Immunologiya

Introduction. Catalogues of common and well-documented HLA alleles are widely used as indicator for ambiguities to be resolved or for checking the universality of any HLA allele in the world. Information about the distribution of HLA alleles in different populations is important, since foreign donors are often involved in unrelated hematopoietic stem cell transplantation. The aim of the study was to establish a catalogue of common and well-documented HLA alleles in the Russian population and compare data with the EFI-CWD catalogue.

Material and methods. The study involved 10 795 potential bone marrow/hematopoietic stem cells donors with self-identification as the Russians, who were typed for the genes HLA-A, -B, -C, -DRB1, -DQB1 with high resolution by the NGS. 5273 donors were recruited in Nizhny Novgorod and the Nizhny Novgorod region; 2577 donors – in Moscow and the Moscow region; 1968 donors – in other regions of the Central Federal District, 977 donors – in different regions of Russia, not included in any of the three above-mentioned samples. HLA alleles are classified as common (detected at least 3 times in each of the first three samples); well-documented alleles (detected five times, or detected three times in unrelated donors sharing a haplotype), and rare.

Results. The first catalogue of common and well-documented HLA alleles of the Russians was established. Of the 412 HLA alleles identified, 101 were common, 90 % of the common alleles in the Russians are shared with the common alleles in EFI-CWD. Some common alleles in the Russians were rare in EFI-CWD, and some common alleles in EFI-CWD were rare in the Russians. So, EFI-CWD catalog cannot be used as a surrogate for the Russian population, although the Russians are HLA-genetically close to other European populations.

Conclusion. Only 90 % of common and 76 % of common and well-documented alleles in the Russian population are shared with those in the EFI-CWD catalogue, which indicates the need to have our own catalog of common and well-documented HLA alleles in the Russians.

Introduction. Specific binding of S. aureus protein A with Fc-fragment of human IgG in a context of possibility of its functioning as soluble Fc-receptor was considered.

Aim – evaluation of cytokine production by human blood cells in presence of human serum γ-globulin and of its metal complexes during equimolar binding them by S. aureus protein A.

Material and methods. The work was made on human peripheral blood cells with use of methods of preparational biochemistry and protein chemistry, of optico-physical and immunochemical methods, of the technologies of cytokine induction.

Results. It has been shown that S. aureus protein A interacted equimolarly with human serum γ-globulin and bound its Fc-fragment that led to formation of characteristical complex and to decrease in induction by γ-globulin of the production of IL-1β, IL-6, TNF-α and IL-10 by human blood cells. Formation of γ-globulin metal complexes caused conformational changes of the protein molecule that led to partial decrease in efficiency of binding the ligand by S. aureus protein A.

Conclusion. Formation of the γ-globulin metal complexes decreased efficiency of its equimolar binding by S. aureus protein A and limited the blocking in mechanisms of cytokine induction activated by interaction of the cell Fc-receptors with specific ligand.

Introduction. Cellular aging is described as the irreversible loss of the ability of cells to proliferate, caused by stressful effects. The accumulation of aging cells has been described in various inflammatory diseases and can be both a consequence of inflammation and contribute to its maintenance, which is due to increased production of proinflammatory cytokines by aging cells.

Aim – to conduct a quantitative and qualitative assessment of senescent subpopulations of blood mononuclear cells (MNCs) in patients with a chronic inflammatory skin disease – atopic dermatitis.

Material and methods. Blood MNCs were isolated from heparinized venous blood of patients and healthy donors in a Ficoll density gradient. To identify senescent cells, MNCs were stained with conjugates of antibodies to surface markers (CD3, CD56, CD14, CD19) and to intracellular proteins – markers of cellular senescence (p21^WAF1/CIP1, p16^INK4A), after which they were analyzed by flow cytometry. Senescence-associated beta-galactosidase activity was measured by flow cytometry after incubation of cells with a specific fluorogenic substrate. Plasma IL-6 levels were measured using ELISA.

