Immunologiya

Modern omics technologies, combined with the latest bioinformatics approaches to data analysis, offer unique opportunities for accurate, detailed or unbiased study of immunological processes. Previously, the scientific community had access to standard mass methods for analyzing a limited number of markers of individual cell populations, entailing significant processes for their isolation, which have recently been replaced by modern technologies for analyzing single cells, which allow simultaneous analysis of hundreds and thousands of markers of traditional cell populations without the need to separate each of the individual cell population. In this review, we describe recent advances in single-cell omics and superplex spatial analysis technologies through their application in immunological research, providing valuable insights for further advances in basic immunology, new strategies, and personalized approaches to selected immune pathologies.

Introduction. The outbreak of coronavirus infection, which has the nature of a pandemic, has not been completed to this day, therefore, research aimed at finding new ways and methods for predicting and treating COVID-19 is becoming relevant.

The purpose of the work is to determine allelic variants of the ACE1 and ACE2 genes, as well as their combinations, in Russian residents of the Chelyabinsk region who having been suffered with bilateral COVID-19 pneumonia.

Material and methods. ACE1 I/D and ACE2 G8790A were determined by Real-Time PCR based on the melting temperature of allele-specific probes bearing a fluorescent label. Analysis of the distribution of markers was carried out taking into account gender, because ACE2 genes are located on the X chromosome.

Results. The ACE2(8790)*GA genotype was significantly less common in sick women compared to the group of comparison. In female patients, the ACE2(8790)*GG genotype and the ACE1*DD/ACE2*G combination were statistically significantly more common than in the control group, associated with decreased ACE2 expression and increased ACE1 expression. In hemizygous men, on the contrary, the ACE2(8790)*A allele and the ACE1*ID/ACE2*A combination associated with higher ACE2 production were more common.

Conclusion. Predisposition factors for COVID-19 of the ACE1–2 system differ in men and women.

Introduction. B cells are an attractive target for gene therapy. With the help of gene transfer to B cells, it is possible to treat diseases associated with genetically determined dysfunction of B cells, as well as lymphoproliferative and autoimmune pathologies. In recent years, adenovirus (Ad)-based vectors have become increasingly popular for gene transfer. This is facilitated by such properties of Ad as high transgene capacity, the ability to produce the vector in high titers, and the ability to transduce terminally differentiated cells.

The aim of the study is to determine the efficiency of transduction of human B cell lines, as well as activated primary B lymphocytes after infection using adenoviral vectors of various serotypes.

Material and methods. Mononuclear cells were isolated from the peripheral blood of healthy donors (n = 11) by Ficoll-Verografin density gradient centrifugation with further enrichment of B lymphocytes using negative selection using magnetic microspheres. B cells were stimulated in vitro with a mixture of lymphokines, which included IL-2, IL-4, BAFF, IL-21 and multimerized CD40L. After 24 h, stimulated B lymphocytes were infected with recombinant adenoviral vectors carrying the green fluorescent protein (GFP) reporter gene. Three serotypes were tested: Ad5, sAd23 and Ad26, as well as a chimeric Ad5 vector containing the corresponding domain of the Ad35 serotype (Ad5/35) at the C-terminus of its fiber. At 48 h postinfection, the per center of transduced B cells and the average fluorescence intensity of the GFP reporter protein were determined. Adenoviral transduction was determined in four B cell lines: NALM-6, Ramos, IM-9, and RPMI-8226, and also in primary B lymphocytes. For comparison with B cells the non-lymphoid line HEK293 was used.

