Single cell transcriptomics in expanded Tregs of APS-1 patients
Aim of study:
Exploring single cell transcriptomics in expanded Tregs of with autoimmune polyendocrine syndrome type 1 (APS-1) patients (global gene expression, immune panel, TCR). N= 9 APS-1 patients and 9 healthy controls
Protocol
1. Regulatory T cells (Tregs) were isolated from EDTA-blood, activated with anti-CD3/CD28 and IL2, expanded in cell culture for 14 days and stored at -150℃ in AB serum or FBS + 5% DMSO until use.
2. Cells were thawed, run through a Live/Dead column and carefully counted. 10000 Tregs were used as input for the 10x protocol.
3. cDNA was generated and amplified using Single Cell VDJ 5’ Gel Beads, Chromium Next GEM Chip K Single cell Kit, Next GEM Single Cell 5’ GEM Kit v2 and 5’ Feature Barcode Kit (all from 10x genomics).
4. Gene expression (GEX) libraries were generated by using the Library Construction Kit from 10x genomics.
5. T-cell receptor (TCR) libraries were generated using the Single Cell Human TCR Amplification Kit and the Library Construction Kit from 10x genomics.
6. 10x Genomics Target Hybridization Kit and Human Immunology Panel was used with GEX libraries. Only 8 patient samples and 8 control samples were used for the immune panel.
7. All libraries have a unique sample index (Dual Index TT Set A).
8. All samples were sequenced on Illumina NovaSeq 6000 on a NovaSeq S2 flow cell.
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II) characterise cellular census of ectopic endometriosis tissue
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This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
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Tocilizumab, an anti-interleukin-6 receptor inhibitor, is recommended in global treatment guidelines for patients hospitalized with severe COVID-19. Using proteomic and transcriptomic analysis, we characterized the immune profile and identified cellular and molecular pathways directly modified by tocilizumab in peripheral blood samples collected from patients enrolled in the COVACTA study, a phase 3, randomized, double-blind, placebo-controlled trial, to assess the efficacy and safety of tocilizumab in hospitalized patients with severe COVID-19 pneumonia. We identified factors predicting disease severity and clinical outcomes, including markers of inflammation, lymphopenia, myeloid dysfunction, and organ injury. Proteomic analysis confirmed a pharmacodynamic effect for tocilizumab. Transcriptomic analysis revealed that tocilizumab treatment leads to faster resolution of lymphopenia and myeloid dysfunction associated with severe COVID-19 thus supporting an underlying anti-inflammatory mechanism of action for the beneficial effects of tocilizumab in patients hospitalized with COVID-19.
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