Reprogramming of myeloid cells of the tumor microenvironment – a new approach in the immunotherapy of malignant neoplasms
Modern therapy of a cancer patient, especially immunotherapy, must take into account and overcome the immunosuppression created inside the tumor and, in general, in the body affected by a malignant neoplasm. Without overcoming immunosuppression, it is impossible to achieve effective elimination of malignant cells by the forces of immune mechanisms.
The immunosuppressive properties of the tumor microenvironment are formed by both malignant and non-malignant cells and the substances they produce. Myeloid cells, tumor-associated macrophages, intratumoral dendritic cells, and myeloid suppressors attracted by the tumor, play a significant role in creating an immunosuppressive milieu inside the tumor. These cells produce soluble inhibitors of immune responses such as IDO, TGF-β, IL-10, prostaglandins, and also exhibit on their surface molecules that have the ability to suppress immune cells, in particular, PD-L1/2 molecules that cause functional paralysis of T cells or even their death.
In this literature review, we consider the immunosuppressive properties of macrophages, dendritic cells, and other tumor-associated myeloid cells aiming to overcome this mechanism of immunosuppression during the treatment of cancer patients. We present an analysis of the literature and our own results of the correction of immunity by targeting of tumor myeloid cells. The presented data suggest that a correction of the tumor myeloid cells’ properties is new effective approach in the immunotherapy of malignant neoplasms.
Immunogenicity of a multi-antigen vaccine made from a lysate of tumor malignant cells or from primary solid tumor tissue
Introduction. Personalized vaccination with tumor antigens is one of the most promising trends in immunotherapy for cancer patients. Various technological platforms are used to create a therapeutic vaccine. The most progressive are vaccines based on synthetic peptides that copy mutant neoantigens, as well as vaccines that are RNA- or DNA-vectors encoding tumor neoantigens. The mentioned technological approaches are very complex and expensive, and require considerable time for their implementation. We are developing an extremely simple, low-cost method for preparing a personalized antitumor vaccine for its wide use in the complex treatment of cancer patients. Our approach is to use tumor tissue as a source of tumor antigens for a personalized vaccine. Vaccines prepared from tumor tissue or cancer malignant cells we define as personalized multi-antigen cancer vaccines.
Aim – to study the immunogenicity of two variants of a multiantigen antitumor vaccine prepared from a tissue lysate of primary solid 4T1 carcinoma or from a pure culture of malignant 4T1 carcinoma cells.
Material and methods. The multi-antigenic vaccines studied in this work were prepared from solid 4T1 mammary carcinoma tissue of BALB/c mice or from a pure population of malignant 4T1 carcinoma cells grown under in vitro cell culture conditions. The homogenate (lysate) of tumor tissue or 4T1 cells was supplemented with molecular immunoadjuvants from the class of PRR-agonists. The resulting compositions were used to immunize BALB/c mice. Immunogens were administered intraperitoneally, four times, with intervals of 2 weeks between injections. Systemic immune responses against 4T1 carcinoma antigens used in the studied immunogens were determined according to the numbers of antitumor T cells in the spleen and the levels of tumor-specific antibodies in the blood serum of mice. Antigen-reactive CD4 and CD8 T memory cells, and T effector cells secreting interferon-γ were analyzed by ELISPOT. Antibodies to 4T1 carcinoma cell surface antigens were analyzed by flow cytometry. Antibo- dies to intracellular antigens of 4T1 carcinoma were studied by enzyme immunoassay.
Results. Multi-antigenic antitumor vaccines based on 4T1 tumor tissue homogenate (Multivac-1) or on 4T1 malignant cell lysate (Multivac-4) demonstrated high immunogenicity. After several injections of these immunogens, BALB/c mice developed intense T cell and antibody responses against 4T1 carcinoma antigens. The total response of CD4 T cells (effector cells + memory cells) in the spleen was about 3000 T cells (per 1 million T cells) after immunization with the Multivac-1 and about 8000 T cells after immunization with the Multivac-4. The total response of CD8 T cells (effector cells + memory cells) was about 3000 T cells after immunization with multivac-1 and about 5000 T cells after immunization with Multivac-4. Both vaccine preparations induced the production of antitumor antibodies. Significantly more antitumor antibodies were produced in response to the multivac-4 vaccine than in response to the Multivac-1 vaccine.