Results. Adenoviruses in low and medium doses had a moderate toxic effect on cells, while the viability remained at least 70 %. The most susceptible to adenovirus infection were RPMI-8226 cells, in which, after transduction, more than 90 % of cells expressed GFP, and in this indicator they were close to non-lymphoid HEK293 cells. The cells most resistant to adenovirus infection were NALM-6, which were transduced to some extent with the Ad26 and Ad35 vectors (65 and 66 %), slightly with the sAd23 vector (18 %), and minimally with the Ad5 vector. Ramos and IM-9 cells were intermediate in sensitivity to infection, with Ramos being transduced somewhat more successfully than IM-9. When comparing different serotypes, Ad26 turned out to be the most infectious, which, even in minimal doses, transduced more than 90 % of Ramos, IM-9 and RPMI-8226 cells. For stimulated primary B lymphocytes, the Ad26 vector had the greatest transducing efficiency. At a virus dose of 2000 PFU per cell, the median proportion of GFP⁺ cells was 35 % (interquartile range 30–40 %). Ad5/35 showed slightly less activity (median value 16 %, interquartile range 13–20 %). Finally, Ad5 and sAd23 had the lowest transducing activity, which consistently exceeded the threshold level only at the highest doses of virus (1000 to 2000 PFU per cell).

Conclusion. Ad serotypes differ in their ability to transduce human B cells. The most efficient transduction is provided by the Ad26 serotype. The Ad5 vector pseudotyped with Ad35 fiber has a slightly reduced efficiency. Transduction of primary B lymphocytes occurs only after their activation.

Introduction. Quantitative determination of chimerism after allogeneic bone marrow transplantation (aBMT) is one of the main methods for assessing the possibility of engraftment of allografts (including transplantation of vascularized composite tissue complexes – VCA) from the same donor. Complete stable chimerism correlates with long-term engraftment of donor tissues. Dynamic studies of chimerism are necessary to assess the effectiveness of transplantation with further withdrawal of immunosuppressive therapy.

Aim – to study hematopoietic chimerism in minipigs after allogeneic hematopoietic stem cell transplantation (aHSCT) during the induction of immunological tolerance to VCA.

Material and methods. The study was performed on minipig hybrids of the Wisenau and Vietnamese pot-bellied pig. The chimerism was detected using the polymerase chain reaction (PCR) on days «0», «+14», «+21» and «+30» using the COrDIS PIG reagent kit (GORDIZ, Russia). PCR results were studied by capillary electrophoresis using an automatic genetic analyzer (ABI PRISM 3500 Thermo Fisher Scientific, USA). The obtained data were analyzed using GeneMapper software (Thermo Fisher Scientific, USA). Alleles were assessed using 15 microsatellite markers for each minipig blood sample, and the percentage of donor and recipient alleles was calculated.

Results. 9 pairs of experimental animals (Donor–Recipient) were studied. In 5 recipients, on day +30 after aBMT, only their own alleles were determined in the bone marrow aspirate. Mixed donor chimerism was observed in 4 recipients. Determination of the sensitivity of the technique showed a sensitivity of at least 5 % in a model experiment. The determination error depends on the number of loci taken for analysis and does not exceed 10 % when the content of donor alleles is 50 % in the test sample.

Conclusion. Determination of donor chimerism in minipigs after aBMT during the induction of immunological tolerance to VCA demonstrated the dynamics of bone marrow transplant engraftment and the maximum expansion of the recipient’s bone marrow by donor cells up to 80–90 %. The method for determining chimerism using STR loci is highly sensitive and can be used in model experiments with minipigs.

Introduction. The mucosal route of immunization is the focus of investigators for many years and COVID-19 is not exception. One of the objective parameters for assessing immunological effectiveness is the study of T-cell sensitization in the blood of recovered COVID-19 patients or healthy volunteers received vaccine prophylaxis. T-cell immunity to SARS-CoV-2 assessment is important not only for risk stratification and identifying potentially protected populations with immunity acquired through infection, but also for determining the immunogenicity and potential effectiveness of vaccines under development. The results of an interim analysis of data obtained as part of a randomized, double-blind, multicenter phase III clinical trial of a two-component vaccine for the prevention of COVID-19 with intranasal and intramuscular routes of administration are presented.

Aim – to evaluate the T-cell response dynamics to various peptides of the SARS-CoV-2 against after intranasal and intramuscular immunization with a two-component vector vaccine to prevent the development of COVID-19.

