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1 Supporting Information Song et al /pnas SI Materials and Methods In Vivo Dislodgment Assays. In vivo dislodgement experiments were performed as previously described (1) using function blocking antibodies to integrins β1 (Ha 2/5 or 9EG7), β3 (2C9.G2), α6 (GoH3), α4 (PS/2), anti leukocyte function associated antigen (LFA)-1 (M17/4), or laminin α5 (2). Six hours after i.v. injection of μg specific antibodies (either alone or in various combinations) or isotype control Igs, whole blood was collected and spleens were harvested. As LFA-1/intracellular adhesion molecule (ICAM)-1 have been shown to act synergistically with α4β1/vascular cell adhesion molecule (VCAM)-1 to mediate binding of marginal zone (MZ) B cells to the MZ (1), integrin β2-null mice (provided by K. Scharfetter-Kochanek, Department of Dermatology and Allergic Diseases, Ulm University, Ulm, Germany) (3) were also used to eliminate LFA-1/ICAM-1 effects. When injected alone or in combination, antibodies to integrins β1 (Ha 2/5, 9EG7), β3 (2C9.G2), α6 (GoH3), and α4 (PS/2) had little or no effect in WT mice (Table S1). Only anti-integrin α4 plus anti LFA-1 (M17/4), or anti-integrin α4 injected into the integrin β2-null mice resulted in a displacement of MZ B cells from the MZ to the circulation (Table S1). Flow Cytometry for Transitional B-Cell Populations. Bone marrow and spleens of the conditional lama5 / and Itga6 / bone marrow chimeric mice were analyzed by flow cytometry: isolated bone marrow cells were stained with a mixture of anti-cd19, anti-aa4.1, and anti-igm, the CD19 + cells were gated, and proportions of pro- and pre-b cells (AA4.1 + IgM ), immature B cells (AA4.1 + IgM + ), and recirculating B cells (AA4.1 IgM ) were measured. Isolated splenic cells were stained with anti- CD19, anti-aa4.1, and anti-cd23, the CD19 + AA4.1 + immature B cells were gated, and proportions of newly formed T1 transistional B cells (CD23 ) and T2+T3 (CD23 + ) transistional B cells were measured. Measurement of Reticular Fiber Diameters. Spleens were frozen in TissueTek (Miles Laboratories) in isopentane at 70 C. Fivemicrometer cryostat sections were fixed for 10 min in 20 C methanol. Immunofluorescence staining was carried out as described previously (2) using the anti-laminin a5 antibody (504). Sections were examined using a Zeiss AxioImager microscope equipped with epifluorescent optics and documented using a Hamamatsu ORCA ER camera or with a Zeiss confocal laser scanning system LSM 700. Volocity software was used to trace and quantify (fluorescence intensity) laminin α5 staining for reticular fiber diameter measurements. Spleens from at least 10 animals were examined; 5 10 sections per animal and two to five areas per section were examined. Statistical Analyses. The data were analyzed by a paired sign t test or Student t test. All P values of 0.05 or less were considered statistically significant. 1. Lu TT, Cyster JG (2002) Integrin-mediated long-term B cell retention in the splenic marginal zone. Science 297(5580): Sorokin LM, et al. (1997) Developmental regulation of the laminin alpha5 chain suggests a role in epithelial and endothelial cell maturation. Dev Biol 189(2): Scharffetter-Kochanek K, et al. (1998) Spontaneous skin ulceration and defective T cell function in CD18 null mice. J Exp Med 188(1): of14

2 Fig. S1. Biochemical and morphological characterization of the reticular fibers of the splenic MZ, red pulp (RP), and B-cell area of the white pulp (WP). (A) Comparison of the immunofluorescent staining patterns for the basement membrane proteins, collagen type IV, perlecan, and laminin α5. Magnification: 100. (B) Immunofluorescent staining for laminin α5 shows differences in the reticular fiber diameters in the WP, MZ, and RP. Magnification: 200. Boxed areas are shown at a higher magnification to the right. Magnification: 400. (C) Image analysis of laminin α5 immunofluorescent staining intensity across the line shown permitted measurement of reticular fiber diameters. (D) Quantification of fiber diameters in the WP, MZ, and RP. Data shown are mean values ± SEM from at least 10 animals; 5 10 sections per animal and two to five areas per section were examined. 2 of 14

