IgH from sequences. aspercent one million usage wascalculated segment orientation with respect to the in D C57BL/6J mice informat methods sorted and subjected to mouse surface IgH CD19, B220, and gene IgM. One expression to two analysis million B as cells and (CD19 young descr IL Spleen cells were prepared from a Supplemental Figure 1 SUPPLEMENTAL INFORMATION % VH sement usage % VH sement usage % VH sement usage 10.0 10.0 10.0 0.0 0.5 1.0 0.0 0.5 1.0 2.0 4.0 6.0 8.0 2.0 4.0 6.0 8.0 0.0 0.5 1.0 2.0 4.0 6.0 8.0 5’ . VH1-85 VH1-85 VH1-85 ion system VH1-84 VH1-84 VH1-84 VH1-82 VH1-82 VH1-82 We used C57BL/6 V VH1-81 VH1-81 VH1-81 VH1-80 VH1-80 VH1-80 VH1-78 VH1-78 VH1-78 VH1-77 VH1-77 VH1-77 Adult wildtype Adult Young wildtype Young Young IL-7Tg Young Adult IL-7 Adult Young wildtype Young VH1-76 VH1-76 VH1-76 wildtype Adult VHgenesdistal to DH-JH VH1-75 VH1-75 VH1-75 VH1-74 VH1-74 VH1-74 - VH1-72 VH1-72 VH1-72 7Tg (n=5) mice and stained with a cocktail of fluorescent antibodies specific for VH1-71 VH1-71 VH1-71 VH8-12 VH8-12 VH8-12 VH1-69 VH1-69 VH1-69 VH1-67 VH1-67 VH1-67 VH1-66 VH1-66 VH1-66 VH8-11 VH8-11 VH8-11 VH1-64 VH1-64 VH1-64 VH1-63 VH1-63 -/- VH1-63 VH1-62-3 VH1-62-3 VH1-62-3 ® VH1-62-2 VH1-62-2 VH1-62-2 VH1-62-1 VH1-62-1 VH1-62-1 were VH 1-61 VH 1-61 VH 1-61 (I VH1-59 VH1-59 VH1-59 VH1-58 VH1-58 VH1-58 VH8-8 VH8-8 VH8-8 MGT VH1-56 VH1-56 VH1-56 VH1-55 VH1-55 VH1-55 . VH1-54 VH1-54 VH1-54 VH8-6 VH8-6 VH8-6 V VH1-53 VH1-53 VH1-53 VH1-52 VH1-52 VH1-52 VH1-50 VH1-50 VH1-50 arranged arranged from H VH8-5 VH8-5 VH8-5 VH1-49 VH1-49 VH1-49 ® VH8-4 VH8-4 VH8-4 VH1-47 VH1-47 VH1-47 gene segmentusage inwildtype, IL VH1-43 VH1-43 VH1-43 ; VH1-42 VH1-42 VH1-42 VH1-39 VH1-39 VH1-39 http://www.imgt.org VH1-37 VH1-37 VH1-37 VH1-36 VH1-36 VH1-36 VH1-34 H VH1-34 VH1-34 VH1-31 VH1-31 VH1-31 VH1-26 VH1-26 VH1-26 VH1-22 VH1-22 VH1-22 dult gene nomenclature from the I VH1-20 VH1-20 VH1-20 H VH1-19 VH1-19 VH1-19 VH1-18 VH1-18 VH1-18 - VH1-17-1 VH1-17-1 VH1-17-1 VH1-15 VH1-15 VH1-15 J VH1-14 VH1-14 VH1-14 H VH1-12 VH1-12 VH1-12 VH1-11 VH1-11 VH1-11 wildtype wildtype (n=4); young wildtype (n=7); adult IL VH1-9 VH1-9 VH1-9 VH15-2 VH15-2 VH15-2 VH1-7 VH1-7 VH1-7 gene region of the IgH locus). The frequency of V VH10-3 VH10-3 VH10-3 VH1-5 VH1-5 VH1-5 VH1-4 VH1-4 VH1-4 VH10-1 VH10-1 VH10-1 5’ to 3’ in the IgH locus (i.e., distal to proximal VH6-7 VH6-7 VH6-7 VH6-6 VH6-6 VH6-6 VH6-5 VH6-5 VH6-5 VH6-4 VH6-4 VH6-4 VHgenesproximal to DH-JH VH6-3 VH6-3 VH6-3 VH12-3 VH12-3 VH12-3 VH13-2 VH13-2 VH13-2 VH3-8 VH3-8 VH3-8 VH9-4 VH9-4 VH9-4 VH3-6 VH3-6 VH3-6 VH13-1 VH13-1 VH13-1 VH3-5 VH3-5 VH3-5 VH3-4 VH3-4 VH3-4 VH7-4 VH7-4 VH7-4 Dickinsonet al., Suppl.Fig.1 VH3-3 VH3-3 VH3-3 ). A VH14-4 VH14-4 VH14-4 VH7-3 VH7-3 VH7-3 VH9-3 VH9-3 VH9-3 VH9-2 VH9-2 VH9-2 VH9-1 VH9-1 VH9-1 VH16-1 VH16-1 VH16-1 VH14-3 VH14-3 VH14-3 ll VH11-2 VH11-2 VH11-2 VH3-2 VH3-2 VH3-2 VH4-2 VH4-2 VH4-2 of VH14-2 VH14-2 VH14-2 VH11-1 VH11-1 VH11-1 VH3-1 VH3-1 VH3-1 VH4-1 VH4-1 VH4-1 