NY-ESO-1 antigen: A promising frontier in cancer immunotherapy.

Significant strides have been made in identifying tumour-associated antigens over the past decade, revealing unique epitopes crucial for targeted cancer therapy. Among these, the New York esophageal squamous cell carcinoma (NY-ESO-1) protein, a cancer/testis antigen, stands out. This protein is presented on the cell surface by major histocompatibility complex class I molecules and exhibits restricted expression in germline cells and various cancers, marking it as an immune-privileged site. Remarkably, NY-ESO-1 serves a dual role as both a tumour-associated antigen and its own adjuvant, implying a potential function as a damage-associated molecular pattern. It elicits strong humoural immune responses, with specific antibody frequencies significantly correlating with disease progression. These characteristics make NY-ESO-1 an appealing candidate for developing effective and specific immunotherapy, particularly for advanced stages of disease. In this review, we provide a comprehensive overview of NY-ESO-1 as an immunogenic tumour antigen. We then explore the diverse strategies for targeting NY-ESO-1, including cancer vaccination with peptides, proteins, DNA, mRNA, bacterial vectors, viral vectors, dendritic cells and artificial adjuvant vector cells, while considering the benefits and drawbacks of each strategy. Additionally, we offer an in-depth analysis of adoptive T-cell therapies, highlighting innovative techniques such as next-generation NY-ESO-1 T-cell products and the integration with lymph node-targeted vaccines to address challenges and enhance therapeutic efficacy. Overall, this comprehensive review sheds light on the evolving landscape of NY-ESO-1 targeting and its potential implications for cancer treatment, opening avenues for future tailored directions in NY-ESO-1-specific immunotherapy. HIGHLIGHTS: Endogenous immune response: NY-ESO-1 exhibited high immunogenicity, activating endogenous dendritic cells, T cells and B cells. NY-ESO-1-based cancer vaccines: NY-ESO-1 vaccines using protein/peptide, RNA/DNA, microbial vectors and artificial adjuvant vector cells have shown promise in enhancing immune responses against tumours. NY-ESO-1-specific T-cell receptor-engineered cells: NY-ESO-1-targeted T cells, along with ongoing innovations in engineered natural killer cells and other cell therapies, have improved the efficacy of immunotherapy.

Single-cell multi-omics reveal stage of differentiation and trajectory-dependent immunity-related gene expression patterns in human erythroid cells.

The role of Erythroid cells in immune regulation and immunosuppression is one of the emerging topics in modern immunology that still requires further clarification as Erythroid cells from different tissues and different species express different immunoregulatory molecules. In this study, we performed a thorough investigation of human bone marrow Erythroid cells from adult healthy donors and adult acute lymphoblastic leukemia patients using the state-of-the-art single-cell targeted proteomics and transcriptomics via BD Rhapsody and cancer-related gene copy number variation analysis via NanoString Sprint Profiler. We found that human bone marrow Erythroid cells express the ARG1, LGALS1, LGALS3, LGALS9, and C10orf54 (VISTA) immunosuppressive genes, CXCL5, CXCL8, and VEGFA cytokine genes, as well as the genes involved in antimicrobial immunity and MHC Class II antigen presentation. We also found that ARG1 gene expression was restricted to the single erythroid cell cluster that we termed ARG1-positive Orthochromatic erythroblasts and that late Erythroid cells lose S100A9 and gain MZB1 gene expression in case of acute lymphoblastic leukemia. These findings show that steady-state erythropoiesis bone marrow Erythroid cells express myeloid signature genes even without any transdifferentiating stimulus like cancer.

Acute blood loss in mice forces differentiation of both CD45-positive and CD45-negative erythroid cells and leads to a decreased CCL3 chemokine production by bone marrow erythroid cells.

