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Blood, Vol. 110, Issue 7, 2381-2389, October 1, 2007
Constitutive alternative NF- B signaling promotes marginal zone B-cell development but disrupts the marginal sinus and induces HEV-like structures in the spleen
Blood Guo et al.
110: 2381
Supplemental materials for Guo et al, Vol. 110, Issue 7, 2381-2389
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 29.7 KB)
- Figure S1. Reduced numbers of peritoneal B1 cells in p100−/− mice (PDF, 97 KB) -
Splenocytes (Sp), peripheral blood leukocytes (PBL), and peritoneal exudate cells (PEC) from 20-day-old wild-type, p100−/−, and nfkb2−/− mice were stained for B220 and CD5 and analyzed by flow cytometry (lymphocyte gate). Percentages indicate B220loCD5lo B1 cells. Representative data from 3 to 4 experiments are shown.
- Figure S2. Increased frequency of T cells and myeloid cells in spleen from p100−/− mice (PDF, 76.2 KB) -
(A) Splenocytes from wild-type and p100−/− mice were stained for B220 and Thy1.2 and analyzed by flow cytometry (lymphocyte gate). (B) Myeloid hyperplasia in spleen from p100−/− mice. Splenocytes from wild-type and p100−/− mice were stained for Gr-1 and analyzed by flow cytometry. Representative data from 5 mice are shown. Similar results were obtained when splenocytes were stained for CD4/CD8 and Mac-1, respectively (data not shown).
- Figure S3. Increased expression of β1 integrin, CD40, and the costimulatory molecule CD80/B7.1 on MZ B cells from p100−/− compared to wild-type spleen (PDF, 46.4 KB) -
Splenocytes from 20-day-old wild-type and p100−/− mice were stained for CD21 and CD23 and expression of  1, CD40, and CD80 on MZ B was analyzed by flow cytometry. No differences in 1, CD40, and CD80 levels were observed between p100−/− and wild-type FO B cells (not shown). Representative data from 3 to 4 experiments are depicted.
- Figure S4. Increased numbers of p100−/− MZ B cells in competitive bone marrow chimeras (PDF, 12.5 KB) -
For competitive chimeras, lethally irradiated Ly5.1+ wild-type (C57BL/6) mice were adoptively transferred with 4 × 106 cells comprising a 1:1 mixture of Ly5.2+ p100−/− and Ly5.1+ wild-type (C57BL/6) bone marrow. After 6 weeks, MZ and follicular (FO) B cells were analyzed by flow cytometry for the expression of CD21 and CD23 together with Ly5.1 or Ly5.2 in spleen of chimeric mice. Absolute numbers per 106 splenocytes are shown. Error bars are SD. MZ B cells derived from p100−/− (Ly5.2+) bone marrow were increased 2.5-fold whereas no significant difference was observed when FO B cell numbers were compared.
- Figure S5. Stamper-Woodruff adhesion assay (PDF, 24.9 KB) -
Lymphocyte binding to HEVs was assessed in Stamper-Woodruff adhesion assays. Briefly, freshly isolated exogenous lymphocytes from C57BL/6 spleens (6 × 106 in 0.2 mL) were incubated on frozen spleen sections from wild-type and p100−/− mice in RPMI-1640 medium, 5% FCS, 20 mM Hepes, pH 7.3 and allowed to adhere for 30 minutes under constant rotation (50 rpm, 7°C). Slides were washed by dipping in cold PBS and adherent cells were fixed in 3% glutaraldehyde/PBS. After washing with deionized water sections were stained overnight with MECA-79 mAb (pink/red) and counterstained with hematoxylin. Adherent lymphocytes are dark blue and above the plane of the section. LN sections (insets) are shown as a control. Images were acquired with an AX70 microscope (Olympus, Hamburg, Germany) equipped with 40×/0.75 objective and an Olympus DP70 digital camera running analySISB Soft Imaging System(Olympus). Images were procesed with Adobe Photoshop 8.0 image processing software (Adobe, San Jose, CA).
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