Conclusion. The use of solid tumor tissue or tumor malignant cells as a source of antigens seems to be a promising technological approach for the preparation of a personalized therapeutic antitumor vaccine. Immunogens prepared from tissue lysate of 4T1 primary solid carcinoma or from a pure culture of malignant 4T1 carcinoma cells, enhanced with molecular adjuvants from the class of PRR-agonists, induce strong antitumor adaptive immune responses such as the generation of CD4 and CD8 effector T cells and memory T cells, and also production of specific antibodies to 4T1 carcinoma antigens.
Study of MAGE-A4 specific TCR-like CAR-T lymphocytes in vitro
Introduction. Currently, one of the promising options for CAR-T technology is the development of TCR-like CAR-T lymphocytes that recognize epitopes of tumor-associated antigens in combination with MHC, which opens up a wide range of possible target antigens of any cellular localization. This work presents characterization in vitro of TCR-like CAR-T cells specific for MAGE-A4, a cancer-germline antigen, which expression is observed only in malignant neoplasms and immune-privileged organs, which makes it a promising target for effective CAR-T cellular therapy with reduced off-target toxicity.
Material and methods. Transduced and non-transduced lymphocytes were studied for the memory subsets (by markers CD45RA and CD62L) and cell activation and exhaustion mar- kers (CD69, CD95, PD-1, TIM3) by flow cytometry. The cytotoxicity of the obtained CAR-T cells was studied colorimetrically by the content of lactate dehydrogenase. The effector function of CAR-T cells was studied after cocultivation with target cells by cell activation markers (4-1BB, CD69, CD40L, FasL) detection using flow cytometry.
Results. Transduced CD4+ and CD8+ lymphocytes were characterized by an increased content of terminal effector cells, as well as activated (according to CD69 marker) and exhausted cells (PD-1+TIM3+ phenotype). However, predominantly, transduced cells were represented by low differentiated memory T cell subsets, and did not carry markers of activation and exhaustion. Transduced cultures exhibited antigen-specific cytotoxicity greater than those of non-transduced cells. At the same time, the cytotoxicity of transduced cells was accompanied by an increase in the number of cells carrying activation immunostimulating molecules 4-1BB, CD69, CD40L.
Conclusion. The MAGE-A4-specific TCR-like CAR-T cells exhibit antitumor response in vitro and can be recommended for further preclinical study in experimental models in vitro and in vivo.
Interaction of dendritic cells with microorganisms capable of colonizing the intestine
Introduction. The use of bacterial vectors is one of the possible ways to deliver vaccine antigens to mucous membranes to induce mucosal immunity. In our opinion, microorganisms suitable for this purpose should cause safe and, preferably, temporary colonization of mucous membranes and effectively induce immune responses, in particular, be well absorbed by antigen-presenting cells and cause their maturation.
The aim of the study – search for microorganisms suitable for use as vectors for live oral vaccines.
Material and methods. 8 different bacteria and yeast capable of permanent or temporary persistence in the gastrointestinal tract were used. We compared the phagocytosis of microorganisms by human monocyte-derived dendritic cells, as well as the effect of these microorganisms on the expression of CD83, CD86, CCR7, and CXCR5 molecules on dendritic cells.
Results. Susceptibility to phagocytosis and the ability to induce the maturation of dendritic cells are independent properties of microorganisms. For example, Escherichia coli actively induce the phenotypic maturation of dendritic cells, but are relatively weakly phagocytosed and brings little microbial material into dendritic cells. Lactiplantibacillus plantarum and Limosilactobacillus fermentum, on the contrary, are well absorbed by dendritic cells, but have little effect on their maturation.
Conclusion. Based on the indicators of phagocytosis and the ability to stimulate the maturation of dendritic cells, Bacillus cereus seems to be the most acceptable candidate for the role of a bacterial vector among the microorganisms used in the work.
Mouse model of respiratory syncytial virus infection mimicking acute human pathology
Introduction. Human respiratory syncytial virus (RSV) is one of the common causes of lower respiratory tract inflammation in children and elderly persons. RSV infection being a serious public health problem is characterized by high morbidity and mortality, especially in children. There are no licensed vaccines and inexpensive medications for RSV prevention and treatment. The lack of universal animal model which mimics all aspects of human pathology is one of the main obstacles to develop new therapy approaches.
