B cells differentiate in human thymus and express AIRE
2016; Elsevier BV; Volume: 139; Issue: 3 Linguagem: Inglês
10.1016/j.jaci.2016.09.044
ISSN1097-6825
AutoresVincent Gies, Aurélien Guffroy, François Danion, Philippe Billaud, Céline Keime, Jean‐Daniel Fauny, Sandrine Susini, Anne Soley, Thierry Martin, Jean‐Louis Pasquali, Frédéric Gros, Isabelle André‐Schmutz, Pauline Soulas‐Sprauel, Anne‐Sophie Korganow,
Tópico(s)Adrenal Hormones and Disorders
ResumoIt has been known for more than a decade that human thymus contains a minor contingent of B cells.1Isaacson P.G. Norton A.J. Addis B.J. The human thymus contains a novel population of B lymphocytes.Lancet. 1987; 2: 1488-1491Abstract PubMed Scopus (201) Google Scholar Some authors suggested that these B cells reflect the homing of peripheral B cells, whereas others wondered whether they could act as antigen-presenting cells (APCs). Recently, 2 important articles approached the role of thymic B cells in murine models, using transfer experiments and deficient lines (eg, autoimmune regulator [Aire] knock-out animals). They demonstrated that murine thymic B cells are potent APCs to autoreactive T cells2Perera J. Meng L. Meng F. Huang H. Autoreactive thymic B cells are efficient antigen-presenting cells of cognate self-antigens for T cell negative selection.Proc Natl Acad Sci U S A. 2013; 110: 17011-17016Crossref PubMed Scopus (72) Google Scholar and that they can harbor different self tissue-restricted antigens (TRAs) induced by Aire expression.3Yamano T. Nedjic J. Hinterberger M. Steinert M. Koser S. Pinto S. et al.Thymic B cells are licensed to present self antigens for central T cell tolerance induction.Immunity. 2015; 42: 1048-1061Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar But only little is known about the fate and function of human B cells in thymus with mostly immunochemistry data.1Isaacson P.G. Norton A.J. Addis B.J. The human thymus contains a novel population of B lymphocytes.Lancet. 1987; 2: 1488-1491Abstract PubMed Scopus (201) Google Scholar Considering this critical issue, we explored the phenotype of B cells from thymuses of young donors undergoing heart surgery (see Table E1 and the Methods section in this article's Online Repository at www.jacionline.org). CD19+ B cells were detected in the medulla and throughout the cortex (Fig 1, A), whereas IgM+ B cells were restricted to the medulla and the cortico-medullary junction (Fig 1, B and C). B cells constituted 0.92% ± 0.23% of thymic lymphocytes (see Fig E1, A, in this article's Online Repository at www.jacionline.org). All developmental stages (see Fig E2 in this article's Online Repository at www.jacionline.org) were represented, from pro-B cells to differentiated B cells, and mature naive B cells were largely predominant (48.53% ± 3.15%; Fig 1, D, and see Fig E1, B). Both switched CD27− and CD27+ memory B cells were present (Fig 1, D). Further analysis of the surface immunoglobulin (sIg) expression showed that they were mainly IgG with less IgA expression. Plasmablast subset was enriched in sIgM with a low level of sIgG or sIgA (Fig 1, E). CD27+ switched B cells are a result of antigen recognition in germinal centers or in mucosal tissues.4Berkowska M.A. Driessen G.J. Bikos V. Grosserichter-Wagener C. Stamatopoulos K. Cerutti A. et al.Human memory B cells originate from three distinct germinal center-dependent and -independent maturation pathways.Blood. 2011; 118: 2150-2158Crossref PubMed Scopus (270) Google Scholar CD27− switched B cells are low mutated and have been suspected to be a reservoir of autoreactive B cells (IgG+) or involved in the maintenance of intestinal homeostasis (IgA+).5Berkowska M.A. Schickel J.N. Grosserichter-Wagener C. Ridder de D. Ng Y.S. Dongen van J.J.M. et al.Circulating human CD27−IgA+ memory B cells recognize bacteria with polyreactive Igs.J Immunol. 