Hemorrhage, a condition that accompanies most physical trauma cases, remains an important field of study, a field that has been extensively studied in the immunological context for myeloid and lymphoid cells, but not as much for erythroid cells. In this study, we studied the immunological response of murine erythroid cells to acute blood loss using flow cytometry, NanoString immune transcriptome profiling, and BioPlex cytokine secretome profiling. We observed that acute blood loss forces the differentiation of murine erythroid cells in both bone marrow and spleen and that there was an up-regulation of several immune response genes, in particular pathogen-associated molecular pattern sensing gene Clec5a in post-acute blood loss murine bone marrow erythroid cells. We believe that the up-regulation of the Clec5a gene in bone marrow erythroid cells could help bone marrow erythroid cells detect and eliminate pathogens with the help of reactive oxygen species and antimicrobial proteins calprotectin and cathelicidin, the genes of which (S100a8, S100a9, and Camp) dominate the expression in bone marrow erythroid cells of mice.

Dendritic cells transfected with DNA constructs encoding CCR9, IL-10, and type II collagen demonstrate induction of immunological tolerance in an arthritis model.

Introduction: Restoring immune tolerance is a promising area of therapy for autoimmune diseases. One method that helps restore immunological tolerance is the approach using tolerogenic dendritic cells (tolDCs). In our study, we analyzed the effectiveness of using dendritic cells transfected with DNA constructs encoding IL-10, type II collagen, and CCR9 to induce immune tolerance in an experimental model of arthritis. Methods: Dendritic cell cultures were obtained from bone marrow cells of Balb/c mice. Dendritic cells (DCs) cultures were transfected with pmaxCCR9, pmaxIL-10, and pmaxCollagen type II by electroporation. The phenotype and functions of DCs were studied using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Migration of electroporated DCs was assessed in vitro. Induction of antigen-collagen induced arthritis (ACIA) was carried out according to the protocol in Balb/c mice. DCs were then administered to ACIA mice. The development of arthritis was monitored by measuring paw swelling with a caliper at different time points. The immunological changes were assessed by analyzing the content of antibodies to type II collagen using enzyme immunoassay. Additionally, a histological examination of the joint tissue was conducted, followed by data analysis. The results are as follows: DCs were obtained, characterized by reduced expression of CD80, CD86, and H-2Db (MHC class I), increased expression of CCR9, as well as producing IL-10 and having migratory activity to thymus cells. Transfected DCs induced T-regulatory cells (T-reg) and increased the intracellular content of IL-10 and TGF-ß in CD4+T cells in their co-culture, and also suppressed their proliferative activity in response to antigen. The administration of tolDCs transfected with DNA constructs encoding type II collagen, IL-10, and CCR9 to mice with ACIA demonstrated a reduction in paw swelling, a reduction in the level of antibodies to type II collagen, and a regression of histological changes. Conclusion: The study presents an approach by which DCs transfected with DNA constructs encoding epitopes of type II collagen, IL-10 and CCR9 promote the development of antigen-specific tolerance, control inflammation and reduce the severity of experimental arthritis through the studied mechanisms: induction of T-reg, IL-10, TGF-ß.

A subpopulation of human bone marrow erythroid cells displays a myeloid gene expression signature similar to that of classic monocytes.

Erythroid cells, serving as progenitors and precursors to erythrocytes responsible for oxygen transport, were shown to exhibit an immunosuppressive and immunoregulatory phenotype. Previous investigations from our research group have revealed an antimicrobial gene expression profile within murine bone marrow erythroid cells which suggested a role for erythroid cells in innate immunity. In the present study, we focused on elucidating the characteristics of human bone marrow erythroid cells through comprehensive analyses, including NanoString gene expression profiling utilizing the Immune Response V2 panel, a BioPlex examination of chemokine and TGF-beta family proteins secretion, and analysis of publicly available single-cell RNA-seq data. Our findings demonstrate that an erythroid cell subpopulation manifests a myeloid-like gene expression signature comprised of antibacterial immunity and neutrophil chemotaxis genes which suggests an involvement of human erythroid cells in the innate immunity. Furthermore, we found that human erythroid cells secreted CCL22, CCL24, CXCL5, CXCL8, and MIF chemokines. The ability of human erythroid cells to express these chemokines might facilitate the restriction of immune cells in the bone marrow under normal conditions or contribute to the ability of erythroid cells to induce local immunosuppression by recruiting immune cells in their immediate vicinity in case of extramedullary hematopoiesis.

