Microscopy data of spleen cells from wild type, 3H9-transgenic and CD40L/3H9 double transgenic mice

<p>Data 2. Fluorescent microscopic analysis of spleen sections from wild type, 3H9-transgenic and CD40L/3H9 double transgenic mice.<br>Spleen sections were obtained from wild type (A), 3H9-transgenic (B), and CD40L/3H9 double transgenic mice (C). Tissue sections were stained with biotin-conjugated rat anti-mouse MOMA-1, Alexa Fluor 647- conjugated streptavidin (red), Pacific Blue-conjugated rat anti-mouse B220 (green) and FITC-conjugated goat anti-mouse λ chain (blue) antibody. Fluorescent images were obtained with a Zeiss LSM 510 META laser scanning confocal microscope.</p

Flow cytometry data of spleen cells from wild type, 3H9-transgenic and CD40L/3H9 double transgenic mice

<p>Data 1. Flow cytometry analysis of spleen cells from wild type, 3H9-transgenic and CD40L/3H9 double transgenic mice.<br>Spleen cells were obtained from wild type (A and D), 3H9-transgenic (B and E), and CD40L/3H9 double transgenic mice (C and F). Cells were stained with Alexa Fluor 647-conjugated (FL 8 channel) rat anti-mouse B220 and Pacific Blue-conjugated (FL 6 channel) anti-mouse Ig λ1(LS-136), and with (D-F) or without (A-C) FITC-conjugated (FL 1 channel) goat anti-mouse Ig λ chain. Lymphoid cells were gated by FSC/SSC dot plots and the B220+ cells were analyzed on a CyAn ADP flow cytometer (Beckman Coulter, USA).</p

The Xid mutation of Btk partially restores abnormalities of IgG-tg B cells.

<p>(A) BCR expression of B cells. Spleen cells from indicated mice were stained for NIP binding, IgG2a, IgM and B220, and analyzed by flow cytometry. Percentages of B cells are indicated. (B) Subsets of spleen B cells. Spleen cells were stained for B220, CD21 and CD23. Percentages of MZ B cells (B220⁺, CD21^hi, CD23^lo/−) and follicular B cells (B220⁺, CD21^inter, CD23^hi) are indicated. (C) Proliferation of B cells. Purified CD23⁺ follicular B cells were labeled with CFSE, and cultured with anti-CD40 antibody for 72 hrs. Cells were analyzed by flow cytometry for CFSE labeling. The numbers of cell division are indicated. (D) BCR ligation-induced tyrosine phosphorylation of cellular substrates. Purified CD23⁺ follicular B cells were stimulated with 0.2 µg/ml NP₁₅-BSA for 2 and 5 min. Phosphorylation of cellular substrates in total cell lysates (upper panel) was analyzed by Western blotting. Western blot analysis of β-tubulin was done as a loading control (lower panel).</p

IgG-tg B cells generate an augmented proliferative response to CD40 ligation but no antigen-induced BCR signaling.

