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, Quantitative PCR was performed using SYBR ® qPCR green and premix Ex Taq (TAKARA)
, post-switch IgG1, membrane and secreted IgM transcripts were quantified after four days of B-cell stimulation with LPS or LPS+IL4. RNA from B-lymphocytes was extracted using GenElute Mammalian RNA miniprep Kit (Sigma Aldrich). cDNA was synthesized using the High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific). Quantitative PCR primers used to study heavy chain expression for the secretory
, All transcripts were normalized to Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) on LPS stimulated sorted CD43 neg spleen B-cells. Briefly, 15×10 6 cells were cross-linked at room temperature for 15 min in 15 mL PBS, 1% formaldehyde. The reaction was quenched with 2, IgM (mIgM qPCR For, mIgM qPCR SEC Rev), membrane IgM (mIgM qPCR for, mIgM qPCR MB Rev)
, chromatin was precleared by rotating for 2h at 4 °C with 50 mL of 50% protein A/G slurry (0.2 mg / mL sheared salmon sperm DNA, 0.5 mg/ mL BSA, 01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, and 167 mM NaCl)
, Merck) or with anti-acetyl-histone H3(Lys27) (Abcam) polyclonal antibodies. Immune complexes were precipitated by the addition of protein A/G. Cross-linking was reversed by overnight incubation (70 °C) in TE References Allen, Cell equivalents (1×10 6 ) were saved as input, and the remaining cell equivalents were incubated overnight with anti-acetyl-histone H3(Lys9), vol.16, pp.3932-3942, 2008.
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, Annexe, vol.8
, Locus Suicide Recombination of the functionally rearranged IgH allele actively occurs in B-cells from inflamed human lymphoid tissues
, Michel Cogné Manuscrit soumis à PLOS Genetics Ce papier montre la détection d'un nouveau type de remaniment qui touche le locus IgH
, expérimentation ayant conduit à la rédaction de cet article (preparation des PBMC à partir du sang total, tri cellulaire, stimulation in-vitro des cellules B humaines, préparation des librairies pour la NGS)
, Locus Suicide Recombination actively occurs on the functionally rearranged IgH allele in B-cells from inflamed human lymphoid tissues
, JeanClaude Aldigier, vol.1, p.4
, 3 Institut Imagine INSERM U1163, vol.87000
, AID promotes affinity maturation of Ig variable regions and class switch recombination (CSR) in mature B lymphocytes. In the IgH locus, these processes are under control by the 3' regulatory region (3'RR) super-enhancer, a region demonstrated in the mouse to be both transcribed and itself targeted by AID-mediated recombination. Alternatively to CSR, IgH deletions joining Sµ to "like-switch" DNA repeats that flank the 3' super-enhancer can thus accomplish so-called "locus suicide recombination" (LSR) in mouse B-cells. We now show that AID-mediated LSR also actively occurs in humans, and provides an activationinduced cell death pathway in multiple conditions of B-cell activation. LSR deletions either focus on the functional IgH allele or are bi-allelic, since they can only be detected when they are ongoing and their signature vanishes from fully differentiated plasma cells or from "resting" blood memory B-cells, but readily reappears when such memory Bcells are re-stimulated in vitro, Abstract: B-cell activation yields abundant cell death in parallel to clonal amplification and remodeling of immunoglobulin (Ig) genes by activation-induced deaminase (AID)
, Manuscript Click here to access/download;Manuscript;dalloul LSR manuscript
, Blood from healthy volunteers was collected, after obtaining written informed consent, from the CHU Dupuytren Hospital, ANSM: 2012-A00630-430) or from the Etablissement Français du Sang
, Blood from septic patients was collected from the Hematology department, CHU Dupuytren. The study was accepted by the Ethics Committee of CHU Dupuytren (n° 119-2013-19). The trial was registered under
, All were operative pieces. Bone marrow aspirates obtained from patients undergoing cardiac surgery. Subjects were recruited under institutional review board approval and informed consent according to the Declaration of Helsinki. Samples from AID-/-patients (3 PBMC samples and 1 sample of sorted tonsil B-cells) were provided by Pr Anne Durandy, ClinicalTrials.gov: NCT01995448. Tonsils were obtained from patients undergoing tonsillectomy performed in CHU de Rennes, while adenoids were obtained from CHU Dupuytren
, Human PBMC were obtained by density gradient centrifugation and human B-cells were negatively sorted from PBMC using EasySep Human B-cell Isolation Kit (StemCell) according to the manufacturer's instructions. Purified B-lymphocytes were seeded at 1 × 10 6 cells/ml in IMDM medium (Lonza) supplemented with 20% fetal bovine serum (Deutscher), 1% penicillin/streptomycin (Gibco) and stimulated for 4 days with 100 ng/ml human recombinant CD40L (Enzo Life Sciences) alone or with 50 ng/ml recombinant human IL-4 (Peprotech) or 2 µg/ml Gardiquimod (InvivoGen) or 2.5 µg/ml CpG oligodeoxynucleotide 2006 (InvivoGen) or 100 ng/ml IL-21 (R&D Systems) or 100ng/ml INF? (R&D Systems) or 50 ng/ml Pam3CSK4 (InvivoGen) or 0
