Figure 1.
Reduced numbers of B cells in the Gnai2 fl/flmb1-cre mice and DKO mice.
A. Flow cytometric analysis of lymphocytes in the spleen and LNs of control and mutant mice. Pooled results from analyzing cells prepared from spleens and LNs from 9 Gnai2fl/fl, 8 Gnai2 fl/flmb1-cre, 3 Gnai3-/-, and 3 DKO mice. B. Flow cytometric analysis of lymphocytes in Peyer’s patches and bone marrow of control and mutant mice. Pooled results from analyzing cells from Peyer’s patches and bone marrow from the same mice used in part A. ND- not detected. C. Flow cytometric analysis of B cell populations in the spleen of control and mutant mice. Pooled results from analyzing cells from the spleens prepared from the same mice used in part A. D. Flow cytometric analysis of immature B cell populations in the spleen. The number of pre-B and T0 B cells pooled and compared to T1 B cells. Data is mean +/- SEM. Statistical significance determined by 2 way ANOVA. ** p<0.001, * p<0.05.
Figure 2.
Disturbed B cell development and trafficking in Gnai2 fl/flmb1-cre and DKO mice.
A. Flow cytometric analysis of bone marrow B cells fractions in the control and mutant mice. Pooled results from analyzing bone marrow from at least 6 mice of each genotype. B. Flow cytometric analysis of lymphocytes and B cell population in the blood of control and mutant mice. Pooled results from analyzing blood from 6 Gnai2fl/fl, 4 Gnai2 fl/flmb1-cre, 4 Gnai3-/-, and 6 DKO mice. C. Flow cytometric analysis of B cell populations in the peritoneum of control and mutant mice. Same mice as used in part B. D. Flow cytometric analysis of spontaneous GCs in control and mutant mice. Pooled results from analyzing cells prepared from spleens and LNs from 9 Gnai2fl/fl, 8 Gnai2 fl/flmb1-cre, 3 Gnai3-/-, and 3 DKO mice. Data is mean +/- SEM. Statistical significance determined by 2 way ANOVA. *** p<0.001, ** p<0.01, * p<0.05.
Figure 3.
Impaired BCR signaling and reduced proliferation of DKO B cells to both anti-IgM and LPS.
A. Intracellular calcium response to anti-IgM using control and mutant B cells. Gnai2fl/fl, Gnai2 fl/flmb1-cre, Gnai3-/-, and DKO B cells were stimulated with anti-IgM and the induced changes in intracellular calcium were monitored over 3 minutes, left panel. The data shown as fluorescent counts and the y-axis labeled as iLm1. Each experimental value is the mean of three determinations. Similar experiment comparing the intracellular calcium responses of B cells prepared from Gnai2fl/fl or Gnai2 fl/flmb1-cre mice to increasing concentrations of anti-IgM, right panel. Experiments repeated three times with similar results. B. Flow cytometry results from the analysis of B220, IgD, and IgM expression on spleen B cells from Gnai2fl/fl, Gnai2 fl/flmb1-cre, Gnai3-/-, and DKO B cells. For each genotype B220 expression is shown on cells in the lymphocyte gate and a second plot of IgM versus IgD expression after gating on B220+ cells. Data is representative of multiple experiments. C. Dye dilution assay to assess the initial proliferative potential of wild type and mutant B cells. B cells from control and mutant strains were stained with Pacific blue and then cultured with the indicated stimulants for 96 h. The percentage of cells that divided in each cultured was determined by the analysis of flow cytometry data. The results are from the analysis of B cells from two mice of each genotype. Statistics are from analysis of the data by 2 way ANOVA. ** p<0.001, * p<0.05.
Figure 4.
Gnai2 fl/flmb1-cre B cells migrate poorly to chemoattractants and DKO B cells are refractory.
A. In vitro chemotaxis assay using splenic B cells from control and mutant mice. Splenocytes were from 3 mice of each genotype and the assay was performed in duplicate. At each concentration the specific migration of Gnai2 fl/flmb1-cre and DKO B cells were statistically different from that of wild type mice. The indicated chemokine concentrations are ng/ml. Data shown as mean +/- SEM. B. In vitro chemotaxis assay using splenic B cells immunostained to allow distinction of various B cells subsets from control and Gnai2 fl/flmb1-cre mice. Splenocytes were prepared from 3 mice of each genotype and the assay performed in duplicate. The specific chemokine and concentration (ng/ml) is indicated. At each concentration the specific migration of Gnai2 fl/flmb1-cre B cells was statistically different from that of control mice. Data shown as mean +/- SEM. The p values are from a paired t test comparing results between B cell subsets from control and Gnai2 fl/flmb1-cre mice. C. In vitro chemotaxis assay was performed using partially purified bone marrow B cells immunostained to allow distinction of different developmental B cell fractions from control and DKO mice. The bone marrow cells were prepared from 3 mice of each genotype. Following depletion of Gr1, Ter119, CD11b, Sca1 and c-kit positive cells, the remaining cells were immunostained for B220, CD43, CD24, BP-1, IgM, and IgD and subjected to a chemotaxis assay. Assay performed in triplicate. The indicated concentrations of CXCL12 were added to the bottom wells. Data shown as mean +/- SEM and the statistics performed by 2 way ANOVA. ** p < 0.001.
