Fig 2: Normal splenic B‐cell development and function upon hematopoietic deletion of the miR‐497/195 cluster. (A) Gating strategy for the identification of different B‐cell subsets in the spleen. CD19+B220+ B2 B cells were divided into mature (AA4.1−) and immature B cells (AA4.1+). The mature B cells were further split into CD1d+ marginal zone B cells and CD1d− follicular B cells. Immature B cells were gated for the different transitional phases T1 (IgM+CD23−), T2 (IgM+CD23+), and T3 (IgM−CD23+). (B) Bar graphs indicate the mean percentages of the indicated B‐cell population of miR‐497/195+/+ Vav‐Cre control (n ≥ 7) and miR‐497/195fl/fl Vav‐Cre mice (n ≥ 8) within the B2, mature, or immature B‐cell pool. Each dot represents the data derived from one mouse. (C, D) Splenocytes of control (n ≥ 5) or miR‐497/195fl/fl Vav‐Cre mice (n ≥ 6) were labeled with a proliferation dye and stimulated with either anti‐IgM or anti‐CD40 antibodies together with IL‐4 for 72 h. The percentage of proliferated B cells (B220+) was quantified by flow cytometric analysis, and the proliferation index was calculated as the total number of divisions normalized to the number of divided cells (C). (E) Serum immunoglobulin levels for IgM, IgG1, and IgA were measured by ELISA and calculated according to the standard curve. The graph for IgG3 depicts the optical density (OD) as measured by the plate reader (n ≥ 6). (F, G) For immunizations, at least six control and miR‐497/195fl/fl Vav‐Cre mice were injected with TNP‐Ficoll (F) or with NP‐CGG (G) at d0. TNP‐specific IgM or IgG3 levels as well as NP‐specific IgM or IgG1 levels were quantified at the indicated time points by ELISA. Error bars depict the standard deviation of the mean.

Fig 3: ABCB7 is required for peripheral B cell proliferation and class switching.(A) Flow cytometry analysis of IgG1, IgG2a, IgG2b, IgG3, and IgA expression on enriched B220+ CD19+ B cells from wild-type (WT) and CD23-cre ABCB7 conditional knockout (cKO) mice after 4 days in culture conditions that induce class switching to the indicated isotypes. Pseudocolor dot plots are representative of five independent experiments (total of five mice/group). (B) Quantification of the proportion (top) and number (bottom) of cells from (A) that class switched to the indicated antibody isotypes. The reported cell number was derived from flow cytometry live CD19+ cells during analysis. (C) Flow cytometry analysis of carboxyfluorescein diacetate succinimidyl diester (CFSE) dilution in cells from (A). Offset histograms are representative of five independent experiments (total of five mice/group). (D) FlowJo proliferation modeling tool was used to quantify the proliferation index (left) and percentage of undivided cells (right) in cells from (C). (E) Intracellular flow cytometric analysis of Ki-67 expression in proliferating B220+ CD19+ cells after 4 days in culture conditions that induce class switching. Contour plots are representative of three independent experiments (total of three mice/group). Quantification of the percentage of undivided, Ki-67- cells is shown on the graph on the right. (B, D, E) Error bars represent SEM, and p-values are indicated above the data. Statistics were obtained by using an unpaired Student’s t-test.

Fig 4: Phenotype of B cells in p110δE1020K-GL mice. a B cell subsets in the spleen and bone marrow from wild-type, p110δE1020K-GL and p110δD910A mice were analyzed by flow cytometry and representative pseudocolor plots with the mean cell proportion are shown. b In the spleen, the number of B1, marginal zone (MZ) and T1 transitional cells B cells were increased in p110δE1020K-GL mice while follicular B cell numbers were normal. These populations were reduced in p110δD910A mice. Analysis of the bone marrow showed normal pro-B cell numbers in p110δE1020K-GL mice with a reduced number of pre-B cells, immature, transitional and mature B cells. Populations of cells are described as follows: Splenic B cells: Total B cells CD19+B220+, B1 cells CD19+B220+CD23-CD21-, Follicular B cells CD19+B220+CD23+CD21+, Marginal zone B cells CD19+B220+CD23-CD21+, Transitional T1 B cells B220+CD93+IgM+CD23-, Transitional T2 B cells B220+CD93+IgM+CD23+, Transitional T3 B cells B220+CD93+IgM−CD23−; Bone marrow B cells - Immature B cells CD19+B220loIgM+, Mature B cells CD19+B220hiIgM+, Pro-B cells B220+IgM−CD19+CD25−, Pre-B cells B220+IgMCD19+CD25+, Transitional B cells B220+IgD+. (Mean cell numbers are shown; combined data from two independent experiments, wild-type n = 10; p110δE1020K-GLn = 12; p110δD910An = 7. Data-points represent individual animals)

Fig 5: ABCB7 is required for pro-B cell development but not peripheral B cell homeostasis.(A) Flow cytometry analysis of B cell development in bone marrow from wild-type (WT), Mb1-cre ABCB7 conditional knockout (cKO), and CD23-cre ABCB7 cKO mice. Pro-B cells were divided into Hardy fractions as follows: Fr. B (B220+ CD19+ CD43+ BP-1-), Fr. C (B220+ CD19+ CD43+ CD24lo BP-1+), and Fr. C’ (B220+ CD19+ CD43+ CD24hi BP-1+), Fr. D (B220+ CD19+ CD43-/low sIgM-), Fr. E (B220+ CD19+ CD43-/low sIgM+), and Fr. F (B220hi CD19+ CD43-/low sIgM+). Contour plots are representative of six independent experiments (total of 6–11 mice/group). (B) Flow cytometry analysis of splenic B cell populations in WT, Mb1-cre ABCB7 cKO, and CD23-cre ABCB7 cKO mice. Populations were identified by gating on CD19+ splenocytes: transitional type 1 (T1; AA4.1+CD21/35- IgM+ CD23-), transitional type 2 (T2; AA4.1+ CD21/35- IgM+ CD23+), transitional type 3 (T3; AA4.1+CD21/35+IgM+), follicular (FO; AA4.1- CD21/35+ IgM+), and marginal zone (MZ; AA4.1- CD21/35hi IgMhi). Contour plots are representative of seven independent experiments (total of 7–12 mice/group). (C) Graph showing the percentage of total live bone marrow cells for each Hardy fraction in (A). (D) Graph showing absolute cell numbers of splenic B cell populations in (B). (C, D) Lines represent the mean ± SEM. Statistics were obtained by using a one-way ANOVA with Dunnett’s test for multiple comparisons.