<p>CD23⁺ follicular B cells were isolated from IgM-tg and IgG-tg spleen by magnet sorting. (A) Purity of CD23⁺ follicular B cells used for in vitro analysis. Expression of B220 and CD23 was analyzed by flow cytometry. Percentages of B220⁺CD23⁺ follicular B cells are indicated. (B and C) Antigen-induced BCR signaling. Purified CD23⁺ follicular B cells were stimulated with 0.2 µg/ml NP₁₅-BSA for 2 and 5 min. Phosphorylation of cellular substrates in total cell lysates (B, upper panel) and that of ERK (B, middle panel) were analyzed by Western blotting. Western blot analysis of β-tubulin was done as a loading control (B, lower panel). Alternatively, cells were loaded with Fluo-4/AM, and intracellular free Ca²⁺ was measured by flow cytometry (C). Cells were added with 0.2 µg/ml NP₁₅-BSA at 25s as indicated by arrows, and measurement of free Ca²⁺ was continued for 200s. Representative data of three experiments are shown. (D) Expression of CD86. Purified CD23⁺ follicular B cells were cultured with or without 0.2 µg/ml NP₁₅-BSA for 48 hrs, and analyzed for expression of CD86 by flow cytometry. Percentage of CD86⁺ cells is indicated. (E) Apoptosis of B cells. Purified CD23⁺ follicular B cells were cultured with indicated reagents for 24 hrs. Percentages of apoptotic cells with hypodiploid DNA were measured by flow cytometry. Data are shown as average of three experiments (±SD). (F) Cell cycle progression of B cells. Purified CD23⁺ follicular B cells were cultured with indicated reagents for 48 hrs, and were pulsed with BrdU for the last 20 min of the culture. Percentages of BrdU-incorporated cells (% S phase) were measured by flow cytometry. Data are shown as average of three experiments (±SD). (G) Division of B cells. Purified CD23⁺ follicular B cells were labeled with CFSE, and cultured with anti-CD40 antibody for 72 hrs. Cells were analyzed by flow cytometry for CFSE labeling. The numbers of cell division are indicated.</p

Flow cytometry analysis of bone marrow and spleen cells from wild type (WT) C57BL/6, IgM-tg and IgG-tg mice.

<p>(A) Analysis of bone marrow cells. Cells were stained for B220, IgM, and CD43. Percentages of pro B cells (IgM⁻, B220⁺, CD43⁺), pre B cells (IgM⁻, B220⁺, CD43⁻), immature B cells (B220^lo, IgM^med), transitional B cells (B220⁺, IgM^hi), and mature B cells (B220^hi, IgM^med) are calculated. Numbers of mice analyzed are indicated, and data are shown as mean (±SD) (bottom). (B) Subsets of spleen B cells. Spleen cells were stained for CD3, B220, CD21 and CD23. Percentages of T cells and B cells (left panel) in total spleen cells, and those of MZ B cells (B220⁺, CD21^hi, CD23^lo/−) and follicular B cells (B220⁺, CD21^inter, CD23^hi) in total B cells (right panel) are calculated. Numbers of mice analyzed are indicated, and data are shown as mean (±SD) (bottom). (C) Expression of BCR on spleen B cells. Spleen cells were stained for NIP binding, IgG2a, IgM and B220. Mean fluorescence intensity (MFI) of NIP binding is indicated. (D) Expression of various molecules on spleen B cells. Spleen cells from IgG-tg (open histograms) and IgM-tg (shaded histograms) mice were analyzed for expression of CD40, CD22, CD19, MHC class II, CD86, CD44, CD23, Fas, and CD93. All histograms are gated on B220⁺ cells.</p

Augmented antibody production in unimmunized IgG-tg mice.

<p>(A). Serum levels of anti-NP IgM, IgG2a, and λ L chain-containing Ig. Sera from 13-wk-old IgM-tg and IgG-tg mice were analyzed by ELISA. Data are shown as mean ± SD. (B). Histological analysis. Frozen spleen sections from upper IgM-tg (left panel), IgG-tg (right upper panel) and wild type (WT) C57BL/6 (lower panes) mice were stained either anti-IgM^a Ab (left upper panel), anti-IgG2a Ab (right upper panel) (red), anti-IgM Ab (left lower panel) or anti-IgG2a (right lower panel) together with anti-CD38 Ab (green).</p

Expression, localization and function of Rasal3.

<p>(A) Expression of Rasal3 mRNA in various organs from C57L/6 mice. Rasal3 mRNA expression was determined by quantitative RT-PCR and normalized relative to β-actin mRNA. (B) Expression of Rasal3 protein in the thymus, spleen, and liver (upper) and sorted thymic or splenic T cell or B cell subpopulations (lower). (C) Subcellular localization of Rasal3 protein in thymocytes. CE, ME, NE, and CB indicate cytoplasmic extract, membrane extract, nuclear extract, and chromatin bound, respectively. (D) GAP activity of Rasal3. DPK cells overexpressing Rasal3 were subjected to a Ras-GTP pull-down assay followed by Western blot analysis. (E) The Q672N mutant Rasal3 could not suppress the increase in GTP-Ras and ERK activation. All data are representative of more than three independent experiments.</p

Decreased naive CD4 and CD8 T cells with increased apoptosis in Rasal3-KO mice.