Figure 5.
Further defects in chemoattractant signaling in the Gnai2 fl/flmb1-cre and DKO mice.
A. Changes in intracellular calcium were monitored over 3 minutes in B cells from control and Gnai2 fl/flmb1-cre mice exposed to increasing concentrations of CXCL12. The data shown as fluorescent counts and the y-axis labeled as iLm1. Each experimental value is the mean of three determinations. Similar results using B cells prepared from 3 mice of each genotype. B. Changes in intracellular calcium were monitored over 3 minutes using B cells from control and mutant mice exposed to CXCL12 or CXCL13. The data shown as fluorescent counts and the y-axis labeled as iLm1. Data from B cells prepared from 2 mice of each genotype. C. In vivo homing assay using B cells from control and mutant mice. B cells from each genotype were fluorescently labeled and adoptively transferred to a wild type recipient. The cells in the blood, spleen, inguinal and axillary LNs (pLN), and the mesenteric LNs were enumerated by flow cytometry. Data shown as mean+/- SEM. Data analyzed by t test. D. Images from multiphoton microscopy of the spleen and inguinal LN following the adoptive transfer of control and DKO B cells. Fluorescently labeled Gnai2fl/fl B cells were adoptively transferred 5 h (blue) and 24 h (red) prior to imaging. The DKO B cells (green) were transferred 5 h prior to imaging. The LN was imaged intravitally while a fresh section was used for the spleen imaging. Evans blue was infused intravenously immediately before the imaging to outline blood vessels. ** p<0.001, * p<0.01.
Figure 6.
Defective antigen induced B cell responses in the Gnai2 fl/flmb1-cre and DKO mice.
A. Flow cytometric analysis to enumerate GC B cells at various sites in control and Gnai2 fl/flmb1-cre mice following sRBC immunization. GC B cells were defined as B220 + CD38-GL7 + Fas+. B. Flow cytometric analysis of TFH cells. TFH cells were defined as CD4+CXCR5 + PD-1+. C. Flow cytometric analysis of antigen induced switched B cells at various sites. The numbers of B220 + CD38-IgG1+ cells were enumerated. D. Flow cytometric analysis of early splenic plasma cells. The numbers of B220 + CD138+ cells were determined. For the above experiments B cells were prepared from the indicated sites from 3 control and 3 Gnai2 fl/flmb1-cre mice and analyzed prior to immunization. Another set of mice were analyzed 9 days after intraperitoneal injection of sRBC. Statistics from an analysis of the data by t test. E. Flow cytometric analysis of antigen induced changes in splenic B cell population in control and DKO mice. Similar experiments as above with the exception that bone marrow plasma cells were assessed. Results are from mice reconstituted with Gnai2fl/fl or DKO bone marrow analyzed prior to and 9 days after sheep RBC immunization. Data is shown as mean +/- SEM and analyzed by t test. *** p<0.001, ** p<0.01, * p<0.05.
Figure 7.
The loss of B cell compartments in the DKO mice.
A. Immunohistochemical and immunofluorescent analysis of slides prepared from control and Gnai2 fl/flmb1-cre mice. Spleen and mesenteric lymph node (bottom right) sections were immunostained as indicated. Immunohistochemistry are 4X images and the immunofluorescent are 10X. Arrowhead delineates marginal zone region (MZ). Scale bars are 50 µm. B. Immunohistochemical analysis of spleen sections prepared from DKO mice. The sections were processed as indicated. Adjacent images 4X and 10X. C. Confocal microscopy using control and DKO mouse spleens immunostained for IgM and IgD. Scale bars are 200 µm. D. Confocal microscopy using sections prepared from the ileum of control or DKO mouse immunostained for Ig. Insert electronically magnified 2.5X. Scale bars are 200 µm. E. Immunohistochemistry of spleen sections from control and Gnai2 fl/flmb1-cre mice (above) or bone marrow re-constituted mice (below) that had been immunized 9 days earlier with sheep RBCs. Sections were immunostained with the indicated antibodies. The IgD/CD35 images were collected with a 4X objective (left) and 10X (right). The images below were collected with a 4X objective. C57/BL6 mice were reconstituted with Gnai2fl/fl or DKO bone marrow 7 weeks prior to immunization. All results are representative of at least 4 mice of the same genotype.
Figure 8.
Hyper-IgM like syndrome in the DKO mice and impaired antibody responses in the Gnai2 fl/flmb1-cre mice.
A. ELISA assay to measure immunoglobulin isotypes in the sera of control and mutant mice. Sera analyzed were from 6 mice for each genotype. Data is shown as mean +/- SEM. Statistics from the analysis of the data using t test. B. ELISA assay measuring specific antibody in the serum of NP-KLH immunized control and Gnai2 fl/flmb1-cre mice. Four mice of each genotype were immunized and serum collected at the indicated time points. Mice were boosted at day 35. Results from individual time points were compared and statistical significance determined by t test. Data shown as mean +/- SEM. C. ELISA assay measuring specific antibody in the serum of NP-Ficoll immunized control and Gnai2 fl/flmb1-cre mice. Four mice of each genotype were immunized and boosted at day 35. Sera collected at the indicated time points. ELISA results from individual time points were compared and statistical significance determined by t test. Data shown as mean +/- SEM. ** p<0.001, * p<0.05.