2015; 195: 1417-1426Crossref PubMed Scopus (62) Google Scholar Thus, it is interesting to note that all these B-cell subsets are present in the thymus in significant proportions. Obviously, thymic B-cell phenotype was different from bone marrow (BM) picture, with a clear predominance of precursors in the BM and of mature naive B cells in the thymus. In addition, switched memory B-cell numbers were very low in the BM, but represented around 15% of thymic B cells. We were able to compare B-cell subpopulations in thymus, BM, and peripheral blood (PB) from a single donor (patient 1). The repartition of the different B-cell subpopulations in the BM and blood for our patient was consistent with literature data. The thymic B-cell picture was also distinct from PB, as there were more precursors and mature naive B cells in the thymus (see Fig E3, A, B, and C in this article's Online Repository at www.jacionline.org). These findings do not support the hypothesis of a peripheral B-cells homing. In thymuses, immature B cells proliferated more than mature subsets (70.96% ± 4.00% vs 46.94% ± 3.05%) (Fig 2, A). Both the proliferation of B-cell progenitors in the thymus and their higher frequency compared with PB argue for in situ B-cell lymphopoiesis. We hypothesized that thymic B cells could harbor an organ-specific function. Mature thymic B cells (CD19+CD10−) expressed higher levels of major histocompatibility class II (MHCII) molecules compared with immature B cells (CD19+CD10+) (Fig 2, B). CD40 expression was detected on 87.6% of mature thymic B cells, whereas being barely detectable on immature B cells (Fig 2, C). CD86 was not expressed on immature B cells but upregulated on 28.9% of CD19+CD10− B cells (Fig 2, D). In further distinction from B cells in other organs, in the same individual, this CD86 upregulation was observed only in mature thymic B cells, but neither in immature nor in mature B cells from BM or PB (see Fig E3, D). In mice, the thymic B-cell licensing is driven through CD40-CD40L signaling and interaction with CD4+ T cells.3Yamano T. Nedjic J. Hinterberger M. Steinert M. Koser S. Pinto S. et al.Thymic B cells are licensed to present self antigens for central T cell tolerance induction.Immunity. 2015; 42: 1048-1061Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 6Fujihara C. Williams J.A. Watanabe M. Jeon H. Sharrow S.O. Hodes R.J. T cell-B cell thymic cross-talk: maintenance and function of thymic B cells requires cognate CD40-CD40 ligand interaction.J Immunol. 2014; 193: 5534-5544Crossref PubMed Scopus (26) Google Scholar Here, mature thymic B cells expressed MHCII molecules and CD86 concomitantly with CD40 expression, which could reflect similar mechanisms of activation. In addition, 18.8% of thymic B cells expressed CD5, with an expression restricted to CD19+CD10− (Fig 2, E), similar to BM B cells, but at a lower level than in PB (see Fig E3, E). In mice, treatment of B2b B cell with anti-IgM and CD40 stimulation induced CD5 expression at low level.7Wortis H.H. Teutsch M. Higer M. Zheng J. Parker D.C. B-cell activation by crosslinking of surface IgM or ligation of CD40 involves alternative signal pathways and results in different B-cell phenotypes.Proc Natl Acad Sci U S A. 1995; 92: 3348-3352Crossref PubMed Scopus (136) Google Scholar A role of CD40-CD40L interaction in the activation of human thymic B cells could explain the CD5 expression on some of them. The fact that (1) human thymic B cells are localized mainly in the medulla and at the cortico-medullary junction and (2) mature thymic B cells strongly express MHCII molecules and CD86 suggest that they could act as APCs. These data prompted us to explore whether they could present auto-antigens to T cells, as recently demonstrated in mice.2Perera J. Meng L. Meng F. Huang H. Autoreactive thymic B cells are efficient antigen-presenting cells of cognate self-antigens for T cell negative selection.Proc Natl Acad Sci U S A. 2013; 110: 17011-17016Crossref PubMed Scopus (72) Google Scholar, 3Yamano T. Nedjic J. Hinterberger M. Steinert M. Koser S. Pinto S. et al.Thymic B cells are licensed to present self antigens for central T cell tolerance induction.Immunity. 2015; 42: 1048-1061Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar Using immunofluorescence analysis, we detected autoimmune regulator (AIRE) protein with typical punctuate nuclear staining in thymic epithelial cells (TECs) (27.05% ± 2.07%) and also, yet with less numerous nuclear dots, in thymic B cells (CD19+: 5.58% ± 1.59%, IgM+: 6.69% ± 0.83%) (Fig 2, F, and see Fig E4 in this article's Online Repository at www.jacionline.org). In addition, the most predominant thymic B-cell subset (CD27−IgM+) expressed 80-fold more AIRE mRNA than did thymocytes (Fig 2, G). If relevant, AIRE expression in thymic B cells may repress or induce the expression of other genes, as the induction of TRAs expression in medullary thymic epithelial cells (mTECs).8Klein L. Kyewski B. Allen P.M. Hogquist K.A. Positive and negative selection of the T cell repertoire: what thymocytes see (and don't see).Nat Rev Immunol. 