Chromosomal rearrangements that lead to the formation of oncogenic gene fusions, such as EML4-ALK, are thought to arise from incorrect repair of double-strand breaks in DNA. However, the mechanisms and factors driving rearrangement formation remain poorly understood, and analysis of these processes is limited by detection methods that are labor-intensive, low-throughput, and not readily quantitative at single-cell resolution. Here, we developed a genetically encoded ALK reporter based on A549 lung adenocarcinoma cells, created by inserting an ALK-P2A-mCherry cassette into the endogenous ALK locus, so that induced EML4-ALK fusion activated mCherry fluorescence. Reporter activation yielded a readily quantifiable mCherry-positive subpopulation that could be measured and enriched by flow cytometry and correlated with EML4-ALK levels. Using this platform, we combined CRISPR-mediated rearrangement induction with knockdown of DNA repair factors using RNA interference. Of the factors involved in base excision repair, homologous recombination-related pathways and canonical non-homologous end joining, knockdown of the APEX1 gene encoding apurinic endonuclease 1 (APE1) selectively increased EML4-ALK levels both in the reporter cell line and in parental A549 cells. Together, this work provides a sensitive, single-cell A549-based ALK reporter platform and a framework for future studies aimed at identifying cellular and environmental factors that modulate oncogenic EML4-ALK rearrangement formation.

SLAMF1 encodes CD150, an immunoregulatory receptor involved in lymphocyte activation, T–B-cell interactions, and humoral immune responses. The SLAMF1 promoter polymorphism rs2295613(G>A) was previously associated with systemic lupus erythematosus (SLE) susceptibility in a Chinese case–control cohort. Here, we investigated the regulatory activity of rs2295613 in the transformed B-cell lines Raji and MP1 and in primary human CD19+ B cells. The rs2295613(A)-containing reporter showed higher promoter activity than the rs2295613(G)-containing reporter in all three cellular systems. Bioinformatic analysis predicted that the G to A substitution strengthens a pre-existing MYC-compatible motif. Substitutions disrupting the motif-containing region attenuated the rs2295613(A)-associated increase in reporter activity and reduced enrichment of the promoter fragment in anti-c-MYC DNA pull-down assays. Partial siRNA-mediated reduction in MYC mRNA also decreased the activity of the rs2295613(A)-containing reporter in Raji cells. Together, these findings identify rs2295613 as a functional SLAMF1 promoter variant in B-cell reporter systems and support a contribution of c-MYC-associated regulation to the enhanced activity of the rs2295613(A)-containing promoter.

For half a century, the quiet work of a specialized immunosuppressive B cell subset has been slowly unveiled, revealing its profound impact on immune balance. This review provides a comprehensive retrospective on the history of regulatory B cell (Breg) investigation, tracing their journey from initial elusive observations to their current recognition as crucial immunomodulators. We explore the paradigm shift from B cells solely as antibody producers to their multifaceted roles in immunosuppression. Key milestones include the earliest suggestions of suppressive B cell activity around 1970, the formal coining of the currently used term "regulatory B cells" in the early 2000s, and the subsequent elucidation of diverse Breg subsets and their suppressive mechanisms. Finally, we discuss contemporary advances, including the application of single-cell multi-omics, the identification of novel markers and metabolic regulators, and the promising yet challenging path toward Breg-based therapeutic strategies. This historical perspective underscores the remarkable progress in Breg biology and illuminates future directions for harnessing their clinical potential.

Recent advances in understanding regulatory B cells (Bregs) as critical modulators of immune homeostasis have opened new avenues for therapeutic intervention across a broad spectrum of immune-mediated diseases. Despite compelling preclinical evidence, Breg-targeted therapies have not yet entered clinical practice. Distinct Breg subpopulations employ diverse mechanisms of immunosuppression, and this heterogeneity allows for more precise selection of proper Breg subsets for therapeutic use in specific pathological contexts. This article provides a comprehensive overview of the current state of Breg field, highlighting their cytokine- and checkpoint-mediated suppressive mechanisms, as well as emerging insights into the role of metabolism in shaping Breg differentiation and function. Strategies for in vivo Breg induction are outlined along with cutting-edge techniques for generating and expanding Bregs ex vivo. Furthermore, we explore how current immunotherapies, such as therapeutic antibodies targeting Breg-associated markers and effector molecules, affect their heterogeneity and functionality, highlighting the potential synergies and antagonisms between these therapies and Breg-mediated immunoregulation. Finally, we discuss current limitations in the field that lie ahead in translating these strategies into clinical reality and highlight future directions for advancing Breg-based immunotherapies.