Material and methods. A total of 137 healthy volunteers with a baseline anti-RBD IgG level not exceeding 100 BAU/ml received immunization by a two-component (Ad26 and Ad5 based) intranasal or intramuscular vaccine administered on day 1 and day 21. Immunogenicity assessment was based on T-cell response data using IGRA-ELISPOT technology on days 21 and 42 after administration of component I.

Results. The proportion of patients with T-cell sensitization to the SARS-CoV-2 significantly increased after intranasal (from the baseline level of 33.9 % to 55.93 % on day 42) and intramuscular (from the baseline level of 30.51 % to 61.02 % on day 42) immunization and was comparable between groups at all visits. There is a statistically significant increase in the number of SARS-CoV-2 spike (S) protein-specific T cells (total CD8⁺ and CD4⁺) and the absence of sensitization of these cells to the N, M, ORF3a and ORF7a proteins of the virus.

Conclusion. The immunogenic potential of a two-component vector vaccine (based on Ad26 and Ad5) with clonal activation of T cells to the spike (S) protein of the SARS-CoV-2 after intranasal or intramuscular administration has been shown.

Introduction. The systematic study of structural and quantitative indicators of sensitization to certain allergens has epidemiological significance. In our country, the profiles of hypersensitivity to inhalant allergens have their own regional differences due to the climatic and geographical component.

Aim – to study of the profile of sensitization to inhalation allergens in the Moscow region.

Material and methods. Using the skin prick test method, 72 people aged from 4 to 62 years (50 men and 22 women) were examined. We used allergenic extracts (AG) of house dust mites Dermatophagoidas pteronyssinus (AG1), D. farinae (AG2), pollen of birch (Betula pendula Roth) (AG3), pollen of alder (Alnus glutinosa) (AG4), pollen of hazel (Corylus avellana) (AG5), pollen of orchard grass (Dactylis glomerata) (AG6), pollen of timothy grass (Phleum pratense) (AG7). Specific polyclonal antisera (PA) were obtained by immunizing rabbits with AG every 28 days for 4 months. PA activity was determined by reverse ELISA.

Results. The number of cases of detection of hypersensitivity decreases in the following series of AG: AG3, AG5, AG4, AG7, AG6, AG2, AG1 (80.56, 72.22, 70.83, 44.44, 43.06, 31.94, 29.17 % respectively). Frequency of cases of detection of sensitization to one (AG2 or AG3, or AG7), two (AG3 + AG4; AG3 + AG5; AG6 + AG7; AG1 + AG2), three (AG3 + AG4 + AG5; AG3 + AG6 + AG7; AG1 + AG2 + AG3), four (AG1 + AG2 + AG6 + AG7; AG3 + AG4 + AG5 + AG7; AG1 + AG2 + AG3 + AG5), five (AG3 + AG4 + AG5 + AG6 + AG7; AG1 + AG2 + AG3 + AG4 + AG5), six (AG2 + AG3 + AG4 + AG5 + AG6 + AG7) and seven AG simultaneously is 4.17, 16.67, 38.88, 5.56, 23.61, 1.39 and 9.72 % respectively. The degree of binding of PA obtained against AG3 with AG4, AG5, AG6, AG7 was 115.15, 70.26, 23.96, 35.91 %, respectively. The degree of binding of PA obtained against AG4 with AG3, AG5, AG6, AG7 was 20.47, 21.46, 2.25, 1.71 %, respectively. The degree of binding of PA obtained against AG5 with AG3, AG4, AG6, AG7 was 60.22, 47.6, 0, 9.98 %, respectively. The degree of binding of PA obtained against AG6 with AG3, AG4, AG5, AG7 was 8.09, 44.71, 8.97, 94.71 %, respectively. The degree of binding of PA obtained against AG7 with AG3, AG4, AG5, AG6 was 12.17, 33.52, 5.16, 89.78 %, respectively. The degree of binding of PA obtained against AG1 with AG2 was 93.89 %. The degree of binding of PA obtained against AG2 with AG1 was 54.74 %.