GD2-targeting therapy: a comparative analysis of approaches and promising directions.

Disialoganglioside GD2 is a promising target for immunotherapy with expression primarily restricted to neuroectodermal and epithelial tumor cells. Although its role in the maintenance and repair of neural tissue is well-established, its functions during normal organism development remain understudied. Meanwhile, studies have shown that GD2 plays an important role in tumorigenesis. Its functions include proliferation, invasion, motility, and metastasis, and its high expression and ability to transform the tumor microenvironment may be associated with a malignant phenotype. Structurally, GD2 is a glycosphingolipid that is stably expressed on the surface of tumor cells, making it a suitable candidate for targeting by antibodies or chimeric antigen receptors. Based on mouse monoclonal antibodies, chimeric and humanized antibodies and their combinations with cytokines, toxins, drugs, radionuclides, nanoparticles as well as chimeric antigen receptor have been developed. Furthermore, vaccines and photoimmunotherapy are being used to treat GD2-positive tumors, and GD2 aptamers can be used for targeting. In the field of cell therapy, allogeneic immunocompetent cells are also being utilized to enhance GD2 therapy. Efforts are currently being made to optimize the chimeric antigen receptor by modifying its design or by transducing not only αß T cells, but also γδ T cells, NK cells, NKT cells, and macrophages. In addition, immunotherapy can combine both diagnostic and therapeutic methods, allowing for early detection of disease and minimal residual disease. This review discusses each immunotherapy method and strategy, its advantages and disadvantages, and highlights future directions for GD2 therapy.

TNFR1 Absence Is Not Crucial for Different Types of Cell Reaction to TNF: A Study of the TNFR1-Knockout Cell Model.

BACKGROUND: One of the mechanisms regulating the biological activity of tumor necrosis factor (TNF) in cells is the co-expression of TNFR1/TNFR2 receptors. A model with a differential level of receptor expression is required to evaluate the contribution of these mechanisms. AIM: The development of a cellular model to compare the effects of TNF on cells depending on the presence of both receptors and TNFR2 alone. METHODS: TNFR1 absence modifications of ZR-75/1 and K-562 cell lines were obtained by TNFR1 knockout. The presence of deletions was confirmed by Sanger sequencing, and the absence of cell membrane receptor expression was confirmed by flow cytometry. The dose-dependent effect of TNF on intact and knockout cells was comparatively evaluated by the effect on the cell cycle, the type of cell death, and the profile of expressed genes. RESULTS: Knockout of TNFR1 resulted in a redistribution of TNFR2 receptors with an increased proportion of TNFR2+ cells in both lines and a multidirectional change in the density of expression in the lines (increased in K562 and decreased in ZR75/1). The presence of a large number of cells with high TNFR2 density in the absence of TNFR1 in the K562 cells was associated with greater sensitivity to TNF-stimulating doses and increased proliferation but did not result in a significant change in cell death parameters. A twofold increase in TNFR2+ cell distribution in this cell line at a reduced expression density in ZR75/1 cells was associated with a change in sensitivity to low cytokine concentrations in terms of proliferation; an overall increase in cell death, most pronounced at standard stimulating concentrations; and increased expression of the lymphocyte-activation gene groups, host-pathogen interaction, and innate immunity. CONCLUSIONS: The absence of TNFR1 leads to different variants of compensatory redistribution of TNFR2 in cellular models, which affects the type of cell response and the threshold level of sensitivity. The directionality of cytokine action modulation and sensitivity to TNF levels depends not only on the fraction of cells expressing TNFR2 but also on the density of expression.

Development of Cell Technologies Based on Dendritic Cells for Immunotherapy of Oncological Diseases.

