The proto-oncogene product Vav is required for receptor clustering, proliferation, and differentiation of T cells, and Vav was identified as a substrate in the TCR and B cell receptor signaling pathway. The role of Vav in B cell responses to Ag challenge in vivo is not known. In this study, we show that Vav regulates B cell proliferation following in vitro activation of Ag receptors, but Vav has no apparent role in CD40-, IL-4-, or LPS-induced B cell activation. Increased degrees of Ag receptor cross-linking can partially reverse the proliferative defect in the anti-IgM response of vav−/− B cells. In vivo, vav−/− mice mounted protective antiviral IgM and IgG responses to infections with vesicular stomatitis virus and recombinant vaccinia virus expressing the vesicular stomatitis virus glycoprotein, which harbor repetitive surface epitopes that directly cross-link the Ag receptor and activate B cells in the absence of T cell help. vav−/− B cells also responded normally to the polyvalent, repetitive hapten Ag trinitrophenyl (TNP)-Ficoll that effectively cross-links B cell receptors. However, vav−/− mice failed to mount immune responses to the nonrepetitive, T cell-dependent hapten Ag (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP)-OVA. These results provide the first genetic evidence on the role of the guanine exchange factor Vav in immune responses to viral infections and antigenic challenge in vivo, and suggest that Vav adjusts the threshold for Ag receptor-mediated B cell activation depending on the nature of the Ag.

The hemopoietic-specific proto-oncogene vav encodes a 95-kDa protein that contains a unique collection of protein interaction motifs, including a calponin homology domain, Dbl homology, and adjacent pleckstrin homology domains, and an SH2 domain flanked by two SH3 domains (1, 2, 3, 4). Vav is rapidly phosphorylated following stimulation of various growth factor receptors and Ag receptors in T and B lymphocytes, and phosphorylated Vav associates with signaling molecules proximal to activated Ag receptors (2, 5, 6). Recent data suggest that Vav functions as a guanine-nucleotide (GDP/GTP) exchange factor for members of the Rho-like small GTPase family members RhoA, Rac1, and CDC42, which regulate cytoskeletal organization and activation of the p21-activated kinase and stress-activated protein kinase/c-Jun N-terminal kinase signaling pathways (7, 8, 9, 10, 11).

Studies in vav−/− mice have shown that vav is essential for TCR capping and actin polymerization in response to Ag receptor activation (12, 13). Moreover, Vav is required for Ag receptor-induced proliferation of B and T cells in vitro and effective T cell selection (12, 13, 14, 15, 16, 17). In T cells, coordinate activation of calcineurin and Vav pathways via the TCR and CD28 is a crucial requisite for IL-2 production, and overexpression of Vav enhances TCR-mediated NF-AT transcriptional activity and IL-2 expression (13, 18, 19, 20). Similar to T cells, Vav is rapidly phosphorylated following Ag receptor activation in B cells. Vav interacts with the B cell costimulatory molecule CD19 and the Bruton’s tyrosine kinase (Btk)3 (21, 22), and it was reported that Vav has an important role in CD19-mediated activation of lipid and protein kinases (23). Moreover, B cells from CD19 mutant mice display reduced Vav tyrosine phosphorylation following IgM ligation (24). Collectively, these observations point to a role of Vav at the interface of Ag-induced receptor signaling and GTPase-controlled actin rearrangements and Ag receptor clustering, and show that Vav is required for normal lymphocyte function (1). The in vivo role of Vav in B cell responses following Ag challenge is not known.

We report in vav−/− mice that Vav expression is important for BCR-induced proliferation, efficient T help-dependent IgG class switching, and Ab responses to T cell-dependent hapten Ags. However, vav−/− mice mount normal B cell responses to T cell-independent repetitive viral and polyvalent hapten Ags, implying that the Vav defect can be overcome by repetitive Ags that effectively cross-link BCR. Moreover, increased degrees of cross-linking can partially reverse the proliferative defect in the anti-IgM response of vav−/− B cells. These results indicate that Vav has an important role in setting the threshold for Ag receptor-mediated stimulation of B lymphocytes.