2014; 14: 377-391Crossref PubMed Scopus (787) Google Scholar Little is known about the nature of TRAs expressed by murine thymic B cells, and there is only a very limited overlap of TRAs between mTECs and extrathymic Aire-expressing cells,3Yamano T. Nedjic J. Hinterberger M. Steinert M. Koser S. Pinto S. et al.Thymic B cells are licensed to present self antigens for central T cell tolerance induction.Immunity. 2015; 42: 1048-1061Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar suggesting that thymic B cells may regulate the expression of a unique set of TRAs. Using the thymic B cells' TRAs (see the Methods section in this article's Online Repository) defined by Yamano et al,3Yamano T. Nedjic J. Hinterberger M. Steinert M. Koser S. Pinto S. et al.Thymic B cells are licensed to present self antigens for central T cell tolerance induction.Immunity. 2015; 42: 1048-1061Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar we detected a significant expression of CDH17 and LAMP3—representative TRAs of the intestinal tract and the lung, respectively—concomitantly with a lower expression of KLRB1, an Aire-repressed gene (Fig 2, H). We were not able to detect GRIK2 or SCEL expression. We performed RNAseq analysis on sorted CD19+IgM+CD27− thymic B cells: this analysis confirmed our quantitative RT-PCR results including AIRE overexpression (fold change, 4.82; P value = 6.53 × 10−5). Moreover, thymic B cells expressed more TRA genes than did peripheral B cells (168 TRA genes upregulated vs 48 repressed). Each TRA is expressed by only 1% to 3% of mTECs at a given time.8Klein L. Kyewski B. Allen P.M. Hogquist K.A. Positive and negative selection of the T cell repertoire: what thymocytes see (and don't see).Nat Rev Immunol. 2014; 14: 377-391Crossref PubMed Scopus (787) Google Scholar In addition, because only 5% of thymic B cells are AIRE positive, the number of TRA genes expressed by AIRE-positive thymic B cells may be even higher. Some of these TRAs have been associated with autoimmune diseases, for example, LAD1 (linear IgA dermatosis), SCG2 (autoimmune hypophysitis), ICA1 (type 1 diabetes), ACHE and AKAP12 (myasthenia gravis), or METRNL (autoimmune hepatitis) (Fig 2, I, and see Table E2 in this article's Online Repository at www.jacionline.org). Interestingly, some molecules of the LAMP family, being associated with the lysosomal membrane, play an essential role in autophagy.9Tanaka Y. Guhde G. Suter A. Eskelinen E.L. Hartmann D. Lüllmann-Rauch R. et al.Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice.Nature. 2000; 406: 902-906Crossref PubMed Scopus (726) Google Scholar This process is involved in providing endogenous self-peptides for MHCII presentation and plays a role in thymocytes selection provided by TECs.8Klein L. Kyewski B. Allen P.M. Hogquist K.A. Positive and negative selection of the T cell repertoire: what thymocytes see (and don't see).Nat Rev Immunol. 2014; 14: 377-391Crossref PubMed Scopus (787) Google Scholar We found equivalent expression of BECN1—initiation of the autophagosome formation—between thymic CD19+CD27−IgM+ and PB CD19+CD27−IgM+, while ATG5 expression—elongation of the autophagosomal membrane—was reproducibly lower in thymus. Similar results were found in RNAseq (not shown) and we cannot conclude at this stage about the involvement of autophagic activity in thymocyte education by B cells. In this study, we confirm the presence of both immature and mature B cells in the thymuses of young donors. Although some B-cell progenitors are present and proliferate likely in the cortex, most B cells in the thymus are mature B cells with an APC phenotype, located to the medulla. The analysis of expression of AIRE and TRA genes support the hypothesis of their involvement in negative selection and suggest a new layer of complexity in the process of human central