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A3.23 Vitamin D Antagonises the Suppressive Effect of Inflammatory Cytokines upon CTLA-4 Expression and Regulatory Function
  1. LE Jeffery1,
  2. K Raza1,2,
  3. DM Sansom1
  1. 1MRC Centre for Immune Regulation, School of Immunity and Infection, Institute of Biomedical Research, University of Birmingham College of Medical and Dental Sciences, Birmingham B15 2TT, UK
  2. 2Department of Rheumatology, Sandwell and West Birmingham Hospitals NHS Trust, Birmingham B18 7QH, UK

Abstract

Background and Objectives The suppressive protein, cytotoxic T lymphocyte antigen–4 (CTLA-4), is constitutively expressed by TRegs and induced in effector T cells upon activation. Its crucial role in adaptive immunity is apparent from the fatal autoimmune pathology of CTLA-4 knockout mice and the association of CTLA-4 genetic variants with autoimmunity. We have recently shown that CTLA-4 functions by depleting antigen-presenting cells of their co-stimulatory ligands, CD80 and CD86 (Qureshi et al, Science 2011). However, little is known about the factors that regulate CTLA-4 expression and function. Since low vitamin D status and elevated Th17 frequencies are evident in many autoimmune conditions, we have investigated the effect of vitamin D and Th17 polarising cytokines upon CTLA-4 expression and function.

Methods Peripheral blood CD4+CD25 T cells were stimulated under Th17 polarising conditions (TGFbeta, IL-1beta, IL-6 and IL-23) with or without active vitamin D (1.25(OH)2D3). FoxP3 and CTLA-4 were measured by flow cytometry and the vitamin D receptor (VDR) by qPCR. To assess CTLA-4 function, T cells were cultured with anti-CD3 and cells expressing GFP-tagged CD86. CD86-GFP acquisition by T cells with or without CTLA-4 blockade was then monitored by flow cytometry. For suppression assays, separately labelled activated T cells and CD4+CD25 responder T cells were co-cultured with dendritic cells in the presence of anti-CD3 and T cell proliferation assessed at five days by flow cytometry.

Results Vitamin D increased CTLA-4 expression and the frequency of FoxP3+CTLA-4+ T cells. By contrast, Th17 polarising cytokines suppressed CTLA-4. Interestingly, when supplied together, Th17 polarising cytokines synergised with vitamin D resulting in significantly higher CTLA-4 expression than with vitamin D alone. This synergy corresponded with increased VDR expression under Th17 conditions. Using a novel assay to test CTLA-4 function, we further confirmed that these changes in CTLA-4 expression correlated with ligand removal. Moreover, in dendritic cell driven stimulations vitamin D-treated T cell blasts showed enhanced CTLA-4-mediated suppression.

Conclusions Vitamin D overrides the inhibitory effect of pro-inflammatory Th17 polarising cytokines upon CTLA-4 expression and function. Given the importance of CTLA-4-mediated suppression in the control of autoimmune diseases, including RA, these data highlight the importance of vitamin D in immune regulation and its potential as a therapeutic agent.

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