Results. Senescent cells with p21⁺p16⁺ phenotype were detected in all studied subpopulations of peripheral blood MNCs in both healthy donors and patients. Significantly higher number of senescent cells was found in CD3⁺CD56⁺-T-cell subpopulation in patients compared to healthy donors. Senescence-associated beta-galactosidase activity in monocyte, T cell, and NK cell subsets in patients correlated with disease severity. Moderate positive correlation of plasma IL-6 levels with age was found in the atopic dermatitis group, which was absent in the healthy donor group.

Conclusion. For the first time was performed quantitative evaluation of senescent cells among blood MNCs in patients with atopic dermatitis. Increased content of senescent cells for patients compared to healthy donors was revealed in CD3⁺CD56⁺-T-cell subpopulation.

Introduction. Allergic airway diseases caused by hypersensitivity to aeroallergens is the most common manifestation of atopy in children and adults. Bronchial asthma (BA) remains one of the urgent medical and social problems of the 21^st century. Today, bronchial asthma is considered one of the most common chronic diseases, which affects more than 348 million people worldwide (GINA, 2023). Urbanization is considered to be a possible reason for this trend. However, the structure of sensitization in the population depends primarily on the allergenic composition of the air environment. Identification of sensitization is a prerequisite for successful treatment of allergic diseases, while the structure of sensitization may have regional characteristics. Despite the numerous epidemiological, clinical, immuno-genetic studies, their results are contradictory, the genetic basis for the formation of bronchial asthma, taking into account the ethnicity and genetic characteristics of patients in Uzbekistan, has not yet been sufficiently studied. This requires the identification of new immunological and hereditary mechanisms of the development of this disease in order to develop effective pharmacotherapeutic measures using personalized therapy. In recent years, the introduction of genetically engineered immunobiological drugs (GEIBD) into the treatment regimen of patients with bronchial asthma contributes to a significant reduction of symptoms, prevention of exacerbations and improvement of the quality of life of patients. The various clinical manifestations of atopic bronchial asthma are based on the release of inflammatory mediators as a result of the interaction of immunoglobulin E associated with mast cell receptors and basophils with the initiating allergen. The creation of omalizumab (anti-IgE) human recombinant monoclonal antibodies against immunoglobulin E has potentially identified the possibility of interfering with the development of an allergic response at the effector phase level.

Aim – evaluate the effectiveness of targeted therapy with Genolar^® (omalizumab) in patients with atopic bronchial asthma, depending on the characteristics of climate and geographic conditions in Uzbekistan.

Material and methods. From January 2022 to March 2023 100 patients with allergic diseases were studied, including 38 children aged 2–18 years, among whom allergic rhinitis (AR) was verified in 20 aged 2–18 years, intermittent BA in 12 children aged 2–18 years, atopic dermatitis (AD) in 6 children aged 2–18 years, and 62 adult patients, including 32 aged 19–59 years with AR, 13 aged 19–59 years with BA, and 17 aged 19–59 years with AD. To evaluate targeted therapy with Genolar^® (omalizumab), patients with atopic bronchial asthma were selected from this cohort of patients. To study the total IgE in blood serum, we used AccuBind ELISA Microwells test system (Monobind, USA); allergen-specific IgE was determined using AllergoIFA-specific IgE test systems («Alcor Bio», Russia); standard diagnostic allergens (JSC SPA «Microgen», Russia) were used for skin scarification testing. In 2020, the Russian company Generium developed the biosimilar of omalizumab named Genolar^® which is received a registration certificate in RF. The clinical study was conducted at the Republican Scientific Specialized Allergological Center of the Ministry of Health of the Republic of Uzbekistan in patients with severe uncontrolled bronchial asthma. Our study included 20 patients in the age group from 10 to 54 years with confirmed uncontrolled severe atopic asthma (ACQ-5, FEV1 < 80 % of the required values) where the diagnosis and severity were established in accordance with the criteria of GINA, 2022, in which polyvalent sensitization was detected, confirmed by molecular allergodiagnostics, skin prick testing and an increased level of total IgE In all patients, in accordance with the inclusion/exclusion criteria, Genolar^® was added to therapy, the dose and frequency of administration of were individually selected taking into account the patient’s body weight and the initial level of total IgE. Based on the improvement in the patient’s condition, which was assessed by changes in respiratory function, peak exhalation rate, the results of the ACQ-5 questionnaire, and a decrease in the number of night and day exacerbations of asthma, the effectiveness of targeted therapy was evaluated. All patients were informed about the drug and gave written consent to participate in a 12-week clinical trial.

