti-IL-10 antibody partially abrogated the suppressive activity of MNC supernatants. Thus, our study demonstrated that inducible regulatory T cells (Tr1) coupled to the naturally occurring СD4+СD25+ regulatory T-cells (Trn) actively contribute to genesis of immune pathology in sepsis.

The study deals with evaluation of immune parameters in healthy fertile women (n = 48) versus female patients with infertility of unknown origin (n = 386). A comparative analysis has shown similar patterns of immune dysfunction in women with primary infertility (n = 179) and recurrent spontaneous abortion (n = 207). The most common markers of immune dysfunction include decreased production of blocking antibodies (86.5% of cases), low proliferative response to paternal alloantigens in mixed leukocyte reaction (MLR) shown in 51.5% of cases, and increased number of activated CD56+CD16+NK-cells (42.0%). Decreased production of MLR-blocking antibodies may be caused by low lymphocyte response, due to the partner's HLA-similarity, or by predominance of Th1/proinflammatory cytokines and low contents of circulating regulatory CD4+CD25+T-cells. The study results demonstrate that development of primary infertility and recurrent spontaneous abortions is associated with a uniform set of immune disorders, thus probably reflecting a failure of suppressor rearrangement within immune system during periovulatory period of menstrual cycle.

Аbstract. Сlinical and immunocorrective efficiency of Roncoleukin® (IL-2) monotherapy applied as subcutaneous injections versus inhalatory mode of treatment, was studied in patients with chronic recurrent infections. It was shown that administration of Roncoleukin® at a total dose of 1.5 mg was accompanied by increased activity of Th1 lymphocytes and reduced in vitro production of IL-10, and does not exert any adverse immunotropic effects associated with Treg expansion. In general, the positive clinical effect was registered in 65% (26/40) of the patients, and its duration ranged between 3 and 7 months (a mean of 4.4±0.2 months). Clinical efficiency of Roncoleukin® inhalations proved to be not inferior to conventional Roncoleukin® immunotherapy performed by subcutaneous injections (resp., 70 and 60% of positive responses), and allows a more significant prolongation of event-free remission. (Med. Immunol., 2011, vol. 13, N 2-3, pp 227-236)