The generation of mice homozygous for the vav mutation has been described previously (12). Mice were analyzed for the vav mutation using PCR (vav+ allele, sense primer, 5′-ATTAGGACCTGATGGGTGCAGCTT-3′, and antisense primer, 5′-GTCCTCGTCTTCCTGTGCGG-3′; vav allele, sense primer, 5′-AAGCGCCTCCCCTACCCGGT-3′, and antisense primer, 5′-GATGGAGCCCAGTGTGTCTGTATA-3′). If not otherwise stated, all mice used for experiments were between 6 and 10 wk of age and backcrossed to a C57BL/6 background for four generations. Mice were kept under pathogen-free conditions in accordance with guidelines of the Canadian Medical Research Council.

Single cell suspensions from thymi, spleen, mesenteric lymph nodes, and bone marrow from vav−/− and vav+/− mice were prepared as described (25), resuspended in immunofluorescence staining buffer (PBS, 4% FCS, and 0.1% NaN3), and incubated with appropriate Abs. The following mAbs were used: anti-B220 (FITC, PE, or biotinylated); anti-CD19 (biotin labeled); anti-CD43 (FITC labeled); anti-CD25/IL-2Rα (biotinylated); anti-H-2Kb (FITC labeled); anti-CD86 (biotinylated); anti-CD44 (PE labeled); anti-FAS (PE or biotinylated); anti-I-Ab (biotinylated); anti-ICAM-1 (biotinylated); anti-CD23 (biotinylated); anti-CD69 (FITC labeled); anti-CD5 (FITC labeled); anti-sIgM (FITC labeled); anti-sIgD (biotinylated) (all above Abs were from PharMingen, San Diego, CA); and anti-CD40 (FITC labeled; Serotec, Oxford, U.K.). All staining combinations were as indicated in the figure and table legends. Biotinylated Abs were visualized using streptavidin-RED670 (Life Sciences, St. Petersburg, FL). Samples were analyzed using a FACScan (Becton Dickinson, Mountain View, CA).

B cells were purified from vav−/− and vav+/− mice, as described (25). FACS analysis revealed that the remaining cells were >90% sIgM+. Cells were placed into a round-bottom 96-well plate (Costar, Cambridge, MA) in IMDM and activated using soluble intact polyclonal goat anti-mouse IgM (Jackson ImmunoResearch, West Grove, PA), soluble polyclonal anti-IgM F(ab′)2 (Jackson ImmunoResearch), the soluble anti-IgM mAb B7.6, murine rIL-4 (Genzyme, Cambridge, MA), soluble anti-CD40 (Serotec), and LPS (Sigma, St. Louis, MO). For super-cross-linking, different concentrations of rabbit anti-goat Ig (Jackson ImmunoResearch) were bound to the plastic of a 96-well plate (4°C for 12 h), washed three times in PBS, and then incubated with a fixed concentration (5 μg/ml) of goat anti-mouse IgM (see above) for 12 h at 4°C. In addition, the same goat anti-mouse IgM Ab was bound to Sepharose beads. B cells were harvested at 1–4 days after a 12-h pulse with 1 μCi [3H]thymidine/well.

For CD40-, anti-IgM-, LPS-, and IL-4-mediated up-regulation of I-Ab, CD86, ICAM-1, and CD23 (26), purified B cells (>90% sIgM+ B cells) were activated with anti-CD40 (1 μg/ml), anti-IgM (20 μg/ml), anti-CD40 (1 μg/ml) plus anti-IgM (2 μg/ml), mouse rIL-4 (10 U/ml), or LPS (10 μg/ml) in IMDM (10% FCS, 37°C). After 24 h of activation, cells were harvested and double stained with Abs reactive against B220 (PE) and ICAM-1 (biotin), CD23 (biotin), I-Ab (rat IgG, followed by goat anti-rat FITC), or CD86 (biotin). Biotinylated Abs were visualized using streptavidin-RED670, and staining of cells was analyzed using a FACScan.

