biostudies-arrayexpressMorten VenøHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15969Introduction: The joint destructive process in rheumatoid arthritis (RA) is highly individual and dominated by complex cellular interactions. In damping the immune activation, the PD-1 pathway is central and highly upregulated in the arthritic joint. Here, we examine how T cells with or without PD-1 influence the function of arthritic mesenchymal cells Methods: We investigated samples from RA synovial fluid and characterized PD-1+ and PD-1- T cells by flow cytometry, ELISA and RNA sequencing (RNA seq). We performed co-culture experiments to investigate the interaction between PD-1+/- cells, osteoclasts, osteoblasts and fibroblasts. We used flow cytometry, ELISA and RNA seq to characterize the RA and HC fibroblasts after co-culture with PD-1+ and PD-1- T cells. Results: We found PD-1+ T cells to express other checkpoint receptors and to have a diminished cytokine production upon CD3/CD28 stimulation. The PD-1+ and PD-1- T cells from the RA synovial fluid clustered differently after RNA seq, and the gene signature of the PD-1+ T shared traits with peripheral T helper cells. Fibroblasts co-cultured with PD-1+ T cells expressed genes associated with collagen synthesis, TGFbeta production and immune regulation. In contrast, the signature in fibroblasts co-cultured with PD-1- cells was associated with immune activation. Conclusion: We demonstrate RA synovial fluid CD3+ T cell subsets can drive healthy fibroblasts into distinct pathological endotypes. When evaluating all PD-1+ T cells, these support fibroblast subsets related to the production of collagen and immune regulation.biostudies-arrayexpressSample Collection - Fibroblasts from HC skin in passage 3 were thawed and cultured in a T175 flask (Sarstedt, cat. no. 83.3911.002) in DMEM low glucose media (VWR, cat. no. 392-0407). When confluency was reached cells were trypsinized and seeded into a 96-well plate and stimulated by adding 20% supernatant from PD-1+/- cultures (n=3), IFNγ (10 µg/ml) (n=3), PD-1+/- T cells (n=3), or no stimulation (n=3). After 24 hours T cells were removed.Library Construction - The SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian (Takara, Japan, cat.no. 634876) was used for library preparation with 200ng of the purified RNA as input following the manufacturer`s protocol. The Bioanalyzer High sensitivity DNA analysis kit (Agilent, cat.no. 5067-4626) was used to determine the size of the library fragments and the Qubit dsDNA High Sensitivity Kit (Invitrogen, cat. no. Q32851) was used to quantify the library.Nucleic Acid Extraction - RNA was purified from the co-cultured fibroblasts after washing with PBS and cell lysis. RNA-purification was done using the RNeasy Mini Kit (Qiagen, cat. no. 74104) (n=1 in triplicates), and the Genomic DNA was removed by using the RNase-Free DNase Set (Qiagen, cat. no. 79254). The RNA integrity number and concentration were measured on Bioanalyzer RNA Nano chip (Agilent, cat.no. 5067-1511).Sequencing - The libraries were pooled in equal amounts and sequenced on a NovaSeq X Plus sequencing machine using a 10B flow-cell for paired-end 150 cycles (Illumina).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Sequencing data were pre-processed by removing the adapter sequence and trimming away low-quality bases with a Phred score below 20 using Trim Galore (0.6.10). Quality control was performed using FastQC (0.12.1), Picard (2.3.1) and Multiqc (1.9) to ensure high-quality data. The filtered RNA-seq data was mapped against the human genome (hg19 / GRCh37) using STAR (2.7.11b) and gene expression was quantified using featureCounts (2.0.0) with gene annotations from Gencode release 37. Raw read counts for each sample are provided as a tab delimited txt file.UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq XRNA-seq of total RNAHomo sapiensStinne Ravn GreisenMorten VenøfalseRheumatoid Synovial Subsets of CD3+ T cells defined by PD-1+ and PD-1- Cells Drive Fibroblasts into Distinct EndotypesIntroduction: The joint destructive process in rheumatoid arthritis (RA) is highly individual and dominated by complex cellular interactions. In damping the immune activation, the PD-1 pathway is central and highly upregulated in the arthritic joint. Here, we examine how T cells with or without PD-1 influence the function of arthritic mesenchymal cells Methods: We investigated samples from RA synovial fluid and characterized PD-1+ and PD-1- T cells by flow cytometry, ELISA and RNA sequencing (RNA seq). We performed co-culture experiments to investigate the interaction between PD-1+/- cells, osteoclasts, osteoblasts and fibroblasts. We used flow cytometry, ELISA and RNA seq to characterize the RA and HC fibroblasts after co-culture with PD-1+ and PD-1- T cells. Results: We found PD-1+ T cells to express other checkpoint receptors and to have a diminished cytokine production upon CD3/CD28 stimulation. The PD-1+ and PD-1- T cells from the RA synovial fluid clustered differently after RNA seq, and the gene signature of the PD-1+ T shared traits with peripheral T helper cells. Fibroblasts co-cultured with PD-1+ T cells expressed genes associated with collagen synthesis, TGFbeta production and immune regulation. In contrast, the signature in fibroblasts co-cultured with PD-1- cells was associated with immune activation. Conclusion: We demonstrate RA synovial fluid CD3+ T cell subsets can drive healthy fibroblasts into distinct pathological endotypes. When evaluating all PD-1+ T cells, these support fibroblast subsets related to the production of collagen and immune regulation.2025-11-27T00:00:00Z2025-11-27T02:01:36.272Z2025-11-03T19:48:19.387ZE-MTAB-15969ERP183615EFO_0002944EFO_0004170EFO_0009653EFO_0005518EFO_0003816EFO_0004184