ANTIBODY-BASED PROXIMITY LIGATION ASSAY COMBINED WITH FLOW CYTOMETRY IS A POWERFUL TOOL TO DETECT ACTIVE IL-7 RECEPTORS IN ARTHRITIS

Background: IL-7 is a leading growth factor for memory T cells and is believed to play a central role in the sustained inflammation observed in many chronic inflammatory diseases, including rheumatoid arthritis (RA). IL-7 signals through the dimeric IL-7 receptor (IL-7R) formed by the IL-7Rα chain and the common γ-chain. The binding of IL-7 to the IL-7Rα chain initiates dimerisation with the common γ-chain leading to receptor activation and intracellular signalling. The IL-7Rα chain is expressed on memory T cells in RA. However, evaluation of the activation of interleukin receptors is challenged by the fact that these often are promiscuous in their dimer associations. We, therefore, combined specific antibodies towards the IL-7Rα chain and the common γ-chain with proximity ligation assay (PLA) as this enables the detection of dimerised receptors with single molecule resolution. Quantitative detection was possible by combining PLA and multicolour flow cytometry. Objectives: The objective was to evaluate the expression of active IL-7Rs on memory T cells in chronic inflammation by quantitative detection of IL-7R dimers on synovial fluid mononuclear cells (SFMCs). Methods: IL-7R dimerisation was investigated by PLA on the human T cell line HPB-ALL and SFMCs isolated from RA patient joint fluid. HPB-ALL has a high surface expression of both IL-7R chains and was used for optimisation of the method. Mouse anti-IL-7Rα (hIL-7R-M21, BD Pharmingen) and rat anti-γc (tUGh4, Biolegend) antibodies were used for PLA. For flow cytometry, cells were stained with LIVE/DEAD (Invitrogen) and fixed. Cells were blocked, followed by incubation with oligonucleotide-conjugated antibodies. Amplification of the signal was done using Detection Reagent Far Red (Olink Biosciences). For SFMCs, fluorescently labelled antibodies to known CD markers were added to distinguish cell populations. For microscopic visualisation, the PLA was done on cytospin preparations. Results: After optimisation of the method, dimerised IL-7Rs on a high fraction of CD4+ HPB-ALL cells were detected by PLA and flow cytometry. By employing PLA on isolated RA SFMCs, signals from dimeric IL-7Rs on CD4+CD45RO+ memory T cells were detected. Thus, PLA combined with flow cytometry makes it possible to examine the role of IL-7 in the survival of these cells in the inflamed synovial environment. We were able to verify these results by visualisation of dimeric IL-7Rs with single molecule resolution in fluorescence microscopy of cytospin preparations of both HPB-ALL and SFMCs. Conclusions: This is the first report of PLA combined with flow cytometry used to quantitatively detect specific interleukin receptor chains in close proximity. The method is a powerful new tool in our examination of the chronic inflammation in RA, as interleukin receptor dimerisation is a direct measure of a high probability of intracellular signalling. References: 1. Hartgring, S. A. et al. (2006). Ann Rheum Dis 65, iii69-74 2. Churchman, S. M. & Ponchel, F. (2008). Rheumatology (Oxford) 47, 753-759 3. Hartgring, S. A. et al. (2009). Arthritis Rheum 60, 2595-2605 4. Soderberg, O. et al. (2006). Nat Methods 3, 995-1000 Disclosure of Interest: None DeclaredCitation: Annals of the Rheumatic Diseases, volume 71, supplement 3, year 2012, page 328Session: Cytokines and inflammatory mediators (Poster Presentations )