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Switched CD21–/low B cells with an antigen-presenting phenotype in the infant thymus

2018; Elsevier BV; Volume: 143; Issue: 4 Linguagem: Inglês

10.1016/j.jaci.2018.11.019

1097-6825

Christina Lundqvist, Alessandro Camponeschi, Marcella Visentini, Esbjörn Telemo, Olov Ekwall, Inga‐Lill Mårtensson,

Immunodeficiency and Autoimmune Disorders

A unique memory B-cell population characterized by the lack of or low expression of CD21, termed CD21–/low, has been described in tonsils and peripheral blood (PB) of healthy individuals.1Thorarinsdottir K. Camponeschi A. Cavallini N. Grimsholm O. Jacobsson L. Gjertsson I. et al.CD21(−/low) B cells in human blood are memory cells.Clin Exp Immunol. 2016; 185: 252-262Crossref PubMed Scopus (50) Google Scholar, 2Ehrhardt G.R. Hsu J.T. Gartland L. Leu C.M. Zhang S. Davis R.S. et al.Expression of the immunoregulatory molecule FcRH4 defines a distinctive tissue-based population of memory B cells.J Exp Med. 2005; 202: 783-791Crossref PubMed Scopus (248) Google Scholar CD21–/low B cells are expanded in patients with chronic infections and autoimmune conditions,3Thorarinsdottir K. Camponeschi A. Gjertsson I. Mårtensson I.L. CD21−/low B cells: a snapshot of a unique B cell subset in health and disease.Scand J Immunol. 2015; 82: 254-261Crossref PubMed Scopus (56) Google Scholar but their role in health and disease is unclear. In the human and mouse thymus, B cells are present at very low percentages.4Yamano 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, 5Perera J. Zheng Z. Li S. Gudjonson H. Kalinina O. Benichou J.I.C. et al.Self-antigen-driven thymic B cell class switching promotes T cell central tolerance.Cell Rep. 2016; 17: 387-398Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 6Gies V. Guffroy A. Danion F. Billaud P. Keime C. Fauny J.D. et al.B cells differentiate in human thymus and express AIRE.J Allergy Clin Immunol. 2017; 139: 1049-1052.e12Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar These cells express tissue-restricted antigens orchestrated by the autoimmune regulator (AIRE) transcription factor and in mice, the thymic B cells are efficient antigen-presenting cells (APCs) and seem to be involved in thymocyte selection and lineage decision. Consistent with previous results,6Gies V. Guffroy A. Danion F. Billaud P. Keime C. Fauny J.D. et al.B cells differentiate in human thymus and express AIRE.J Allergy Clin Immunol. 2017; 139: 1049-1052.e12Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 7Isaacson 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 we found that B cells in the thymus of young children represented less than 1% of total lymphocytes (see Fig E1, A and B, in this article's Online Repository at www.jacionline.org) and were located exclusively in the medulla (Fig 1, A). Although their distribution and frequency were similar to those of the medullary thymic epithelial cells (Fig 1, B and C), the latter showed a 10 times larger surface area (Fig 1, D). The thymic B cells displayed a nuclear expression pattern of AIRE that was similar to that of medullary thymic epithelial cells, supported by previous findings showing that thymic B cells express AIRE6Gies V. Guffroy A. Danion F. Billaud P. Keime C. Fauny J.D. et al.B cells differentiate in human thymus and express AIRE.J Allergy Clin Immunol. 2017; 139: 1049-1052.e12Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar (Fig 1, E). B cells in early stages express CD10 and low levels of CD21. Unexpectedly, we found that half (43% ± 5%) of the thymic B cells were CD21–/low, and mainly negative for CD10. This mature phenotype is found in the adult PB, but in contrast the 10% (9.5% ± 3%) CD21–/low B cells in PB from children were mainly CD10+ (Fig 1, F and G). The proportions of early B-cell stages in the thymus were low, with less than 1% pro-B (CD19+CD10+CD34+CD24hiCD38hiIg–) and pre-B (CD19+CD10+CD34–CD24hiCD38hiIg–) and 15% immature B (CD19+CD10+) cells (Fig E1, C-E). To confirm and extend previous analyses,4Yamano 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, 5Perera J. Zheng Z. Li S. Gudjonson H. Kalinina O. Benichou J.I.C. et al.Self-antigen-driven thymic B cell class switching promotes T cell central tolerance.Cell Rep. 2016; 17: 387-398Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 6Gies V. Guffroy A. Danion F. Billaud P. Keime C. Fauny J.D. et al.B cells differentiate in human thymus and express AIRE.J Allergy Clin Immunol. 