Supplementary Figure S1

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1 Supplementary Figure S1 Supplementary Figure S1. CD11b expression on different B cell subsets in anti-snrnp Ig Tg mice. (a) Highly purified follicular (FO) and marginal zone (MZ) B cells were sorted from spleen (gated on CD19 + cells). (b) Sorted FO B cells were stained with mabs against CD3, F4/80, Gr-1, NK1.1 and corresponding isotype control mabs. (c) Cell lysates of FO B cells (12 x 10 6 ) and MZ B cells (3.5 x10 6 ) from CD11b WT or KO mice were prepared and western blot analysis was performed to detect CD11b expression (~160Kd). (d) CD11b expression assessed by RT-PCR analysis. (e) The percentages of FO and MZ B cells in spleen (n=7) and B1 B cells (n=10) from peritoneal cavity of CD11b WT and KO anti-snrnp Ig Tg mice. Data are means ± s.e.m.

2 Supplementary Figure S2 Supplementary Figure S2. FO B cells, but not MZ B cells from CD11b-deficient mice exhibit enhanced proliferation and activation upon BCR crosslinking. (a) Purified FO B cells and MZ B cells (n=3) were stimulated with anti-igm for 48 h. B cell proliferation was assessed by [ 3 H] thymidine incorporation assay. Data are means ± s.e.m. **p<0.01 (unpaired two-tailed student s t test). (b) sigm expression by FO and MZ B cells was analyzed by flow cytometry. (c) Purified B cells were stimulated with anti-igm (40 μg/ml) for 48 h. The surface molecule expression was analyzed using flow cytometry. Means ± s.e.m of the MFI of the specific molecules are shown (n=3 or 4). **p<0.01, ***p<0.001 (unpaired two-tailed student s t test). (d) BrdU in vivo incorporation in B cells as determined after 5 days of in vivo exposure to BrdU (n=5). Percentages of BrdU + B cells out of CD19 + cell population from spleen are shown. Data are means ± s.e.m. **p<0.01 (unpaired two-tailed student s t test).

3 Supplementary Figure S3 Supplementary Figure S3. CD11b deficiency on BM environment does not influence autoreactive B cell activation but CD11b deficiency on B cells promotes autoab production. (a) B cells were purified from BM chimeric mice (n=5) as indicated and stimulated with anti- IgM (40 μg/ml) for 48 h. B cell proliferation was assessed by [ 3 H] thymidine incorporation assay. Data are shown as the means ± s.e.m. * p<0.05; ** p<0.01 (unpaired two-tailed student s t test). (b) B cell in vitro survival as determined by Annexin V/7-AAD staining. B cells were cultured in medium or stimulated with anti-igm (40 μg/ml) for 72 h. Viable cells were identified by staining with Annexin V and 7-AAD. Data are shown as the means ± s.e.m. ** p<0.01; *** p<0.001 (unpaired two-tailed student s t test). (c) Rag1-deficient mice (n =7 or 8) transferred with B cells from CD11b WT or KO anti-snrnp Ig Tg mice along with T cells were immunized with apoptotic cells. FACS analysis of CD138 expression on B cells from immunized mice. The percentage of CD138 + cells (gated on CD19 + population) and serum ANA levels in immunized mice are shown. Data are shown as the means ± s.e.m. * p<0.05; (unpaired two-tailed student s t test). (d) Anti-snRNP level in sera of immunized B cell-deficient μmt mice (n=5) adoptively transferred with B cells (15 x 10 6 ) from CD11b WT or KO mice. Data are shown as the means ± s.e.m. * p<0.05 (unpaired two-tailed student s t test).

4 Supplementary Figure S4 Supplementary Figure S4. Generation of stable B cell lines expressing WT or R77H mut human CD11b. (a) CD11b expression on the surface of K46-17/CD11bWT, K46-17/CD11bMut, and K46-17/Control B cell lines as determined by flow cytometry. (b) Cell lysates from three cell lines were analyzed by western blot for CD11b and Flag tag expression. (c) Surface and intracellular expression of CD18 on three cell lines were analyzed by flow cytometry. (d) K46-17 cells do not express CD11b as assessed by flow cytometry. A histogram of each cell line is overlaid with a gray-filled histogram of isotype control.

5 Supplementary Figure S5 Supplementary Figure S5. Co-localizations of B cell-associated molecules in CD11b WT and R77H Mut-transfected B cell lines in the presence or absence of BCR ligation. Representative histograms showing co-localizations of BCR-CD22, BCR-p-Lyn, and BCR-SHP- 1 (a) and co-localizations of CD11b-CD22, CD11b-p-Lyn, CD11b-SHP-1, and BCR-CD11b (b). Numbers indicate percent of co-localization with similarity score >2. CD11b transfected B cell lines were stimulated with or without anti-igm (5 μg/ml) at indicated time points and stained with mabs against BCR, CD11b, CD22, SHP-1, and phospho-lyn. Cells were captured by the Amnis Imagestream System. Co-localization was analyzed using IDEAS software. The similarity feature within IDEAS software was used for the quantitative co-localization of molecules. Cells with a similarity score (S) of 2 or higher were classified as co-localized cells.

6 Supplementary Figure S6 Supplementary Figure S6. List of full blots. (a) Full blots of SFN, Bcl-xL, and Bcl-2 as shown in Figure 3b. (b) Full blots of phospho-syk (p-syk), p-akt, p-p38, p-erk1/2, and p-lyn as shown in Figure 5b and 5c. β-actin was used as normalization controls. (c) Full blots of total CD22, SHP-1 and SHIP-1 as shown in Figure 5d. (d) Full blots of p-cd22, SHP-1, and Lyn after CD22 immunoprecipitation (IP) as shown in Figure 5e. (e) Full blots of CD11b (flag) and CD22 in wildtype or R77H-mutated CD11b-transfected K46-17 B cell lines after CD22 IP as shown in Figure 7f.

7 Supplementary Table S1 Genes Forward Reverse CD11b TGGAAGCTTGCCACCATGGCTCTC AGAGTCCTTCTGTTAACAGCCTTG TCGTCTAGACTGGGGTTC GGCCCCCGGGGGACCCCC CD11b mutation CTCATGCGAGCCCATCCACCTGC AGGTCCCCGTGGAGGCC GGCCTCCACGGGGACCTGCA GGTGGATGGGCTCGCATGAG SFN ATGAAGACATGGCAGCTTTC GCTCGGTCTCTACCTTCTCC CD11b CCTTCATCAACACAACCAGAGTGG CGAGGTGCTCCTAAAACCAAGC (RT- PCR) GAPDH CCTTCCGTGTTCCTACCC GCCTGCTTCACCACCTTC STMN-1 AAGAATCTGTCCCCGATTTC GCCATCTTGCTGAAGTTGTT STIM-1 GCTAGCTCCGGGGCGACTTCT TCCACAGGTCCTCCACGCTGA Supplementary Table S1. Primers for Cloning and Gene Expression