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Blood, Vol. 112, Issue 10, 4139-4147, November 15, 2008
WASP confers selective advantage for specific hematopoietic cell populations and serves a unique role in marginal zone B-cell homeostasis and function
Blood Westerberg et al.
112: 4139
Supplemental materials for: Westerberg et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 1.15 MB)
- Figure S1. Identification of central and peripheral B-cell subsets (JPG, 88.1 KB)
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B-cell lineage cells in the BM and spleen were defined by surface and intracellular markers, labeled with anti-WASP antibodies and analyzed by flow cytometry. (A) BM cells were labeled with B220, CD24, CD43, CD95, IgD, and IgM and developmental stages defined according to Hardy’s classification (Fr A-F). (B) Spleen cells were labeled with CD21, CD23, and CD24 to identify T1, T2-FO, FO, T2-MZP, and MZ B-cell developmental stages. Abbreviations: Fr, Fraction; FO, follicular; MZ, marginal zone; T1/T2, transitional 1/2 B cell; T2-FO, transitional 2 follicular precursor B cells; T2-MZP, transitional 2 marginal zone precursor B cell.
- Figure S2. Selective advantage of WASP-expressing cells in response to sheep red blood cells (SRBC) (JPG, 75.2 KB)
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WASP+/− and WASP−/− mice when 4 weeks old were injected intravenously with SRBC diluted 1:100 in phosphate buffer saline. (Upper panel) The proportion of WASP-expressing GC B cells (PNA+CD95/Fas+) was determined at day 6 post-immunization. (Lower panel) WASP expression in GC B cells was compared to that of non-GC B cells (PNA−CD95−) and follicular B cells (CD23+CD21+IgM+, lower panel). ***, p < 0.005.
- Figure S3. Reduced MZ B cells and MZM in WASP-deficient mice on C57BL/6 background (JPG, 116 KB)
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(A) FO and MZ B cells were defined based on CD21, CD23, and IgM expression. Results from two WT and two WASP−/− mice are shown. (B) Immunohistochemistry analysis of spleen sections from WT (left panel) and WASP−/− (right panel) mice stained for B220+ B cells and MARCO+ MZM. (C) Quantification of MZM from spleen sections labeled as in B. Mean value (±SD) of triplicates is shown for each mouse (n=3).
- Figure S4. WASP is involved in signaling pathways that regulate MZ B-cell development (JPG, 87.7 KB)
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Analysis of FO and MZ B cells in WT, Btkxid, WASP−/−, and WASP−/−/Btkxid mice is shown. (A) Proportion of FO and MZ B cells. (B) Absolute numbers of FO and MZ B cells. *, p < 0.05.
- Figure S5. Immune response to high dose TNP-Ficoll and increased spontaneous secretion of natural IgM antibodies in WASP-deficient mice (JPG, 77.1 KB)
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(A) WT and WASP−/− mice when 12–14 weeks old were injected intravenously with 25 µg TNP-Ficoll. Anti-TNP IgM and IgG3 antibody titers were determined at day 7 post-immunization by ELISA. Each group represents mean values (±SD) from n = 5 mice and were corrected for background binding. (B) Anti-TNP IgM antibody and (C) anti-phosphorylcholine (PC) antibody titers in non-immunized mice determined by ELISA. Note that WASP−/− mice have increased spontaneous secretion of anti-TNP and anti-PC IgM relative to WT mice. *, p < 0.05; **, p < 0.01 and ***, p < 0.005.
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