Immunotherapy using dendritic cell-based vaccination is a natural approach using the capabilities and functions inherent in the patient's immune system to eliminate tumor cells. The development of dendritic cell-based cell technologies evolved as the disorders of dendritic cell differentiation and function in cancer were studied; some of these functions are antigen presentation, priming of cytotoxic T-lymphocytes and induction of antigen-specific immune responses. At the initial stage of technology development, it was necessary to develop protocols for the in vitro generation of functionally mature dendritic cells that were capable of capturing tumor antigens and processing and presenting them in complex with MHC to T-lymphocytes. To achieve this, various forms of tumor-associated antigen delivery systems were tested, including lysates, tumor cell proteins (peptides), and DNA and RNA constructs, and it was shown that the use of DNA and RNA constructs was the most effective method, as it made it possible not only to deliver the most immunogenic epitopes of tumor-associated antigens to dendritic cells, but also to enhance their ability to induce antigen-specific cytotoxic T-lymphocytes. Currently, cell therapy based on dendritic cells is a modern basis for antigen-specific immunotherapy of cancer due to the simplicity of creating DNA and RNA constructs encoding information about both target tumor antigens and regulatory molecules. The potential development of cell technologies based on dendritic cells aims to obtain antigen-specific cytotoxic T-lymphocytes induced by dendritic cells, study their functional activity and develop cell-based therapy.

Knockouts of TNFRSF1A and TNFRSF1B Genes in K562 Cell Line Lead to Diverse Long-Lasting Responses to TNF-α.

This research delves into the intricate landscape of tumor necrosis factor-alpha (TNF-α) signaling, a multi-functional cytokine known for its diverse cellular effects. Specifically, we investigate the roles of two TNF receptors, TNFR1 and TNFR2, in mediating TNF-α-induced transcriptional responses. Using human K562 cell lines with TNFR1 and TNFR2 knockouts, we explore changes in gene expression patterns following TNF-α stimulation. Our findings reveal distinct transcriptional profiles in TNFR1 and TNFR2 knockout cells, shedding light on the unique contributions of these receptors to TNF-α signaling. Notably, several key pathways associated with inflammation, apoptosis, and cell proliferation exhibit altered regulation in the absence of TNFR1 or TNFR2. This study provides valuable insights into the intricate mechanisms governing TNF-α signaling and its diverse cellular effects, with potential implications for targeted therapeutic strategies.

Phenotypic Alterations in Erythroid Nucleated Cells of Spleen and Bone Marrow in Acute Hypoxia.

Hypoxia leads to metabolic changes at the cellular, tissue, and organismal levels. The molecular mechanisms for controlling physiological changes during hypoxia have not yet been fully studied. Erythroid cells are essential for adjusting the rate of erythropoiesis and can influence the development and differentiation of immune cells under normal and pathological conditions. We simulated high-altitude hypoxia conditions for mice and assessed the content of erythroid nucleated cells in the spleen and bone marrow under the existing microenvironment. For a pure population of CD71+ erythroid cells, we assessed the production of cytokines and the expression of genes that regulate the immune response. Our findings show changes in the cellular composition of the bone marrow and spleen during hypoxia, as well as changes in the composition of the erythroid cell subpopulations during acute hypoxic exposure in the form of a decrease in orthochromatophilic erythroid cells that are ready for rapid enucleation and the accumulation of their precursors. Cytokine production normally differs only between organs; this effect persists during hypoxia. In the bone marrow, during hypoxia, genes of the C-lectin pathway are activated. Thus, hypoxia triggers the activation of various adaptive and compensatory mechanisms in order to limit inflammatory processes and modify metabolism.

Murine Bone Marrow Erythroid Cells Have Two Branches of Differentiation Defined by the Presence of CD45 and a Different Immune Transcriptome Than Fetal Liver Erythroid Cells.