Conclusion. The frequency of detection of polysensitization is 95.86 %, while for different complexes of aeroallergens it decreases in the following order: AG3 + AG4 + AG5; AG3 + AG4 + AG5 + AG6 + AG7; AG1 + AG2 + AG3 + AG4 + AG5 + AG6 + AG7; AG6 + AG7; AG1 + AG2; AG1 + AG2 + AG3 + AG4 + AG5 (33.33, 18.06, 9.72, 6.94, 6.94, 5.56 %). Compared to 2012 data, the number of people with hypersensitivity to pollen hypertension increased by 15.52 %. The prevalence of hypersensitivity to house dust mites has decreased by almost half compared to 2009 data.

Introduction. Blockade of immune checkpoint receptors (immune checkpoint blockade, ICB) is one of the effective approaches of modern immunotherapy for patients with malignant neoplasms. Clinicians use blocking antibodies to the PD1, PD-L1 and LAG3 receptor proteins exposed on various types of cells, including T-, NK-, B-cells, as well as myeloid cells of the tumor microenvironment and malignant cells. Extensive experience with ICB has shown significant differences in the clinical effectiveness of different checkpoint blocking drugs and their combinations. The need of development of a wide range of new drugs for ICB has become obvious. In turn, the development of new checkpoint blockers requires convenient and efficient experimental models suitable for the screening of existing and newly developed drugs.

Aim. This paper is devoted to the development and detailed study of two experimental models for the detection and characterization of new checkpoint blockers.

Material and methods. To analyze the blockade of the PD1 → PD-L1 signaling axis, transduced HEK293-PD1 cells that stably express the PD1 receptor protein on their surface were used. One of two experimental models developed examines the inhibition of the interaction of soluble recombinant biotin-labeled PD-L1-Fc protein with HEK293-PD1 cells. In a second experimental model, we analyze the inhibition of interaction of a fluorochrome-labeled anti-PD1 antibody with HEK293-PD1 cells. The labeled ligands to cell binding was studied by flow cytometry. Inhibition of binding was assessed according to changes in the fluorescence intensity of labeled cells.

Results. In the developed experimental models, the blocking properties of therapeutic antibodies to PD1 and PD-L1, as well as soluble recombinant PD-L1-Fc and PD1-Fc proteins were studied. The proposed experimental models shown to be highly sensitive, detecting not only the blocking properties of any substance with respect to the PD-L1-to-PD1 interaction, but also determining the specific activity of PD-L1 and PD1 blockers with half-maximal inhibition constant (K₅₀) in the range of nanomolar concentrations of the test substance. It is claimed that the proposed approach can be used to create experimental models with any cellular receptors as molecular targets of inhibition.

Introduction. The treatment of malignant tumors is one of the most urgent tasks of modern medicine. Reprogramming of tumor-associated macrophages into antitumor effector cells is a promising direction in immunotherapy of cancer. Strong inducers of the antitumor activity of macrophages are signals from the pattern-recognition receptors (PRR) of innate immunity. Previously, we showed that the combination of NOD1 and TLR4 agonists effectively induces macrophage activity against erythromyeloid leukemia cells K562 [1].

The aim of the work was to study activity of human macrophages stimulated by a NOD1 agonist in combination with a TLR4 or a TLR7/8 agonist against cell lines derived from disseminated and solid tumors, as well as to analyze the mechanisms of antitumor activity of reprogrammed macrophages.

Material and methods. Erythromyeloid K562 cells, human epithelioid cervical carcinoma HeLa cells and human colorectal carcinoma HT29 cells carrying green fluorescent protein (GFP) gene were used as targets for macrophages. Tumor cells were coincubated with macrophages in the presence of NOD1, TLR4, TLR7/8 agonists separately and in combination. Tumor cells without macrophages were used as controls. To analyze the antiproliferative properties of macrophages, the cell cycle of tumor cells was analyzed using flow cytometry. The number of dead cells was determined by staining with annexin-V and propidium iodide. TNF mRNA expression in macrophages was evaluated by real-time reverse transcript PCR.

Results. Combinations of NOD1+TLR4 and NOD1+TLR7/8 agonists effectively induced macrophage activity against K562, HeLa and HT29 cells. The activity of agonist combinations in all cases was higher than the activity of individual agonists. Macrophages activated by a combination of NOD1+TLR4 agonists inhibited the proliferation of K562 tumor cells and enhanced their death.

Discussion contains an analysis of the data obtained and an assessment of the applicability of the proposed models of reprogramming macrophages in relation to various cell lines.

Conclusion. Combinations of NOD- and Toll-like receptor agonists effectively induce macrophage activity against tumor cell lines derived from disseminated and solid tumors.

Introduction. The expansion of the spectrum of molecular prognostic and predictive markers of tumors is a modern trend aimed at increasing the effectiveness of drug therapy for tumors of various origins, including ovarian cancer, the results of which remain unsatisfactory. In this regard, the cytoskeleton protein TUBB3, which is expressed in epithelial tumors, has significant potential. Basic research has proven that TUBB3 overexpression increases the metastatic potential of tumor cells and induces resistance to a wide range of drugs. However, in translational studies with a semi-quantitative analysis of TUBB3 expression, estimates of the contribution of this protein to the aggressiveness of ovarian cancer and the effectiveness of chemotherapy are ambiguous.

The aim of the study was a quantitative assessment of TUBB3 expression parameters in ovarian cancer tissue and determination of the correlation of the identified parameters with the clinical characteristics of the neoplasm.

Material and methods. Surgical samples of ovarian cancer tissue were examined (n = 51). Primary monoclonal antibodies to TUBB3 (clone EP1569Y, Abcam, England) and secondary antibodies conjugated with DyLight650 dye (ab98510, Abcam, England) were used. Fluorescence was measured using a Navios flow cytometer (Beckman Coulter, USA). The number of stained cells was determined in the FlowJo 10.0.8 program (USA) by the Kolmogorov–Smirnov method. Three indicators of TUBB3 expression were calculated: level (%), intensity (units), index (units). Statistical analysis of the normality of the distribution of TUBB3 expression indices, sample comparisons and correlation estimates were carried out in the GraphPad Prism 6.0 program (GraphPad Software, USA).

Results. In the studied sample of tumors, TUBB3 was detected in 100 % of cases with significant differences in quantitative indicators (up to 10 times) in different patients, which «coincides» with clinical data on the heterogeneity of the patient’s response to drug therapy and the overall survival time. There was a weak association between the level and intensity of TUBB3 expression and the absence of differences in indicators depending on the stage of ovarian cancer and the degree of tumor differentiation. In a comparative study in the same patients with primary and metastatic ovarian cancer (tumor cells from ascitic fluid in peritoneal carcinomatosis) The stability of the molecular phenotype has been demonstrated in less than 40 % of cases. In the remaining patients, the level of TUBB3 expression in tumor cells increased in half of the cases, decreased in half.

Conclusion. Quantitative immunofluorescence analysis of TUBB3 expression in ovarian cancer tissue revealed this protein in 100 % of cases with a median TUBB3 expression level of 55 % and pronounced heterogeneity of indicators in different patients. There were no differences in the level, intensity and expression index of TUBB3 depending on the stage of the disease and the degree of differentiation of the tumor. Multidirectional changes in the expression level of TUBB3 were noted in the cells of metastatic ovarian cancer compared with the primary tumor of the same patients. The generated database will allow for an accurate analysis of the predictive significance of TUBB3 expression in tumors and to determine quantitative indicators that contribute to drug resistance.

The study is devoted to the collection, analysis, integration and interpretation of data on approaches and design validation of cell line phenotyping techniques using flow cytometry from the works of various groups of authors over the last two decades. Based on the studied scientific data, the work presents the results in the form of an algorithm for cell line phenotyping method validation, including steps to describe the validation strategy and its implementation, obtaining and analyzing data, as well as determining acceptance criteria.