Mice were immunized with VSV-Indiana (2 × 106 PFU, i.v.) or recombinant vaccinia virus expressing VSV glycoprotein (Vacc-G; 2 × 106 PFU, i.v.). At the indicated time points, sera were collected, and neutralizing IgM and IgG Ab titers were determined, as described (27). In brief, 1/2 dilutions of 40-fold prediluted and heat-inactivated sera were incubated with VSV for 90 min. The presence of remaining infectious virus was determined by incubating the VSV serum samples with fibroblasts for another 24 h. Serum dilutions that reduced the number of viral plaques by 50% were taken as specific titers. IgG titers were determined after preincubation of sera with 2-β-ME, a procedure that eliminates IgM (27).

Mice were immunized with 50 μg of the T-dependent haptenated protein NIP-OVA s.c. at the base of the tail and i.p. (100 μg total per mouse), or with the polyvalent T-independent hapten TNP-Ficoll (10 μg/mouse, i.p.) (28). NIP-specific serum IgG1 and IgG2a titers were determined 8 and 15 days later by ELISA on NIP-BSA-coated plates (Nunc-Immuno Plate; Nunc, Naperville, IL). NIP-OVA and NIP-BSA were kind gifts of A. Rolink (Basel Institute for Immunology, Basel, Switzerland). TNP-specific IgM and IgG3 Ab titers were determined by ELISA on day 0 and days 5 and 7 after immunization (28).

Previously, it has been shown in vav−/−rag−/− blastocyst complementation studies that Vav has a role in the development of conventional and peritoneal CD5+ B1 B lymphocytes (16, 17). Fig. 1 shows that vav−/− mice exhibit normal numbers of B220+sIgM+ B cells and B220+CD43+ B cell precursors in the bone marrow and peripheral lymphoid organs. B cell development also proceeded normally in the bone marrow, as measured by the expression of the early B cell differentiation markers CD25 and heat stable Ag (data not shown). Moreover, splenic B cells from vav−/− mice expressed normal levels of sIgD, CD19, CD23, CD40, CD44, ICAM-1, CD95 (FAS), and H-2Kb on the cell surface (data not shown), implying that Vav has no apparent role in the development of conventional B cells. However, the numbers of peritoneal CD5+ B1 cells were 50–75% reduced in vav−/− mice as compared with vav+/− and vav+/+ littermate controls (data not shown).

FIGURE 1.

B cell development in vav−/− mice. Immunofluorescence analysis of splenic B cells (upper panels) and bone marrow B cell precursors (lower panels). Cells were isolated from 6-wk-old vav+/− and vav−/− mice and stained with anti-B220 PE and anti-CD43 FITC (bone marrow), or anti-B220 PE and anti-sIgM FITC (spleen). Percentages of positive cells within a quadrant are indicated. It should be noted that ∼4–5% of bone marrow B220+CD43 B cells expressed IgM on the cell surface in vav+/− and vav−/− mice. In this experiment, total cell numbers were: vav+/− bone marrow (one femur), 1.2 × 107; vav+/− spleen, 1.4 × 107; vav−/− bone marrow (one femur), 1.4 × 107; vav−/− spleen, 1.3 × 107. One result representative of five experiments is shown.

FIGURE 1.

B cell development in vav−/− mice. Immunofluorescence analysis of splenic B cells (upper panels) and bone marrow B cell precursors (lower panels). Cells were isolated from 6-wk-old vav+/− and vav−/− mice and stained with anti-B220 PE and anti-CD43 FITC (bone marrow), or anti-B220 PE and anti-sIgM FITC (spleen). Percentages of positive cells within a quadrant are indicated. It should be noted that ∼4–5% of bone marrow B220+CD43 B cells expressed IgM on the cell surface in vav+/− and vav−/− mice. In this experiment, total cell numbers were: vav+/− bone marrow (one femur), 1.2 × 107; vav+/− spleen, 1.4 × 107; vav−/− bone marrow (one femur), 1.4 × 107; vav−/− spleen, 1.3 × 107. One result representative of five experiments is shown.

