Remarkably, we found that TL1A significantly reduces proliferation of suboptimally activated B cells

Remarkably, we found that TL1A significantly reduces proliferation of suboptimally activated B cells. subfamily members, DR3 shows the highest homology to TNFR1 [3], [4]. However, unlike TNFR1 that shows a ubiquitous expression, DR3 expression is restricted to lymphocyte-enriched tissues, including peripheral blood leukocytes, thymus and spleen, and it has been shown to be especially up-regulated in activated T cells [2], [6]. The ligand for DR3 is TNF-like ligand 1A (TL1A), a member of the TNF superfamily [7]C[10]. TL1A is expressed in a variety of cell types, including activated endothelial cells, monocytes, macrophages, dendritic cells, and T cells [7], [11]C[15]. Like other TNF members, TL1A contains a predicted transmembrane domain and a bioactive, proteolytically cleaved truncated form that can be released as a soluble factor [7], [8]. TL1A expression is highly regulated and induced by inflammatory stimuli [7], [11], [15], [16]. The TL1A/DR3 axis has been shown to costimulate T cells to produce a wide variety of cytokines and promote cell proliferation of activated T cells and by the B cell receptor (BCR) stimulation express DR3 molecule. Further, DR3 was expressed in antigen-stimulated B cells of tonsil germinal centers (GC). Remarkably, we found that TL1A significantly reduces proliferation of suboptimally activated B cells. Our data suggest a novel role for the TL1A/DR3 axis in modulating proliferation of activated B cells. Materials and Methods Cell and Tissue Samples Cryopreserved peripheral blood mononuclear cells (PBMC) from 10 human blood buffy coats and formalin-fixed paraffin-embedded human Tenofovir hydrate tissue tonsil (n?=?4) and spleen (n?=?3) sections were used in this study. Buffy coats were collected at the Hematology Unit, Azienda Ospedaliera Universitaria Integrata (AOUI) in Verona (Italy); tonsil specimens were obtained from hyperplastic tonsils of subjects undergoing tonsillectomy and collected at the Pathological Anatomy Unit, AOUI, Verona (Italy); spleen specimens were obtained from normal spleen removed after traumatic injuries and collected at the Pathological Anatomy Unit, AOUI, Verona (Italy). PBMCs were isolated by Ficoll-hypaque centrifugation (Lymphoprep, Tenofovir hydrate Nicomed, Oslo, Norway) and suspended in freezing medium for storage in liquid nitrogen. Upon thawing, cell viability consistently exceeded 95% in all samples. Cells were washed twice in PBS and then resuspended in the appropriate buffer or medium. PBMC-derived B cells were isolated by negative selection using the Human B-Cell Enrichment Kit (without CD43 depletion; Stem Cell Technologies, Vancouver, Canada). After separation, B cells were washed twice and counted. Cell purity as assessed with CD19 staining was routinely above 98%. Ethics Statement Blood and tissue samples were collected under a protocol approved by the local Ethics Committee (Comitato Etico per la Sperimentazione C AOUI) and data were analyzed anonymously. In accordance with the Declaration of Helsinki, all blood donors provided written informed consent for the collection and use of their blood samples for research purposes. For the use of tissue samples, the local Ethics Committee (Comitato Etico per la Sperimentazione C AOUI) approved the anonymous retrospective use of samples consisting of diagnostic remnants without written consent release, as also specifically stated in the Italian law, according to the directive issued on Tenofovir hydrate March 1st 2012 from the Italian Privacy Authority (Deliberazione n. 85) (12A03185) (complying with EU directives). Cell Stimulation Peripheral blood (PB) purified B cells were stimulated by incubating with sulfate latex beads (2.3 m diameter) (Interfacial Dynamics Corporation, Portland, OR) [27] coated with goat F(ab)2 anti-human IgM (20 g/ml) (Southern Biotech, Birmingham, AL) in 24-well plates, at 5106 cells/ml, for the indicated time. At the end of the incubation, the cells were subjected to flow cytometry or biochemical analysis. Flow Cytometry Analyses PB purified B cells stimulated or not with sulfate latex beads coated with anti-IgM for 24 h were harvested, washed, resuspended Tenofovir hydrate in PBS and incubated with either PE-conjugated anti-human DR3 mAb (clone JD3, BioLegend, London, UK) or isotype control antibody on ice for 45 min. The cells were then stained with APC-conjugated anti-CD19 mAb (BD Biosciences, San Jose, CA) and 7-amino-actinomycin (7AAD, BD Biosciences) for COL4A5 15 min on ice. Approximately 1104 gated events were acquired for each sample on FACSCanto cytometer (Becton Dickinson, San Jose, CA). Flow cytometry data were gated using the FlowJo software (TreeStar, Ashland, OR)..