2017; 139: 1049-1052.e12Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar we investigated the presence of immunoglobulin-switched B cells in the thymus and found IgG- and IgA-expressing cells (Fig 1, H). Most CD21+ B cells were unswitched IgM+IgD+ (87% ± 10%), whereas a third of the CD21–/low cells were switched IgG+ (22% ± 11%) or IgA+ (9% ± 4%). Although similar to CD21–/low B cells in adult PB, this finding was in sharp contrast to PB from the same children in whom switched cells were nearly absent (Fig 1, I, and Fig E1, F and G). Moreover, a few of the switched thymic B cells were positive for IgE or IgG4 (Fig E1, H), which suggests that all immunoglobulin isotypes are present on the thymic B cells. To exclude the possibility of cell microchimerism as the origin of the switched B cells, we analyzed the sex chromosomes of the thymic B cells from 2 male children. All B cells contained 1 X and 1 Y chromosome (Fig 1, J; see Table E3 in this article's Online Repository at www.jacionline.org), disproving the hypothesis of maternal origin. Furthermore, except for a few classical memory (CD27+CD38–) and plasma (CD27highCD38high) cells, the thymic B cells were mainly negative for the CD27 memory marker (Fig E1, I and J). Thus, in contrast to infant PB, almost a quarter in the thymus are switched CD27–CD21–/low B cells. Both CD21+ and CD21–/low B cells from adult and child PB were small in cellular size and expressed low levels of the activation markers CD69, CD95, and CD86, except for a bimodal expression of CD95 in adult CD21–/low B cells (Fig 2, A). In contrast, thymic CD21+ and CD21–/low B cells showed a bimodal expression of all markers, with the highest percentage of positive cells among the latter. MHC class II (HLA-DR) and CD40 levels were similar in CD21+ and CD21–/low B cells independent of origin. Comparing CD21–/low B cells from thymus and adult PB confirmed a larger cellular size as well as elevated levels of CD69, CD95, CD86, and CD40 in the former, whereas those of HLA-DR were similar (Fig 2, B). These results suggest that thymic CD21–/low B cells are activated and may function as APCs. CD21–/low B cells have been found to express an unusual pattern of inhibitory and homing receptors, such as CD11c, T-bet, CXCR3, and FcRL4.2Ehrhardt G.R. Hsu J.T. Gartland L. Leu C.M. Zhang S. Davis R.S. et al.Expression of the immunoregulatory molecule FcRH4 defines a distinctive tissue-based population of memory B cells.J Exp Med. 2005; 202: 783-791Crossref PubMed Scopus (248) Google Scholar, 3Thorarinsdottir K. Camponeschi A. Gjertsson I. Mårtensson I.L. CD21−/low B cells: a snapshot of a unique B cell subset in health and disease.Scand J Immunol. 2015; 82: 254-261Crossref PubMed Scopus (56) Google Scholar, 8Rubtsova K. Rubtsov A.V. Cancro M.P. Marrack P. Age-associated B cells: a T-bet-dependent effector with roles in protective and pathogenic immunity.J Immunol. 