Close modal

To determine the requirement of Vav for B cell activation, we measured proliferation of B cells in response to various stimuli. vav−/− B cells displayed impaired proliferative responses to cross-linking using a polyclonal goat anti-mouse IgM Ab, but responded normally to LPS, IL-4, and anti-CD40 (Fig. 2,A). Whereas vav−/− and vav+/− B cells up-regulated MHC class II (I-Ab) (Fig. 2,B), CD86 (B7.2) (Fig. 2,C), ICAM-1, and CD23 (data not shown) upon activation with anti-CD40, LPS, or IL-4, vav−/− B cells failed to up-regulate these molecules following anti-IgM cross-linking. Stimulation of vav−/− B cells with anti-CD40 plus anti-IgM partially rescued the BCR proliferation defect (Fig. 2,A) and induced up-regulation of MHC class II molecules (Fig. 2 B). Although these results imply that Vav has no apparent role in LPS- and CD40-mediated B cell activation in vitro, our data do not preclude that Vav has an important function in CD40 and LPS signaling. Importantly, Vav expression is required for cell cycle progression and MHC class II expression following BCR engagement.

FIGURE 2.

B cell stimulation in vav−/− mice. A–C, Activation of splenic B cells. Purified splenic B cells (105/well) from vav−/− and vav+/− littermate mice were seeded in medium containing no added stimulus (control), soluble polyclonal anti-IgM Ab (20 μg/ml), soluble anti-CD40 (1 μg/ml), IL-4 (10 U/ml), soluble anti-IgM Ab (2 μg/ml) plus soluble anti-CD40 (1 μg/ml), and LPS (10 μg/ml). After 24 h, the cells were pulsed for 12 h with 1 μCi [3H]thymidine/well (A), and stained for surface expression of I-Ab and CD86 (B and C). [3H]Thymidine uptake of triplicate cultures is shown in cpm ± SD. I-Ab and CD86 surface expression of triplicate samples is shown as mean fluorescence of anti-I-Ab and anti-CD86 Ab staining ± SD. One result representative of three experiments is shown. D–F, Super-cross-linking of the B cell Ag receptor. Purified splenic B cells (105/well) from vav−/− and vav+/− littermate mice were seeded in medium containing no added stimulus (Control), LPS (2 μg/ml) as positive control, different concentrations of soluble polyclonal anti-IgM F(ab′)2 Abs, and different concentrations of the soluble anti-IgM mAb B7.6 (D); or seeded in medium containing no added stimulus (Control), different doses of polyclonal intact anti-IgM coupled to beads (anti-IgM-beads), and plate-bound polyclonal intact goat anti-IgM (5 μg/ml) super-cross-linked with different concentrations of rabbit anti-goat Ig (IgM-X-link) (E and F). [3H]Thymidine uptake of triplicate cultures is shown in cpm ± SD after 48-h (D and E) and 72-h (F) culture periods. One result representative of five experiments is shown.

FIGURE 2.

B cell stimulation in vav−/− mice. A–C, Activation of splenic B cells. Purified splenic B cells (105/well) from vav−/− and vav+/− littermate mice were seeded in medium containing no added stimulus (control), soluble polyclonal anti-IgM Ab (20 μg/ml), soluble anti-CD40 (1 μg/ml), IL-4 (10 U/ml), soluble anti-IgM Ab (2 μg/ml) plus soluble anti-CD40 (1 μg/ml), and LPS (10 μg/ml). After 24 h, the cells were pulsed for 12 h with 1 μCi [3H]thymidine/well (A), and stained for surface expression of I-Ab and CD86 (B and C). [3H]Thymidine uptake of triplicate cultures is shown in cpm ± SD. I-Ab and CD86 surface expression of triplicate samples is shown as mean fluorescence of anti-I-Ab and anti-CD86 Ab staining ± SD. One result representative of three experiments is shown. D–F, Super-cross-linking of the B cell Ag receptor. Purified splenic B cells (105/well) from vav−/− and vav+/− littermate mice were seeded in medium containing no added stimulus (Control), LPS (2 μg/ml) as positive control, different concentrations of soluble polyclonal anti-IgM F(ab′)2 Abs, and different concentrations of the soluble anti-IgM mAb B7.6 (D); or seeded in medium containing no added stimulus (Control), different doses of polyclonal intact anti-IgM coupled to beads (anti-IgM-beads), and plate-bound polyclonal intact goat anti-IgM (5 μg/ml) super-cross-linked with different concentrations of rabbit anti-goat Ig (IgM-X-link) (E and F). [3H]Thymidine uptake of triplicate cultures is shown in cpm ± SD after 48-h (D and E) and 72-h (F) culture periods. One result representative of five experiments is shown.