VH14-1 VH14-1 VH14-1 the VH7-2 VH7-2 VH7-2 VH7-1 VH7-1 VH7-1 VH2-9 VH2-9 VH2-9 VH5-17 VH5-17 VH5-17 VH5-16 VH5-16 VH5-16 VH5-15 VH5-15 VH5-15 113 VH2-7 VH2-7 VH2-7 VH2-6-8 VH2-6-8 VH2-6-8 VH2-9-1 VH2-9-1 VH2-9-1 VH5-12-4 VH5-12-4 VH5-12-4 nternational nternational ImMunoGeneTics VH5-9-1 VH5-9-1 VH5-9-1 - VH2-6 VH2-6 VH2-6 7 VH5-12 VH5-12 VH5-12 VH2-5 VH2-5 VH2-5 + functional functional V - VH5-9 VH5-9 VH5-9 / VH2-4 VH2-4 VH2-4 - VH5-6 VH5-6 VH5-6 B220 VH2-3 VH2-3 VH2-3 and IL VH5-4 VH5-4 VH5-4 3’ VH2-2 VH2-2 VH2-2 VH5-2 VH5-2 VH5-2 ibed in materials and + IgM 7Tg mice. 7Tg mice. H +/ gene - ) were FACS - s 7 egments - / - (n=7) H Supplemental Figure 2. Characterization of phenol-chloroform extracted B1355 dextran. To eliminate TLR ligand contamination, whole peritoneal cavity cells of naïve C57BL/6J mice were incubated either with various concentrations of crude bacterial B1355 preparation or with B1355 obtained from three rounds of Phenol-extraction as previously described (37). As a positive control, LPS (100ng/ml, TLR4 agonist) Dickinsonwas used. et.After al., 18 Suppl-24 hours, Fig. t he2 culture supernatants were subjected to IL-6 ELISA as a readout to measure the degree of TLR ligand contamination. The data is representative of two independent experiments. A. C57BL/6J 100 T 34±4100 B1a 110±17100 B1b 77±12100 B2 71±9 80 1704±57 80 94±9 80 65±8 80 62±6 60 60 60 60 40 40 40 40 % of Max 20 20 20 20 0 0 0 0 2 3 4 5 0 102 103 104 105 0 10 10 10 10 0 102 103 104 105 0 102 103 104 105 B. BALB/cJ 100 T 54±4100 B1a 75±22100 B1b 42±22100 B2 43±17 80 1124±229 80 75±19 80 41±21 80 41±16 60 60 60 60 40 40 40 40 % of Max 20 20 20 20 0 0 0 0 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 IL-7Rα Supplemental Figure 3. Mature B cells do not express IL-7Rα. Peritoneal cavity cells of adult naïve C57BL/6J (n=5) or BALB/cJ (n=5) mice were stained with antibodies specific for surface IgM, CD19, Mac1, CD5, and IL7Rα, and analyzed by flow cytometry to assess IL7Rα surface expression onDickinson T cells (CD19 et. al.,-, IgM Suppl.-, CD5 Fig.high ),3 B1a (CD19+, IgM+, CD5+, Mac1+), B1b (CD19+, IgM+,CD5-, Mac1+), and B2 (CD19+, IgM+, CD5-, Mac1-) subsets in the peritoneal cavity(plots not shown). Histograms depicting IL-7Rα expression (black line) in each cell subset compared to isotype control (gray lines) are provided. Mean fluorescent intensity ± standard deviation of IL-7Rα-specific antibody binding is provided in each plot (black number) as compared to unstained control (gray number). Supplemental Figure 4. α1,3-dextran-specific polysaccharide-specific B cell expansion does not require IL-7. Expansion of α1,3-dextran-specific (EB3-7+Dickinson) peritoneal B et.cell al.,subsets Suppl in MK7 Fig.-immunized 4 -/- VHJ558 Tg x IL-7 mice was measured by flow cytometry as described in the Figure 7 legend and absolute cell counts were calculated. Statistical significance was determined using Mann- Whitney test with p values shown in the plot. .