Cdc42 is a key regulator of B cell differentiation and is required for antiviral humoral immunity

Abstract

The small Rho GTPase Cdc42, known to interact with Wiskott-Aldrich syndrome (WAS) protein, is an important regulator of actin remodeling. Here, we show that genetic ablation of Cdc42 exclusively in the B cell lineage is sufficient to render mice unable to mount antibody responses. Indeed Cdc42-deficient mice are incapable of forming germinal centers or generating plasma B cells upon either viral infection or immunization. Such severe immune deficiency is caused by multiple and profound B cell abnormalities, including early blocks during B cell development; impaired antigen-driven BCR signaling and actin remodeling; defective antigen presentation and in vivo interaction with T cells; and a severe B cell-intrinsic block in plasma cell differentiation. Thus, our study presents a new perspective on Cdc42 as key regulator of B cell physiology.

Figure 1.

Cdc42 is important for early B cell development in the bone marrow. (A) Genetic approach used to ablate Cdc42 specifically in B cells. (B and C) Bone marrow from WT and Cdc42 KO mice was analyzed by flow cytometry. The gating strategies are shown on the left. B cell progenitors were divided into 3 populations on the basis of CD43 and B220 expression levels (B220⁺CD43⁺, B220⁺CD43⁻, and mature recirculating cells [B220hiCD43⁻]) as shown in B. They were further subdivided on the basis of CD24, BP-1, IgM, and IgD expression levels into populations A(B220⁺CD43⁺CD24⁻BP-1⁻), B(B220⁺CD43⁺CD24⁺BP-1⁻), C(B220⁺CD43⁺CD24⁺BP-1⁺), D(B220⁺CD43⁻IgM⁻IgD⁻), E(B220⁺CD43⁻IgM⁺IgD^−/Int), and nonrecirculating (IgD^hiB220⁺CD43⁻IgM^+/IntIgD⁺) as shown in C. Quantifications are shown in the right-hand column and indicate percentage of cells in the indicated gates. Data were pooled from at least 4 independent experiments with at least 2 mice in each group. (D) B cell progenitors (A–E) were sorted from the bone marrow and protein expression of Cdc42 (first row) and actin (second row) were analyzed by Western blot. Densitometric analysis was used to quantify the signal intensity of Cdc42 normalized to actin and is shown in the chart below. (E) In vivo labeling of bone marrow progenitors. Intravenously injected anti CD45.2-PE was used to distinguish cells in BM sinusoid (PE⁺) from cells in BM parenchyma (PE⁻). The first two columns show a representative example of one WT mouse (blue) and one Cdc42 KO mouse (red). Populations D, E, and E’ are shown. Quantification charts are shown in the two right-hand columns; data were pooled from three independent experiments with five mice in each group. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Figure 2.

Cdc42 is essential to establish the mature B cell compartment. (A) Splenic WT and Cdc42 KO B cells were isolated and protein expression levels of Cdc42 (top row) and actin (bottom row) were analyzed by Western blot. Densitometric analysis was used to quantify the signal intensity of Cdc42 normalized to actin and is shown in the chart below. (B and C) Spleen (B) and inguinal LNs (C) from WT and Cdc42 KO mice were analyzed by flow cytometry. Gated populations are as follows: B cells (first row in B and C, B220⁺), marginal zone B cells (second row, B220⁺CD21^hiCD23^low), and double negative B cells (third row, B220⁺CD21⁻CD24⁻). Quantification charts are shown in the right-hand column and represent percentage of live cells (first row in B and C) or percentage of B220⁺ cells (second and third rows in B). (D–E) Frozen sections from spleens (D) or inguinal LNs (E) from WT and Cdc42 KO mice were stained with antibodies against B220, TCRβ, and F4/80 (D) or CD169 (E), and tiled images were acquired with a confocal microscope. Bars, 800 µm. Data were pooled from 3 experiments with n = 8 WT mice and n = 4 Cdc42 KO mice. Mean and SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.0001; ****, P < 0.0001.

Figure 3.

Cdc42 is essential for antibody responses to NP-KLH. (A) Serum from WT and Cdc42 KO mice was collected and Ig (IgM, IgG1, IgG2b, and IgG2c) levels were measured by ELISA. Data are representative of more than two independent experiments with at least three mice in each group. (B and C) WT and CDc42 KO mice were immunized with NP-KLH in alum. NP₂₃-specific IgM (B) or IgG1, IgG2b, IgG2c, and IgG3 (C) titers were measured by ELISA. Graphs represent NP-specific titers in WT (black dots) or Cdc42-KO (clear dots). (D) ELISA analysis showing affinity maturation of total IgG and IgG1, IgG2b, or IgG2c isoforms in response to NP-KLH in WT and Cdc42 KO mice, expressed as the ratio of NP3 to NP23. Data are representative of 2 independent experiments with at least 3 animals in each group. Mean and SEM. Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.0001; ****, P < 0.00001.

Figure 4.