Results. All patients treated with Genolar^® noted a significant decrease in the severity of BA symptoms during omalizumab therapy. After 1 month from the beginning of the therapy, patients demonstrated a decrease in night and daytime seizures, a decrease in shortness of breath, and the need for SABA. The average ACQ-5 score of the test after 1 month compared with the baseline data was 2.46 points with a tendency to further decrease in the continuation of therapy and reached 0.95 points by the 3^rd month.

Conclusion. When assessing the volume of anti-asthmatic basic therapy used on the background of Genolar^®, it became possible to escalate the dose of SGCs up to complete withdrawal in 9 patients, dose reduction was initiated 4 weeks after the start of therapy according to an individual scheme, depending on the initial dose and subject to maintaining control over the symptoms of asthma. In 17 patients, they managed to get away from an additional control drug (montelukast). Due to improved symptom control, patients did not need unscheduled medical attention for BA, did not need emergency medical calls, and were not hospitalized for an exacerbation of BA. Among the patients of the studied group, a high degree of sensitization to pollen and household allergens was found in both children and adults, reflecting the climatic and geographical features of the Republic of Uzbekistan.

Introduction. The tissue fibrosis is an element not only of reparation but also of many pathologic states in particular of uterine leiomyoma, in view of what one of the most actual vectors of investigation is the clarification of the mechanisms of regulation by macrophages of the fibrotic process.

The aim of the study was to assess the influence of endometrial macrophages upon collagen synthesis and production in the primary cultures of autologous leiomyocytes.

Material and methods. 25 women of reproductive age with symptomatic intramural uterine leiomyoma participated in the study. Collagen concentration in the supernatants of 24 and 72 hour myofibroblast cultures, cocultured with autologous endometrial macrophages was assessed by ELISA method. Col 1A1 mRNA expression was assessed by real time RT-PCR.

Results. Collagen production by myofibroblasts from myomatous nodes in 24 and 72 hour culture supernatants decreased under the influence of autologous endometrial macrophages. Though the increase of collagen production and synthesis on the third day of incubation under the influence of macrophages allow suggesting, that the effect of suppression had transitional character and is directly connected with the regulation of activity by macrophages.

Conclusion. Endometrial macrophages are able to suppress collagen I type synthesis and production by myofibroblasts from myomatous nodes.

Introduction. The ability of immune cells to destroy tumor cells due to the recognition of tumor antigens by the T-cell receptor (TCR) and the transmission of activating signals inside the cell underlies modern immunotherapy of oncological diseases. The development of technology for isolating and cloning T-cell receptors and genetic engineering has made it possible to create patient T-cells encoding TCR for tumor antigens, capable of effectively destroying tumor cells.

Aim – production of genetically modified TCR-T cells aimed at recognizing epitopes of antigens associated with melanoma by induction of antigen-specific T cells and sequencing of single cell RNA.

