CYTOTOXIC ACTIVITY OF DENDRITIC CELLS AGAINST ACTIVATED CD4+ AND CD8+T-LYMPHOCYTES IN THE CULTURE IN VITRO

The aim of the present study was a comparative assessment of the cytotoxic activity of IFNα-induced dendritic cells (IFN-DCs) and IL-4-induced DCs (IL4-DCs) against allo-MLC- activated CD4+ and CD8+T-lymphocytes. It was shown the level of AnnexinV+/PI- cells corresponding to early apoptosis among CD4+T-lymphocytes was lower in cultures with IFN-DCs compared with IL4-DCs. At the same time the relative number of AnnexinV+/ PI+ cells (late phase of apoptosis/necrosis) among CD4+T-lymphocytes activated by allogenic IFN-DCs and IL4-DCs increased in an equal degree. It was demonstrated that IFN-DCs possesed similar ability to induce apoptosis of CD3+CD8+T-lymphocytes

CORRECTION OF INTERFERON-α-INDUCED DENDRITIC CELL DYSFUNCTIONS IN IMMUNOCOMPROMISED PATIENTS

The correction of impaired functions of in vitro generated interferon-alpha-induced dendritic cells (IFN-DCs) in patients with malignant brain tumors (n = 8), malignant lymphomas (n = 14) and pulmonary tuberculosis (n = 26) was analyzed in the present study. The principal possibility of restoring reduced functional activity of patient DCs was shown. The addition of recombinant human IL-2, complex of pro-inflammatory cytokines, double-stranded human DNA or polyoxidonium to IFN-DC cultures at the stage of maturation could significantly increase the allostimulatory activity of DCs in patients with malignant lymphomas and pulmonary tuberculosis, as well as enhance the cytotoxic activity of cancer patient DCs against NK- and TRAIL-resistant tumor cell line HEp-2. These findings offer new approaches to the correction of DC dysfunctions in the pathology and justify ways of improving the treatment of infectious diseases and cancer using dendritic cell vaccines

INTERLEUKIN-10 REGULATES PD-1-B7-H1-MEDIATED CYTOTOXIC ACTIVITY OF DENDRITIC CELLS

In this investigation the phenotypic and functional properties of healthy donor and patient with pulmonary tuberculosis (PT) dendritic cells (DCs) were characterized. We also studied the influence of IL-10 on the phenotype and apoptosis-inducing activity of healthy donor DCs. 60 patients with pulmonary tuberculosis with different proliferative response to antigens of M. tuberculosis (purified protein derivative, PPD) and 40 healthy donors were enrolled in this study. It was revealed that DCs, generated in vitro from PT patient's blood monocytes with GM-CSF+IFN-α, were characterized by increased B7-H1 expression, up-production of IL-10 and reducing of allostimulatory activity in mixed lymphocyte culture (MLC). The endogenous IL-10 production by DCs was correlated with expression of B7-H1 in the general group of persons. It was revealed that addition of IL-10 to semi-mature DCs of healthy donor results in increasing of B7-H1 expression, diminishing of allostimulatory activity and enlargement of pro-apoptogenic activity of DCs

VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR-1 SIGNALING AS A NOVEL MECHANISM OF T CELL SUPPRESSION IN TUMOR NEOANGIOGENESIS

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

EFFECT OF DEXAMETHASONE ON INTERFERON-α-INDUCED DIFFERENTIATION OF MONOCYTES TO DENDRITIC CELLS

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

INFLUENCE OF DEXAMETHASONE-MODIFIED DENDRITIC CELLS GENERATED WITH IFNα UPON AUTOLOGOUS T LYMPHOCYTE FUNCTIONS IN THE PATIENTS WITH RHEUMATOID ARTHRITIS

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

Expression of M2-associated molecules in circulating monocyte subsets in fertile non-pregnant women and pregnant women with uncomplicated 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

SPONTANEOUS AND LPS-INDUCED PRODUCTION OF 26 CYTOKINES SECRETED BY BLOOD CELLS OF PATIENTS WITH LIVER CIRRHOSIS DURING OF CELL THERAPY