Cdc42 is essential to achieve responses against viral infection WT and Cdc42 KO mice were infected with Influenza A virus and immune responses were analyzed at day 9 after infection. (A) Influenza-specific antibody titers were measured by ELISA. Serial dilutions of the sera are shown. (B, D, and F) Mediastinal LNs from WT and Cdc42-KO mice were analyzed by flow cytometry 9 d after infection. Plasmablasts (B; IgD^lo CD138^Int), plasma cells (B; IgD^lo CD138^hi), germinal center cells (D, B220⁺CD95⁺GL7⁺) or Tfh cells (F, PD-1⁺CXCR5⁺) are shown. (C–E) Tile images of spleen sections were acquired and show extrafollicular plasma cells (C; intracellular anti-κ staining) or germinal center cells (E; Bcl6⁺ cells). Quantification charts of spleen sections are shown in the right-hand columns of C and E. Bar, 200 µm. Data are representative of three independent experiments with at least 3 mice in each group. (G) Mixed bone marrow chimeras (either 50:50 WT CD45.1:WT CD45.2 or 50:50 WTCD45.1:Cdc42 KO CD45.2) were infected with Influenza virus, and mLNs were analyzed by flow cytometry. Germinal center (B220⁺CD95⁺GL7⁺) and Tfh (PD-1⁺CXCR5⁺) cells are shown. Quantification charts are shown in the right-hand column and indicate the ratio between CD45.1 and CD45.2 cells in each population.

Figure 5.

Cdc42 plays a role in early BCR signaling. (A) Purified splenic Cdc42 KO and WT B cells were settled on TIB120-coated coverslips. Representative scanning electron microscopy images (top row) and direct stochastic optical reconstruction microscopy for IgM nanoscale organization are shown (bottom row). Quantifications were performed on at least 50 cells and are shown in the right-hand columns and show the percentage of cells with more than 30 protrusions and the H function (see text). Data are representative of two independent experiments. (B) Purified splenic WT and Cdc42 KO B cells were stained intracellularly with phalloidin and analyzed by flow cytometry. Data are representative of 3 independent experiments. (C) Purified splenic WT B cells were stimulated with 10 µg.ml⁻¹ anti-IgM F(ab’)₂ for different times and lysed. Nonstimulated lysates incubated with GTPgS (positive) or GDP (negative) were used as controls. Lysates were incubated with GST-PAK beads (binding to Cdc42-GTP) and both total lysate and the bound fraction were analyzed by Western blot. Cdc42-GTP (top row) and actin (bottom row) protein levels are shown. Densitometric analysis was used to quantify the signal intensity of Cdc42-GTP normalized to actin. Data are representative of at least 3 independent experiments. (D and F) Flow cytometry analysis for levels of pSrc and pSyk (D), or pErk and pAkt (F) in purified splenic WT (blue lines) or Cdc42 KO (red lines) B cells at steady state (gray shaded histogram) or 2 min after stimulation with 10 µg ml⁻¹ anti-κ light chain (colored lines). Quantifications are shown in the right column and were calculated as the difference between the geometric mean of the fluorescence between stimulated and nonstimulated samples (ΔMFI). Data are representative of >3 independent experiments. (E) Intracellular Ca²⁺ influx in purified splenic WT (blue lines) or Cdc42 KO (red lines) B cells after stimulation with either anti-IgM F(ab’)₂ (top; 1 µg ml⁻¹ [dotted lines], 10 µg ml⁻¹ [solid lines]) or latrunculin A (bottom; 1 µmol/liter⁻¹). Data are representative of two independent experiments. (G) Western blot analysis showing protein levels of phospho-CD19 (top) and ERK (bottom) in splenic WT or Cdc42 KO B cells after stimulation with 10 µg.ml⁻¹ anti-κ light chain antibody. Densitometric analysis was used to quantify the signal intensity of pCD19 normalized to ERK, referred to the respective controls. Quantification is shown in the chart below. (H) Representative images by total internal reflection microscopy (TIRF) of splenic WT (left column) or Cdc42 KO B cells (right column) settled on planar lipid bilayers loaded with Alexa Fluor 633-streptavidin and anti-κ light chain antibody. Representative images were taken at 1 and 15 min after settling and show the spreading (top row) and contraction (bottom row) phases. Quantification chart in the right-hand column indicates maximum spreading area. Data are representative of 3 independent experiments. (I) Representative TIRF (top row) and SEM (bottom row) of WT (left column) and Cdc42 KO (right column) splenic B cells stimulated on glass coverslips coated with anti-κ light chain antibody for 10 min, before fixation and staining with phalloidin for TIRF or processing for SEM. Quantifications (right column) were performed on >100 cells and indicate circularity index calculated with Image J (top right) and percentage of fully spread cells (bottom right). Data are representative of 3 independent experiments. (J) Internalization of soluble anti-IgM antibody measured by flow cytometry in WT (blue line) or Cdc42 KO splenic B cells (red line) after 15 min of stimulation with biotinylated anti-IgM antibody. Gray shaded histograms indicate basal IgM levels. The right-hand chart shows internalization kinetics. Data are representative of >3 independent experiments. (Bars, 3 µm). Error bars are mean ± SEM. Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.0001; ****, P < 0.00001. All charts are representative of at least 3 independent experiments.

Figure 6.

Cdc42 is essential for antigen presentation. (A) Representative confocal microscopy (with a 63× objective) images showing internalized Alexa Fluor 647 anti-IgM antibody in WT (left) or Cdc42 KO (right) splenic B cells. Bars, 3 µm. Quantification (bottom) indicates percentage of cells with polarized antigen per field of view, each image containing at least 10 cells. Data are representative of 3 independent experiments. (B) Antigen presentation assay showing presentation of Eα peptide on MHCII measured by flow cytometry. Splenic WT (left, blue line) and Cdc42 KO (right, red line) B cells were loaded with beads coated with anti-IgM antibody and Eα peptide and cultured for 3 h (top) or 5 h (bottom). Quantification (right) indicates ratio between mean Eα fluorescence and total MHCII fluorescence. Data are representative of 2 independent experiments. (C) Proliferation profiles of B (left column) and T cells (right column) by flow cytometry. OTII T cells were co-cultured for 3 d with purified WT (blue line) or Cdc42 KO (red line) B cells loaded with anti-IgM- and ovalbumin-coated beads (at the concentrations noted). Data are representative of 2 independent experiments. (D) Representative two-photon microscopy image of a whole fixed popliteal LN after adoptive transfer of HEL-WT B cells (green), HEL-Cdc42 KO B cells (cyan), and OTII T cells (red) into WT mice, and subsequent immunization with Hel- and OVA-coated beads. B cell follicles and T cell area were highlighted in magenta and gray for illustrative purposes. The proportion of cells in B cell follicles versus T cell area were measured and quantification is shown in the chart (bottom right). Bars, 150 µm. Data are representative of 2 independent experiments. (E) Representative image of the visualization of homeostatic movement of WT (green) and Cdc42 KO (cyan) cells by two-photon microscopy. Representative tracks are shown on the image. Quantifications are shown below and indicate the mean speed over the span of the video as well as the mean displacement of cells. Data are representative of 2 independent experiments. (F) Representative images of the whole field of view (left) as well as higher magnification images (7×) of the visualization of B–T cell interactions in explanted LNs by two-photon microscopy. SNARF-1–labeled OTII T cells (red), CFSE-labeled HEL-WT (green), and CTV-labeled MD4⁺ Cdc42 KO (cyan) B cells were adoptively transferred into WT recipients, which were then immunized intra-footpad with Hel- and OVA-coated beads. Popliteal LNs were explanted and imaged 15 h after immunization. Quantifications are shown below and represent the percentage of B cells interacting with T cells over the span of the video, as well as the mean length and the distribution of the contact time of B–T cell interactions. Data are representative of 3 independent experiments. Mean and SEM. Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.0001; ****, P < 0.00001.

Figure 7.

Plasma cell differentiation requires Cdc42. (A–E) CTV-labeled splenic WT and Cdc42 KO B cells were stimulated for 4 d with LPS or CD40L in the presence of IL-4 and IL-5. Flow cytometry was used to measure class switch recombination (IgG1⁺ cells; A), proliferation (CTV dilution; B), and plasma cell differentiation (CD138^hi; C). Secreted IgM in culture supernatants was measured by sandwich ELISA (D). Cultured cells were sorted on the basis of IgG1 and CD138 expression as well as CTV dilution. Cells were then prepared for transmission electron microscopy (TEM). (E) Representative TEM images of IgG1⁺ cells; plasmablasts (IgG1⁻CTV^loCD138^int), and plasma cells (IgG1⁻CTV^loCD138^hi) are shown. Quantitation of percentage of cells with expanded or nonexpanded ER is shown in graphs at the bottom. Data are representative at least 4 experiments with one mouse in each. Mean and SEM. Student’s t test, *, P < 0.05; **, P < 0.01.

Figure 8.

Cdc42 is required to initiate the plasma cell differentiation program. (A) Splenic WT and Cdc42 KO B cells were stimulated with CD40L for 30 min. Western blot analysis shows phosphorylation of Akt, FoxO1, Erk, STAT3, and PAK1. Quantifications shown in charts under blots indicate intensity of proteins relative to total Erk as determined by densitometry. (B and C) qRT-PCR for mRNA of indicated genes of WT or Cdc42 KO B cells stimulated for 0 –2 h (B) or for 4 d (C) with CD40L (B and C) or LPS (C) in the presence of IL-4 and IL-5. mRNA expression was normalized against the levels of carboxypeptidase H (CPH) and is presented as fold increase over the no stimulated condition (B) or cells treated with IL-4 and IL-5 only (C). Data are representative of 3 independent experiments.