2015; 195: 1933-1937Crossref PubMed Scopus (148) Google Scholar In adult PB, almost half of the CD21–/low B cells expressed CD11c, T-bet, and CXCR3, whereas only a small proportion expressed FcRL4. Of the thymic CD21–/low B cells, a quarter were positive for CD11c, CXCR3, and FcRL4, whereas less than 10% expressed T-bet (Fig 2, C; see Fig E2, A, in this article's Online Repository at www.jacionline.org). As a comparison, only low frequencies of the CD21+ B cells from PB and thymus expressed these markers. Finally, in support of a role in T-cell selection, the thymic CD21–/low B cells showed the highest levels of AIRE (Fig 2, D). To corroborate these findings, we sought to test the functionality of the thymic B cells as APCs in a thymic environment. We isolated and cocultured the CD3+ thymocytes with CD21+ or CD21–/low B cells from the same thymus, and after 24 and 72 hours assessed the expression of the activation marker CD25 on the thymocytes. As controls, we cultured part of the thymocytes without B cells in the absence or presence of phorbol 12-myristate 13-acetate. As expected, all thymocytes were positive for CD25 in the presence of phorbol 12-myristate 13-acetate, whereas in its absence around 20% were positive. After 72 hours, 30% of the thymocytes cocultured with either B-cell subset were CD25+, a percentage observed already after 24 hours but only in those cocultured with the CD21–/low B cells (Fig 2, E). This suggests that the CD21–/low B cells interact more rapidly with the thymocytes, possibly because of the higher expression of CD40 and even more so CD86 on the CD21–/low B cells than the CD21+. Under chronic immune stimulation, CD21–/low B cells exhibit reduced B-cell receptor (BCR)-induced phosphorylation of the extracellular signal-regulated kinase,9Visentini M. Marrapodi R. Conti V. Mitrevski M. Camponeschi A. Lazzeri C. et al.Dysregulated extracellular signal-regulated kinase signaling associated with impaired B-cell receptor endocytosis in patients with common variable immunodeficiency.J Allergy Clin Immunol. 2014; 134: 401-410Abstract Full Text Full Text PDF Scopus (17) Google Scholar which is important for proliferation and cell survival. We found that only half of the CD21–/low B cells from the thymus responded to BCR triggering, compared with those from adult PB of which almost all responded (Fig 2, F and G). The fact that half of the thymic CD21–/low B cells were unresponsive and expressed high levels of CD95 (Fig 2, A and B), an activation marker but also a death receptor, prompted us to investigate whether the CD21–/low cells were prone to undergo apoptosis. Fifty percent of thymic and 40% of adult PB CD21–/low B cells showed spontaneous apoptosis in vitro, as assessed by staining positive for active Caspases (CaspGLOW), whereas only a small percentage of CD21+ B cells were apoptosis prone (Fig 2, H, and Fig E2, B). The bimodal functional pattern of thymic CD21–/low B cells, with half of the cells responding efficiently to BCR stimulation together with the large size and the activated phenotype, indicated that at least part of these cells were proliferating (Fig 2, A and B). Consistent with this notion, 50% of the thymic CD21–/low B cells expressed high levels of the proliferation marker Ki67, compared with the markedly lower expression in adults and CD21+ B cells (Fig 2, I, and Fig E2, B). Collectively, these results show that around half of the thymic CD21−/low B cells are cycling and half are prone to undergo apoptosis. In this study, we demonstrate that half of the B cells residing in the infant thymus are mature CD21–/low cells that are large in size and express high levels of AIRE and typical activation markers. Despite being CD27–, almost half are switched B cells that, as expected, were nearly absent in the PB from the same infants. In mice, switching is probably facilitated by cognate interactions with thymocytes.5Perera J. Zheng Z. Li S. Gudjonson H. Kalinina O. Benichou J.I.C. et al.Self-antigen-driven thymic B cell class switching promotes T cell central tolerance.Cell Rep. 2016; 17: 387-398Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar The localization of the human CD21–/low B cells in the medulla suggests a role in T-cell selection, and their role as APCs is supported by their high expression of CD40, CD86, and HLA-DR, and by their ability to activate the thymocytes. These findings, together with our observations that half of the thymic CD21–/low B cells are apoptosis prone and half proliferating, suggest that this population is composed of APCs with a high turnover. Altogether, our data provide evidence that thymic B cells are heterogeneous, with subsets reminiscent of those in health and under conditions of chronic immune stimulation.1Thorarinsdottir K. Camponeschi A. Cavallini N. Grimsholm O. Jacobsson L. Gjertsson I. et al.CD21(−/low) B cells in human blood are memory cells.Clin Exp Immunol. 