Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Comparison of gene expression in NOD versus B6 splenic B cell subsets.

ABSTRACT: NOD mice are an inbred strain that display enhanced MZ B cell differentiation from an early age. Interestingly, several lines of evidence implicate MZ B cells in this strain as important contributors to the T cell mediated beta cell destruction associated with the development of type 1 diabetes (T1D). In order to develop a better understanding of the underlying causes for augmented MZ B cell production in NOD mice, we obtained the transcriptional profiles of FO and MZ subsets and TR precursors from NOD mice and compared them to those of the B6 strain. Red cell depleted splenocyte suspensions from from eight pooled NOD or B6 mice were stained with biotinylated anti-CD3Îµ (145-2C11) and CD11b (M1/70, BD Biosciences) antibodies followed by anti-biotin Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Separation of unlabeled B cells from samples was achieved to a >93% purity (as determined by flow cytometry) by negative depletion with an autoMACS cell sorter (Miltenyi Biotec). These cells were then stained with a cocktail of: non-stimulatory Affinipure goat anti-mouse-IgM-FITC Fab fragments from Jackson Immunoresearch; anti-CD21/35-PE (7G6), anti-CD24-biotin (30-F1; with streptavidin-PerCP secondary), B220-APC-Cy7 (RA36B2) from BD Biosciences; anti-CD93-APC (AA4.1), anti-CD23-PE-Cy7 (B3B4) from eBiosciences. T1, T2FO, T3, FO, T2MZ and MZ subsets were then separated with a FACSAria Cell Sorter (BD Biosciences). This process was repeated in an independent experiment in order to generate a duplicate sample of each B cell subset from NOD and B6 mice. Labelled cRNA from B cell samples were hybridized to Mouse Genome 430 2.0 Arrays.

ORGANISM(S): Mus musculus

SUBMITTER: Pablo Silveira

PROVIDER: E-GEOD-43396 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: identify differentially expressed genes between ex-Treg obtained 5, 10 or 15 days post-adoptive transfer into a lymphopenic environment compared to ex vivo Treg in order to identify Treg subset with heightened instability. Method: Treg from Foxp3YFP-Cre Rosa26RFP (PMID: 18387831, PMID: 17171761) were co-injected with congenically-disparated naive T cells in a 1:1 ratio into Rag1KO mice and ex-Treg were sorted at 15 days, 10 days or 5 days post-transfer. As a control, ex vivo Treg were sorted from Foxp3YFP-Cre Rosa26RFP (d0 sample). Next, samples were stained with multiplexing antibodies using the BD Single-Cell Multiplexing Kit (BD biosciences), then pooled and stained with the following oligonucleotide-conjugated antibodies: anti‑CD25 (PC61), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-GITR (DTA-1), anti-CD4 (RM4-5), anti‑TCRb (H57-597), anti‑CD69 (H1.2F3), anti‑TIGIT (1G9), anti‑CD95 (Jo2), anti‑CD122 (TM‑1β), anti‑Tim-3 (5D12), anti‑CCR7 (4B12), anti‑CD103 (M290), anti‑CD279 (J43), anti‑CD274 (MIH5), anti‑CD233 (C9B7W), anti‑CD71 (C2), anti‑CD278 (7E7G9), anti‑ITGB7 (M293), anti‑CD137 (1AH2), anti‑CD40 (3/23), anti‑CD3e (145-2C) from BD Biosciences. Single-cell capture and cDNA library preparation was performed using the BD Rhapsody Single-cell analysis system (BD Biosciences), according to the manufacturer’s instructions, using a custom gene panel (591 genes). Sequencing was performed on an Illumina NextSeq500 instrument using a Mid‑Output kit v2.5 (150 cycles, paired-end). Sample demultiplexing, barcode processing, alignment, filtering, UMI counting were done using the standard BD Biosciences Rhapsody analysis pipeline on Seven Bridges (www.sevenbridges.com). Result: In this study, we showed that ex-Treg downregulate Treg core-specific genes immediatly upon adoptive transfer into a lymphopenic environment. Further, we identified a Treg population enriched for unstable Trg clones. Conclusion: Our study was able to identify a Treg subset enriched for unstable Treg with plastic potential. This Treg subset appears highly enriched for naïve peripheral-induced Treg.

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Comparison of gene expression in NOD versus B6 splenic B cell subsets.

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