A certain degree of chromatin openness is necessary for the activity of transcription-regulating regions within the genome, facilitating accessibility to RNA polymerases and subsequent synthesis of regulatory element RNAs (regRNAs) from these regions. The rapidly increasing number of studies underscores the significance of regRNAs across diverse cellular processes and diseases, challenging the paradigm that these transcripts are non-functional transcriptional noise. This review explores the multifaceted roles of regRNAs in human cells, encompassing rather well-studied entities such as promoter RNAs and enhancer RNAs (eRNAs), while also providing insights into overshadowed silencer RNAs and insulator RNAs. Furthermore, we assess notable examples of shorter regRNAs, like miRNAs, snRNAs, and snoRNAs, playing important roles. Expanding our discourse, we deliberate on the potential usage of regRNAs as biomarkers and novel targets for cancer and other human diseases.

Wound healing is a complex process involving a coordinated series of events aimed at restoring tissue integrity and function. Regulatory B cells (Bregs) are a subset of B lymphocytes that play an essential role in fine-tuning immune responses and maintaining immune homeostasis. Recent studies have suggested that Bregs are important players in cutaneous immunity. This review summarizes the current understanding of the role of Bregs in skin immunity in health and pathology, such as diabetes, psoriasis, systemic sclerosis, cutaneous lupus erythematosus, cutaneous hypersensitivity, pemphigus, and dermatomyositis. We discuss the mechanisms by which Bregs maintain tissue homeostasis in the wound microenvironment through the promotion of angiogenesis, suppression of effector cells, and induction of regulatory immune cells. We also mention the potential clinical applications of Bregs in promoting wound healing, such as the use of adoptive Breg transfer.

Interleukin 10 (IL10) is a major anti-inflammatory cytokine that acts as a master regulator of the immune response. A single nucleotide polymorphism rs3024505(C/T), located downstream of the IL10 gene, is associated with several aggressive inflammatory diseases, including systemic lupus erythematosus, Sjögren’s syndrome, Crohn’s disease, and ulcerative colitis. In such autoimmune pathologies, IL10-producing B cells play a protective role by decreasing the level of inflammation and restoring immune homeostasis. This study demonstrates that rs3024505 is located within an enhancer that augments the activity of the IL10 promoter in a reporter system based on a human B cell line. The common rs3024505(C) variant creates a functional binding site for the transcription factor STAT3, whereas the risk allele rs3024505(T) disrupts STAT3 binding, thereby reducing the IL10 promoter activity. Our findings indicate that B cells from individuals carrying the minor rs3024505(T) allele may produce less IL10 due to the disrupted STAT3 binding site, contributing to the progression of inflammatory pathologies.

Tumor necrosis factor (TNF) is one of many cytokines - protein molecules responsible for communication between the cells of immune system. TNF was discovered and given its grand name because of its striking antitumor effects in experimental systems, but its main physiological functions in the context of whole organism turned out to be completely unrelated to protection against tumors. This short review discusses "man-made" mouse models generated by early genome-editing technologies, which enabled us to establish true functions of TNF in health and certain diseases as well as to unravel potential strategies for improving therapy of TNF-dependent diseases.

The complement inhibitor CD55/DAF is expressed on many cell types. Dysregulation of CD55 expression is associated with increased disease severity in influenza A infection and vascular complications in pathologies that involve excessive activation of the complement system. A luciferase reporter system was used to functionally analyze the single nucleotide polymorphism rs2564978 in the U937 human promonocytic cell line. The polymorphism is in the promoter of the CD55 gene, and its minor allele T is associated with a severe course of influenza A(H1N1)pdm09. A decreased activity of the CD55 promoter carrying the minor rs2564978(T) allele was observed in activated U937 cells, which provide a cell model of human macrophages. Using bioinformatics resources, PU.1 was identified as a potential transcription factor that may bind to the CD55 promoter at the rs2564978 site in an allele-specific manner. The involvement of PU.1 in modulating CD55 promoter activity was verified by a PU.1 genetic knockdown with small interfering RNAs under specific monocyte activation conditions.

B cells play a crucial role in the pathogenesis of various diseases, such as autoimmune disorders, cancers, and infections. Unlike regulatory T cells, the anti-inflammatory capabilities of B cells have only recently garnered attention. Cytokines IL-10 and TGF-β are among the key secreted immunosuppressive factors, therefore studying the characteristics of their transcriptional regulation in B cells appears to be a relevant task. This study focuses on characterizing the promoter regions of IL10 and TGFB1 genes in immortalized B cell lines representing different developmental stages – Reh and Raji. To achieve this, we identified potential promoter regions guided by the epigenetic features of functional regulatory regions determined by bioinformatics methods of ChIP-Seq data analysis of chromatin marks in CD19+ lymphocytes. We examined the activity of selected promoters using reporter analysis in B cells. Additionally, we studied the impact of a single nucleotide polymorphism rs1800469 in the TGFB1 promoter, which is associated with the development of colorectal cancer, chronic obstructive pulmonary disease, and the risk of radiation fibrosis. Our results showed increased promoter activity of IL10 and TGFB1 in the Reh pro-B cells compared to the Raji mature B cells upon stimulation. Interestingly, the presence of the minor allele of rs1800469 led to enhanced TGFB1 promoter activity in the Reh cells. Higher activity of IL10 and TGFB1 promoters in acute lymphoblastic leukemia Reh cells may be associated with the increased immunosuppression, which is characteristic of this pathology. It is also possible that activation of pro-B cells Reh induces their differentiation into monocyte-like cells, which can be polarized into alternatively activated (M2) macrophages by autocrine TGF-β and IL-10. M2 macrophages can function as tumor-associated macrophages and contribute to the development of colorectal cancer. Moreover, increased levels of TGF-β in tissues increase the risks of fibrosis and decrease inflammation levels in chronic obstructive pulmonary disease.