Material and methods. Peripheral blood mononuclear cells from healthy donors with the HLA-A02 genotype were used to generate dendritic cells loaded with peptides to target antigens and subsequent clonal expansion of antigen-specific T cells. TCR sequence analysis was performed using single cell RNA sequencing. Bioinformatic analysis using different approaches allowed us to obtain TCR sequences for subsequent cloning and expression in a lentiviral vector. The obtained vectors were used for T cell transduction and evaluation of cytotoxic activity against melanoma cell lines with the expression of PRAME and MART-1 [SK-Mel-5 and SK-Mel-37, provided by Professor H. Shiku from the collection of the Graduate School of Medicine of the University of Mie (Japan)]. The transduced T cells were cultured with tumor cells in a 10 : 1 ratio, and the cytotoxic effect was assessed by the level of the enzyme lactate dehydrogenase, which is released from the lysed cells.

Results. As a result of clonal expansion, an almost 1000-fold enrichment of the initial population of antigen-specific cells was obtained. Cells with stable expansion of specific TCR and proliferative activity were used for transcriptome analysis on the BD Rhapsody platform. To determine the optimal strategy for selecting antigen-specific TCR, we used the ERGO-II neural network, which predicts the affinity of TCR to the target peptide of the PRAME antigen and determines the dominance of the clonotype for TCR recognizing MART-1. The transduction of T-lymphocytes by lentiviral constructs encoding the obtained TCR resulted in the lysis of at least 30 % of target cells on the first day of interaction with tumor cells.

Conclusion. We have achieved significant enrichment of the target antigen-specific cell population in quantities sufficient for single cell transcriptome analysis and T cell clonotypes. Using single cell RNA sequencing technology, we obtained detailed information about the sequence of each TCR and the immune transcriptome of a single T cell, which made it possible to accurately design the resulting TCR clones, assess the functional state of the cells, and improve the selection of TCR candidate clones. The obtained sequences for TCR were used to develop lentiviral constructs that have shown their effectiveness in the interaction of T cells transduced by these constructs with target cells.

Introduction. The adoptive transfer of ex vivo chimeric antigen receptor (CAR)-modified autologous T cells represents a promising approach to the treatment of hematological malignancies, but the treatment of solid tumors remains a challenge. Disialoganglioside (GD2) is a potential target for CAR T cell therapy of solid tumors, due to the high degree of homology between human and murine antigens. The syngeneic murine model with homologous antigen, as an additional preclinical approach, offers significant potential for the optimization of CAR-T technology, and, in turn, may facilitate the successful clinical trials for solid tumors.

Aim of the study was to evaluate the phenotypic and cytotoxic properties of murine GD2-specific CAR T cells in vitro, and their antigen-specific antitumor activity in vivo in the syngeneic B78-21 melanoma model.

Material and methods. The study was conducted using mice of the C57Bl/6 line. Murine CD3⁺ T cells isolated by magnetic separation were transduced with a retroviral vector enco­ding the GITR ligand (GITRL) and a GD2-specific CAR. The phenotype of GD2-specific CAR T cells was analyzed by flow cytometry with regard to the T cell memory markers CD44 and CD62L, and the T cell depletion markers PD-1 and TIM-3. The in vitro cytotoxic characteristics of transduced cells co-cultivated with target cells (GD2⁺ cells B78-21 and GD2^– cells B78-P4) were evaluated by the assay of lactate dehydrogenase content in conditioned media and by flow cytometry to determine the presence of activation markers (CD69, CD137, CD154) and cytotoxicity markers (CD107a, CD178). The in vivo antitumor activity of the transduced cells was investigated using the GD2-positive syngeneic B78-21 melanoma model and a single administration of GD2-specific CAR T cells at a dose of 5 - 10⁶ cells per mouse.

Results. The generation of murine CAR T cells through retroviral transduction resulted in the production of the population of CD8⁺ cells that was twice the size of the CD4⁺ cells proportion. This population is dominated by effector memory CD44⁺CD62L^– cells, but transduction resulted in an increase in the proportion of central memory CD44⁺CD62L⁺ cells and cells expressing the TIM-3 exhaustion marker in both population. The interaction with GD2⁺ target cells resulted in a significant increase in the proportion of transduced cells expressing the activation markers CD69, CD137, CD154, the cytotoxicity marker CD107a, and a discernible tendency for an increase in the marker CD178. The in vitro cytotoxicity assay demonstrated that the transduced T cells not only exhibited cytotoxic potential, but also demonstrated antigen specificity for GD2⁺ tumor cells. The in vivo antitumor activity of murine CAR T cells was demonstrated by their capacity to inhibit tumor node growth, and the survival rate of tumor-bearing mice was 85 days. The median overall survival of mice following infusion of transduced cells was 61,5 days, which is twice as long as the control groups.

