SRA

Analysis of follicular helper and follicular regulatory T cell subsets suggests that acquasition of nuclear proteins by antigen-specififc B cells promotes an increase in follicular regulatory T cells with immunosupressive gene expression profile Overall design: Four C57BL/6J mice were subcutaneously immunized with SA-DEL (streptavidin linked to biotinylated Duck Egg Lysozyme) in Ribi adjuvant. At 8 d.p.i. two mice were boosted with SA-NucPrs (streptavidin linked to biotinylated nuclear proteins, including bovine nucleosomes, Jo-1, Scl-70, Ro, SmRNP) in Ribi and two mice boosted with SA in Ribi for control. Follicular T cells (CD4+PD1hiCXCR5hi) were sorted from draining lymph nodes of mice at day 11 after immunization into PBS + 2% FBS. Single cell suspensions were subjected to counting and viability checks on the LUNA Fx7 Automated Cell Counter (Logos Biosystems) and diluted to a concentration of 700 -1000 cells/ul. Single cell libraries were generated using the 10x Genomics Chromium Controller with Immune Profiling reagents following the manufacturer's protocol (10x Genomics). Final library quality was assessed using the LabChip GX (PerkinElmer). Libraries were subjected to paired-end sequencing according to the manufacturer's protocol (Illumina NovaSeq 6000). Bcl2fastq2 Conversion Software (Illumina) was used to generate de-multiplexed Fastq files and the CellRanger Pipeline (10x Genomics) was used to align reads and generate count matrices. Barcodes with mitochondrial reads > 5% were removed. Preprocessing, clustering, and dimensionality reduction were performed using Loupe Browser and Loupe V(D)J browser (10x genomics, Cell Ranger). Graph-based clustering of Tfrs were visualized in 2D using UMAP algorithm.