The aim of the study was to develop the model of RSV infection using BALB/c mice and purified and concentrated virus, which allows reproducing the main features of human pathology.
Material and methods. Mice were divided into 4 groups (n = 10). Animals of the 1st group (RSV hd) were intranasally infected with purified RSV strain A2 at a high dose of 5 - 106 pfu/mouse in a volume of 50 mcl of phosphate buffered saline. The 2nd group (RSV ld) received non-purified virus at low dose 105 pfu/mouse. Мice of the 3rd group (RSV-UV) were treated with the same dose (5 - 106 pfu/mouse) and volume (50 mcl) of the UV-inactivated RSV. Animals of the 4th group (Intact) were left untreated. The following indicators were measured: the airway hyperreactivity, cell composition in broncho-alveolar lavage (BAL) samples, the severity of inflammation and levels of pro-inflammatory cytokines in lungs. The body weight of the animals was monitored daily throughout the experiment. In separate experiments mice infected with high dose (5 - 106 pfu/mouse) of purified RSV received ribavirin orally twice a day in dose 85 mg/kg for 5 days.
Results. The virus was detected in the respiratory tract of animals for 5 days after the infection. Additionaly, a 12% body weight loss was observed on day 3, indicating successful infection. Presented protocol allows to induce many manifestations of the human pathology such as: increased airway hyperreactivity (AHR), mucus secreting goblet cell metaplasia in the bronchial epithelium and lung inflammation associated with the proinflammatory cell infiltration. Proinflammatory cytokine genes (Ifng and Tbet) expression was increased compared to intact mice. At the same time, the expression of Th2 cytokines gens (Il4, Il13 and Gata3) didn’t change significantly after infection with RSV. These data indicate the ability of RSV productively replicate in the respiratory tract, induce pulmonary inflammation and shift the immune response toward Th1-type. We also investigated the effect of ribavirin treatment on the replication of RSV in the current model and how it correlated with lung inflammation and airway hyperreactivity. In the experiment, oral daily administration of ribavirin significantly reduced viral load in lungs and lymphocytes number in the BAL. In addition, improvement in airway hyperreactivity was observed after ribavirin-mediated suppression of RSV replication. Thus, this model is sensitive to known antiviral drugs and may be useful for testing new drugs against RSV.
Conclusion. We described the experimental model of RSV infection in mice. This model mimics the main features of human pathology. The described model can be useful for the testing of novel anti-RSV drugs and further understanding of RSV infection immunopathogenesis.
Features of postinfectious immunity formation in a patient with common variable immune deficiency and severe COVID-19 course: a description of a clinical case
Background. Common variable immune deficiency (CVID) is a complicating comorbid background of COVID-19. Post-infectious immunity formation to SARS-CoV-2 during a pandemic is of particular relevance for such patients.
Aim of the study – to present the features of the development of postinfectious humoral immune response in a patient with CVID.
Material and methods. Patient K., 49 years old, the diagnosis of CVID verified at the age of 33, has been receiving regular replacement therapy with intravenous immunoglobulins for the last 10 years. After intrafamily contact and infection with SARS-CoV-2 due to the progressive deterioration of the clinical course of COVID-19, he was admitted to a monoinfection hospital.
Results. During the treatment of the severe clinical case of COVID-19 a patient with CVID proved to be effective therapy combining anti-cytokine drugs and additional courses of replacement therapy with intravenous immunoglobulins. 6 weeks later from the development of the clinic, the patient was detected specific antibodies to SARS-CoV-2 antigens – IgM (CP 4.73) and IgG (43 BAU), in 4 months the corresponding parameters were 3.55 (CP IgM) and 487 BAU (CP IgG). Comparative analysis of immunophenotyping of the patient’s B lymphocytes before the disease, during periods of early and late convalescence showed the dynamics of changes in the number of naive B-lymphocytes, unswitched and switched B-memory cells, plasmablasts, B-reg and B-lymphocytes expressing intercellular cooperation molecules.
Conclusion. In the patient with CVID the development of a specific humoral immune response to SARS-CoV-2 after a COVID-19 infection is accompanied by an increase in the proportion of B-memory cells, increased maturation of B-lymphocytes, coordinated dynamics of B-cell suppression and activation parameters.