3 Fig. S2. Immunofluorescence staining of adult spleen sections from C57BL/6, CD19 /, and agrin / mice for laminin α5, IgM, and IgD showing reduced laminin α5 localization in the MZ of CD19 / only and associated reduction in the IgM high /IgD low MZ B-cell population. Staining of serial sections shows no differences in agrin expression in the CD19 / mice compared with C57BL/6 controls; VCAM-1 and ICAM-1 distribution does not differ between the three strains. WP, white pulp. Magnification: 150, except agrin, of14

4 Fig. S3. Laminin α5 conditional KO mouse: (A) Targeting strategy for generation of floxed laminin α5 gene, showing insertion of loxp sites flanking the exons encoding domains VI, V, and IV of laminin α5; exons appear as green boxes. (B) Southern blot analyses of transfected ES cell clones. (C) Immunofluorescence analysis of laminin α5 conditional KO mice generated by crossing laminin α5 floxed mouse with Tie-2-cre mice. Skin sections from the ears of a KO and a WT littermate were doubled stained with anti-laminin α5 chain (green) and anti-platelet endothelial cell adhesion molecule (PECAM) antibodies (red) showing specific deletion of laminin α5 from the endothelial basement membranes, i.e., there is no colocalization of laminin α5 and PECAM staining in the KO animals. Laminin α5, however, remains in basement membranes of the epidermis and the smooth muscle layer of larger blood vessels of KO mice. Magnification: 75 (top panels); 100 (bottom panels). 4of14

5 Fig. S4. Double immunofluorescent staining for laminin α5 and VE-cadherin (A) or laminin α5 and PDGF-receptor-β (PDGFrecβ) (B), showing the expression of the endothelial cell marker, VE-cadherin, in the marginal zone stromal cells and in the sinus lining vessels and the expression of perivascular cell marker, PDGFrecβ, at the marginal sinus and in the RP. (C) Double immunofluorescent staining for another endothelial cells marker, mouse endothelial cell antigen (MECA)-32, and mucosal addressin cell adhesion molecule (MAdCAM) as a marker of the sinus lining cells, shows MECA32 on sinus lining vessels and RP vessels, but also on MZ stromal cells. Boxed areas are shown at high magnification to the right. Magnification: 100 (A C); 400 (boxed areas). 5of14

6 Fig. S5. Immunofluorescence and quantification of flow cytometry analyses of spleens of conditional Lama5 / mice and WT littermates. (A) Immunofluorescence staining of adult spleen sections from conditional Lama5 / mice and WT littermates; WP, white pulp. Magnification: 150. Stainings for ICAM-1 and VCAM-1 were performed on spleen sections from mice injected with anti-integrin α4 plus anti leukocyte function associated antigen(lfa)-1 to displace MZ B cells. (B) Quantification of flow cytometry data shows no difference in numbers of total B220+ B cells, MOMA-1+ sinus lining macrophages, and ERTR9+ MZ macrophages; data shown are means ± SEM from three experiments with n = 5 in each experiment. (C) Representative flow cytometry for CD19+ B cells and CD21highCD23low MZ B cells in the blood of Lama5 / mice and WT littermates showing no differences. 6 of 14

7 Fig. S6. Representative flow cytometry of immature B cells in the bone marrow (BM) (A) and transitional B-cell populations (T1, T2, T3) in the spleen (B) of adult conditional Lama5 / mice and WT littermates. Quantification of T1, T2, and T3 populations are shown in the bar graphs. Data shown are mean values ± SEM from at least three separate experiments with n = 6 for each treatment in each experiment. **P < 0.01; ***P < of14

8 Fig. S7. (A) Representative flow cytometry of cell surface integrin expression on MZ B cells (black) and follicular (FO) B cells (red), with antibody isotype controls shown in green and yellow, respectively. (B) In vitro adhesion of MZ and FO B cells to VCAM-1, laminin 511/521, or laminin α5 domain IVa (all 30 μm) in the presence or absence of function blocking antibodies to integrins α4 (PS/2), α6 (GoH3), β1 (Ha2/5), β3 (2C9.G2), or cyclic-rgd or RGE (control pep) peptides. Hamster IgG is an isotype control for anti-integrin β1 and β3 antibodies; rat IgG is an isotype control for anti-integrin α4 and α6 antibodies; 100% is binding in the presence of isotype control antibody. Adhesion data shown are mean values ± SEM from at least three separate experiments with n = 6 for each treatment in each experiment. *P < 0.05; **P < of14