tolerance. Indeed, it will be of great interest to go further, but it is a challenging issue considering the low percentages of B cells in this organ. Primary immunodeficiencies affecting B cells include autoimmune manifestations in their clinical presentation. The mechanisms of this autoimmunity remain unclear and can be organ specific. In view of our results, it is reasonable to assume that in some cases central T-cell tolerance mediated by B cells could be affected. We thank C. Herouard-Molina (IGBMC, Illkirch, France) for expert technical assistance for the whole transcriptome sequencing. Sequencing was performed by the GenomEast platform, a member of the "France Génomique" consortium (ANR-10-INBS-0009). We thank E. Meffre (Department of Immunobiology, Yale University School of Medicine, New Haven, Conn) and M. Van Zelm (Department of Immunology and Pathology, Central Clinical School, Monash University, Melbourne, Australia) for helpful discussion. The study was conducted in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from the parents or legal wards of the patients, and the study protocol was approved by the Clinical Research Ethics Committee of Strasbourg's University Hospital. Anticoagulated venous blood from 4 healthy donors was collected and was subjected to density gradient centrifugation with LSM1077 (GE Healthcare Life Sciences, Velizy-Villacoublay, France) to get PBMCs and remove dead cells. Thymus glands were collected after thymectomy of infants undergoing corrective heart surgery in the Department of Pediatric Surgery (Strasbourg's University Hospital). Patients affected by diseases or receiving treatments that may alter the immunological system were excluded, namely, primary immunodeficiencies, active bacterial or viral infections, chronic viral infections, chemotherapy, or neoplasms. None of them was affected with Down syndrome or other evident dysmorphic features. Their ages ranged from 1 week to 26 weeks. The main characteristics of patients are described in Table E1. Pieces of thymuses from patient 1 were embedded in O.C.T. (Tissue-Tek, Sakura, Torrance, Calif) and stored at −80°C for immunofluorescence microscopy experiments. Thymus cellular suspensions were obtained by grinding the organ on a 40-μm cell strainer (BD Falcon, Franklin Lakes, NJ). Thymic and BM cells were subjected to density gradient centrifugation with LSM1077 to get mononuclear cell suspensions and remove dead cells. Thymus was cut into pieces of 3 mm2 and washed 4 times in RPMI 1640 medium (Invitrogen, Carlsbad, Calif). Thymus pieces were digested in medium A (RPMI 1640, collagenase 0.12% [from Clostridium histoliticum, Sigma-Aldrich], DNAse I 0.1% [Sigma-Aldrich]) for 15 minutes in a warm bath at 37°C. The supernatant was stored at 4°C and the pieces of thymuses were once again incubated with medium A. This step was renewed 2 times. The 3 supernatants were pooled in the same tube and centrifugated at 1500 rpm for 5 minutes. The pellet was resuspended and incubated, with magnetic stirring, in medium B (RPMI 1640, collagenase 0.12% [from Clostridium histoliticum], dispase 1.2 mg/mL [StemCell Technologies, Grenoble, France], DNAse I 0.1%) for 30 minutes in a warm bath at 37° and then centrifugated at 1500 rpm for 5 minutes. The pellet was resuspended with MACS buffer (0.5% BSA, EDTA 2 mmol/L pH 8, PBS pH 7.2), incubated for 10 minutes at 4°C, and filtered through 70-μm filters. The cells were then sorted to eliminate CD45+ cells (Miltenyi Biotec, Bergisch Gladbach, Germany). The magnetic separation from autoMACS separator (Miltenyi Biotec) was used. Sections of thymuses (6 μm) were cut from frozen samples, air dried, and fixed in acetone. Then, before each step, slides were washed 3 times using PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4), unless otherwise specified by protocol. After rehydratation in PBS, endogenous peroxidases were quenched with 0.5% H2O2 in PBS for 20 minutes. Endogenous biotins were blocked using Endogenous Biotin-Blocking Kit (Molecular Probes, Eugene, Ore) following the manufacturer's protocol. Sections were blocked, using Cyanine 3 tyramide signal amplification kit (PerkinElmer, Woodbridge, Ontario, Canada), and labeled with biotin anti-human CD19 antibody (clone HIB19) from