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R. Lee, C. Dayan, M. Norman, A. Dick; Development of a Flow Cytometric Assay to Predict an Individual's CD4 Lymphocyte Response to Immunosuppression . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2389.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Reduced T–cell sensitivity to the suppressive action of glucocorticoids is a common problem which complicates the management of many autoimmune diseases, including posterior segment intraocular inflammation (uveitis). Steroid sensitivity may be influenced by IL–2 signalling and we have modified an in–vitro assay to explore the effects of a number of agents, each of which acts at a different point in the IL–2 pathway, upon T–cell proliferation and steroid responsiveness. This has potential to predict response to, and optimise, immunosuppressive therapy. Methods: PBMCs from 4 healthy human volunteers were labelled with CFSE and cultured for 5 days with CD3 / CD28 dynabeads to induce T–cell expansion. The effect of increasing concentrations of Dexamethasone (Dex), Tacrolimus (Tac; calcineurin inhibitor), Basiliximab (Bas; IL–2r mAb), Daclizumab (Dac; IL–2r mAb) and AG490 (JAK kinase inhibitor), upon the ability of CD4+ and CD8+ T–cells to proliferate was then evaluated using 4 colour flow cytometry. Cell death was quantified using 7–AAD. Results: Increasing concentrations of each immunosuppressive agent reduced the proportion of lymphocytes going into cycle, and those that did underwent fewer divisions. A subset of CD4+ T cells still proliferated despite supra–therapeutic doses of Dex. This proportion (8%) was higher than with supra–therapeutic doses of Bas (2%), Dac (6%) or Tac(4%). Proliferating CD8+ T cells underwent more divisions than CD4+ T cells. Greater CD4+ T cell suppression was achieved when Dex was combined with IL–2 inhibition than with either agent alone, and equivalent suppression was achieved at lower concentrations of each agent in combination than when used in isolation. Conclusions: This in–vitro assay can reliably quantify the proliferation of lymphocyte sub–groups in response to immunosuppressive drugs. Greater CD4+ suppression is achieved with IL–2 inhibition than with Dex, and even greater inhibition is achieved with combination therapy. Further investigation is required to evaluate the steroid–sensitising effect of IL–2 inhibition on CD4+ cells from patients with steroid–resistant disease and to assess the potential of this assay to predict treatment response for individual patients.
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