Currently, numerous associations between genetic polymorphisms and various diseases have been characterized through the Genome-Wide Association Studies. Majority of the clinically significant polymorphisms are localized in non-coding regions of the genome. While modern bioinformatic resources make it possible to predict molecular mechanisms that explain influence of the non-coding polymorphisms on gene expression, such hypotheses require experimental verification. This review discusses the methods for elucidating molecular mechanisms underlying dependence of the disease pathogenesis on specific genetic variants within the non-coding sequences. A particular focus is on the methods for identification of transcription factors with binding efficiency dependent on polymorphic variations. Despite remarkable progress in bioinformatic resources enabling prediction of the impact of polymorphisms on the disease pathogenesis, there is still the need for experimental approaches to investigate this issue.

PDF - 395KB, Supplementary Figure 1: Structure of MO-TES391 and KIAA1864 transcripts.

PDF - 60KB, Supplementary Table 1: Serological reactivity of MO-TES391 assessed by SMARTA.

PDF - 49KB, Supplementary Table 3: Epitope prediction for HLA-A*02 binding peptides derived from KIAA1864 and MO-TES391.

Single-nucleotide polymorphism rs71327024 located in the human 3p21.31 locus has been associated with an elevated risk of hospitalization upon SARS-CoV-2 infection. The 3p21.31 locus contains several genes encoding chemokine receptors potentially relevant to severe COVID-19. In particular, CXCR6, which is prominently expressed in T lymphocytes, NK, and NKT cells, has been shown to be involved in the recruitment of immune cells to non-lymphoid organs in chronic inflammatory and respiratory diseases. In COVID-19, CXCR6 expression is reduced in lung resident memory T cells from patients with severe disease as compared to the control cohort with moderate symptoms. We demonstrate here that rs71327024 is located within an active enhancer that augments the activity of the CXCR6 promoter in human CD4+ T lymphocytes. The common rs71327024(G) variant makes a functional binding site for the c-Myb transcription factor, while the risk rs71327024(T) variant disrupts c-Myb binding and reduces the enhancer activity. Concordantly, c-Myb knockdown in PMA-treated Jurkat cells negates rs71327024’s allele-specific effect on CXCR6 promoter activity. We conclude that a disrupted c-Myb binding site may decrease CXCR6 expression in T helper cells of individuals carrying the minor rs71327024(T) allele and thus may promote the progression of severe COVID-19 and other inflammatory pathologies.

PDF - 61KB, Supplementary Table 2: Primers used in the study.

PDF - 453KB, Supplementary Figure 2: Mapping of major immunogenic region of MO-TES391.

B lymphocytes play an important role in the regulation of immune response in both normal and pathological conditions. Traditionally, the main functions of B cells were considered to be antibody production and antigen presentation, but in recent decades there have been discovered several subpopulations of regulatory B lymphocytes (Bregs), which maintain immunological tolerance and prevent overactivation of the immune system. Memory (mBregs, CD19+CD24hiCD27+) and transitional (tBregs, CD19+CD24hiCD38hi) subpopulations of Bregs are usually considered in the context of studying the role of these B cells in various human pathologies. However, the mechanisms by which these Breg subpopulations exert their immunosuppressive activity remain poorly understood. In this work, we used bioinformatic analysis of open-source RNA sequencing data to propose potential mechanisms of B cell-mediated immunosuppression. Analysis of differential gene expression before and after activation of these subpopulations allowed us to identify six candidate molecules that may determine the functionality of mBregs and tBregs. IL4I1-, SIRPA-, and SLAMF7-dependent mechanisms of immunosuppression may be characteristic of both Breg subsets, while NID1-, CST7-, and ADORA2B-dependent mechanisms may be predominantly characteristic of tBregs. In-depth understanding of the molecular mechanisms of anti-inflammatory immune response of B lymphocytes is an important task for both basic science and applied medicine and could facilitate the development of new approaches to the therapy of complex diseases.

Regulatory B lymphocytes (Bregs) have the ability to suppress the function of effector cells using a diverse range of immunosuppressive mechanisms, making them a promising candidate for adoptive cell therapy in conditions with hyperactive immune responses. Our research focuses on generation of functional Bregs from human peripheral blood B cells ex vivo.