NGS data analysis algorithm for evaluating repertoire of T-cell receptors involved in the antitumor immune response
Introduction. The repertoire of T- and B cellular receptors is represented by a large number of sequences. Therefore, the characterization of the immune receptor repertoire is a technically difficult task. The most advanced method of analyzing the immune receptor repertoires is NGS sequencing. To reduce the risk of error when working with sequences, computational biology methods are used, which simplify the processing of large amounts of data.
Aim of study - development and testing of NGS data analysis algorithm for comparative evaluation of the T-cell receptor repertoire in syngeneic and allogeneic responses to the tumor.
Material and methods. As a part of this work we have created a chain of program scenarios that allow to increase the efficiency of analyzing the repertoire of T-cell receptors. The developed scenarios allow us to automatically start processing of «raw» data in MIGEC software, merge sequences within groups, and analyze clonal composition of T-cell populations taking into account the specificity of the immune response. The conditions and parameters of the analysis have been tested in a biological experiment in comparison with the antitumor response in groups of mice immunized by EL-4 line tumor cells.
Tumor cells of EL-4 line were grafted into C57BL/6 mice to evaluate the syngeneic response, and tumor cells of B10.D2 (R101) line were grafted into B10.D2 (R101) mice to evaluate the allogeneic response. To exclude allospecific component in allogeneic response to the tumor, an additional group of animals of B10.D2 (R101) line was immunized with normal thymocytes of C57BL/6 mice.
Results. When comparing the repertoire of T-cell receptors in the examined groups, it was found that the antitumor T-cell response is characterized by a decrease in diversity and an increase in the clonality of the immune response to the tumor, most pronounced in the allogeneic group of B10.D2 (R101) mice. The analysis of antitumor clones in the syngeneic and allogeneic models showed reliable differences in qualitative composition of T-cell repertoire. Using the developed program scenarios, the clonal composition of T-cell populations was analyzed, taking into account the allospecific component of the immune response, and specific clones providing antitumor response in the syngeneic and allogeneic models were identified.
Conclusion. As a result of the research, an algorithm for analyzing the data of NGS-sequencing processed in MIGEC software was created. The conditions and parameters of the analysis were tested in a biological experiment in comparison with the antitumor response in groups of mice immunized by EL-4 line tumor cells. T-cell receptor sequencing of α- and β-chains in immunized mice and control animals were performed. A comparative analysis of repertoire of T-cell receptors in mice in a syngeneic and allogeneic model in the development of antitumor immune response was carried out. It was found that the syngeneic and allogeneic antitumor response, as well as allogeneic response to intact tissue, differ from each other. The structure of the immune response in the allogeneic model seems to contain tumor-specific clones that are absent in the syngeneic model.
Analysis of main and small populations of lymphocytes of rhesus macaque peripheral blood using the method of flow cytometry
Introduction. When assessing the immune status of humans and animals, along with the main populations of lymphocytes, in some cases, it is necessary to take into account data on small subpopulations of lymphocytes and pools of activated cells.
The aim - determination presence and intervals of main and small lymphocytes populations distribution in the peripheral blood of healthy rhesus macaque using multicolored cytometric analysis and comparison of the date obtained with the corresponding characteristics of humans.
Material and methods. Venous blood of 17 healthy Macaca mulatta males 2.0-2.5 years old was used in the experiments. Whole blood of animals was stained with fluorochrome-conjugated mouse anti-human monoclonal antibodies that cross-react with rhesus monkey lymphocyte antigens. Determination of the populations of monkey lymphocytes was carried out using the flow cytometry method. The results obtained were compared with similar data for children and adolescents published in Russian scientific journals.
Results. We identified 20 major and small populations of rhesus monkey lymphocytes, studied the spontaneous expression level of markers of early (CD25) and late (HLA-DR) lymphocyte activation on T-cell subpopulations. In rhesus monkeys, a significantly lower relative content of T-lymphocytes is observed; NKT cells, T-helpers, cytotoxic T-lymphocytes, double negative T-lymphocytes, T-lymphocytes expressing an early activation marker, T-helpers expressing an early activation marker, T-regulatory cells, but higher - B-lymphocytes, natural killer cells, CD8 positive natural killer cells, double-positive T-lymphocytes, T-lymphocytes expressing a late activation marker, T-helpers expressing a marker of late activation, and cytotoxic T-lymphocytes expressing a marker of late activation (p < 0.05).