Mouse erythropoiesis is a multifaceted process involving the intricate interplay of proliferation, differentiation, and maturation of erythroid cells, leading to significant changes in their transcriptomic and proteomic profiles. While the immunoregulatory role of murine erythroid cells has been recognized historically, modern investigative techniques have been sparingly applied to decipher their functions. To address this gap, our study sought to comprehensively characterize mouse erythroid cells through contemporary transcriptomic and proteomic approaches. By evaluating CD71 and Ter-119 as sorting markers for murine erythroid cells and employing bulk NanoString transcriptomics, we discerned distinctive gene expression profiles between bone marrow and fetal liver-derived erythroid cells. Additionally, leveraging flow cytometry, we assessed the surface expression of CD44, CD45, CD71, and Ter-119 on normal and phenylhydrazine-induced hemolytic anemia mouse bone marrow and splenic erythroid cells. Key findings emerged: firstly, the utilization of CD71 for cell sorting yielded comparatively impure erythroid cell populations compared to Ter-119; secondly, discernible differences in immunoregulatory molecule expression were evident between erythroid cells from mouse bone marrow and fetal liver; thirdly, two discrete branches of mouse erythropoiesis were identified based on CD45 expression: CD45-negative and CD45-positive, which had been altered differently in response to phenylhydrazine. Our deductions underscore (1) Ter-119's superiority over CD71 as a murine erythroid cell sorting marker, (2) the potential of erythroid cells in murine antimicrobial immunity, and (3) the importance of investigating CD45-positive and CD45-negative murine erythroid cells separately and in further detail in future studies.

T-helper cells flexibility: the possibility of reprogramming T cells fate.

Various disciplines cooperate to find novel approaches to cure impaired body functions by repairing, replacing, or regenerating cells, tissues, or organs. The possibility that a stable differentiated cell can reprogram itself opens the door to new therapeutic strategies against a multitude of diseases caused by the loss or dysfunction of essential, irreparable, and specific cells. One approach to cell therapy is to induce reprogramming of adult cells into other functionally active cells. Understanding the factors that cause or contribute to T cell plasticity is not only of clinical importance but also expands the knowledge of the factors that induce cells to differentiate and improves the understanding of normal developmental biology. The present review focuses on the advances in the conversion of peripheral CD4+ T cells, the conditions of their reprogramming, and the methods proposed to control such cell differentiation.

TCR-Engineered Lymphocytes Targeting NY-ESO-1: In Vitro Assessment of Cytotoxicity against Tumors.

Adoptive T-cell therapies tailored for the treatment of solid tumors encounter intricate challenges, necessitating the meticulous selection of specific target antigens and the engineering of highly specific T-cell receptors (TCRs). This study delves into the cytotoxicity and functional characteristics of in vitro-cultured T-lymphocytes, equipped with a TCR designed to precisely target the cancer-testis antigen NY-ESO-1. Flow cytometry analysis unveiled a notable increase in the population of cells expressing activation markers upon encountering the NY-ESO-1-positive tumor cell line, SK-Mel-37. Employing the NanoString platform, immune transcriptome profiling revealed the upregulation of genes enriched in Gene Ontology Biological Processes associated with the IFN-γ signaling pathway, regulation of T-cell activation, and proliferation. Furthermore, the modified T cells exhibited robust cytotoxicity in an antigen-dependent manner, as confirmed by the LDH assay results. Multiplex immunoassays, including LEGENDplex™, additionally demonstrated the elevated production of cytotoxicity-associated cytokines driven by granzymes and soluble Fas ligand (sFasL). Our findings underscore the specific targeting potential of engineered TCR T cells against NY-ESO-1-positive tumors. Further comprehensive in vivo investigations are essential to thoroughly validate these results and effectively harness the intrinsic potential of genetically engineered T cells for combating cancer.

Exploring TCR-like CAR-Engineered Lymphocyte Cytotoxicity against MAGE-A4.