BD Biosciences (San Jose, Calif) or biotin anti-human IgM antibody (clone TIBI 82) from Sera-lab (West Sussex, United Kingdom) following the manufacturer protocol. Nuclei were stained with 4′-6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Sigma-Aldrich, St Louis, Mo). Thymus sections were incubated with 4% paraformaldehyde in PBS for 30 minutes. Coverslips were mounted in DAKO (Sigma-Aldrich) and sealed with nail polish. Images were acquired using a spinning disk confocal microscope (Zeiss Axio Observer Z1 Yokogawa spinning disk system, objective: Zeiss Plan-Apochromat 20X/0.8). No bleed-through was detected between channels. Images were stitched to get an overview of the whole tissue and were analyzed using National Institutes of Health Image J software.E15Schneider C.A. Rasband W.S. Eliceiri K.W. NIH Image to ImageJ: 25 years of image analysis.Nat Methods. 2012; 9: 671-675Crossref PubMed Scopus (34981) Google Scholar In thymic sections, cortico-medullary borders were distinguished by the cellular density visualized by DAPI staining. Mononuclear thymic cell suspensions were collected in PBS, after a density gradient centrifugation with LSM1077 to remove dead cells. Unfixed cells (3 × 105) were allowed to adhere for 45 minutes at 37°C in multiwell culture slides (BD Falcon) coated with poly-l-lysine (Sigma-Aldrich). Then, before each step, slides were washed 3 times using PBS-1% BSA (Sigma-Aldrich), unless otherwise specified by protocol. The cells were blocked with PBS-1% BSA for 30 minutes at +4°C. Except for wells containing TECs, the cells were labeled with biotin anti-human CD19 antibody (clone HIB19) from BD Biosciences or biotin anti-human IgM antibody (clone TIBI 82) from Sera-lab in BSA-1% BSA for 45 minutes at room temperature followed by an incubation with streptavidine AlexaFluor 647 (Thermo Fisher Scientific, Illkirch, France) in PBS-1% BSA for 30 minutes at +4°C. Cells were then fixed with 2% paraformaldehyde in PBS for 30 minutes and permeabilized with 0.05%Triton X-100 (GE Healthcare Life Sciences) in PBS-1% BSA for 30 minutes. Samples were incubated with anti-AIRE antibody (clone D-17, SantaCruz, Santa Cruz, Calif) or goat IgG (polyclonal, SouthernBiotech, Birmingham, Ala), as isotype control, in PBS-1% BSA-0.05% Triton-X at 4°C overnight. After 3 washes with PBS-1% BSA-0.05% Triton-X-100, cells were incubated with AlexaFluor 488 donkey antigoat IgG (polyclonal, Thermo Fisher Scientific) in PBS-1% BSA- 0.05% Triton-X for 1 hour at room temperature. Nuclei were then stained with DAPI (Sigma-Aldrich). Coverslips were mounted in DAKO (Sigma-Aldrich) and sealed with nail polish. Images were acquired using either a spinning disk confocal microscope (Zeiss Axio Observer Z1 Yokogawa spinning disk system, objective: Zeiss Plan-Apochromat 20X/0.8 and Plan-Apochromat 100X/1.4) or a laser scanning microscope (Zeiss Axio Observer Z1, LSM 780 confocal head, objective: Zeiss Plan-Apochromat 63X/1.4). No bleed-through was detected between channels. Images were stitched to get an overview and were analyzed using National Institutes of Health Image J software.E15Schneider C.A. Rasband W.S. Eliceiri K.W. NIH Image to ImageJ: 25 years of image analysis.Nat Methods. 2012; 9: 671-675Crossref PubMed Scopus (34981) Google Scholar Cell viability was assessed by incubation of cells with Fixable Viability Dye eFluor 780 (eBioscience, San Diego, Calif) following the manufacturer's protocol. BM and thymic cells were washed twice in PBE (PBS, 0.5% BSA, 2 mM EDTA) and resuspended in PBE. Classical surface staining was performed in the dark for 15 minutes at 4°C. Intracellular staining was performed, for Ki67 staining, using Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent Kit (eBioscience) following the manufacturer's protocol. For PB, BM, and thymic B cells of the same individual (patient 1), the staining was performed using OptiLyse C Lysis Solution (Beckman Coulter, Fullerton, Calif) according to manufacturer protocol. The cells were washed twice in PBE buffer and analyzed on a Gallios flow cytometer (Beckman Coulter). Living cells were identified by forward scatter and side scatter gating. Isotypic controls were performed. Data analysis was performed using Kaluza software (Beckman Coulter) with "logicle" compensation.E16Tung J.W. Heydari K. Tirouvanziam R. Sahaf B. Parks D.R. Herzenberg L.A. et al.Modern flow cytometry: a practical approach.Clin Lab Med. 2007; 27 (v): 453-468Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar For the analysis of protein expression level, the geometric mean fluorescence intensity was used. And considering the low numbers of B cells in the thymus, we roughly separated immature and mature B cells on the basis of their CD10 expression. A comprehensive list of antibodies used for this experiment includes the following: CD19 APC-AlexaFluor700 (clone J3-119) purchased from Beckman Coulter; Ki67 FITC (clone B56), CD3 PC-Cy7 or APC (clone UCHT1), CD5 FITC (clone UCHT2), CD10 PerCP-Cy5.5 (clone HI10a), CD24 FITC or PE (clone ML5), CD27 PE or APC (clone M-T271), CD34 PC7 (clone 581), CD38 PE-CF594 or PC-Cy7 (clone HIT2), CD40 FITC (clone 5C3), CD86 FITC (clone 2331 [FUN-1]), CD138 AlexaFluor647 (clone MI15), HLA DR/DQ/DP FITC (clone Tu39), IgD PerCP-Cy5.5 (clone IA6-2), IgD FITC (clone IA6-2), IgM FITC (G20-127), and IgM PE-CF594 (G20-127) purchased from BD Biosciences; CD5-APC-AlexaFluor750 (clone BL1a) purchased from Beckman Coulter; and IgG PE-Vio770 (clone IS11-3B2.2.3), IgA PerCP-Vio700 (clone IS11-8E10), and IgE PE (clone MB10-5C4) purchased from Miltenyi Biotec. As previously described, a protein coding gene was defined as coding for a TRA if its mRNA expression was above 5 times the median (the median expression over all tissues) in fewer than 5 tissues.E17Pinto S. Michel C. Schmidt-Glenewinkel H. Harder N. Rohr K. Wild S. et al.Overlapping gene coexpression patterns in human medullary thymic epithelial cells generate self-antigen diversity.Proc Natl Acad Sci U S A. 2013; 110: E3497-E3505Crossref PubMed Scopus (53) Google Scholar Each of the following group was considered as a single tissue: (1) all nervous central tissues, (2) colon and small intestine, and (3) all testis tissues. Fetal tissues were excluded from the analysis. In addition, a gene was excluded from the TRAs list if its expression was above 5 times the median in the B-cell lineage (CD19+ B cells or B lymphoblast). Identification of TRAs was performed using the Human U133A/GNF1H Gene Atlas public databaseE18Su A.I. Wiltshire T. Batalov S. Lapp H. Ching K.A. Block D. et al.A gene atlas of the mouse and human protein-encoding transcriptomes.Proc Natl Acad Sci U S A. 2004; 101: 6062-6067Crossref PubMed Scopus (2848) Google Scholar at www.biogps.org.E19Wu C. Orozco C. Boyer J. Leglise M. Goodale J. Batalov S. et al.BioGPS: an extensible and customizable portal for querying and organizing gene annotation resources.Genome Biol. 2009; 10: R130Crossref PubMed Scopus (1093) Google Scholar, E20Wu C. MacLeod I. Su A.I. BioGPS and MyGene.info: organizing online, gene-centric information.Nucleic Acids Res. 2013; 41: D561-D565Crossref PubMed Scopus (228) Google Scholar According to the list of genes that are induced/repressed in murine B cells, published by Yamano et al,E21Yamano T. Nedjic J. Hinterberger M. Steinert M. Koser S. Pinto S. et al.Thymic B cells are licensed to present self antigens for central T cell tolerance induction.Immunity. 2015; 42: 1048-1061Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar the following human genes were selected as AIRE-related genes for quantitative real-time RT-PCR analysis: SCEL, GRIK2, LAMP3, and CDH17 for AIRE-induced genes and KLRB1 for AIRE-repressed genes. CD3+CD19− and CD3−CD19+CD27−IgM+ populations were sorted (FACSAria II, BD Biosciences) from thymic mononuclear cell suspensions of infants undergoing corrective heart surgery, and CD3−CD19+CD27−IgM+ were also sorted from PBMCs of healthy donors. Cell viability was assessed with DAPI (Sigma-Aldrich). Purity was controlled after sort (see Fig E4). RNA was isolated using RNeasy Mini kit (Qiagen, Valencia, Calif) following the manufacturer's protocol, including a DNase digest. Reverse transcription was done using High Capacity cDNA Reverse transcription Kit (Applied Biosystems, Foster City, Calif) following the manufacturer's protocol, and was performed on a T100 Thermal cycler (BioRad, Hercules, Calif). Preamplification of 10 ng of cDNA was performed, with TaqMan PreAmp Master Mix Kit (Applied Biosystems) on a