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02.32 Exploring the role of bcl-3 in cd4+ t cells
  1. Natasha L. West1,
  2. Amy E. Anderson1,
  3. Arthur G. Pratt1,
  4. Nisha Nair2,
  5. Andrew Skelton1,
  6. Anne Barton2,
  7. Ruaidhri J Carmody3,
  8. Andrew D Rowan1,
  9. John D Isaacs1
  1. 1National Institute for Health Research Newcastle Biomedical Research Centre, Newcastle upon Tyne Hospitals NHS Foundation Trust and Newcastle University, Newcastle upon Tyne, UK
  2. 2NIHR Manchester Musculoskeletal Biomedical Research Unit, Manchester Academic Health Science Centre, Central Manchester Foundation Trust, Manchester, UK
  3. 3Institute of Infection, Immunity and Inflammation, University of Glasgow, UK

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 controls1. 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.

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