9 Fig. S8. (A) Quantification of flow cytometry analysis of host (CD ) and donor (CD ) NF B cells (CD19 + CD21 low CD23 low ) and FO B cells (CD19 + CD23 high / CD21 low ) in the spleens of WT and Lama5 / mice at 16 h after i.v. transfer of donor cells. Transfers were performed in the presence or absence of integrin α6 blocking antibody (GoH3). Data shown are mean values ± SEM from at three separate experiments with n = 3 for each treatment in each experiment. ***P < (B) 5 /6 -carboxytetramethylrhodamine (TAMRA)-labeled NF B cells were injected i.v. into WT or Lama5 / recipients and spleens were analyzed at 16 h after injection. Immunofluorescent staining for laminin α5 reveals that in WT mice, TAMRA + NF B cells localize in the MZ (double arrows) but also in the follicle and in the RP, whereas in the Lama5 / mice TAMRA + NF B cells home predominantly to the follicle. Magnification: 100. Bar graph shows quantification of the ratio of MZ/ FO localized TAMRA + cells. Data are means ± SEM from two experiments with four mice in each category. **P < of14

10 Fig. S9. Representative flow cytometry of immature B cells in the bone marrow (BM) (A) and transitional B-cell populations (T1, T2, T3) in the spleen (B) of adult conditional Itga6 / mice and WT littermates. Quantification of T1, T2, and T3 populations are shown in the bar graphs. Data shown are mean values ± SEM from at least three separate experiments with n = 6 for each treatment in each experiment. **P < of 14

11 Fig. S10. Competitive bone marrow reconstitution studies were performed with a 1:1 ratio of CD WT: CD Itga6 / bone marrow cells injected i.v. into WT irradiated mice and subsequent analysis of CD (WT) or CD45.1 (Itga6 / ) cells in pro-pre (B220 + IgM AA4.1 + ), immature (B220 + IgM + AA4.1 + ), and recirculating (B220 + IgM + AA4.1 ) B-cell compartments (A) and CD11b + myeloid cells (B), showing a comparable development of WT and Itga6 / B cells and myeloid cells. Data shown are one representative example. 11 of 14

12 Fig. S11. (A) Qualitative RT-PCR (qrt-pcr) for antiapoptotic (Bcl-XL, Bcl-2) and proapoptoic (Bim) genes in NF, MZ, and FO B cells in the spleens of Lama5 / mice. Data shown are fold differences from WT mice, n = 3 experiments, with triplicates in each. (B) qrt-pcr for Deltex-1 (Dtx-1) in NF, MZ, and FO B cells sorted from the spleens of Lama5 / mice, showing up-regulation in NF B cells. Data shown are fold differences from WT mice, n = 3 experiments. (C) Forward scatter (FSC) of sorted NF, MZ, and FO B cells from the spleens of Lama5 / mice reveals a population of small Deltex-1 negative NF B cells and larger cells of similar to mature MZ B cells that are Deltex-1 positive. Bar graph shows fold differences compared with FO B cells, n = 3 experiments, with triplicates in each. *P < 0.05; **P < of 14

13 Fig. S12. NF and MZ B cells before and after 48-h culture in the presence of medium, laminin 511 (LM 511), LM 511 plus B cell activating factor (BAFF), and BAFF alone. (Left) Flow cytometry shows the CD21/ CD1d profile of the cells before culture. (Right) Flow cytometry shows the proportions of surviving 7AAD neg cells after 48-h culture. Data shown are one representative experiment of three independent experiments performed. Fig. S13. Representative flow cytometry of sorted CD19 + CD21 low CD23 low NF B cells after 48-h culture in the presence of BAFF, laminin 511, agrin, or different combinations thereof, in the presence or absence of GoH3. Bar graphs show quantitative data from all experiments performed; n = 3 experiments, with triplicates in each; *P < 0.05, **P < 0.01, ***P < of 14

14 Table S1. Mouse strain injected Summary of in vivo MZ B-cell dislodgement experiments Antigen specificity of injected antibody/antibodies Displacement of MZ B cells from spleen* Displacement of MZ B cells to circulation WT LFA-1 No No Integrin α4 No No LFA-1 and α4β1 Yes Yes Integrin β1 No No Integrin β3 No No Integrins β1 and β3 No No Integrin β1/β3/lfa-1 No No Integrins α6 and α4 No No Integrins α6 and β3 No No Integrins α6, β3, and LFA-1 No No Laminin α5 No No Laminin α5 and LFA-1 No No Laminin α5 and integrin α4 No No Integrin β2 / α4β1 Yes Yes Integrin β1 No No Integrin β3 No No Integrins β1 and β3 No No Integrin α6 No No Integrins α6, β1, and β3 No No *Normal % MZ B cells in spleen is 5 6%. MZ B cells are normally not found in the circulation. All experiments involved at least six mice per treatment per experiment, and each experiment was repeated at least three times. 14 of 14