Fig 1.
Development of B cells in the bone marrow of female and male Ly-6A/Sca-1 deficient mice.
Bone marrow cells from female and male Ly-6A//Sca-1 and wild-type Ly-6A/Sca-1+/+ mice were gated based on their size and density to exclude dead cells (gating strategy shown in S1 Fig) and then analyzed for expression of B220 and IgM as shown in dot bots (upper panels). a) Percentage of developing B cells in the bone marrow of each genotype and sex, gauged by B220 (R6) expression is shown. The data shown is cumulative of at least 5 mice/genotype/gender. b) Percentage of living cells in the bone marrow at various stages of B cell development. Four different stages of B cell development were identified based on expression of B220 and IgM: IgM- and B220low Pre/Pro B cells (R2), IgMhigh B220low immature B cells (R3), IgMhigh B220high re-circulating B cells (R4) and a transitional stage expressing IgMlow B220low between pre B cells and immature B cells (labeled pre-immature) (R5). Data presented as a percentage of live bone marrow cells from sex and genotype combinations. Data represents the mean with SEM. n = 5–7 per genotype/gender.
Fig 2.
Absolute numbers of developing B cells in the bone marrow of female and male Ly-6A/Sca-1-/- mice.
Bone marrow cells from female and male Ly-6A/ Sca-1-/- and wild-type Ly-6A/Sca-1+/+ mice were gated based on their size and density to exclude dead cells (gating strategy shown in S1 Fig) and then analyzed for expression of B220 and IgM (as in Fig 1). a) Total number (x106) of developing B cells in the bone marrow of each genotype and gender, gauged by B220 expression is enumerated. The data shown is cumulative of at least 5 mice/genotype/gender. b) Total number of living cells in the bone marrow at various stages of B cell development. Four stages of B cell development were identified based on expression of B220 and IgM: IgM- and B220low Pre/Pro B cells (R2), IgMhigh B220low immature B cells (R3), IgMhigh B220high re-circulating B cells (R4) and a transitional stage expressing IgMlowB220low between pre B cells and immature B cells (labeled pre-immature) (R5). Data presented as a percentage of live bone marrow cells from sex and genotype combinations. Data represents the mean with SEM. n = 5–7 per treatment. Percentage representation of each of developmental subsets based on the expression of B220 and IgM was similar to what is presented in Fig 1.
Table 1.
B lymphocyte subsets in peritoneum of young (5–8 week) and older (8.5–33 week) old wild-type and Ly-6A/Sca-1-/- mice.
Fig 3.
Anti-cOVA IgG1, IgA and IgG2a antibody response generated in Ly-6A/Sca-1-/-and wild- type female mice.
Wild-type, Ly-6A/Sca-1+/+ (open bars) and Ly-6A/Sca-1knockout (hatched) animals were injected with TiterMax Gold (control), 100 μg OVA mixed with TiterMax Gold (Ova). Serum from blood drawn from the immunized mice on day-21 post-immunization (4 animals/genotype/experimental group) was examined for anti-cOVA antibody of IgG1, IgA and IgG2a isotype antibodies by ELISA. Each sample was tested in duplicate. Data were pooled, and graphs display average optical density (OD) with standard deviation of all samples (n = 4 per group, per genotype).
Fig 4.
Analysis of serum immunoglobulin isotypes.
Serum from Ly-6A/Sca-1-/- and wild-type (WT) female mice was analyzed via Cytometric Bead Array that specifically detect immunoglobulin heavy and light (κ & λ) chains by flow cytometer. A representative bead array dot blot is shown in panel a. Immunoglobulin isotypes with the κ or λ light chain were detected with both the isotype and light chain- specific beads for semi-quantitative analysis using mean fluorescence intensity (MFI). Each dot represents one individual mouse per genotype (b). Seven mice were analyzed per genotype. * denotes statistical significance (IgA λ p = 0.003; IgG2b κ p = 0.04).
Table 2.
λ and κ light chain expression on B lymphocyte in Peyer’s patch of 5–8 week old wild-type and Ly-6A/Sca-1-/- mice.
Fig 5.
Flow cytometry analysis of immunoglobulin light chain expression on B220+ cells in the lymph node, spleen and bone marrow of Ly-6A/Sca-1-/- and Ly-6A/Sca-1+/+ wild-type mice.
a). Percentage of λ and κ light chain positive cells within the B220+ cells from the lymph node is shown. b). Percentage of λ and κ light chain positive cells within the B220+ cells from the spleen is shown. c). Percent λ and κ light chain positive cells within the B220+ cells from the bone marrow is shown. Data represents the mean with SEM. n = 4–5 mice per genotype/gender.
Fig 6.
Detection of IgA (λ & κ) and λ light chain lamina propria of Ly-6A/Sca-1-/- and wild-type C57bl/6 mice.
Ileum from the wild-type and Ly-6A/Sca-1-/- mice were cut with a microtome to obtain 5–8 μm thick sections which were then stained with biotinylated anti-IgA and anti-Ig λ primary antibodies followed by ABC (anti-biotin-biotin complex) kit from Vector Laboratories. Sections were then stained with H2O2 and chromogen Di-Amino Benzedine (DAB) followed by counterstaining with hematoxylin QS (Vector Laboratories). Images of ileum tissue sections were taken at 100X magnification. a) A representative staining for the wild-type and Ly-6A/Sca-1-/- mice is shown. b) Numbers of IgA λ+ light chain clusters. Clusters of cells stained within the ileum tissue sections were counted and data was represented as the average number of clusters from four mice/genotype. One way ANOVA was used to determine statistical significance among the groups (n = 22, F1,21 = 14.489, p = 0.001). Error bars were taken across all tests.