PI3 kinase signals BCR-dependent mature B cell survival

Abstract

Previous work has shown that mature B cells depend upon survival signals delivered to the cells by their antigen receptor (BCR). To identify the molecular nature of this survival signal, we have developed a genetic approach in which ablation of the BCR is combined with the activation of specific, BCR dependent signaling cascades in mature B cells in vivo. Using this system, we provide evidence that the survival of BCR deficient mature B cells can be rescued by a single signaling pathway downstream of the BCR, namely PI3K signaling, with the FOXO1 transcription factor playing a central role.

Figure 1. Constitutively active PI3K rescues BCR negative B cells

(A) Schematic representation of the experimental strategy. (B) Representative FACS analysis of spleen and bone marrow lymphocytes. Numbers represent percentages of cells within the lymphocyte gate or of cells in the gate indicated in the FACS plot. Expression of P110* leads to accumulation of BCR^neg mature B cells (B220+, AA4.1−) in the spleen that are GFP positive. The histogram plot in the bone marrow section compares GFP expression in the progenitor gate (B220 low, IgM^neg) and the IgM^neg mature B cell gate (B220 high, IgM^neg) from P110*/+; CD21-cre; B1-8f/J_HT mice. (C) FACS analysis of lymphocytes from peritoneal cavity (PEC, top two rows) and lymph nodes (LN, bottom two rows). (D) Comparison of B cell numbers from the spleen of B1-8f/J_HT (white bars), CD21-cre; B1-8f/J_HT mice (grey bars) and P110*/+; CD21-cre; B1-8f/J_HT mice (filled bars), mean and SD of a minimum of 6 mice per genotype.

Figure 2. P110* rescued IgM^neg B cells are arranged in follicles in the spleen and are similar to IgM^pos B cells

(A) Hematoxylin and Eosin staining of sections of spleen from a wild type C57Bl/6 (B6) and P110*/+; CD21-cre; B1-8f/J_HT (PI3K, Fetal liver chimera) mice. (B) Immuno-fluorescent staining of sections of spleen showing part of a single follicle from B1-8f/J_HT and P110*/+; CD21-cre; B1-8f/J_HT mice. B220, marking all B cells is stained in green, IgM is in red and MOMA1, marking metallophilic macrophages is in blue. (C) Comparison of cell size (FSC), and surface expression of B cell markers B220, CD19, CD23 and CD21 between IgM^pos (grey) and IgM^neg (black) follicular B cells from spleen of CD21-cre; B1-8f/J_HT (top row) and P110*/+; CD21-cre; B1-8f/J_HT (bottom row) mice.

Figure 3. P110* rescued IgM^neg B cells are resting B cells dependent on BAFF for survival

(A) Comparison of surface expression of activation markers on IgM^pos (grey histograms) and IgM^neg (black histograms) mature follicular B cells (gated on B220+, AA4.1− and CD23 high) from the spleen of CD21-cre; B1-8f/J_HT (left column) and P110*/+; CD21-cre; B1-8f/J_HT (right column) mice. (B) Representative FACS plots showing BrdU incorporation in B lymphocytes from spleen after 20 days of BrdU feeding of mice of indicated genotype. The numbers indicate the percentage of BrdU positive or negative cells within the mature B cell fraction (B220+, AA4.1−). (C) Graphical representation of data in B. Also shown is incorporation of Brdu in the IgM^pos mature B cells from CD21-cre; B1-8f/J_HT and P110*/+; CD21-cre; B1-8f/J_HT fetal liver chimeric mice. (D) P110* rescued IgM^neg B cells are dependent on BAFF for survival in vitro. IgM^pos and IgM^neg mature follicular B cells from B1-8f/J_HT (B1-8f), CD21-cre; B1-8f/J_HT (B1-8cre) and P110*/+; CD21-cre; B1-8f/J_HT (P110*) mice were cultured in vitro in the presence (filled markers) or absence (open markers) of BAFF. Data is plotted as percent recovery of viable cells.