Flow cytometric assay for the detection of anti-SARS-CoV-2 Spike protein antibodies in serum of vaccinated volunteers
Introduction. The determination of antibodies against the Spike (S) protein of the novel coronavirus is widely used to confirm current or past infection with SARS-CoV-2, and as an indicator of the effectiveness of vaccination against COVID-19. The most common method for detecting anti-S-antibodies is enzyme-linked immunosorbent assay (ELISA), which uses a recombinant S-protein. Immunofluorescence followed by flow cytometry provides an alternative approach to detect anti-S-antibodies, where a protein in the native transmembrane conformation is used as the S-antigen.
The aim of the study was to develop a method for determining anti-S-antibodies using flow cytometry, and to select the most appropriate method for processing experimental data.
Material and methods. The study involved 22 volunteers (7 men and 15 women aged 25 to 70 years, median 48). All volunteers were vaccinated with two doses of the «Sputnik V» vaccine between January and February 2021. Donor sera samples were collected before vaccination with «Sputnik V» and 3 months after vaccination. 5 volunteers had already had a mild form of COVID-19 before the time of vaccination. The remaining 17 volunteers did not encounter the SARS-CoV-2. Antibodies against S-protein were determined by immunofluorescence with registration on a flow cytometer. HEK293 cells were transiently transfected with a plasmid encoding the wild type S-protein which was used as target. Transfection was performed by the calcium phosphate method. Cells were incubated with serially diluted sera and then stained with anti-IgG-PE and anti-IgM-FITC secondary antibodies. The fluorescence level was measured using a flow cytometer. As a measurement result, the mean fluorescence intensity (MFI) obtained at 1:18 serum dilution, or the area under the titration curve (area under curve, AUC) was used. Anti-RBD-antibodies were determined using enzyme immunoassay, and virus-neutralizing activity using pseudotyped or surrogate virus-neutralization analysis (pVNA and sVNA).
Results. Using the developed method, the formation of anti-S antibodies of the IgG and IgM isotypes was shown 3 months after immunization with the «Sputnik V» vaccine. In a simplified version of the method, the relative concentration of antibodies was determined at a single dilution of the test serum by measuring the mean fluorescence intensity (MFI) of the target cells. More reliable results were obtained by construction the titration curve and calculating the area under the curve (AUC). The results thus obtained correlated well with the detection of anti-RBD antibodies by ELISA, as well as with virus neutralization data in pseudotyped and surrogate assays.
Conclusion. Flow cytometry is a convenient method for the simultaneous determination of anti-S antibodies of IgG and IgM isotypes in human serum. The advantages of the method include the fact that the S-protein is presented in a native transmembrane conformation. After minor modification, the established method can be used to determine the level of anti-S-antibodies against mutant variants of SARS-CoV-2.
«Storm» of soluble differentiation molecules in COVID-19
The review presents data on changes in the content, on the prognostic and monitoring significance of soluble differentiation molecules of immune system cells in COVID-19. Among them are soluble differentiation molecules mediating innate immune responses, soluble differentiation molecules that are activation markers of lymphocytes, soluble markers of endothelial dysfunction, soluble forms of adhesion molecules, soluble differentiation molecules related to immune response checkpoints. At development of COVID-19 in blood of patients the level of the soluble molecules CD14, CD25, CD163, TREM-1, CD177, ST-2, RAGE, ICAM-1, P- and E-seleсtin, 14 soluble control points of the immune response including HLA-G dramatically increases. The level of soluble CD26 molecules, on the contrary, decreases. Changing the level of soluble differentiation molecules has monitoring and prognostic significance. The ideas about the «storm» of soluble differentiation molecules in the severe course of COVID-19 were formulated, complementing the ideas about the «cytokine storm».
The proteins of γ-globulin fraction, that bind metal ions, in physiological immune regulation. Polarization of the responses and rational limitation of inflammation
The data are summarized in the paper allowing to describe effector properties of human γ-globulin fraction proteins that chelate copper and zinc ions, and due to their conformational changes translate the signals into the immune cells during activation of the cells and initiation of inflammation. It could be assumed that biochemical processes dealing with transport and exchange of metal ions in the cell environment, and involving the γ-globulin fraction proteins as translating macromolecules formed the basis for mechanism of rational limitation of inflammation and immunogenesis that in parallel with induction of cellular immunity seems to be evident to exclude pathological reactions towards own normal cells of the body by three levels of functional interactions.