(A) Splenic CD4 T (TCRβ⁺CD4⁺), Fo, T2-MZP and MZ B cells (phenotypted as for Fig. 1) from WT mice were purified by FACS. CNR2 gene expression was determined by quantitative real-time PCR. Relative CNR2 expression was calculated from a GAPDH standard curve. Data are cumulative from three independent experiments each comprised of pooled cells from 2–3 mice. (B) CD45.1 WT BM (white bar) was mixed with CD45.2 WT (gray bar) (upper left panel) or CD45.2 CB2^−/− (black bar) (lower panels) BM in a 1:1 (leftt panels) or 1:4 (lower right panel) ratio and transplanted into lethally irradiated µMT mice. After 10–12 weeks of reconstitution the percentages of CD45.1 and CD45.2 cells were determined in the Fo, T2-MZP and MZ B cell populations. Data shown are the mean ± SEM of one representative experiment of three with four-five mice per group. **p <0.01.

RBC and macrophage-depleted splenocytes (1 × 10⁶) from WT or CB2^−/− mice were placed in the upper chamber of a five µm Transwell and allowed to migrate towards medium + vehicle or 2-AG (1–10 µM) in the lower chamber for 3h. Migrated cells were collected from the lower chamber and Fo, T2-MZP and MZ B cells were detected by flow cytometry as for Fig. 1. Migration was measured as the percentage of input cells. Data are shown as the mean ± SEM of five independent experiments. **p <0.01.

(A,B) Anti-CD21-PE (0.25–1 µg) was i.v. administered to WT and CB2^−/− mice and after 20 min splenocytes were isolated and stained to identify T2-MZP and MZ B cells (as for Fig. 1) that had localized to the MZ (CD21PE⁺). (A) Representative histograms show relative frequencies of CD21-PE⁺ (right gate) T2-MZP (left panels) and MZ (right panels) B cells. Background fluorescence was determined by mixing CD45.1⁺ splenic tissue from unmanipulated mice to splenic tissue of CD21-PE-labeled (CD45.2⁺) mice prior to processing and staining. Background fluorescent gates (left gate) were set on CD45.1⁺CD21-PE⁻ T2-MZP or MZ B cells. Numbers on the plots represent the percentage of cells in the corresponding gate. (B) Percentages of CD21-PE⁺ (left panel) and CD21-PE⁻ (right panel) WT (white bar) and CB2^−/− (black bar) T2-MZP and MZ B cells. Data shown are the mean ± SEM of four independent experiments (n=10). (C,D) Peripheral blood from WT and CB2^−/− mice was stained and analyzed for IgM^hiCD21^hi (T2-MZP + MZ) B cells. (C) Representative contour plots showing the relative frequencies of IgM^hiCD21^hi cells in the peripheral blood. Numbers on the plots represent the percentage of cells in the corresponding gate. (D) The absolute number of WT (white bar) and CB2^−/− (black bar) IgM^hiCD21^hi cells/ml in the peripheral blood. Data shown are the mean ± SEM of two independent experiments each with three mice per group. **p <0.01; ***p <0.005

(A, B) Splenocytes from WT and CB2^−/− mice were stained for B220, IgM, CD21, CD23 and CD93 to identify T1 (B220⁺IgM^hiCD21^lo/−CD23⁻CD93⁺), T2 (B220⁺IgM^hiCD21^lo/−CD23⁺CD93⁺), Fo (B220⁺IgM^intCD21^intCD23⁺CD93⁻), T2-MZP (B220⁺IgM^hiCD21^hiCD23⁺), and MZ B cells (B220⁺IgM^hiCD21^hiCD23⁻) by flow cytometry. (A) WT (white bar) and CB2^−/− (black bar) B cells were gated for B220 and the percentage (upper panel) and absolute number (bottom panel) of the indicated B cell subsets is shown. (B) Representative contour plots show relative frequencies of B220-gated IgM^hiCD21^hi (T2-MZP + MZ) B cells (left panel) and T2-MZP and MZ B cells separately (middle panels) from WT (top panels) or CB2^−/− (bottom panels) mice. Numbers on the plots represent the percentage of cells in the corresponding gate. The cumulative percentage and absolute number of T2-MZP and MZ B cells is shown (right panel). (A,B) Data are the mean ± SEM from five independent experiments (n=10 mice/group). (C) Seven µm frozen spleen sections from WT and CB2^−/− mice were stained with anti-IgM (red) and anti-IgD (green) to indicate MZ B cells (IgM^hi, thick arrows) and B cell follicles (IgD^hi, arrowheads), respectively. Magnification is 100x. Data is representative of two independent experiments (n=2). *p <0.05; ***p <0.005.