The objective of the present study was to assess the level of 26 cytokines secreted by peripheral blood cells of the patients with liver cirrhosis (LC; n = 20) during the cell therapy (CT). All the patients were administered with intravenously injected autologous bone marrow-derived mononuclear cells (MNCs) in a dose of 1.3±0.3 × 109 (Ме 1.0 × 109 ) followed by 14 days later intravenous injection of ex vivo generated mesenchymal stromal cells (MSCs) in a dose of 22.3±5.0 × 106 (Ме 16.0 × 106 ). The patients were examined before the CT, 2-3 days after the administration of MNCs and, then, 2-3 days after the introduction of MSCs. Cytokine-secretory function of peripheral blood cells was evaluated in a 24-hour whole blood cultures both in the absence of any stimulation and in response to lipopolysaccharide (LPS). The control group consisted of 10 healthy donors. The administration of patients’ bone marrow cells (both MNCs and MSC) was safe and well tolerated and caused no any adverse (toxic or allergic) events. Compared with donors, LC patients (especially, with class B+C by Child-Pugh) differed by an initially increased production of several cytokines and chemokines. Actually, there was a statistically significant increase of the spontaneous production of IL-9, MIP-1β, and IP-10, as well as a distinct trend to an increase in TNFα, IL-1ra, IL-4, IL-5, IL-6, IL-13, of MCP-1, MIP-1α, RANTES and Eotaxin. Moreover, the blood cells of LC patients were susceptible to the stimulatory effect of LPS, and the LPS-induced production of 11 out of 26 cytokines (IL-1ra, IL-6, IL-9, IL-15, IL-17, IL-7, IL-8, IP-10, MIP-1α, MIP-1β, Eotaxin) significantly exceeded the normative values. Transplantation of bone marrow MNCs had minimal impact on cytokine production. Meanwhile, the MSCs introduction resulted in a significant decrease in spontaneous and LPS-induced production of, respectively, 20 and 18 analytes including pro-/anti-inflammatory and immunoregulatory cytokines, chemokines and growth factors. The normalization of cytokine-secretory function following transplantation of MSCs revealed in the patients with LC indicates the weakening of an inflammatory activity of circulating blood cells and the decrease in their reactivity to endotoxin. MSC suppressive effect on cytokine production was dose-dependent, and most pronounced in patients with decompensated LC (class B+C by Child-Pugh) of viral etiology

