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European Journal of Immunology Supporting Information for DOI 10.1002/eji.201747195 Alsya J. Affandi, Sandra C. Silva-Cardoso, Samuel Garcia, Emmerik F. A. Leijten, Tessa S. van Kempen, Wioleta Marut, Joel A. G. van Roon and Timothy R. D. J. Radstake CXCL4 is a novel inducer of human Th17 cells and correlates with IL-17 and IL-22 in psoriatic arthritis
C 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag
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www.eji-journal.eu
Supporting Information CXCL4 is a novel inducer of human Th17 cells and correlates with IL-17 and IL-22 in psoriatic arthritis Alsya J. Affandi1,2, Sandra C. Silva-Cardoso1,2, Samuel Garcia1,2, Emmerik F.A. Leijten1,2, Tessa S. van Kempen1,2, Wioleta Marut1,2, Joel A. G. van Roon1,2#, Timothy R. D. J. Radstake1,2#* 1
Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands 2
Department of Rheumatology and Clinical Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands #
Shared last co-authors
* Correspondence: Prof. Timothy R. D. J. Radstake, MD, PhD
[email protected] Index 1
Supplementary Materials and Methods .......................................................................................... 2
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Supplementary Figures ................................................................................................................... 3
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Supplementary Tables .................................................................................................................... 9
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Supplementary Materials and Methods
RNA extraction and gene expression analysis Total RNA was isolated from cell lysates using the RNeasy Mini Kit (#74106 Qiagen), according to the manufacturer’s instructions. Gene expression was analysed by quantitative PCR using a 3 ng RNA-equivalent after retrotranscription with iScript cDNA Synthesis Kit (#1708891 Bio-Rad). Reactions were conducted using the SYBR Select Master Mix with 500 nM specific primer pairs on a StepOnePlus Real-Time PCR System (ThermoFisher Scientific). Fold change was calculated using the comparative cycle threshold (
Ct) method and values were normalized to the expression of
B2M. All primers used are listed in Table S2. Naïve CD4 T cell isolation Peripheral blood mononuclear cells from healthy donors were isolated by Ficoll gradient (#17-144002, GE Healthcare). Cells were first enriched using magnetic beads for CD4 T cells (#130-096-533, Miltenyi Biotec) on autoMACS Pro Separator (Miltenyi Biotec) according to manufacturer’s instructions. After staining with antibodies for 30 min at 4°C, naïve CD4 T cells (CD3+ CD4+ CD127+ CD25- CD27+ CD45RO-) were further purified using fluorescence-activated cell sorting on BD FACSAria (BD Biosciences). Antibodies used are listed in Table S3.
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Supplementary Figures
Figure S1. CXCL4 increases IL-17 mRNA in stimulated human CD4 T cells. CD4 T cells were isolated from healthy individuals and cultured with CD3/CD28 Dynabeads and 2 µg/ml CXCL4 for 24 h. RNA was isolated, followed by cDNA synthesis, and gene expressions were assessed. The effect of CXCL4 on IL17A, IFNG, and IL22 mRNA in CD4 T cells was determined by quantitative PCR. Mean and values from each donor are shown and paired t-test was used for statistical analysis. * p