TCR-like chimeric antigen receptor (CAR-T) cell therapy has emerged as a game-changing strategy in cancer immunotherapy, offering a broad spectrum of potential antigen targets, particularly in solid tumors containing intracellular antigens. In this study, we investigated the cytotoxicity and functional attributes of in vitro-generated T-lymphocytes, engineered with a TCR-like CAR receptor precisely targeting the cancer testis antigen MAGE-A4. Through viral transduction, T-cells were genetically modified to express the TCR-like CAR receptor and co-cultured with MAGE-A4-expressing tumor cells. Flow cytometry analysis revealed a significant surge in cells expressing activation markers CD69, CD107a, and FasL upon encountering tumor cells, indicating robust T-cell activation and cytotoxicity. Moreover, immune transcriptome profiling unveiled heightened expression of pivotal T-effector genes involved in immune response and cell proliferation regulation. Additionally, multiplex assays also revealed increased cytokine production and cytotoxicity driven by granzymes and soluble Fas ligand (sFasL), suggesting enhanced anti-tumor immune responses. Preliminary in vivo investigations revealed a significant deceleration in tumor growth, highlighting the therapeutic potential of these TCR-like CAR-T cells. Further investigations are warranted to validate these revelations fully and harness the complete potential of TCR-like CAR-T cells in overcoming cancer's resilient defenses.

Erythroid Cells as Full Participants in the Tumor Microenvironment.

The tumor microenvironment is an important factor that can determine the success or failure of antitumor therapy. Cells of hematopoietic origin are one of the most important mediators of the tumor-host interaction and, depending on the cell type and functional state, exert pro- or antitumor effects in the tumor microenvironment or in adjacent tissues. Erythroid cells can be full members of the tumor microenvironment and exhibit immunoregulatory properties. Tumor growth is accompanied by the need to obtain growth factors and oxygen, which stimulates the appearance of the foci of extramedullary erythropoiesis. Tumor cells create conditions to maintain the long-term proliferation and viability of erythroid cells. In turn, tumor erythroid cells have a number of mechanisms to suppress the antitumor immune response. This review considers current data on the existence of erythroid cells in the tumor microenvironment, formation of angiogenic clusters, and creation of optimal conditions for tumor growth. Despite being the most important life-support function of the body, erythroid cells support tumor growth and do not work against it. The study of various signaling mechanisms linking tumor growth with the mobilization of erythroid cells and the phenotypic and functional differences between erythroid cells of different origin allows us to identify potential targets for immunotherapy.

TCR Sequencing in Mouse Models of Allorecognition Unveils the Features of Directly and Indirectly Activated Clonotypes.

Allorecognition is known to involve a large number of lymphocytes carrying diverse T-cell receptor repertoire. Thus, one way to understand allorecognition and rejection mechanisms is via high-throughput sequencing of T-cell receptors. In this study, in order to explore and systematize the properties of the alloreactive T-cell receptor repertoire, we modeled direct and indirect allorecognition pathways using material from inbred mice in vitro and in vivo. Decoding of the obtained T-cell receptor genes using high-throughput sequencing revealed some features of the alloreactive repertoires. Thus, alloreactive T-cell receptor repertoires were characterized by specific V-gene usage patterns, changes in CDR3 loop length, and some amino acid occurrence probabilities in the CDR3 loop. Particularly pronounced changes were observed for directly alloreactive clonotypes. We also revealed a clustering of directly and indirectly alloreactive clonotypes by their ability to bind a single antigen; amino acid patterns of the CDR3 loop of alloreactive clonotypes; and the presence in alloreactive repertoires of clonotypes also associated with infectious, autoimmune, and tumor diseases. The obtained results were determined by the modeling of the simplified allorecognition reaction in inbred mice in which stimulation was performed with a single MHCII molecule. We suppose that the decomposition of the diverse alloreactive TCR repertoire observed in humans with transplants into such simple reactions will help to find alloreactive repertoire features; e.g., a dominant clonotype or V-gene usage pattern, which may be targeted to correct the entire rejection reaction in patients. In this work, we propose several technical ways for such decomposition analysis, including separate modeling of the indirect alloreaction pathway and clustering of alloreactive clonotypes according to their ability to bind a single antigen, among others.

Parameters of TNF receptor co-expression in allergic and autoimmune processes: Differences and diagnostic significance.

The authors used a method quantitative estimation density of TNFR1/TNFR2 on cells by flow cytometry with calibration particles, which allowed them to estimate the absolute number of receptors on cells regardless of the type of flow cytometer. The TNF receptor expression parameters were used to determine their association with the fact of disease and to build diagnostic models. The proposed methodological approach using a combination of flow cytometry and mathematical modeling techniques represents a promising direction for testing the diagnostic and prognostic significance of the studied biomarkers. The multifactorial regression analysis constructed on the basis of this approach made it possible to refine and supplement diagnostic schemes for determining the probability of rheumatoid arthritis and bronchial asthma in patients.