Close modal

Because Vav regulates receptor clustering in T and B cells (12, 13), we analyzed whether the defect of BCR-mediated activation could be reversed by increased degrees of cross-linking. Indeed, increased doses of the polyclonal goat anti-mouse IgM F(ab′)2 Ab could partially restore proliferation in vav−/− B cells (Fig. 2,D). Increased doses of the anti-IgM mAb B7.6 could not restore proliferation (Fig. 2,D), suggesting that enhanced cross-linking via polyclonal anti-IgM Abs, but not a monoclonal anti-IgM Ab, can overcome the proliferative defect in vav−/− B cells. Importantly, super-cross-linking of an anti-IgM Ab increased proliferation of vav−/− B cells in a dose-dependent fashion. In addition, increased degrees of cross-linking using different doses of anti-IgM coupled to beads enhanced proliferation of vav−/− B cells (Fig. 2, E and F). Similar to negative regulation of B cell proliferation in wild-type and vav+/− B cells using the intact goat anti-mouse anti-IgM Ab, super-cross-linking of the goat anti-mouse IgM F(ab′)2 Ab induced significantly higher proliferation in vav−/− B cells than super-cross-linking of the intact goat anti-mouse IgM Ab, indicating that this mechanism of negative regulation is still operational in vav−/− B cells. These data show that increased degrees of cross-linking can partially reverse the proliferative defect in the anti-IgM response of vav−/− B cells.

To examine the requirement for Vav in B cell responses in vivo, we challenged vav−/− and vav+/− mice with VSV, which has highly repetitive surface epitopes (29). VSV infections are controlled exclusively by neutralizing Abs (30). All neutralizing Abs are directed against the VSV glycoprotein that is present in a highly repetitive form in the viral envelope. Due to this repetitiveness, neutralizing IgM Abs are induced in complete absence of T cell help (31). However, the isotype switch from IgM to IgG is Th cell dependent (32, 33). Moreover, production of VSV-neutralizing IgG Abs and the formation of VSV-specific germinal centers are dependent on CD28 expression (25, 34). In vav+/− mice, VSV infections induced rapid, T cell-independent IgM production, followed by a Th cell and CD28 costimulation-dependent IgG response (Table I). The T cell-independent IgM response induced by VSV was not affected by the absence of Vav, indicating that efficient cross-linking mediated by highly repetitive Ags can overcome the defect in vav−/− B cells. However, the T cell-dependent VSV-specific IgG response was reduced in vav−/− mice, indicating that the VSV-specific Th cell response is partially impaired in the absence of Vav expression. This IgG response was protective, because mice survived for more than 4 wk after VSV infection (data not shown). Whereas VSV-specific germinal centers were completely absent in CD28−/− mice after challenge with VSV (25), vav−/− mice developed germinal centers with normal morphology and normal distribution of T cells, B cells, macrophages, and follicular dendritic cells (data not shown).

Table I.

Neutralizing anti-VSV response in vav−/− mice

GenotypeTiters of Neutralizing Activities (log2)a
Day 4Day 6Day 21, IgG
IgMIgGIgMIgG
vav+/− 11 11 10 
 12 12 10 
vav−/− 11 
 10 
CD28−/− ND 
 ND 
GenotypeTiters of Neutralizing Activities (log2)a
Day 4Day 6Day 21, IgG
IgMIgGIgMIgG
vav+/− 11 11 10 
 12 12 10 
vav−/− 11 
 10 
CD28−/− ND 
 ND 
a

Sera were isolated from mice after i.v. infection with VSV (2 × 106 pfu). Neutralizing IgM and IgG titers were determined as described in Materials and Methods. Titers represent 2-fold dilution steps of sera starting with 1:40.