Conclusion. Determination by flow cytometry of indicators of the main, small and activated lymphocytes of rhesus monkeys will allow, when simulating human diseases in animals, to assess not only the period of the onset of the immune response and its nature, but also make it possible to predict the course of the disease, carry out laboratory monitoring of therapy, and in the future will allow the use of lymphocytes as targets of therapeutic action.
Сhain-centricity of TCR phenomenon – opportunities and problems of application in medicine
Crystallographic studies of TCR interactions with MHC molecules formed the general conception that a TCR acquire diagonal orientation on an MHC molecule to provide interaction of an antigen peptide, presented by this MHC molecule with the most variable CDR3 regions of α- and β-chains of the TCR. Using alanine mutagenesis, X-ray crystallography, and methods of plasmon resonance researches demonstrated that amino acid residues of the peptide, α-helices and β-sheets of MHC that form the peptide-binding groove, and all three variable regions of both TCR chains take part in TCR-MHC/peptide interaction. Recent studies reveal a novel unusual mode of the specific recognition of HLA/peptide complexes by TCRs. The unique feature of such recognition is in the leading role of the α-chain TCR in interaction with HLA/ peptide while the β-chain TCR plays a passive role, influencing the overall avidity of the TCR-MHC interaction but not its specificity. Such an unusual way of recognition could completely change the practical application of T-cell receptors in medicine. The possibility to identify one TCR chain, dictating the specificity allowed us to avoid long-term and labor-consuming T-cell cloning and apply NGS-sequencing to analyze α-chains in the polyclonal populations of T-cell and their response to the antigen. This approach could significantly fasten selection of therapeutic α-chains and open perspectives for personalized therapy for cancer patients and operative formation of immune resistance to infections.
Functional activity of bronchial granulocytes in the cytokine profile formation in asthma patients during airway reaction to cold stimulus
Introduction. The expression of cytokine genes for Th2 and Th1 types and epithelial dysfunction associated with the production of information molecules of inflammation and oxidative stress by granulocytes are indicators of the functional activity of bronchial granulocytes in patients with bronchial asthma (BA).
Aim - to study the activity of granulocytes in the formation of the cytokine profile and structural signs of epithelial dysfunction in asthma patients with cold airway hyperresponsiveness (CAHR).
Material and methods. 22 patients with persistent asthma with cold airway hyperresponsiveness (1st group) and 20 asthma patients with no reaction to cold air (2nd group) were examined. Lung function indices were recorded by spirometry, a 3-minute test by isocapnic hyperventilation with cold (-20°C) air (IHCA) was performed. In sputum collected before and after IHCA, cytosis, composition, degree of destruction and intensity of cytolysis of cellular elements, cytokine content (IL-1β, IL-8, TNFα, IL-18, IL-5, IL-10, IL-13) were determined.
Results. In the sputum of patients of 1st group, in response to the IHCA, an increase in cytosis, a decrease in the number of bronchial epithelial cells due to destruction and cytolysis were observed. After the IHCA in 1st group, in relation to the 2nd group, an increase in the concentrations of IL-1β, IL-8 and TNFα was recorded. A close relationship was found between the concentration of IL-1β and IL-8 in sputum and a high level of cytosis after the IHCA, as well as between the level of IL-18 and the level of cytosis before and after the IHCA.
Conclusion. The processes of destruction and cytolysis of neutrophils, accompanied by an increase in the production of proinflammatory cytokines, as well as structural signs of epithelial dysfunction during the reaction of bronchi of asthma patients to a cold stimulus, occupy an important place in the formation of cold airway hyperresponsiveness.
Analysis of changes in cytokine concentrations and neutrophil activity in patients with essential arterial hypertension depending on the affected target organs against the background of antihypertensive therapy
Introduction. It is known that the assessment of cytokine levels and neutrophil activity in the peripheral blood in patients with essential arterial hypertension will reveal differences depending on the target organ affected.
The aim of the study was to assess changes in cytokine concentrations and neutrophil activity in peripheral blood in patients with essential arterial hypertension and subclinical damage to target organs during antihypertensive therapy.