2016; 185: 252-262Crossref PubMed Scopus (50) Google Scholar, 3Thorarinsdottir K. Camponeschi A. Gjertsson I. Mårtensson I.L. CD21−/low B cells: a snapshot of a unique B cell subset in health and disease.Scand J Immunol. 2015; 82: 254-261Crossref PubMed Scopus (56) Google Scholar Further untangling of the functional properties of thymic B cells may lead to a better understanding of their role in central tolerance and autoimmune diseases. We thank the Clinical Genetics Department at the Sahlgrenska Hospital for performing the Fluorescence in situ Hybridization (FISH) analysis. We also acknowledge all help and assistance by the staff at Drottning Silvias Children's Hospital, and we thank the patients and their families for the contribution to the study. Thymic tissue samples and matched PB were obtained from children undergoing corrective cardiac surgery at Sahlgrenska University Hospital, Gothenburg, Sweden, where the thymus is surgically removed to gain access to the heart. Parents gave informed consent, and the study was approved by the Regional Ethical Board at the University of Gothenburg (no. 217-12, 2012-04-26). Demographic characteristics and the congenital heart defect of patients included in the study are presented in Table E1. The children did not have any known chronic infections or inflammatory conditions. Adult PB was obtained from healthy blood donors. No informed consent was needed because no personal information about the donors was recorded (Swedish law 2003: 460, paragraphs 4 and 13). The thymic tissue was collected immediately in cold PBS. Pieces of tissue were embedded in optimal cutting temperature compound (Histolab Products AB, Västra Frölunda, Sweden) for immunohistochemistry and snap frozen in isopentane precooled with liquid nitrogen. Thymic lymphocytes were extracted by injecting tissue with PBS, cutting the tissue into small pieces (1 mm3), and gently pressing against a 40-μm cell strainer. PBMCs were obtained after separation on Ficoll (GE Healthcare, Little Chalfont, UK), according to the manufacturer's protocol. Cells were filtered using a 40-μm filter and stained immediately or frozen for later analysis. Thymus tissue was enzymatically (DNase I, Worthington, Lakewood, NJ, and Liberase TH, Roche, Risch-Rotkreuz, Switzerland) and mechanically digested with a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). For ImageStream analysis, the single-cell suspension was density centrifuged with Percoll 1.07 g/mL (GE Healthcare Life Sciences, Chicago, Ill) to enrich thymic epithelial cells. Cells were stained at a concentration of (10-50) × 106 cells/mL in a volume of 100 μL. The antibodies and dilutions used are presented in Table E2. Mouse and rat sera were used to inhibit unspecific binding. For flow cytometry, the cells were acquired on a FACSVerse (BD Biosciences, San Diego, Calif), and data were analyzed using FlowJo software (TreeStar, Inc, Ashland, Ore). For ImageStream analysis, the cells were acquired and analyzed on an ImageStream X Mark II imaging flow cytometer (Amnis, Seattle, Wash). Thymic lymphocytes were negatively depleted with anti-CD3 Dynabeads (Invitrogen, Carlsbad, Calif) and sorted into 2 populations: CD3–CD19+CD21+, CD3–CD19+CD21–/low B cells. CD3+ thymocytes were sorted from nondepleted samples and cultured in RPMI 1640 supplemented with l-glutamine, nonessential amino acids, sodium pyruvate, penicillin, streptomycin, β-mercaptoethanol, and 10% FBS (complete medium), with and without phorbol 12-myristate 13-acetate 1 μg/mL (InvivoGen, Toulouse, France). Part of the CD3+ thymocytes were