biostudies-arrayexpressCharline BEGUINHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15881We developed a humanized mouse model of graft-versus-host disease in which Treg injected two days after PBMC persist in the blood of infused mice and can be characterized. This model provides in vivo assessment of manipulated Treg. We used that model to evaluate the impact of ex vivo priming of Treg with TNF-alpha on their ability to prevent xenogeneic GVHD. Indeed, it had been demonstrated that Treg function can be enhanced through stimulation of TNFR2. scRNAseq analysis was performed to assess the impact of TNF priming on Treg transcriptomics. Peripheral blood mononuclear cells were isolated from the blood of three healthy donors. Treg were isolated through immuno-magnetic selection then cultured for 24 hours with IL-2 at 10 ng/mL with or without TNF at 100 ng/mL. After 24 hours, cells were collected and washed. We performed cell multiplexing to label samples individually, then pooled them for single cell RNA sequencing. For each donor (named donors 3, 4 and 7), half cells were in the control group (no TNF) and half cells were in the experimental group (with TNF). 10x Genomics 3’ Single Cell Gene Expression with CellPlex multiplexing (Set A). Cells were labeled with Cell Multiplexing Oligos prior to pooling and library preparation.biostudies-arrayexpressSample Treatment - Treg were isolated as previously described and cultured for 24 hours under two conditions: Treg with IL-2 at 10 ng/mL alone or Treg with IL-2 at 10 ng/mL and TNF-α at 100 ng/mL.Nucleic Acid Extraction - 1) Cell multiplexing As described in the kit protocol, 1 mL of PBS with BSA 0.04% (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) were added to each sample (approximately 1 million cells per sample) which were then centrifuged at 400g for 5 minutes at RT. The supernatant was removed and cells were resuspended in 100 μL of Cell Multiplexing Oligo (one oligonucleotide per sample, 3’ CellPlex Kit 502 Set A, 10x Genomics B.V.) by gently pipetting 10-15 times. Cells were incubated at RT for 5 minutes and mixed by gently pipetting with 1.9 mL of Wash and Resuspension buffer (composed of PBS and BSA 1%). Cells were centrifuged at 400g for 5 minutes at 4°C, the supernatant was removed, and two additional washes were made with 2 mL of the Wash and Resuspension Buffer at 400g for 5 minutes at 4°C (total of three washes). The supernatant was removed and cells were resuspended in the Wash and Resuspension Buffer to be counted (volume according to protocol) and assessed for viability using a Countness™ 2 Automated Cell Counter (Thermo Fischer Scientific). Next, labeled cells were pooled and the number of cells in the final mixture was counted. 2) Library preparation A total of 20,000 equimolarly pooled, CellPlex-labeled cells were processed using the Chromium Next GEM Single Cell 3ʹ Reagent Kit v3.1 with Cell Multiplexing (10x Genomics, protocol CG000390) on a Chromium iX instrument with Chip G (10x Genomics). 3) Single-cell Gene Expression Matrices (GEMs) Generation GEMs were generated by combining barcoded Single Cell 3ʹ v3.1 Gel Beads, a Master Mix, and CellPlex-labeled cells with Partitioning Oil on a Chromium Chip G (10x Genomics). Reverse transcription was performed using a Veriti™ Thermal Cycler (Thermo Fisher Scientific) with the following conditions: 53°C for 45 minutes, 85°C for 5 minutes, and holding at 4°C. 3) cDNA Amplification and Purification Following emulsion breakage, pooled fractions were purified using DynaBeads MyOne Silane Beads (10x Genomics). cDNA and CellPlex barcodes were amplified via PCR with a 12-cycle reaction module: 98°C for 3 minutes, 98°C for 15 seconds, 63°C for 20 seconds and 72°C for 1 minute, followed by a final extension at 72°C for 1 minute. Feature cDNA Primer 3 was used to amplify both cDNA and CellPlex (primer included in the kit). The cDNA reaction was purified using magnetic SPRI Beads (Beckman Coulter, Indiana, United States). The supernatant, containing barcoded CellPlex amplicons, was separated and purified again for CellPlex library preparation.Sample Collection - PBMC from three healthy donors (Belgian Red Cross) were used for that experiment. Treg were isolated through immuno-magnetic selection and cultured for 24 hours under two conditions: Treg with IL-2 at 10 ng/mL alone or Treg with IL-2 at 10 ng/mL and TNF-α at 100 ng/mL. Cell concentration