Switched CD21–/low B cells with an antigen-presenting phenotype in the infant thymus Christina Lundqvist, MSc, Alessandro Camponeschi, PhD, Marcella Visentini,

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Switched CD21–/low B cells with an antigen-presenting phenotype in the infant thymus Christina Lundqvist, MSc, Alessandro Camponeschi, PhD, Marcella Visentini, MD, PhD, Esbjörn Telemo, PhD, Olov Ekwall, MD, PhD, Inga-Lill Mårtensson, PhD Journal of Allergy and Clinical Immunology Volume 143, Issue 4, Pages 1616-1620.e7 (April 2019) DOI: 10.1016/j.jaci.2018.11.019 Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 A and B, CD19 (green) and Keratin 5 (red) expression in thymic medulla (m) and cortex (c). Hoechst (gray), scale bars = 200 μm. C, Percentage of B cells and TECs among total thymic cells (n = 3). D, Area covered by Keratin 5 and CD19. Scale bar = 100 μm. E, AIRE (red) expression in mTECs and B cells. CD19/EpCAM (yellow), Hoechst (blue). Scale bar = 7 μm. F and G, Percentage of CD21–/low among total B cells in adult PB (n = 4), and child thymus and PB (n = 5). H, Switched B cells in thymic medulla. IgG/IgA (red), Hoechst (gray). Scale bar = 50 μm. I, Percentage of immunoglobulin isotype-positive among total CD19+ B cells in child thymus and PB (n = 5). J, FISH of male thymic switched and unswitched B cells. X chromosome (green), Y chromosome (red), DAPI (blue). (****P < .0001). DAPI, 4'-6-Diamidino-2-phenylindole, dihydrochloride; mTEC, medullary thymic epithelial cell; TEC, thymic epithelial cell. Journal of Allergy and Clinical Immunology 2019 143, 1616-1620.e7DOI: (10.1016/j.jaci.2018.11.019) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 A and B, Activation and APC marker-expression on CD21+ and CD21–/low B cells from thymus and PB. B and C and F-I, Indicated B cells from adult PB and child thymus. C, Percentage of CD11c, T-bet, CXCR3, and FcRL4 (n ≥ 3). D, Percentage of CD25 on CD3+ thymocytes with no B cells, cocultured with CD21+ and CD21–/low B cells, and stimulated with PMA after 24 and 72 hours. E, AIRE MFI fold-change in indicated cells relative to T cells. F, pERK expression in unstimulated and BCR-stimulated B cells. G-I, Percentage of pERK, CaspGLOW, and Ki67-positive B cells (n = 3). FSC, Forward scatter; MFI, mean fluorescence intensity; pERK, phosphorylated extracellular signal-regulated kinase; PMA, phorbol 12-myristate 13-acetate. *P < .05, **P < .01, ***P < .001, ****P < .0001. Journal of Allergy and Clinical Immunology 2019 143, 1616-1620.e7DOI: (10.1016/j.jaci.2018.11.019) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 A and B, Representative fluorescence activated cell sorting (FACS) plots of CD19 vs CD3, and percentages of CD19+ B cells in child thymus and PB (n = 5), and adult PB (n = 4). C, Representative FACS plots of CD19 vs CD34. D and E, Percentage of pro-B, pre-B, and immature B cells in the thymic CD19+ B-cell population (n = 5). F-J, B cells from child thymus and PB (n = 5), and adult PB (n = 4) as indicated. F and G, Representative FACS plots of immunoglobulin isotype-expression. H, IgE/IgG4 (red) expression in thymic medulla. Hoechst (gray) and scale bar = 50 μm. I, Percentage of CD27–, CD27+, and CD27hiCD38hi (Plasma cells, PCs). J, Percentage of CD27– and CD27+ B cells, and PCs. (**P < .01, ****P < .0001). Journal of Allergy and Clinical Immunology 2019 143, 1616-1620.e7DOI: (10.1016/j.jaci.2018.11.019) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 A 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. Journal of Allergy and Clinical Immunology 2019 143, 1616-1620.e7DOI: (10.1016/j.jaci.2018.11.019) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 A 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. Journal of Allergy and Clinical Immunology 2019 143, 1616-1620.e7DOI: (10.1016/j.jaci.2018.11.019) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions