Research ArticleHUMORAL IMMUNITY

R-Ras2 is required for germinal center formation to aid B cells during energetically demanding processes

See allHide authors and affiliations

Science Signaling  29 May 2018:
Vol. 11, Issue 532, eaal1506
DOI: 10.1126/scisignal.aal1506
  • Fig. 1 Nonredundant role of R-Ras2 in GC formation.

    (A) Flow cytometry analysis of the frequency of marginal zone (MZ) phenotype (CD21highCD23low) or follicular (CD21lowCD23high) B cells within B220+ splenic B cells from mice of the indicated genotype. Representative two-color contour plots (top) and means of the total number of each B cell population ± SEM (bottom) are from five mice per group. (B) Flow cytometry analysis of the frequency of GL7+CD95+ GC B cells within B220+ splenic B cells from mice of the indicated genotype at 7 days after SRBC immunization. Representative two-color contour plots (left) and means of the GC B cell percentage ± SEM (right) are from at least four mice per group. (C) Flow cytometry analysis of the percentage of GC B cells in Peyer’s patches from nonimmunized mice. Representative two-color contour plots (left) and means of the GC B cell percentage ± SEM (right) are from at least two mice per group. (D) Flow cytometry analysis of the frequency of PD-1+CXCR5+ TFH within CD4+B220 splenic T cells from mice of the indicated genotype at 7 days after SRBC immunization. Representative two-color contour plots (left) and means of the TFH percentage ± SEM (right) are from at least two mice per group. All data are representative of at least three independent experiments. One-way analysis of variance (ANOVA) was applied to compare the different groups. Dunnett’s multiple comparison test was used to derive P values. *P < 0.05, **P < 0.005 by two-tailed Student’s t test. ns, not significant; p.i., postinjection.

  • Fig. 2 B cell–intrinsic role of R-Ras2 in the GC response.

    (A) Flow cytometry analysis of the percentage of GC B cells (GL7+CD95+) within CD19+NP+ B cells in Rras2−/− or WT transgenic B1-8hi mice at 7 days after immunization with NP-CGG. Representative two-color contour plots (left) and means of the TFH cells percentage ± SEM (right) are from at least three mice per group. (B) Enzyme-linked immunosorbent assay (ELISA) analysis of NP-ovalbumin–reactive antibodies in sera taken from Rras2−/− or WT B1-8hi mice at 7 and 14 days p.i. with NP-CGG. Data are means ± SEM from at least two mice per group. (C) Flow cytometry analysis of the frequency of NP antigen-specific IgG1+ GC (B220+NIP+IgG1+CD38) and memory (B220+NIP+IgG1+CD38+) B cells at 12 days after immunization with NP-CGG. Representative two-color contour plots (left) and means of TFH cells percentage ± SEM (right) are from at least two mice per group. (D) ELISA analysis of NP-reactive antibodies IgM, IgG, and IgG1 in sera from mice of the indicated genotypes at different time points after immunization with NP-CGG. Data are means ± SEM from at least two mice per group. (E) Flow cytometry analysis of the frequency of donor CD45.2+ Rras2−/− or WT B cells in host CD45.1+ mice at the indicated time points after immunization with SRBC. Representative two-color dot plots (left) and means of the total numbers of donor cells ± SEM (right) are from at least four mice per group. (F) Flow cytometry analysis of the frequency of CD95+GL7+ GC B cells within donor CD45.2+ Rras2−/− or WT B cells in host CD45.1+ mice at the indicated time points after immunization with SRBC. Representative two-color dot plots (left) and means of total numbers of donor (CD45.2+) and host (CD45.2) cells ± SEM (right) are from at least four mice per group. All data are representative of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-tailed Student’s t test.

  • Fig. 3 R-Ras2–deficient GC B cells predominantly present a DZ B cell phenotype.