T100 Thermal cycler (Biorad). Quantitative real-time PCR reactions were performed on a StepOnePlus realtime thermal cycler (Applied Biosystems) using the TaqMan Gene Expression Master Mix (Applied Biosystems) following the manufacturer's protocol. Assays were plated in triplicate. Thermal cycling conditions were set at 2 minutes at 50°C and 10 minutes at 95°C, then 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. Relative expression levels were calculated with the comparative Ct method using the mean of the Ct between GAPDH and C1orf43 for normalization.E22Eisenberg E. Levanon E.Y. Human housekeeping genes, revisited.Trends Genet. 2013; 29: 569-574Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar CD3+ thymic cells or CD19+CD27−IgM+ from PBMCs were used as calibrator samples. The following list of TaqMan probes (Applied Biosystems) was used: GAPDH (Hs02758991_g1), C1orf43 (Hs00367486_m1), AIRE (Hs00230829_m1), KLRB1 (Hs00174469_m1), CDH17 (Hs00900408_m1), GRIK2 (Hs00222637_m1), LAMP3 (Hs00180880_m1), SCEL (Hs01557103_m1), BECN1 (Hs00186838_m1), ATG5 (Hs00355492_m1). CD3−CD19+CD27−IgM+ populations were sorted (FACSAria II, BD Biosciences) from thymic mononuclear cell suspensions of infants undergoing corrective heart surgery (n = 4) and from PBMCs of healthy donors (n = 4). Cell viability was assessed with DAPI (Sigma-Aldrich). Purity was controlled after sort (see Fig E4). RNA was isolated using RNeasy Mini kit (Qiagen) following the manufacturer's protocol, including a DNase digest. Sample quality was assessed using Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, Calif). Transcriptome libraries preparation and sequencing were performed in the sequencing facility of the IGBMC (Strasbourg, France) using Illumina sequencing platform (Illumina, San Diego, Calif). Image analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14. Reads were mapped onto the hg38 assembly of the human genome using Tophat v2.0.14E23Kim D. Pertea G. Trapnell C. Pimentel H. Kelley R. Salzberg S.L. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions.Genome Biol. 2013; 14: R36Crossref PubMed Scopus (8748) Google Scholar and the bowtie v2.1.0 aligner. Gene expression was quantified using HTSeq v0.6.1E24Anders S. Pyl P.T. Huber W. HTSeq–a Python framework to work with high-throughput sequencing data.Bioinforma Oxf Engl. 2015; 31: 166-169Crossref PubMed Scopus (10941) Google Scholar and gene annotations from Ensembl release 83. Read counts were normalized across libraries with the method proposed by Anders and Huber.E25Anders S. Huber W. Differential expression analysis for sequence count data.Genome Biol. 2010; 11: R106Crossref PubMed Scopus (10310) Google Scholar Comparison between thymic B cells and peripheral B-cell gene expression was performed using the method proposed by Love et alE26Love M.I. Huber W. Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol. 2014; 15: 550Crossref PubMed Scopus (32370) Google Scholar implemented in the DESeq2 Bioconductor library (v1.6.3). Adjustment for multiple testing was performed with the Benjamini and HochbergE27Benjamini Y. Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing.J R Stat Soc Series B Stat Methodol. 1995; 57: 289-300Google Scholar method. In human thymic B cells, 912 genes were induced (168 were TRAs) and 364 genes were repressed (48 were TRAs) (read normalized and divided by gene length in kb >50; fold-change >4; P value adjusted for multiple testing <.05). Statistical comparison between groups was carried out using nonparametric 2-tailed Mann-Whitney test. A P value of less than .05 was considered significant. All data were presented as mean ± SEM. Tests were applied using Prism 5.0 (GraphPad Software Inc, San Diego, Calif).Fig E2B-cell ontogeny and markers used for B-cell subsets gating strategy.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3A, Flow cytometry analysis of CD19+ B cells in thymus, BM, and PB of P1. B and C, Frequency of immature B cells (Fig E3, B) and mature B cells (Fig E3, C) in P1. D and E, The panels represent, respectively, B cells' MFI of CD86 (Fig E3, D) and CD5 (Fig E3, E) in PB (red), BM (blue), and thymus (black) for P1. MFI, Mean fluorescence intensity; P1, patien
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