02.32 Exploring the role of bcl-3 in cd4+ t cells. (1st March 2017)

• Record Type: Journal Article
• Authors: West, Natasha L.; Anderson, Amy E.; Pratt, Arthur G.; Nair, Nisha; Skelton, Andrew; Barton, Anne; Carmody, Ruaidhri J; Rowan, Andrew D; Isaacs, John D
• Is Part Of: Annals of the rheumatic diseases. Volume 76(2017)Supplement 1
• Page Start: A21
• Page End: A22
• Publication Date: 2017-03-01
• Subjects: Rheumatism -- Periodicals; 616.723005
• DOI: 10.1136/annrheumdis-2016-211050.32
• Languages: English
• ISSNs: 0003-4967

Abstract

Abstract : Background: Rheumatoid arthritis (RA) is a disease of immune dysregulation whose pathogenesis remains incompletely understood. We previously identified an IL-6 mediated, STAT3 target gene-enriched 'signature' in circulating CD4 + T cells of untreated arthritis patients that predicted progression to RA. BCL-3 was the most differentially expressed of these genes between RA patients and disease controls 1 . This atypical IκB molecular product appears to be important in murine T-cell biology. In this project we aim to explore the function of BCL-3 in human primary CD4 + T cells, and its potential relevance to RA pathogenesis. Materials and methods: Highly purified CD4 + T-cells were isolated from human peripheral blood using magnetic beads. Phospho-STAT3 (pSTAT3) and paired BCL-3 gene expression were measured in cells from 161 untreated arthritis patients using flow cytometry and microarray technology, respectively. To study BCL-3 kinetics, CD4 + T-cells isolated from healthy volunteers were stimulated with cytokines or TCR ligands; gene and protein expression were analysed by Taqman Real-Time PCR and Western Blotting respectively. To overexpress BCL-3 two methods were compared: use of our previously developed murine mimetic peptide, and lentiviral transduction. The phenotype of CD4 + T-cells by means of proliferation, activation and intracellular cytokines were assessed by CFSE labelling, surface and intracellular flow cytometry respectively. Results: Intracellular pSTAT3 correlates strongly with paired BCL-3 gene expression in circulating CD4 + T-cells of early arthritis patients. In vitro kinetics experiments show its expression is induced by IL-6, but becomes maximal after TCR stimulation for 72 hours; protein peaks later at 5 days. A limited effect of the mimetic peptide on cultured CD4 + T-cell phenotype likely reflects incomplete homology of murine and human peptides. Transduction of CD4 + T-cells with rLV-hBCL3-IRES-ZsGreen1 lentivirus results in 6-fold upregulation of BCL-3 gene expression that remains detectable 6 days post transduction. Conclusions: Enhanced STAT3 signalling accounts for increased BCL-3 expression in circulating CD4 + T cells of early RA patients, and the kinetics of IL-6- and CD3/28-mediated BCL-3 induction in these cells have been defined, Having optimised a robust means of over-expressing BCL-3 by lentiviral transduction of primary human CD4 + T cells, its specific functional role will now be explored.