<p>(A) Flow cytometry profiles for CD4 and CD8 of splenocytes from WT and Rasal3-KO mice (left). The bar graphs show the number of total splenocytes and number and frequency of splenic CD4⁺ TCRβ⁺ and CD8⁺ TCRβ⁺ cells (n = 19 mean ± s.e.) (right). (B and C) Profiles of CD44 and CD62L in splenic CD4⁺ TCRβ⁺ (B) or CD8⁺ TCRβ⁺ (C) cells (left). The bar graphs show the number and frequency of naive (CD44^lo CD62L^hi) cells, effector memory (CD44^hi CD62L^lo) cells and central memory (CD44^hi CD62L^hi) cells (n = 17, mean ± s.e.) (right). (D) Annexin V and 7AAD staining of CD4⁺ TCRβ⁺ CD44^lo, CD8⁺ TCRβ⁺ CD44^lo and CD4^- CD8^- TCRβ^- spleen cells freshly isolated from WT or Rasal3-KO mice. (E) The bar graphs show the frequency of Annexin V⁺ 7AAD^- and Annexin V⁺ 7AAD⁺ cells depicted in panel D (n = 5, mean ± s.e.).</p

Rasal3 deficient T cells were activated normally upon agonistic anti-TCR antibody stimulation.

<p>TCR-induced phosphorylation of ERK in thymocytes (A) and splenocytes (B). (A) Total thymocytes were stimulated with anti-CD3 Ab (5 μg/ml) for 1 min. (B) Total splenocytes were stimulated with anti-CD3 (30 μg/ml) and CD28 (12 μg/ml) Abs for 1min. (C) Upregulation of CD69, 25, and 44 upon stimulation. Sorted naive CD4 T cells (CD4⁺ CD44^lo CD62L^hi CD25^-) were activated by immobilized anti-CD3 Ab (5 μg/ml) and soluble anti-CD28 Ab (1 μg/ml) for 24 hours. Expression of CD69, CD25 and CD44 were analyzed by FACS. (D) IL-2 production from naive CD4 T cells. The cells were activated by immobilized CD3 (5 μg/ml) for 24 hours and IL-2 production was measured by ELISA. (E) and (F) Calcium influx triggered by TCR stimulation. Anti-CD3 plus anti-CD28 (Ab) and ionomycin (IM) were added at the points indicated by arrows. (G) CFSE-labeled splenic CD4 T cells were activated by immobilized anti-CD3 Ab (5 μg/ml) and soluble anti-CD28 Ab (1 μg/ml) for 72 hours. CFSE dilution by cell division was measured by flow cytometry. These data are representative of more than three experiments. Solid and dashed lines indicate WT and Rasal3-KO, respectively. The shadow indicates non-stimulated cells as a control.</p

T cell development in Rasal3-deficient mice.

<p>(A) Flow cytometry profiles for CD4 and CD8 of thymocytes from WT and Rasal3-KO mice. (B) Proportions of TCRβ⁺ CD69⁺ post-selected cells in DP thymocytes. (C) Flow cytometry profiles for CD4 and CD8 of thymocytes from OT-II, OT-I, and female HY TCR-Tg RAG2^-/- mice. (D) Flow cytometry profiles for CD4 and CD8 of male HY Tg RAG2^-/- thymocytes (left). The bar graph shows the number of DP thymocytes (n = 3–4, mean ± s.e.) (right). (E) Analysis of developmental stage of DN thymocytes. Representative cytometry profiles for CD25 and CD44 expression in DN thymocytes are shown. (F) Expression of differentiation makers (CD2, CD5 and CD69) on CD4-SP or CD8-SP thymocyte. Solid and dashed lines indicate WT and Rasal3-KO, respectively.</p