    (A) The total number of splenic total B (B220+) and GC B cells (B220+GL7+CD95+) from SRBC-immunized WT and Rras2−/− mice at the indicated time points was determined by flow cytometry. Data are means ± SEM and are from at least three mice per group. (B) Flow cytometry analysis of cell cycle phase in GC B cells (B220+GL7+CD38) from WT and Rras2−/− mice at the indicated times after immunization with SRBC. Representative two-color dot plots (left) and means of the frequency of GC cells in apoptosis (7AAD), G0-G1 (7AADloBrdU), G2-M (7AAD+BrdUlo), and S phase (BrdU+) ± SEM (right) are from at least four mice per group. (C) Flow cytometry analysis of the frequency of centroblasts (CXCR4+CD86) and centrocytes (CXCR4CD86+) within GC B cells in WT and Rras2−/− mice at the indicated times after SRBC immunization. Two-color dot plots (left) are representative of at least seven mice. Quantification of the frequency of centroblasts, centrocytes, and their relative ratio are means ± SEM (right) from three independent experiments. (D) Flow cytometry analysis of cell cycle phase in CXCR4+CD86 centroblasts (left) and CXCR4CD86+ centrocytes (right) within B220+GL7+ CD38 GC B cells from WT and Rras2−/− mice at the indicated times after immunization with SRBC. Quantification of the frequency of cells in apoptosis (7AAD), G0-G1 (7AADloBrdU), G2-M (7AAD+BrdUlo), and S phase (BrdU+) are means ± SEM (right) from at least three mice per group. Data in (A), (B), and (D) are representative of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-tailed Student’s t test.

  • Fig. 4 Defective proliferative response and activation of the PI3K-Akt pathway by BCR or CD40 in the absence of R-Ras2.

    (A) Proliferation of purified naïve B cells of the indicated genotype stimulated for 3 days with anti-IgM or anti-CD40 was measured by 3H-thymidine incorporation. Data are means ± SEM of at least three mice and representative of three independent experiments. cpm, counts per minute. (B and C) Western blot analysis of phosphorylated PDK1, Akt, p70S6K, and S6 at the indicated time points on lysates from purified naïve B cells stimulated with either anti-IgM (B) or anti-CD40 (C). Representative blots (left) and mean quantified densitometry data ± SEM (right) are from three independent experiments. (D) Coimmunoprecipitation of Rras2 with anti-CD40 from lysates of purified total splenic B cells from WT mice and treated with or without anti-CD40 for 5 min. Blots are representative of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-tailed Student’s t test.

  • Fig. 5 Defective activation of the PI3K-Akt pathway in GC B cells in the absence of R-Ras2.

    (A) Flow cytometry analysis of Akt and ERK phosphorylation after either anti-IgM or anti-CD40 stimulation of GL7+CD95+ GC B cells from WT and Rras2−/− mice at 7 days after SRBC immunization. Representative histogram plots (left) and mean fluorescence intensity (MFI) ± SEM are from three mice per group. (B) Flow cytometry analysis of p70S6K phosphorylation after either anti-IgM stimulation of total GC B cells (B220+CD95+CD38), centroblasts (B220+CD95+CD38CXCR4+CD86), or centrocytes (B220+CD95+CD38CXCR4CD86+) from WT and Rras2−/− mice at 7 days after immunization with SRBC. Representative histogram plots (left) and MFI ± SEM are from three mice per group. (C) Flow cytometry analysis of FoxO1 phosphorylation after either anti-IgM or anti-CD40 stimulation of total GC B cells (B220+CD95+GL7+) from WT and Rras2−/− mice at 7 days after immunization with SRBC. Representative histogram plots (left) and MFI ± SEM are from three mice per group. (D) Flow cytometry analysis of total FoxO1 abundance in total GC B cells (B220+CD95+CD38) from WT and Rras2−/− mice at 7 days after immunization with SRBC. Shaded histograms show FoxO1 levels in nonstimulated cells. Dashed histograms show FoxO1 levels in IgM-stimulated cells. Representative histogram plots (left) and normalized MFI ± SEM are from three mice per group. (E) Reverse transcription (RT)–qPCR for the expression of the indicated FoxO1 target genes relative to hypoxanthine phosphoribosyltransferase control in FACS (fluorescence-activated cell sorting)–sorted GC B cells (B220+CD95+GL7+) from WT and Rras2−/− mice at day 7 after SRBC immunization. All data are representative of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-tailed paired Student’s t test.

  • Fig. 6 Impaired mitochondrial function in GC B cells in the absence of R-Ras2.

    (A to C) Microarray analysis of the transcriptomes from WT and R-Ras2–deficient GC B cells isolated at day 7 after SRBC immunization. Differentially expressed transcripts are depicted as green circles on a SAM plot (A). GSEA identified transcripts that correlate with the “GC vs. naïve B cell up-regulation” signature (B). Nine differentially expressed genes were identified and mitochondria-encoded tRNAs are highlighted in red (C). Data are representative of four mice per group. p0, the estimated prior probability that a gene is not differentially expressed; FDR, false discovery rate; d(i), statistics “d” for each gene i. (D) RT-qPCR for the differentially expressed mitochondria-encoded tRNAs in GC B cells from WT and RRas2−/− mice at day 7 after SRBC immunization. Data are means ± SEM from three mice per group. (E) qPCR quantification of mtDNA content in FACS-sorted GC B cells (CD19+CD95+GL7+) from WT and RRas2−/− mice at day 7 after SRBC immunization (pool of four mice per group). Data are means of the ratio of CO1 mitochondrial-encoded DNA to 18S nuclear-encoded DNA ± SEM from three mice per group. (F) RT-qPCR for the indicated mitochondrial genes in FACS-sorted GC B cells (CD19+CD95+CD38) from WT and RRas2−/− mice at day 7 after SRBC immunization. Data are means ± SEM from three mice per group. (G) Representative transmission electron micrographs of WT and R-Ras2–deficient GC B cells (CD19+CD95+GL7+) indicate mitochondria morphology (red arrowheads). Representative micrographs (left) and mean number of number of mitochondria with normal or atypical morphology per cell per section ± SEM (right) are from 13 cells per group. (H) Mitochondrial health and ROS activity in WT and R-Ras2–deficient GC B cells (B220+CD95+CD38) were measured by flow cytometry. The ratio of MMP to MM (left) and fluorescence of ROS-reactive CM-H2DCFDA (right) are means ± SEM from at least three mice per group. All data are representative of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student’s t test.

  • Fig. 7 R-Ras2 deficiency prevents increased mitochondrial respiration upon activation.

    (A) The Oxygen consumption rate (OCR) of WT and R-Ras2–deficient purified B cells stimulated with anti-IgM (left) or anti-CD40 (right) for 24 hours. Sequential addition of the indicated inhibitors was used to define the indicated respiratory parameters. Representative OCR measurements (top) and quantified values as means ± SEM are from an experiment performed in quintuplicate. (B) ATP cell content of WT and Rras2−/− purified B cells in basal conditions or stimulated with anti-IgM for 24 hours. Data are means ± SEM from at least four mice per group. (C) The fluorescence of ATP-sensitive quinacrine was measured by flow cytometry in quinacrine-treated GC (B220+CD95+GL7+) B cells from WT and Rras2−/− mice at day 7 after SRBC immunization. Data are means ± SEM from at least four mice per group. (D) Our proposed model describing the role of R-Ras2 downstream of BCR and CD40 and controlling mitochondrial activity through the PI3K-Akt-mTORC1 pathway. All data are representative of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-tailed Student’s t test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/532/eaal1506/DC1

    Fig. S1. Nonredundant role of R-Ras2 in B cell function.

    Fig. S2. R-Ras2–deficient mice show a defective GC reaction, but GC B cells and TFH cells have normal amounts of surface receptors.

    Fig. S3. Defective activation of the PI3K-Akt pathway in GC B cells in the absence of R-Ras2.

    Fig. S4. GSEA data.

    Fig. S5. R-Ras2 deficiency causes dysfunctional mitochondria.

    Table S1. Antibodies and other materials.

  • Supplementary Materials for:

    R-Ras2 is required for germinal center formation to aid B cells during energetically demanding processes

    Pilar Mendoza, Nuria Martínez-Martín,* Elena R. Bovolenta, Diana Reyes-Garau, Pablo Hernansanz-Agustín, Pilar Delgado, Manuel D. Diaz-Muñoz, Clara L. Oeste, Isabel Fernández-Pisonero, Ester Castellano, Antonio Martínez-Ruiz, Diego Alonso-Lopez, Eugenio Santos, Xosé R. Bustelo, Tomohiro Kurosaki, Balbino Alarcón*

    *Corresponding author. Email: balarcon{at}cbm.csic.es (B.A.); nmartinez{at}cbm.csic.es (N.M.-M.)

    This PDF file includes:

    • Fig. S1. Nonredundant role of R-Ras2 in B cell function.
    • Fig. S2. R-Ras2–deficient mice show a defective GC reaction, but GC B cells and TFH cells have normal amounts of surface receptors.
    • Fig. S3. Defective activation of the PI3K-Akt pathway in GC B cells in the absence of R-Ras2.
    • Fig. S4. GSEA data.
    • Fig. S5. R-Ras2 deficiency causes dysfunctional mitochondria.
    • Table S1. Antibodies and other materials.

    [Download PDF]


    © 2018 American Association for the Advancement of Science

Navigate This Article