1. Surrogate-Light-Chain Silencing Is Not Critical for the Limitation of Pre-B Cell Expansion but Is for the Termination of Constitutive Signaling 
Pieter Fokko van Loo, Gemma M. Dingjan, Alex Maas, Rudi W. Hendriks 
Immunity 
Volume 27, Issue 3, Pages (September 2007)
DOI: /j.immuni
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Structure and Expression of CD19-λ5 and CD19-Vpreb Transgenes
(A) Map of the mouse Igll1-Vpreb1 locus and the CD19-λ5 and CD19-Vpreb Tg constructs. Exons of Igll1 (encoding λ5) and Vpreb1 genes (pointed out by numbers) are indicated.
(B) Cytoplasmic (c) λ5 (Igll1) and Vpreb expression in the indicated B lineage cell populations from WT and λ5#2;Vpreb#1 Tg mice. Pro-B (CD19+cμ-), large pre-B (CD19+cμ+CD2-), small pre-B (CD19+cκ-CD2+), immature B (CD19+cκ+) fractions from BM, and mature B (CD19+cκ+) fractions from spleen were gated and analyzed for intracellular expression of SLC components.
(C) Flow cytometric analysis of mebrane λ5 and Vpreb expression on CD19+cκ- pre-B cells from WT and λ5#2;Vpreb#1 mice.
(D) Examination of membrane expression of λ5 and Vpreb on gated CD19+ splenic B cells from WT mice and SLC Tg mice. Results are displayed as histogram overlays. Coexpression of κLC and SLC components is shown at the bottom as dot plots. The percentages of cells within the indicated quadrants are given. Plots are representative for four mice of each genotype.
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2 Enforced SLC Expression Affects B Cell Development
(A) Flow cytometric analysis of total lymphoid fractions in BM, spleen, mesenteric lymph nodes (LN), and peritoneal cavity (PC) of indicated mice. Expression profiles of the indicated markers are shown as dot plots and percentages of cells within the indicated gates are given. Data shown are representative of 9–11 animals per genotype.
(B) Absolute numbers of total B cell (B220+CD19+), pro-;pre-B cell (B220+Igμ-), immature B cell (B220lowIgμ+), and mature B cell (B220highIgμ+) fractions per hind leg. Bars represent average values and the standard error of the mean (SEM) (×106) of 9–11 animals per genotype. Total numbers of BM B cells were significantly reduced in λ5#2 and Vpreb#1 (p < 0.02) and in λ5#2;Vpreb#1 (p < ) mice, when compared with WT mice. Likewise, immature B cell fractions were reduced in Vpreb#1 (p < 0.002) and λ5#2;Vpreb#1 (p < 0.001) mice and mature B cells in all three groups of SLC mice (p < ).
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3 Pre-B Cell Differentiation of SLC Tg Mice
(A) Flow cytometric analysis of B220 and cytoplasmic κ on CD19+ gated B cells in BM (top panel). The B220+cκ− pro-;pre-B cell fractions were gated and analyzed for cytoplasmic μ, CD2, and CD25 expression. Data are displayed as dot plots, and numbers indicate the percentages of cells within the quadrants and are representative of more than five animals per group.
(B) Proliferation of developing B-lineage cells in vivo in WT and λ5#2;Vpreb#1 mice. Mice were intra peritoneal (i.p.) injected with a single dose BrdU, and after 4 hr, percentages of BrdU+ cells in the BM B lineage cell fractions were determined by flow cytometry. Analyzed B cell population were pro-;large pre-B cell (CD19+CD2−cκ−), small pre-B cell (CD19+CD2+cκ−), and immature B cells (CD19+cμ+cκlow/+). The average and SEM of three animals per group are shown.
(C) Flow cytometric analysis of intracellular λ5 and Vpreb expression in the large pre-B cell (CD19+cμ+CD2−) and small pre-B cell (CD19+cκ−CD2+) fractions and of membrane λ5 and Vpreb expression on CD19+cκ− pro-;pre-B cells from WT and mice heterozygote and homozygote for λ5#2;Vpreb#1 Tg. Results are displayed as histograms with overlays of the indicated Tg mice. Plots are representative for four to five mice of each genotype.
(D) In vivo proliferation of pre-B cells in heterozygous or homozygous λ5#2;Vpreb#1 Tg mice. Four hours after BrdU injection, the percentages of BrdU+ cells in pre-B cell fractions were determined by flow cytometry. The average and SEM of three animals per group are shown.
(E) Proliferative responses of total BM cells to IL-7, measured by [3H]-thymidine incorporation after 4 days of culture. Bars represent average counts per minute (cpm) values and SEM of 5–11 animals per group.
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4
Arrested B Cell Development at the Immature B Cell Stage in SLC Tg Mice
(A) Analysis of surface BCR expression on immature B cells from WT and SLC Tg mice by flow cytometry. B220lowcμ+ pre-;immature B cell fractions were gated and examined for cytoplasmic (cκ) and surface (Igκ) expression (top panel). Cytoplasmic κ+ cells were gated and analyzed for surface Igκ. Results are displayed as histogram overlays on top of profiles of WT mice (lower panel). Data shown are representative of more than six animals per group.
(B) Kinetics of the developmental progression of B lineage cells in the BM of WT and λ5#2;Vpre#1 mice. Mice were injected with a single dose of BrdU. After 24, 48, or 72 hr, the percentages of BrdU+ cells in BM B lineage cell fractions were determined by flow cytometry. Pro-;large pre-B cells were CD19+CD2−cκ−, small pre-B cells were CD19+CD2+cκ−, and CD19+CD2+ immature B cells were divided into cκlow and cκhigh fractions. Error bars represent SEM values of n = 3 animals per group, and asterisks indicate statistically significant differences (p < 0.05).
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions

6. Figure 5
SLC Expression Induces Slp65-Dependent BCR Internalization and Secondary Ig LC Rearrangement in Immature B Cells
(A) Analysis of surface BCR expression on immature B cells from the indicated mouse groups by flow cytometry. Cytoplasmic and surface κ expression was determined in the B220lowcμ+ pre-;immature B cell fractions (top panel). Subsequently, cytoplasmic κ+ cells were gated and analyzed for surface Igκ. Results are displayed as histogram overlays on top of profiles of WT mice (lower panel). Data shown are representative of more than four animals per group.
(B) Flow cytometric analysis of splenic lymphoid populations of 3-83μδ Tg and low-copy-number λ5#1;Vpreb#3;3-83μδ Tg mice. Numbers indicate percentages of Igμ+Igδ+ B cells within the total lymphoid fractions (upper panel). CD19+ cells were gated and analyzed for surface expression of 54-1 idiotype 3-83μδ BCR and Igλ (lower panel). Percentages of cells within the quadrants are given, and the data shown are representative of more than three animals per group.
(C) Flow cytometric analysis of specified surface markers in BM and spleen from the indicated four groups of mice. The top panel shows B220,Igμ profiles of gated total lymphoid BM fractions. Total B cell fractions (CD19+B220+) and immature B cell fraction (CD19+B220low) were gated and analyzed for Igμ,Igδ or Igκ,Igλ profiles, respectively. Total splenic lymphoid compartments were examined for CD19 and Igμ expression. Data shown are representative of more than five animals per group.
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions

7. Figure 6 Constitutive Activation of SLC Tg B Cells in the Spleen
(A) Analysis of splenic B cells of SLC Tg mice by flow cytometry. CD19+ B cells were gated and analyzed for the indicated markers; results are displayed as dot plots or histogram overlays on top of profiles of WT mice. The given numbers represent the percentages of gated cells within total CD19+ B cell population. Data shown are representative of move than six animals per group.
(B) Absolute numbers of CD5− and CD5+ B cells per spleen (average and SEM of more than six animals per group) in the four groups of mice. Asterisks show statistically significant differences (p < 0.01) within the CD5+ population.
(C) Examination of protein tyrosine phosphorylation in extracts of untreated and anti-IgM treated WT and λ5#2;Vpreb#1 purified splenic B cells, analyzed by immunoblot analysis with a phosphotyrosine (pY)-specific antibody. The membrane was reblotted with anti-Erk1,2 as a loading control.
(D and E) Kinetics of the constitutive (D) and ligand-mediated (E) Igμ internalization by WT total splenic B cells and λ5#2;Vpreb#1 CD5− and CD5+ B cells determined with anti-Igμ Fab' and anti-Igμ F(ab')2, respectively. Constitutive Igμ internalization by λ5#2;Vpreb#1 CD5− B cells was significantly (p < 0.025) increased from T = 3 min onward, when compared with WT B cells. Data shown are average values ± SEM of six WT and five λ5#2;Vpreb#1 mice.
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions

8. Figure 7
Increased IgM+ Plasma Cell Formation and Altered In Vivo Ig Subclass Production in SLC Tg Mice
(A) Immunohistochemical analysis of IgM (brown, for IgM+ B cells and plasma cells) and MOMA1 (blue, staining metallophillic marginal zone macrophages) in spleen of the indicated mice.
(B) Splenic cytoplasmic μ+ cells were analyzed for the cytoplasmic μHC and surface CD138. Numbers indicate the percentages of cμ+CD138- B cells and cμhighC138+ plasma cells within cμ+ splenic cell population. Data shown are representative of more than six animals per group.
(C) Percentages of splenic IgM+ plasma cells (gated as cμhighCD138+) within in the total splenic cμ+ cell population in WT and SLC Tg mice. Each symbol indicates an individual animal; asterisks indicate significant differences (p < 0.05).
(D) Numbers of IgM-producing cells per 105 cells in spleen and BM, determined by ELISPOT assay for the indicated genotypes. Each symbol indicates one individual animal.
(E) Serum concentration of Ig subclasses in indicated mice, determined by ELISA. Each symbol indicates an individual animal. IgM concentration was significantly increased in λ5#2 (p < 0.025), Vpreb#1 (p < 0.04) and λ5#2;Vpreb#1 (p < 0.015) mice, when compared to WT. IgG1 concentration was decreased in λ5#2 (p < 0.006) and λ5#2;Vpreb#1 (p < ) mice, when compared to WT. Likewise, IgG3 concentration was reduced in λ5#2 (p < 0.005) and λ5#2;Vpreb#1 (p < ) mice.
(F) TI-II immune responses, determined by ELISA for TNP-specific IgM and IgG3 antibodies, 7 days after TNP-Ficoll injection. Each point represents the mean optical density of three to five mice per group.
(G) TD immune responses, determined 7 days after TNP-KLH booster. Each point represents the mean optical density of three to five mice per group.
(H) Immunohistochemical analysis of WT and λ5#2;Vpreb#1 spleen, 7 days after TNP-KLH booster. Sections were stained with anti-IgD (blue), PNA (brown, indicated by arrow head) for germinal center (GC) B cells, anti-IgM (brown) for IgM+ B cells and plasma cells, MOMA1 (blue) for staining metallophillic marginal zone macrophages, and anti-IgG1 (blue) for IgG1+ GC B cells and plasma cells.
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions