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

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

The Risk G Allele of the Single-Nucleotide Polymorphism rs928413 Creates a CREB1-Binding Site That Activates IL33 Promoter in Lung Epithelial Cells

Cytokine interleukin 33 (IL-33) is constitutively expressed by epithelial barrier cells, and promotes the development of humoral immune responses. Along with other proinflammatory mediators released by the epithelium of airways and lungs, it plays an important role in a number of respiratory pathologies. In particular, IL-33 significantly contributes to pathogenesis of allergy and asthma; genetic variations in the IL33 locus are associated with increased susceptibility to asthma. Large-scale genome-wide association studies have identified minor “G” allele of the single-nucleotide polymorphism rs928413, located in the IL33 promoter area, as a susceptible variant for early childhood and atopic asthma development. Here, we demonstrate that the rs928413(G) allele creates a binding site for the cAMP response element-binding protein 1 (CREB1) transcription factor. In a pulmonary epithelial cell line, activation of CREB1, presumably via the p38 mitogen-activated protein kinases (MAPK) cascade, activates the IL33 promoter containing the rs928413(G) allele specifically and in a CREB1-dependent manner. This mechanism may explain the negative effect of the rs928413 minor “G” allele on asthma development

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

Interfollicular channel region of mouse mesenteric lymph node.

<p>MLN was stained with anti-clusterin and ER-TR7 antibodies after immunization with SRBC. Note clear immunopositivity of conduits (arrows) for clusterin. Lower row represents the close up of the indicated square region. Data is representative of at least 2 experiments. Scale bar: 100 µm.</p

Changes in sCLU protein level in spleen and MLN of WT mice at day 8 after immunization with SRBC.

<p>Western blot of total tissue homogenates shows an increase in sCLU amount in spleen but not MLN. Data is representative of 2 independent experiments.</p

Western blot analysis of CLU isoform.

<p>(A) Electrophoresis of stromal proteins from spleen and mesenteric lymph nodes (MLN) was performed in reducing and non-reducing conditions. Immunopositive bands mobility corresponds to secreted CLU isoform (sCLU). (B) Quantitative comparison of splenic sCLU expression in wild type (WT) and LTβR-deficient (LTβR-KO) mice. Data is normalized to the average WT expression and represented as mean±SD. * – Difference from the wild type is significant at <i>p</i><0.05.</p

Relative mRNA levels of known and potential LTβR target genes in various mouse tissues.

<p>Real-time PCR data on mRNA levels in various tissues from wild type, TNFR1-KO, and LTβR-KO mice of selected genes down-regulated in LTβR-KO splenic stroma. Data was normalized to GAPDH, which expression level was taken as 100%. Note high expression of clusterin in wild type spleen and its dramatic reduction in spleen upon LTβR knockout. Data is represented as mean±SD. * – Difference from the wild type is significant at <i>p</i><0.05.</p

Clusterin expression in activated MEF.

<p>(A) MEF were incubated with either Reh cells bearing surface LT (“LT+”) or Jurkat cells not expressing LT on their surface (“LT−”) for indicated time periods, and clusterin mRNA was measured by real-time RT-PCR. Data was normalized to mouse β-actin. (B) Physical interaction of MEF with lymphoid cells in culture. Data is represented as mean±SD.</p