Breg-Mediated Immunoregulation in the Skin.

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.

Differentially activated B cells develop regulatory phenotype and show varying immunosuppressive features: a comparative study.

Regulatory B lymphocytes (Bregs) are B cells with well-pronounced immunosuppressive properties, allowing them to suppress the activity of effector cells. A broad repertoire of immunosuppressive mechanisms makes Bregs an attractive tool for adoptive cell therapy for diseases associated with excessive activation of immune reactions. Such therapy implies Breg extraction from the patient's peripheral blood, ex vivo activation and expansion, and further infusion into the patient. At the same time, the utility of Bregs for therapeutic approaches is limited by their small numbers and extremely low survival rate, which is typical for all primary B cell cultures. Therefore, extracting CD19+ cells from the patient's peripheral blood and specifically activating them ex vivo to make B cells acquire a suppressive phenotype seems to be far more productive. It will allow a much larger number of B cells to be obtained initially, which may significantly increase the likelihood of successful immunosuppression after adoptive Breg transfer. This comparative study focuses on finding ways to efficiently manipulate B cells in vitro to differentiate them into Bregs. We used CD40L, CpG, IL4, IL21, PMA, and ionomycin in various combinations to generate immunosuppressive phenotype in B cells and performed functional assays to test their regulatory capacity. This work shows that treatment of primary B cells using CD40L + CpG + IL21 mix was most effective in terms of induction of functionally active regulatory B lymphocytes with high immunosuppressive capacity ex vivo.

rs71327024 Associated with COVID-19 Hospitalization Reduces CXCR6 Promoter Activity in Human CD4+ T Cells via Disruption of c-Myb Binding.

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.

Novel Potential Mechanisms of Regulatory B Cell-Mediated Immunosuppression.

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.

CRISPR/Cas9 genome editing demonstrates functionality of the autoimmunity-associated SNP rs12946510.

Genome-wide association studies (GWAS) map genetic associations of complex traits with precision limited to a linkage disequilibrium group. To translate GWAS results into new understanding of disease mechanisms, individual causative polymorphisms and their target genes should be identified. CRISPR/Cas9 genome editing can be used to create isogenic cell lines bearing alternative genotypes of candidate single-nucleotide polymorphisms to test their causality and to reveal gene targets. An intergenic polymorphism rs12946510 is associated with multiple sclerosis, inflammatory bowel disease and asthma. We created sublines of the T-helper cell line bearing alternative genotypes of rs12946510 and showed that its risk ("T") allele is associated with lower expression of IKZF3 and ORMDL3 genes and reduced cell activation. Our editing procedure can become an effective tool for discovering new genes involved in pathogenesis of complex diseases.

EGR1 and RXRA transcription factors link TGF-ß pathway and CCL2 expression in triple negative breast cancer cells.

Transforming growth factor beta (TGF-ß) is the main cytokine responsible for the induction of the epithelial-mesenchymal transition of breast cancer cells, which is a hallmark of tumor transformation to the metastatic phenotype. Recently, research demonstrated that the chemokine CCL2 gene expression level directly correlates with the TGF-ß activity in breast cancer patients. CCL2 attracts tumor-associated macrophages and is, therefore, considered as an important inductor of breast cancer progression; however, the precise mechanisms underlying its regulation by TGF-ß are unknown. Here, we studied the behavior of the CCL2 gene in MDA-MB-231 and HCC1937 breast cancer cells representing mesenchymal-like phenotype activated by TGF-ß. Using bioinformatics, deletion screening and point mutagenesis, we identified binding sites in the CCL2 promoter and candidate transcription factors responsible for its regulation by TGF-ß. Among these factors, only the knock-down of EGR1 and RXRA made CCL2 promoter activity independent of TGF-ß. These factors also demonstrated binding to the CCL2 promoter in a TGF-ß-dependent manner in a chromatin immunoprecipitation assay, and point mutations in the EGR1 and RXRA binding sites totally abolished the effect of TGF-ß. Our results highlight the key role of EGR1 and RXRA transcription factors in the regulation of CCL2 gene in response to TGF-ß pathway.

Minor C allele of the SNP rs7873784 associated with rheumatoid arthritis and type-2 diabetes mellitus binds PU.1 and enhances TLR4 expression.

Toll-like receptor 4 (TLR4) is an innate immunity receptor predominantly expressed on myeloid cells and involved in the development of various diseases, many of them with complex genetics. Here we present data on functionality of single nucleotide polymorphism rs7873784 located in the 3'-untranslated region (3'-UTR) of TLR4 gene and associated with various pathologies involving chronic inflammation. We demonstrate that TLR4 3'-UTR strongly enhanced the activity of TLR4 promoter in U937 human monocytic cell line while minor rs7873784(C) allele created a binding site for transcription factor PU.1 (encoded by SPI1 gene), a known regulator of TLR4 expression. Increased binding of PU.1 further augmented the TLR4 transcription while PU.1 knockdown or complete disruption of the PU.1 binding site abrogated the effect. We hypothesize that additional functional PU.1 site may increase TLR4 expression in individuals carrying minor C variant of rs7873784 and modulate the development of certain pathologies, such as rheumatoid arthritis and type-2 diabetes mellitus.

Multi-dimensional immunoproteomics coupled with in vitro recapitulation of oncogenic NRASQ61R identifies diagnostically relevant autoantibody biomarkers in thyroid neoplasia.

Tumor-associated antigen (TAA)-specific autoantibodies have been widely implicated in cancer diagnosis. However, cancer cell lines that are typically exploited as candidate TAA sources in immunoproteomic studies may fail to accurately represent the autoantigen-ome of lower-grade neoplasms. Here, we established an integrated strategy for the identification of disease-relevant TAAs in thyroid neoplasia, which combined NRASQ61R oncogene expression in non-tumorous thyroid Nthy-ori 3-1 cells with a multi-dimensional proteomic technique DISER that consisted of profiling NRASQ61R-induced proteins using 2-dimensional difference gel electrophoresis (2D-DIGE) coupled with serological proteome analysis (SERPA) of the TAA repertoire of patients with thyroid encapsulated follicular-patterned/RAS-like phenotype (EFP/RLP) tumors. We identified several candidate cell-based (nicotinamide phosphoribosyltransferase NAMPT, glutamate dehydrogenase GLUD1, and glutathione S-transferase omega-1 GSTO1) and autoantibody (fumarate hydratase FH, calponin-3 CNN3, and pyruvate kinase PKM autoantibodies) biomarkers, including NRASQ61R-induced TAA phosphoglycerate kinase 1 PGK1. Meta-profiling of the reactivity of the identified autoantibodies across an independent SERPA series implicated the PKM autoantibody as a histological phenotype-independent biomarker of thyroid malignancy (11/38 (29%) patients with overtly malignant and uncertain malignant potential (UMP) tumors vs 0/22 (p = 0.0046) and 0/20 (p = 0.011) patients with non-invasive EFP/RLP tumors and healthy controls, respectively). PGK1 and CNN3 autoantibodies were identified as EFP/RLP-specific biomarkers, potentially suitable for further discriminating tumors with different malignant potential (PGK1: 7/22 (32%) patients with non-invasive EFP/RLP tumors vs 0/38 (p = 0.00044) and 0/20 (p = 0.0092) patients with other tumors and healthy controls, respectively; СNN3: 9/29 (31%) patients with malignant and borderline EFP/RLP tumors vs 0/31 (p = 0.00068) and 0/20 (p = 0.0067) patients with other tumors and healthy controls, respectively). The combined use of PKM, CNN3, and PGK1 autoantibodies allowed the reclassification of malignant/UMP tumor risk in 19/41 (46%) of EFP/RLP tumor patients. Taken together, we established an experimental pipeline DISER for the concurrent identification of cell-based and TAA biomarkers. The combination of DISER with in vitro oncogene expression allows further targeted identification of oncogene-induced TAAs. Using this integrated approach, we identified candidate autoantibody biomarkers that might be of value for differential diagnostic purposes in thyroid neoplasia.

Functional SNPs in the Human Autoimmunity-Associated Locus 17q12-21.

Genome-wide association studies (GWASes) revealed several single-nucleotide polymorphisms (SNPs) in the human 17q12-21 locus associated with autoimmune diseases. However, follow-up studies are still needed to identify causative SNPs directly mediating autoimmune risk in the locus. We have chosen six SNPs in high linkage disequilibrium with the GWAS hits that showed the strongest evidence of causality according to association pattern and epigenetic data and assessed their functionality in a local genomic context using luciferase reporter system. We found that rs12946510, rs4795397, rs12709365, and rs8067378 influenced the reporter expression level in leukocytic cell lines. The strongest effect visible in three distinct cell types was observed for rs12946510 that is predicted to alter MEF2A/C and FOXO1 binding sites.