cultured without phorbol 12-myristate 13-acetate but with the addition of either CD21+ or CD21–/low B cells. To support thymocytes survival, IL-2 20 ng/mL (R&D Systems, Minneapolis, Minn) and IL-7 0.2 ng/mL (Peprotech, Rocky Hill, NJ) were added to the cultures. Thymocytes CD25 expression was analyzed after 24 and 72 hours. The cells were acquired with a FACSlyric (BD Biosciences) and data were analyzed using FlowJo software. Cells were sorted on a SH800Z cell sorter (Sony Biotechnology, San Jose, Calif) with postsort purities of greater than or equal to 95% (Fig E3, A and B). For this assay, freshly isolated cells from PB and thymus were used. The phosphorylation of extracellular signal-regulated kinase was studied using the BD PhosFlow Protocol for Human PBMCs (Becton-Dickinson Biosciences, Franklin Lakes, NJ). After isolation, cells were resuspended in 100 μL of complete medium at a concentration of 2 to 10 × 106 cells and then split into 2 vials and left to equilibrate at 37°C for at least 20 minutes. An equal volume of prewarmed complete medium, with and without (unstimulated control) 20 μg/mL of F(ab')2 anti-human IgM/G/A (Jackson Immunoresearch Laboratories, Bar Harbor, Me), was then added, and then the cells were incubated for 10 minutes at 37°C. The samples were then fixed by the addition of an equal volume of prewarmed BD Cytofix Fixation Buffer for 10 minutes at 37°C, washed twice in Phosflow Perm/Wash Buffer I, split into 2 vials, and stained for 60 minutes at room temperature protected from light either with anti–phosphorylated extracellular signal-regulated kinase 1/2 Alexa Fluor 647 or with a mouse IgG Alexa Fluor 647 as control. Surface stainings with other mAbs were performed as requested by the experimental design. The samples were washed and resuspended in Phosflow Perm/Wash Buffer I and finally acquired with a flow cytometer. Freshly isolated cells from PB and thymus were resuspended in complete medium supplemented with penicillin and streptomycin and then cultured in 96-well plates at 2 × 105 cells/well. The cells were then harvested after 24 hours and the apoptotic cells detected by incubation with zVAD-FMK-FITC (CaspGLOW; eBioscience, Waltham, Mass) in RPMI 1640 for 1 hour at 37°C, according to manufacturer's protocol. Surface stainings with other mAbs were performed as requested by the experimental design. The cells were then washed and analyzed by flow cytometer. Optimal cutting temperature compound–embedded thymus pieces were cut to 7-μm sections, air dried, and fixed with acetone for 5 minutes. Sections were rehydrated in PBS and blocked with Protein block (Dako, Santa Clara, Calif). The tissues were stained for 1 hour at 4°C with primary antibody. Antibodies and dilutions are listed in Table E2. The sections were incubated for 30 minutes at 4°C with secondary antibodies and Hoechst 34580 (Thermo Fisher Scientific, Waltham, Mass). If the secondary antibodies were biotinylated, the slides were incubated at 4°C for 30 minutes with Streptavidin Alexa Fluor 488 (Thermo Fisher Scientific). Sections were mounted with ProLong Gold Antifade Mountant (Thermo Fisher Scientific) and acquired using an LSM700 confocal microscope (Carl Zeiss AG, Oberkochen, Germany). Image analysis was performed with ImageJ software (Rasband WS, ImageJ, US National Institutes of Health, Bethesda, Md) measuring area with threshold set to 35, 255. Immunoglobulin switched and unswitched cells were sorted from 2 thymuses from 4-day old males with a Sony SH800 cell sorter and cytospinned to a glass slide. The slides were placed in hypotone solution (0.3% NaCl) in deionized water with increased concentration of fixation solution added (99.5% ethanol and ice cold acetic acid, 3:1) stepwise to dehydrate cells. FISH was performed using probes for X chromosome centromere and Y chromosome centromere (Vyvis CEP X DXZ1 Spectrum Green, Y DYZ3 Spectrum Orange, Abott Molecular, Inc, Chicago, Ill). All switched cells were counted and