Material and methods. The study included 104 patients with grade II essential arterial hypertension, grade 2, aged 30 to 50 years with left ventricular myocardial hypertrophy (n = 33), with predominantly vascular lesions in the form of asymptomatic atherosclerosis (n = 35) and with predominant kidney damage in the form of the development of stage 3 chronic kidney disease (n = 36).
Results. The greatest changes in the concentration of cytokines (TNFα, IL-1α, IL-6, IL-8, IL-10, IL-1Ra, IL-2) in patients with essential arterial hypertension are determined in asymptomatic atherosclerosis. Disturbances of functional and metabolic activity (NBT-test spontaneous and stimulated) of peripheral blood neutrophils are more pronounced in patients with left ventricular myocardial hypertrophy. The immunocorrective efficacy of antihypertensive therapy in patients with essential arterial hypertension is more effective in patients with asymptomatic atherosclerosis and is least effective in patients with chronic kidney disease.
Conclusion. Determination of the concentration of cytokines and functional-metabolic activity of neutrophils in patients with essential arterial hypertension in patients with essential arterial hypertension will reveal differences depending on the affected target organ and thereby serve as a search for predictors of the development of irreversible changes in target organs with the development of associated clinical conditions and their complications.
Molecular mechanisms of viral evasion from immune surveillance in autophagy
Autophagy is a conservative evolutionary established cellular process functioning to maintain cell homeostasis. Furthermore, the cellular autophagy program acts as the primary mechanism of intracellular defense in the cells infected with viruses or other pathogens. However, many viruses are able to modulate the cellular autophagy program by induction or inhibition its individual stages through various mechanisms. Autophagy can function both, as proviral and antiviral mechanism, in the pathogenesis of infection depending on the virus and cell type. This review discusses relevant studies of the mechanisms of interaction between the cellular program of autophagy and viral replication, in particular, flaviviruses, paramyxoviruses, coronaviruses and others. The study of these mechanisms may be important for a better understanding of the pathogenesis of viral infectious diseases and the identification of new therapeutic targets.
Red blood cells as bactericidal cells, participants and regulators of inflammation
The study of the biology of red blood cells has led to revolutionary changes in the understanding of its physiological role. It turned out that these «simple oxygen carriers» are direct participants and regulators of innate immunity reactions. Red blood cells have a unique electrochemical bactericidal mechanism - oxycytosis - based on their ability to acquire an additional triboelectric charge in the mobile medium of the bloodstream and agglutinate circulating bacteria. At the same time, binding to the surface of red blood cells causes the release of reactive oxygen species with antibacterial activity from oxygenated hemoglobin. The bacteria killed in this way cease to be retained on the surface of the red blood cell, return to the blood plasma, after which they are captured by Kupffer cells in the liver and macrophages in the spleen. The participation of red blood cells in immune system reactions is also associated with the ability to bind endogenous and exogenous inflammatory molecules, including chemokines, nucleic acids and components of pathogens, to secrete lipid mediators, etc. The participants in the reactions of innate immunity are the multiscale internal structures of red blood cells (hemoglobin, heme, globins, various alarmins) that can induce and control the development of inflammatory protective and pathological reactions. Under certain conditions, the implementation of protection mechanisms initiated by red blood cells can also cause unwanted destructive responses to infection. In general, depending on the microenvironment, red blood cells can either support anti-inflammatory conditions or promote immune activation. The information presented in this review provides an understanding of the role of red blood cells in the generalization of infections, the pathogenetic role of hemolysis and extracellular hemoglobin in conditions of ischemia/reperfusion. Further research in this area will help identify new therapeutic targets and increase the effectiveness of the treatment of many serious, life-threatening conditions and diseases. Literature was searched using the original publications and databases PubMed, MedLine, Scopus, Google Scholar, etc.
Application of chitosan and its derivatives in immunotherapy of malignant neoplasms
The article provides an overview of the effect of chitosan and its derivatives chitooligomers on the components of the immune system. The main attention is paid to the use of chitosan in immunotherapy of malignant neoplasms. The physicochemical properties of chitosan make it promising for the development of immunotropic drugs, adjuvants for vaccines, and a pharmaceutical basis for the creation of delivery vehicles for medicinal or immunoactive substances. Targeted platforms based on chitosan are of great interest for practical medicine. The successful application of the indicated approaches in the treatment of oncopathology will lead to an increase in the therapeutic efficiency of existing treatment methods and a potential increase in the quality of life of patients.