Figure 4. Expression of constitutively active IKK2 (IKK2CA) causes limited rescue of IgM^neg B cells

(A) Representative FACS analysis of lymphocytes from Spleens of CD21-cre; B1-8f/J_HT (left column) and IKK2CA/+; CD21-cre; B1-8f/J_HT mice. Numbers represent percentages of cells within the lymphocyte gate or of cells in the gate indicated in the FACS plot. (B) Comparison of B cell numbers from CD21-cre; B1-8f/J_HT (open bars) and IKK2CA/+; CD21-cre; B1-8f/J_HT (filled bars) mice showing limited rescue of IgM^neg B cells by the IKK2CA transgene, mean and SD of 4 mice per genotype. (C) Comparison of rescue of IgM^neg B cells by activation of PI3K signaling (left panel) or canonical NF-kB signaling (right panel) in the spleen. Data is plotted as percent of mature B cells (B220+, AA4.1−).

Figure 5. Deletion of Pten rescues BCR^neg B cells

Representative FACS analysis of lymphocytes from spleen (A), bone marrow (B), inguinal lymph node (iLN, C) and peritoneal cavity (PEC, D) from CD21-cre; B1-8f/J_HT and PtenΔ/f; CD21-cre; B1-8f/J_HT mice showing rescue of IgM^neg mature B cells by Pten deletion. Numbers represent percentages of cells within the lymphocyte gate or of cells in the gate as indicated in the FACS plot. (E) IgM^neg cells rescued by Pten deletion are not activated. Comparison of surface expression of activation markers CD69 and MHC class II on IgM^pos (grey histograms) and IgM^neg (black histograms) cells from the spleen of CD21-cre; B1-8f/J_HT and PtenΔ/f; CD21-cre; B1-8f/J_HT mice. (F) Western blot analysis for Pten (top panel) and Actin (bottom panel) in sorted IgM^pos and IgM^neg mature B cells from spleen of PtenΔ/f; CD21-cre; B1-8f/J_HT mice. (G) Immuno-fluorescent staining of sections of spleen showing a single follicle from PtenΔ/f; CD21-cre; B1-8f/J_HT mice. B220, marking all B cells is stained in green, IgM is in red and MOMA1, marking metallophilic macrophages is in blue.

Figure 6. P110* rescued IgM^neg cells do not up-regulate FOXO targets

Quantitative RT-PCR analysis of FOXO targets in sorted IgM^pos and IgM^neg mature B cells from spleens of CD21-cre; B1-8f/J_HT and P110*/+; CD21-cre; B1-8f/J_HT mice. Transcripts shown include BCL2L11 (A), P27/KIP1 (B), AICDA(C) and Rag1 (D). Lanes as indicated in the figure, mean and SD of triplicate experiment are shown.

Figure 7. Survival of resting B cells through BCR is mediated by PI3K-FOXO pathway

(A) FACS analysis of lymphocytes from spleens of B1-8f/J_HT (left column), CD21-cre; B1-8f/J_HT (middle column) and Foxo1Δ/f; CD21-cre; B1-8f/J_HT mice (right column) showing rescue of IgM^neg mature B cells by deletion of Foxo1. (B) Comparison of splenic B cell numbers of the mice as indicated in the figure, mean and SD of a minimum of 4 mice per genotype are shown. (C) Immuno-fluorescent staining of splenic sections from Foxo1Δ/f; CD21-cre; B1-8f/J_HT mice. (D) FACS analysis of lymphocytes from lymph nodes of the mice as indicated in the figure showing limited rescue of IgM^neg B cells in the lymph node upon deletion of Foxo1. (E) Comparison of surface expression of CD62L on IgM^pos (grey histogram) and IgM^neg (black histogram) B cells from the lymph nodes of mice as indicated in the figure. (F) Western blot analysis of sorted IgM^pos and IgM^neg B cells from mice as indicated in the figure (Lanes 5–12) for Phospho- Akt (Ser473, Top panel), Akt (middle panel) and Actin (bottom panel). Lanes 1–4 represents mature B cells sorted from B1-8f/J_HT mice and contains two fold less protein than lanes 5–12. For a detailed description of this experiment see the Experimental Procedures section.