was 80,000 cells per 200 μL in a 96-well U bottom plate. After 24 hours, cells were collected and washed with 1 mL of PBS. The 10x Genomics 3’ CellPlex Kit (10x Genomics B.V., Leiden, the Netherlands) was used to perform cell multiplexing: our six samples were labelled individually with cell multiplexing oligonucleotides. This allowed us to pool samples and read them together.Library Construction - 1) Library Construction for 3’ Gene Expression For 3ʹ Gene Expression libraries, amplified cDNA was fragmented, end-repaired, A-tailed and ligated to sequencing adapters. Libraries were then amplified by PCR (indexing PCR) using Dual Index TT primers plate (10x Genomics). 2) CellPlex Library Construction Purified CellPlex amplicons were processed in an indexing PCR using Feature SI Primers 2 (included in the kit) with dual single index primers (Dual Index Kit NN Set A, 10x Genomics).Sequencing - 1) Library Quality Control and Sequencing Both the 3ʹ Gene Expression and CellPlex libraries were quality-checked on QIAxcel Advanced System (QIAGEN, Hilden, Germany) using a DNA screening cartridge. Final dual-indexed libraries were quantified by qPCR with KAPA Library Quantification Kit including Illumina standard (Roche, Basel, Switzerland). Libraries were sequenced on an Illumina NovaSeq 6000 (Illumina, San Diego, California, USA) using a S4 flow cell for paired-end 150 cycles, and demultiplexed with read 1: 28 cycles; read 2: 91 cycles; index i7: 10 cycles; index i5:10 cycles. Sequencing depth was set to ~20,000 reads per cell for gene expression libraries and 5,000 reads per cell for CellPlex libraries. 2) Raw data preparation Raw sequencing data were demultiplexed using sample-specific indexes, filtered for quality, and converted into FASTQ files for downstream analysis, using the bcl2fastq software (Illumina). Sequencing data quality was assessed using the FastQC tool (Illumina) for individual reports per sample, and with MultiQC (Illumina) to generate a report comprising all samples.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - The scanpy (sc) package (v. 1.9.3) implemented in Python software v.3.10.9 (Python Software Foundation, Beaverton, Oregon, USA) was used to perform the dimensional reduction of Treg. To normalize raw data, we used the “sc.pp.normalize_total” function, specifying that we wanted a total of 10,000 Counts Per Million (CPM). The “sc.pp.highly_variable_genes” function was used to extract the most variable genes for each sample. We then calculated clusters using the Leiden algorithm by the “sc.tl.leiden” function, with resolutions between 0.4 and 0.8, and integrated with the UMAP algorithm “sc.tl.umap” with the following parameters: min_dist=0.5, gamma=1, alpha=1, negative_sample_rate=5, init_pos=”spectral”.UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNA from single cellsHomo sapiensCharline BEGUINOswin KWANfalseDevelopment of a humanized mouse model of graft-versus-host disease to assess human regulatory T cell functionWe developed a humanized mouse model of graft-versus-host disease in which Treg injected two days after PBMC persist in the blood of infused mice and can be characterized. This model provides in vivo assessment of manipulated Treg. We used that model to evaluate the impact of ex vivo priming of Treg with TNF-alpha on their ability to prevent xenogeneic GVHD. Indeed, it had been demonstrated that Treg function can be enhanced through stimulation of TNFR2. scRNAseq analysis was performed to assess the impact of TNF priming on Treg transcriptomics. Peripheral blood mononuclear cells were isolated from the blood of three healthy donors. Treg were isolated through immuno-magnetic selection then cultured for 24 hours with IL-2 at 10 ng/mL with or without TNF at 100 ng/mL. After 24 hours, cells were collected and washed. We performed cell multiplexing to label samples individually, then pooled them for single cell RNA sequencing. For each donor (named donors 3, 4 and 7), half cells were in the control group (no TNF) and half cells were in the experimental group (with TNF). 10x Genomics 3’ Single Cell Gene Expression with CellPlex multiplexing (Set A). Cells were labeled with Cell Multiplexing Oligos prior to pooling and library preparation.2025-11-20T00:00:00Z2025-11-20T02:02:22.56Z2025-10-27T21:08:11.051ZE-MTAB-15881ERP183149EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_0003816EFO_0004184EFO_0003969