Фактор роста плаценты модулирует ответ активированных in vitro T-клеток

Background. Recent studies demonstrated immunosuppressive properties of vascular endothelial growth factor (VEGF-A) and identified VEGF-A as a key mediator of tumor-induced immunosuppression. Placental growth factor (PlGF) is another member of VEGF family in which dramatic elevation is associated with effective immune adaptation in successful pregnancy, whereas low concentrations are related to pregnancy complications resulting from the activation of immune system. Previously, we have shown that activated T cells express VEGF receptor type 1 (VEGFR-1), and PlGF inhibits T cell proliferation in VEGFR-1–dependent manner.The aim of the present study was to further characterize PlGF effects on T cell responses in vitro.Materials and methods. Peripheral blood mononuclear cells (PBMC) from healthy donors were stimulated with anti-CD3 monoclonal antibodies (a-CD3) in the absence or presence of PlGF and assessed for IL-10 production, programmed cell death and the expression of inhibitory receptors (PD-1, CTLA-4, Tim-3) in CD4+ and CD8+ T cell subsets.Results. The addition of PlGF to PBMC cultures activated with a-CD3 resulted in increased percentages of IL- 10-producing CD4+ and CD8+ T cells. Besides, PlGF promoted CD8+ T cells apoptosis while did not affect programmed cell death within CD4+ T cells. Notable, T cell activation with a-CD3 in the presence of PlGF was accompanied by the enhancement of PD-1-expressing cells in CD8+ T cell subset and Tim-3-expressing cells in both CD4+ and CD8+ T cells, and by the increased expression of PD-1 and Tim-3 on T cells.Conclusion. Our in vitro findings indicate that PlGF can inhibit T cell responses due to the increasing interleukin-10 (IL-10) production, promoting CD8+ T cell apoptosis and enhancing the expression of PD-1 and Tim-3 inhibitory receptors. Given the elevated levels of PlGF in successful pregnancy and its decrease in gestation complications, the obtained data also suggest that PlGF-mediated suppression may be implicated into the governing immune evasion in pregnancy.Актуальность. Недавние исследования выявили иммуносупрессивные свойства фактора роста эндотелия сосудов (VEGF-A) и его ключевую роль в опухоль-индуцированной иммуносупрессии. Плацентарный фактор роста (PlGF) является еще одним представителем семейства VEGF, резкое возрастание которого ассоциировано с эффективной иммунной адаптацией при успешной беременности, тогда как низкие  концентрации PlGF являются предиктором гестационных осложнений на фоне активации  иммунной системы. Ранее нами показано, что активированные Т-клетки экспрессируют рецепторы VEGF 1-го типа (VEGFR-1) и PlGF через связывание с VEGFR-1 ингибирует пролиферацию Т-клеток.Цель. Дальнейшее изучение влияния PlGF на T-клеточный ответ in vitro.Материалы и методы. Мононуклеарные клетки (МНК) периферической крови здоровых доноров стимулировали моноклональными анти-CD3-антителами (a-CD3) в отсутствие и присутствии рекомбинантного PlGF и оценивали продукцию интерлейкина-10 (IL-10), уровень апоптоза и экспрессию ингибиторных рецепторов (PD-1, CTLA-4, Tim-3) в  субпопуляциях CD4+ и CD8+ T-клеток. Результаты. Активация МНК a-CD3 в присутствии PlGF приводила к возрастанию относительного содержания CD4+ и CD8+ T-клеток, продуцирующих IL-10. Кроме того, PlGF усиливал апоптоз активированных CD8+ T-лимфоцитов, не влияя значимо на уровень программированной клеточной гибели CD4+ Т-клеток. Характерно, что активация Т-клеток a-CD3 в присутствии PlGF сопровождалась возрастанием PD-1  экспрессирующих клеток в субпопуляции CD8+ Т-клеток и Tim-3-экспрессирующих клеток среди CD4+ и CD8+ Т-клеток, а также повышением уровня экспрессии PD-1 и Tim-3 на Т-клетках.Заключение. PlGF способен ингибировать Т-клеточный ответ посредством усиления продукции IL-10 и активационно-индуцированного апоптоза CD8+ Т-клеток, а также экспрессии ингибиторных рецепторов. Учитывая повышенный уровень PlGF при физиологической беременности и его снижение при гестационных осложнениях,  полученные данные позволяют предполагать, что ингибиторный эффект PlGF на Т-клеточный ответ может являться еще одним механизмом, обеспечивающим защиту плода  от иммунной системы матери

Отдаленные результаты применения комбинированной иммунотерапии в комплексном лечении больных со злокачественными глиомами головного мозга

The problem of search and development of new approaches to integrated treatment of patients with malignant brain glioma remain one of the most urgent issues for contemporary neurosurgery and neurooncology. In spite of the applied efforts the results of a complex therapy for malignant glioma, especially for glioblastoma, are utterly unsatisfactory. Immunotherapy is one of the directions searching for a generation of cytotoxic cells able to lyse the tumor. Combination of various immunotherapeutic approaches is regarded the most perspective way. Since 1999 Neurosurgical Clinic of Novosibirsk Research Institute of Traumatology and Orthopaedics was carrying out clinical trials of combined immunotherapy within complex therapy of patients with malignant brain glioma in compliance with two protocols.Проблема поиска и разработки новых подходов в комплексном лечении больных со злокачественными глиомами головного мозга остается одним из актуальнейших вопросов современной нейрохирургии и нейроонкологии. Несмотря на усилия, предпринимаемые в этой области, результаты комплексной терапии злокачественных глиом, особенно глиобластом, остаются крайне неудовлетворительными. Одним из направлений является иммунотерапия, направленная на генерацию цитотоксических клеток, способных лизировать опухоль. При этом наибольшие перспективы связывают с комбинацией различных иммунотерапевтических подходов. В клинике нейрохирургии НИИ травматологии и ортопедии (г. Новосибирск) с 1999 г. проводились клинические испытания комбинированной иммунотерапии в комплексном лечении больных со злокачественными глиомами головного мозга по двум протоколам