, Sorting of memory, transitional, and naive B-cells, and generation of in vitro activated plasmablasts from cultured naive B-cells
, PBMCs from healthy volunteers were obtained after density centrifugation. Naive B-cells (NBCs) were purified by negative selection using magnetic cell separation (Naive B-cell Isolation Kit II
, Purified NBCs were labeled with 1 ?M CFSE (Thermofisher) at 37°C for 10 min and washed in complete medium consisting of RPMI 1640 supplemented with 10% FCS and antibiotics (all from Gibco, Thermofisher), Miltenyi Biotec), following the manufacturer's instructions, with the AutoMACS deplete-sensitive program. Purity of isolated CD19+CD27? NBCs was routinely >99%
, At day-6, cells were separated by cell sorting (FACSAria cell sorter, BD Biosciences) into undifferentiated CFSE hi CD38 lo (bystander lymphocytes, 100 ng/ml recombinant human soluble CD40L (NCI), 2 ?g/ml CpG oligodeoxynucleotide 2006 (Miltenyi Biotec), and 50 U/ml recombinant IL-2 (SARL Pharmaxie)
, Miltenyi Biotec), we also purified transitional, naive and memory Bcells which were sorted as CD27+CD38 lo CD24 lo , CD27-CD38 lo CD24 lo and CD27-CD38 hi CD24 hi subsets, respectively. Isolated cells were stimulated 4 days with different combinations of 100 ng/ml CD40L, After B-cell isolation (B-cell isolation kit II
, CD38 hi /CD138+ mature plasma cells were purified from bone marrow aspirates. RNAseq on activated human B-cells Human naive B-cells were purified and activated in vitro as described, Actively differentiating plasmablasts from tonsils were purified as previously described [21]as CD19+IgD-CD10-CD38 hi cells, 2017.
, Quality of sequencing data was monitored by FastQC. Residual adapters from sequencing were trimmed using Cutadapt 1.0. Potential PCR duplicates were removed using SAMtools 1.3. Reads were then aligned CSR-seq experiments DNA from B-cells was extracted using GenElute Mammalian Genomic DNA Miniprep Kit (Sigma), S?/S? junctions were amplified in triplicate by nested PCR with 200 ng DNA (Herculase II Fusion DNA Polymerase, Agilent
, Genomics) using the following consensus primers which amplify all four classes of human IgG junctions: PCR1: Sµ1a 5'-CCAGGTAGTGGAGGGTGGTA-3' and IgGa consensus reverse 5'-GGTCACCACGCTGCTGAG-3, Sµ1b forward 5'-CAGGGAACTGGGGTATCAAG-3' and IgGb consensus reverse: 5', vol.2
, S?/3'RR junctions were amplified under the same conditions as for Sµ/S? junctions using different primers located at different positions within the 3' RR. The forward primer located in the E? region (E?H1) was used with a reverse primer located downstream of Hs4 (3'FarHhs4 reverse) or with a primer located in Hs1.2 (Hs1.2 reverse1) or in Hs3 (Hs3 reverse 1)
, ) for a survey of direct repeats in window range set over 20bp, with a minimal identity threshold set at 90% (Fig. 1A). Five to six human LS regions stand in each of the human IgH 3'RR superenhancers. These LS regions are each 0.5 to 2 kb long, and are interspersed with core enhancer elements, which they do not overlap. Human 3'RR LS sequences additionally show 35-50% G-richness on one or the other DNA strand, and analysis using the G4, Architectures of human 3'RR1 and 3'RR2 elements were analyzed for the presence of direct and inverted repeats (Fig1)
, This is in contrast with IgH constant genes (see for example C? and the preceding S? region on Fig 1B), which are predominantly transcribed in the sense orientation. LSR occurs at multiple sites within the whole extent of both human 3'RR As previously described for mouse LSR, we identified human LSR junctions by nested PCR in DNA from B-cell samples [4]. The specificity of the read-out for these PCR assays was improved by replacing the final hybridization step of PCR products, initially done with specific probes[4]), with high-throughput sequencing adapted from our recently described CSR evaluation algorithm, CSReport [22]. Sequence determination thus precisely mapped the breakpoint sites. CSReport mapped LSR junctions linking Sµ to either the human 3'RR1 downstream of C?1 or 3'RR2 downstream of C?2. Although such an "LSR-seq" DNA amplification method, When checking RNA-seq data from activated human B-cells, we noticed that 3'RR transcription occurred in both the sense and antisense orientation in reference to IgH constant genes, and that, quantitatively, it revealed equivalent amounts of transcripts corresponding to either orientation (Fig. 1B)
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