compared with unswitched cells as presented in Table E3. Outliers were removed by using the Robust regression and Outlier removal (ROUT) method, with Q set to 5%. Shapiro-Wilk test was used to evaluate normality. Data were analyzed by 1-way ANOVA followed by uncorrected Fisher Least Significant Difference (LSD) multiple comparison test, using Graph-Pad Prism version 7 (La Jolla, Calif). Statistical significance was set as P < .05. *P < .05, **P < .01, ***P < .001, and ****P < .0001.Fig E2A and B, Samples from adult PB and child thymus. A, Representative histograms for expression of CD11c, T-bet, CXCR3, and FcRL4 on CD21–/low B cells. B, Representative histograms of CaspGLOW and Ki67 in CD21+ and CD21–/low B cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3A and B, Gating strategy and purity control for the in vitro stimulation assay. The gating and purity for sorting (Fig E3, A) CD21+ and CD21–/low thymic B cells and (Fig E3, B) CD3+ thymocytes. FSC-A, Forward scatter-area; SSC-A, side scatter-area.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Demographic characteristics and cause for cardiac surgery of included patientsPatient no.Congenital heart defectSexAge at time of surgery (d)Analyzed results presented in figure12E1E2E31TGA, VSDM3C, EE-HB2APWM4F, G, I, JA-CA, B, F, G, I, JA, B3TGA, VSDM4D, F, G, I, JA-CA, B, F, G, I, JA, B4TGA, VSDM4CF-H5HLHSF5C-E6VSDM8E7IAA, TAF8C-E8DORVM11C-E9PA, VSD, DORVM12C10VSDF63C-E11VS, PSM123C, D, I12VSDM152C-E13VSDF169F-IA-D, IA, B, F, G, I, JA, B14ASDF170F-H15VSDM177C-E16FallotF195C17VSDF268C-E18FallotM376H19ASD, PDA, VSDF520F, G, IA-D, IA, B, F, G, I, JA, B20AS, PS, WSM726F, G, IA-CA, B, F, G, I, JA, B21SVDF1923EA, BAPW, Aortopulmonary window; AS, aorta stenosis; ASD, atrial septal defect; DORV, double outlet right ventricle; Fallot, Tetralogy of Fallot; F, female; HLHS, hypoplastic left heart syndrome; IAA, interrupted aortic arch; M, male; PA, pulmonary atresia; PDA, patent ductus arteriosus; PS, pulmonary stenosis; SVD, sinus venosus atrial defect; TA, truncus arteriosus; TGA, transposition of the great arteries; VSD, ventricular septal defect; WS, Williams syndrome. Open table in a new tab Table E2Antibodies used in the studyAntibodyCloneCompanyDilutionFlow cytometry CD3-APC-H7SK7BD50 CD10-APCHI10aBD20 CD11c-FITCB-ly6BD20 CD19-BV510HIB19Biolegend40 CD21-PerCP-Cy5.5Bu32Biolegend50 CD24-PE-Cy7ML5BD40 CD25-APC2A3BD50 CD27-PEL128BD40 CD34-PerCP-Cy5.58G12BD10 CD38-BV421HIT2BD20 CD40-PE5C3BD20 CD69-FITCL78BD10 CD86-APC2331 (FUN-1)BD20 CD95-bioDX2BD10 CD193(CXCR3)-PE1C6BD20 EpCAM-PE9C4Biolegend25 ERK1/2-AF48820ABD20 FcRL4-PE413D12BioLegend20 HLA-DR-bioL243BD50 IgD-PEIA6-2BD40 IgD-FITCIA6-2BD50 IgM-FITCCat nr F0058DAKO200 IgG-PEG18-145BD25 IgA-FITCCat nr F0057DAKO150 T-bet-AF4884B10BioLegend20Confocal microscopy CD19SP110Invitrogen200 CD19HIB19BD100 Cytokeratin 5Cat nr PRB-160PBioLegend100 IgA6E2C1Dako20 IgECIA-E-7.12Dako100 IgGH10015Medac200 IgG4HP6025SouthernBiotech100 IgM-bioG20-127BD100 Anti-rabbit-AF488A11034Life Technologies200 Anti-mouse-AF555A21422Life Technologies400 Anti-rabbit-bioCat nr E0432Dako400 Anti-mouse bioBA-2000Vector Laboratories200ImageStream X CD19-PEHIB19BD10 EpCAM-PE9C4Biolegend25 AIRE-bioTM-724eBioscience100 Rat IgG2a-bioeBR2aeBioscience100 Open table in a new tab Table E3Chromosomal FISH analysis shows that the switched B cells in the thymus are not derived from the motherB cell stateXXXYTotal no. of counted cellsSwitched04343Unswitched0270270Switched03434Unswitched0500500 Open table in a new tab APW, Aortopulmonary window; AS, aorta stenosis; ASD, atrial septal defect; DORV, double outlet right ventricle; Fallot, Tetralogy of Fallot; F, female; HLHS, hypoplastic left heart syndrome; IAA, interrupted aortic arch; M, male; PA, pulmonary atresia; PDA, patent ductus arteriosus; PS, pulmonary stenosis; SVD, sinus venosus atrial defect; TA, truncus arteriosus; TGA, transposition of the great arteries; VSD, ventricular septal defect; WS, Williams syndrome.