Conclusion. The resulting murine CAR T cells have the phenotype of cytotoxic T lymphocytes and exhibit antigen-specific properties both in vitro, against GD2-positive tumor cell lines, and in vivo, in the syngeneic B78-21 melanoma model.

The review presents the collected material on the induction of tolerance to allografts in large animal models (pigs and non-human primates) in preclinical studies. Since progress in clinical transplantology is impossible without the use of step-by-step studies in preclinical models on large animals, experiments on these models are still relevant.

The technique of tolerance induction described in this review involves the formation of chimerism in recipients after hematopoietic stem cell transplantation based on the in vivo removal of newly formed donor-reactive T lymphocytes during their interaction in the thymus with donor antigen-presenting cells, with parallel development of tolerance to the organ transplanted from the same donor.

The presented data demonstrate that hematopoietic stem cell transplantation is a real alternative to lifelong use of immunosuppressive drugs. It is necessary to continue research in order to understand the immunological mechanisms underlying tolerance based on mixed chimerism in order to use potential targets and necessary manipulations for future strategies of tolerance induction. Knowledge gained from large animal models may have direct implications for various human donor transplant and will greatly enhance the implementation of tolerance induction protocols to prevent rejection of donor organs and tissue assemblies.

Introduction. Due to the spread of antibiotic resistance, the search for new antibacterial vaccines and adjuvants becomes relevant. One of the promising directions is the creation of immunotropic drugs based on lipopolysaccharide (LPS) of gram-negative bacteria, which is a powerful immunostimulant and leads to the production of protective antibodies. However, the clinical use of LPS is limited by its extremely high toxicity, which can be reduced by modifying the structure of lipid A. Currently, detoxified LPS (d-LPS) and lipids A (d-LA) have been obtained, which are widely used in vaccine preparations: d-LPS – as an active substance, d-LA – as an adjuvant. In addition, the possibility of using d-LPS and d-LA as built-in adjuvants is being studied.

Structure and biological activity of LPS of gram-negative bacteria. LPS is composed of three different fragments – lipid A, core oligosaccharide and O-specific polysaccharide. The carbohydrate part of LPS provides serological specificity of different strains, lipid A activates the TLR4/MD-2 complex located on the surface of immunocompetent cells. The biological activity of lipid A is closely related to its structure: with a decrease in the number of fatty acid residues and phosphate groups, the toxicity of LPS is reduced. Thus, the classic, highly toxic lipid A of E. coli is composed of two glucosamine residues, to which 6 fatty acid residues and 2 phosphate groups are attached.

Methods for obtaining detoxified lipids A. Obtaining d-LA involves two steps – reduction of the number of fatty acid residues while retaining both phosphate groups or removal of the phosphate group while retaining the fatty acid composition. Both approaches have been successfully implemented to produce clinically applicable d-LA, OM-174 (d-LA with 3 fatty acid residues and 2 phosphate groups) and MPL (d-LA with 6 fatty acid residues and 1 phosphate group).

Methods for obtaining detoxified LPS. Obtaining d-LPS also involves removing fatty acid residues or phosphate groups by various approaches – chemical, enzymatic and genetic engineering.

Conclusion. Thus, preparations based on d-LPS and d-LA are a promising platform for the creation of new, highly immunogenic antibacterial vaccines and other immunotropic drugs. Since the production of these compounds is associated with a number of disadvantages (duration, high cost, toxicity of the reagents used), the development of new methods for obtaining d-LPS and d-LA becomes relevant.