DOI: 10.25405/data.ncl.30884033.v1
title: Gene expression of CD4+ T cells of rheumatoid arthritis patients primed by mature or tolerogenic dendritic cells

description: Tolerogenic dendritic cells (tolDC) are currently in clinical trials for the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. The methods for producing therapeutic tolDC vary widely, with little being known about the commonalities and differences of distinct cell products in terms of their regulatory actions on CD4<sup>+</sup> T cells. We generated human monocyte-derived tolDC with vitamin D3 alone or in combination with dexamethasone, and compared their regulatory actions on T cells to the actions of mature moDC. We co-cultured these DC types with CD4<sup>+</sup> T cells from four treatment-naïve early arthritis female patients, age range 46-68, with a confirmed diagnosis of RA, and analysed the gene expression profile of the responding T cells, using the nCounter NanoString Immune Exhaustion panel. Cryopreserved PBMC from early arthritis patients were thawed and labelled with CellTrace<sup>™ </sup>as above and co-cultured with freshly generated allogeneic healthy donor moDC at a 1:10 DC:PBMC ratio (1x10<sup>5 </sup>moDC and 1x10<sup>6</sup> PBMC/well) in a 24-well plate in RF10 at 37°C, 5% CO<sub>2. </sub>On day 6, the cells were harvested and stained for fluorescence activated cell sorting (FACS) using viability dye Zombie NIR<sup>™ </sup>and the following mouse anti-human antibodies: CD3-BUV395 (UCHT1), CD4-PE-Cy7 (RPA-T4), CD11c-PE/Dazzle™ 594 (BU15) and CD14-FITC (M5E2) in staining buffer supplemented with human IgG (Hizentra®). Cells were strained through a 35μm cell strainer and acquired at 4°C on the BD FACSAria<sup>™ </sup>II using a 70μm nozzle. Events were gated sequentially as follows: cells, single cells, live cells (Zombie NIR<sup>-</sup>), proliferated cells (CellTrace<sup>-</sup>), CD11c<sup>-</sup>, CD14<sup>-</sup> and CD3<sup>+</sup>CD4<sup>+</sup>. Approximately 3 x 10⁵ cells were sorted into cold RF10 per sample. Sorted CD4<sup>+</sup> T cells that had proliferated in response to matDC (T-MatDC), DexVD<sub>3</sub>DC (T-DexVD<sub>3</sub>DC) or VD<sub>3</sub>DC (T-VD<sub>3</sub>DC) were lysed in RLT buffer containing 1% β-mercaptoethanol, passed through a QIAshredder column (Qiagen, Hilden, Germany) and stored at -80°C. Total RNA was extracted using the RNeasy® Micro Kit (Qiagen) according to manufacturer’s instruction. RNA concentration was determined using Qubit (Invitrogen) and 100ng RNA per sample was analysed on the NanoString nCounter® platform using the Immune Exhaustion panel (NanoString Technologies, Seattle, WA, USA).<b>Ethical approval: </b>RA blood samples were obtained with informed consent from the Northeast Early Arthritis Clinic (NEAC) (Musculoskeletal Unit, Freeman Hospital, Newcastle-upon-Tyne Hospital Trust). Ethical approval was given by the Newcastle & North Tyneside 2 Research Ethics Committee under the project titled ‘Prognostic and therapeutic biomarkers in an observational inception cohort: the Northeast Early Arthritis Cohort’ (REC reference 12/NE/0251). Instruments, hardware and software used: BD FACSAria<sup>™ </sup>II for cell isolation, NanoString nCounter®for gene expression analysis, R software for data analysisDate(s) of data collection: January 2022Geographic coverage of data: Newcastle upon Tyne, UKData validation: <i>A</i><b> </b>pseudo-count of 1 was added to all raw counts to enable subsequent log transformation of the data in cases where zero-counts are present. The data was normalised to the most stable housekeeping genes determined using the “geNorm” method within the NormqPCR package (Vandesompele et al., 2002). Further data analysis was carried out as described in the published paper (Barran et al, doi: 10.3389/fimmu.2025.1698413), and the analysed datasets are included as Supplementary Data Tables in that paper.<br><br>