Immunoregulatory properties of erythroid nucleated cells induced from CD34+ progenitors from bone marrow.

CD 71+ erythroid nucleated cells have pronounced immunoregulatory properties in normal and pathological conditions. Many populations of cells with immunoregulatory properties are considered candidates for cellular immunotherapy for various pathologies. This study characterized the immunoregulatory properties of CD71+ erythroid cells derived from CD34-positive bone marrow cells under the influence of growth factors that stimulate differentiation into erythroid cells. CD34-negative bone marrow cells were used to isolate CD71+ erythroid nuclear cells. The resulting cells were used to assess the phenotype, determine the mRNA spectrum of the genes responsible for the main pathways and processes of the immune response, and obtain culture supernatants for the analysis of immunoregulatory factors. It was found that CD71+ erythroid cells derived from CD34+ cells carry the main markers of erythroid cells, but differ markedly from natural bone marrow CD71+ erythroid cells. The main differences are in the presence of the CD45+ subpopulation, distribution of terminal differentiation stages, transcriptional profile, secretion of certain cytokines, and immunosuppressive activity. The properties of induced CD71+ erythroid cells are closer to the cells of extramedullary erythropoiesis foci than to natural bone marrow CD71+ erythroid cells. Thus, when cultivating CD71+ erythroid cells for clinical experimental studies, it is necessary to take into account their pronounced immunoregulatory activity.

Murine Placental Erythroid Cells Are Mainly Represented by CD45+ Immunosuppressive Erythroid Cells and Secrete CXCL1, CCL2, CCL3 and CCL4 Chemokines.

Erythroid cells are emerging players in immunological regulation that have recently been shown to play a crucial role in fetomaternal tolerance in mice. In this work, we set ourselves the goal of discovering additional information about the molecular mechanisms of this process. We used flow cytometry to study placental erythroid cells' composition and BioPlex for the secretome profiling of 23 cytokines at E12.5 and E19.5 in both allogeneic and syngeneic pregnancies. We found that (1) placental erythroid cells are mainly represented by CD45+ erythroid cells; (2) the secretomes of CD71+ placental erythroid cells differ from the ones in syngeneic pregnancy; (3) CCL2, CCL3, CCL4 and CXCL1 chemokines were secreted on each day of embryonic development and in both types of pregnancy studied. We believe that these chemokines lure placental immune cells towards erythroid cells so that erythroid cells can induce anergy in those immune cells via cell-bound ligands such as PD-L1, enzymes such as ARG1, and secreted factors such as TGFß-1.

TNFα Causes a Shift in Gene Expression of TNFRSF1A and TNFRSF1B Isoforms.

Alternative splicing is a part of mRNA processing that expands the diversity of proteins encoded by a single gene. Studying the full range of proteins-products of translation of alternatively spliced mRNA is extremely important for understanding the interactions between receptor proteins and ligands since different receptor protein isoforms can provide variation in the activation of signaling pathways. In this study, we investigated the expression of isoforms of TNFR1 and TNFR2 receptors before and after exposure to TNFα in two cell lines that had previously demonstrated diverse effects on cell proliferation under TNFα incubation using RT-qPCR. We found that after incubation with TNFα: (1) expression of isoform 3 of the TNFRSF1A gene was increased in both cell lines; (2) the cell line with increased proliferation, K562, had decreased expression of isoforms 1 and 4 of the TNFRSF1A gene and expression of isoform 2 of TNFRSF1B gene was absent at all; (3) the cell line with decreased proliferation-MCF-7 had significantly increased expression of isoform 2 of TNFRSF1B gene. Thus, we can conclude that TNFα exposure to the K562 and MCF-7 cell lines leads to changes in the expression of TNFα receptor isoforms, which, in turn, can appear via diverse proliferative effects.