(A) Splenocytes from WT (white bar) and CB2^−/− (black bar) mice were stained with anti-B220, IgM, CD21, CD23 and CD93 antibodies to identify T2-MZP and MZ B cells (as for Fig. 1). Intracellular staining was performed to determine the percentage of cells containing active-caspase-3. Data shown are the mean ± SEM of two experiments (n=6) (B,C) BrdU (0.8 mg/ml) was administered in the drinking water for 16 h (B) or continuously for nine days (C) to WT and CB2^−/− mice and the percentage of T2-MZP and MZ B cells that incorporated BrdU was analyzed on the indicated days. Data represent a single experiment with three-four mice per group. All data are shown as the mean ± SEM. * p < 0.05. (D) Splenocytes from WT (gray line) and CB2^−/− (black line) mice were stained to identify T2 B cells and loaded with Indo-1 AM for 30 min. After base line establishment, B cells were stimulated with 10 µg of F(ab’)₂ fragment goat anti-mouse IgM in the absence or presence of 100 nM or 1 µM 2-AG. Calcium mobilization was evaluated by measuring the fluorescence ratio of Indo-1 AM (405 nm/530 nm). Two independent experiments of four are shown (n = 4). (E) Splenic Fo, T2-MZP and MZ B cells from WT (white bar) and CB2^−/− (black bar) mice were purified by FACS and the expression of RBP-J, Hes-1 and Deltex-1 genes was quantified by real-time PCR. Relative gene expression was calculated from a GAPDH standard curve. Data are shown as the mean ± SEM of three independent experiments with each containing cells pooled from 2–3 mice.

(A, B) WT and CB2^−/− B cells were labeled with Cell Tracker Violet or CFSE, respectively, mixed in 1:1 ratio (input cells) and adoptively transferred into WT recipients. A fraction of the input cells was labeled for Fo, T2-MZP and MZ B cells (as in Fig. 1) and analyzed by flow cytometry to determine the ratio of CB2^−/− to WT cells in each B cell subset (Input ratio) (A). After 4 h of adoptive transfer, 1 µg of CD21-PE was administered (i.v.) to recipient mice for 20 min to specifically label MZ-residing B cells. Subsequently, spleens were processed and stained to distinguish Fo, T2-MZP and MZ B cells. In each CD21-PE⁺ B cell subset, the proportion of WT (Cell Tracker Violet⁺) and CB2^−/− (CFSE⁺) was determined by flow cytometry. The same assay was also carried out with reversely labeled WT and CB2^−/− B cells, and the data are combined. (A) Representative dot plots show proportions of WT and CB2^−/− cells in the input (upper panel), and recipient (bottom panel) T2-MZP or MZ B populations. (B) Homing ratio of CB2^−/−/WT cells in the CD21-PE⁺ T2-MZP (gray bar) and MZ (black bar) B cell fractions, as calculated by dividing the ratio of CB2^−/−/WT cells post-homing by the input ratio. Data are cumulative from three independent experiments with 3 mice per group. The homing ratio is indicated. (C–E) WT mice were treated (i.v.) with 10 µg of SR144528 (black bar) or vehicle (1% ethanol) (white bar) for 4 h, and B cells from the spleen and blood were analyzed by flow cytometry. (C) Representative contour plots show relative frequencies of B220-gated IgM^hiCD21^hi (T2-MZP + MZ) B cells (left) and T2-MZP and MZ B cells separately (right panels) within B220-gated cells after vehicle (top panels) or SR144528 (bottom panels) treatment. Numbers on the plots represent the percentage of cells in the corresponding gates. (D) Percentage of B220⁺ (upper panel) and the subsequent absolute number (bottom panel) of Fo, T2-MZP and MZ B cell subsets is shown. (E) The absolute number of IgM^hiCD21^hi (T2-MZP + MZ) B cells/ml in the blood. Data shown are the mean ± SEM from two independent experiments each with 3–4 mice per group. *p <0.05; **p <0.01.

(A) Sera was collected from 8–12 week WT (open symbol) and CB2^−/− (closed symbol) mice and total serum IgM, IgG₁, IgG_2b, IgG_2c, IgG₃, IgA levels were measured by ELISA. Each symbol represents a single mouse. (B, C) WT (open symbol) and CB2^−/− (closed symbol) mice were immunized (i.p.) with 30 µg of NP-Ficoll (B). Blood was collected on days 0, 7 and 14 and NP-specific IgM and IgG₃ titers were determined from 1:2 serially diluted sera by ELISA. Data shown are the mean ± SEM of one representative of three independent experiments each with 3–5 mice per group. (C) Spleens were collected 6–7 days post-immunization and NP-specific IgM secreting B cells in the spleen were enumerated by ELISPOT. Data shown are the mean ± SEM of three independent experiments (n = 9). (D) WT (white bar) and CB2^−/− (black bar) mice were immunized (i.p.) with 23 µg of Pneumovax 23. Blood was collected on days 0, 7 and 14 and Pneumovax-specific IgM levels were measured from serially diluted sera (1:25 to 1:1875; O.D. from 1:625 dilution is shown) by ELISA. Data shown are the mean ± SEM of two independent experiments (n = 6). *p <0.05; **p <0.005