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Blood, Vol. 112, Issue 10, 4158-4169, November 15, 2008
Wiskott-Aldrich syndrome protein deficiency in B cells results in impaired peripheral homeostasis
Blood Meyer-Bahlburg et al.
112: 4158
Supplemental materials for: Meyer-Bahlburg et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 1.74 MB)
- Figure S1. B-cell phenotype in WASp deficient mice (JPG, 59.3 KB)
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(A) BM was stained for expression of B220, CD43, IgM, BP1, CD24, CD19 and IgD and developmental stages determined according to the Hardy classification. Percentages of each fraction (Fr) A-F, are shown for wt vs. WASp−/− mice (n=12). (B) Splenocytes were stained for expression of B220, CD19, CD24, CD21, CD23 and CD1d and B-cell subsets analyzed. Percentages of each splenic B-cell subset from wt vs. WASp−/− mice (n=12) are shown. (D) Percentage of BM B1 B-cell progenitors in wt (n=13) vs. WASp−/− mice (n=12).
- Figure S2. The WASp−/− B-cell phenotype reflects a cell intrinsic defect (JPG, 56.4 KB)
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BM from wt or WASp−/− was transplanted into sublethally irradiated µMT mice. Recipient mice were bled and sacrificed 6 weeks after transplantation. B cells in peripheral blood, BM and spleen were analyzed. Shown are percentages of each B-cell subset in (A) BM (n=9) and (B+C) spleen (n=7).
- Figure S3. WASp+ B cells do not exhibit a selective advantage during early BM B-cell development (JPG, 123 KB)
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(A) BM was stained for expression of B220, CD43, IgM, BP1, CD24, and IgD and cell fractions (Fr) determined according to the Hardy classification. An example of the gating criteria used in one wt and one WASp−/− mouse is shown. Numbers refer to relative percentage of live cell cells within each gate. (B–C) BM from female heterozygote mice was stained for WASp in addition to cell surface markers as in (A). (B) Relative WASp expression in Hardy Fraction B vs. C. (C) Percentage of WASp positive cells within each of the BM fractions (n=12).
- Figure S4. Altered Hes1 expression in WASp−/− MZ B cells reflects a B-cell extrinsic defect (JPG, 51.4 KB)
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(A) Expression of Hes1 and Deltex1 transcripts was determined by Q¬PCR using sort purified FM and MZ B cells from wt vs. WASp−/− mice. Average data (n=2 for each strain) from one of two similar experiments is shown. (B) BM from wt or WASp−/− mice was transplanted into sublethally irradiated µMT mice. Recipient mice were sacrificed 6–10 weeks after transplant. Expression of Hes1 was determined in sorted FM vs. MZ B cells (n=3 for wt and n=4 for ko). (C) Reduced expression of S1P3 in WASp−/− MZ B cells. S1P1 and S1P3 expression levels were determined by Q-PCR using sort purified FM and MZ B cells from wt vs. WASp−/− mice. Representative data from one of two experiments are shown.
- Figure S5. Analysis of BCR and BAFF-receptor survival signaling in wt vs. WASp−/− mice (JPG, 138 KB)
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(A) BrdU chase experiment. Mice were fed BrdU for 7 days. Mice were sacrificed 14 and 21 days after the beginning of BrdU administration and percentage of BrdU+ determined in MZP/MZ and FM B cells. Data shown are representative for 3 mice per time point and group. (B) Splenic B cells were stained intracellularly for Bcl-2 in conjunction to cell surface markers and Bcl-2 expression was determined within each B-cell subset in wt vs. WASp−/− mice. (C) Sort purified T1, T2, FM and MZP/MZ B cells were stimulated with BAFF, anti-IgM (1 or 10ug/ml) or left unstimulated as indicated and incubated for 24 hrs. Survival was determined by gating live cells. Data shown are the average results with standard deviation of 6 mice of each strain from 3 independent experiments. (D) Sort purified FM B cells were stimulated with BAFF or left unstimulated and expression of p100 and p52 determined by western blotting. (E) Splenic B cells were stained for BAFF-R in addition to surface staining. Expression of BAFF-R in each B-cell subset from wt vs WASp−/− mice is shown. (F) Splenic B cells were stimulated with 15µg/ml IgM for 10min, fixed, surface stained with various markers and subsequently stained for intracellular phospho-AKT. Phosphorylation of AKT is shown before and after stimulation in each B-cell subset from wt vs. WASp−/− mice.
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