VSV glycoprotein (VSV-G) in the viral envelope behaves as a T cell-independent type 1 Ag due to its high degree of organization (31). To assess whether vav−/− B cells could also be stimulated by a less repetitive form of VSV-G, vav+/− and vav−/− littermate mice were immunized with a recombinant vaccinia virus expressing VSV-G (Vacc-G). This form of VSV glycoprotein has been shown to act as a type 2 T cell-independent Ag (29, 30). As observed after infection with VSV, vav−/− mice mounted normal VSV-G-specific IgM responses after immunization with Vacc-G (Table II). The T cell-dependent VSV-G IgG response was reduced significantly in vav−/− mice, albeit clearly detectable (Table II). Moreover, immunization with the type 2 T cell-independent hapten TNP-Ficoll, a polyvalent Ag that can effectively cross-link the BCR (28), showed that the levels and kinetics of anti-TNP-specific IgM and IgG3 production are comparable among vav+/− and vav−/− mice, albeit slightly lower in vav−/− mice (Fig. 3). Our in vivo results show that vav−/− B cells are able to respond to repetitive type 1 and type 2 T cell-independent viral and hapten Ags. However, Th cell-dependent neutralizing IgG responses to viral Ags are reduced in vav−/− mice.

Table II.

Neutralizing Vacc-G response in vav−/− mice

GenotypeTiters of Neutralizing Activities (log2)a
Day 6Day 8Day 21, IgG
IgMIgGIgMIgG
vav+/− 
 
vav−/− 
 
GenotypeTiters of Neutralizing Activities (log2)a
Day 6Day 8Day 21, IgG
IgMIgGIgMIgG
vav+/− 
 
vav−/− 
 
a

Sera were isolated from mice after i.v. infection with recombinant vaccinia virus expressing VSV-G (2 × 106 pfu). Neutralizing IgM and IgG titers were determined as described in Materials and Methods. Titers represent 2-fold dilution steps of sera starting with 1:40.

FIGURE 3.

vav−/− mice respond to the polyvalent hapten TNP-Ficoll. vav+/− (○) and vav−/− (▴) mice were immunized with 10 μg TNP-Ficoll (i.p.). TNP-specific IgM (A) and TNP-specific IgG3 (B) responses are shown in arbitrary units at day 5 and day 7 following initial immunization. The mean values and SDs of this experiment were vav+/−, n = 5; vav−/−, n = 4. Day 0: vav+/−, IgM = 6.1 ± 1.4; vav−/−, IgM = 18.1 ± 5.2; vav+/−, IgG3 < 1; vav−/−, IgG3 < 1. Day 5: vav+/−, IgM = 108 ± 36; vav−/−, IgM = 62.5 ± 38; vav+/−, IgG3 = 73.1 ± 23.3; vav−/−, IgG3 = 7.2 ± 6.2. Day 7: vav+/−, IgM = 124.2 ± 37; vav−/−, IgM = 67 ± 36; vav+/−, IgG3 = 474 ± 160; vav−/−, IgG3 = 48.5 ± 44. The differences in IgG3 production were statistically significant between the vav+/− and vav−/− groups on days 5 and 7 (Student’s t test, p < 0.05). One result representative of three experiments is shown.

FIGURE 3.

vav−/− mice respond to the polyvalent hapten TNP-Ficoll. vav+/− (○) and vav−/− (▴) mice were immunized with 10 μg TNP-Ficoll (i.p.). TNP-specific IgM (A) and TNP-specific IgG3 (B) responses are shown in arbitrary units at day 5 and day 7 following initial immunization. The mean values and SDs of this experiment were vav+/−, n = 5; vav−/−, n = 4. Day 0: vav+/−, IgM = 6.1 ± 1.4; vav−/−, IgM = 18.1 ± 5.2; vav+/−, IgG3 < 1; vav−/−, IgG3 < 1. Day 5: vav+/−, IgM = 108 ± 36; vav−/−, IgM = 62.5 ± 38; vav+/−, IgG3 = 73.1 ± 23.3; vav−/−, IgG3 = 7.2 ± 6.2. Day 7: vav+/−, IgM = 124.2 ± 37; vav−/−, IgM = 67 ± 36; vav+/−, IgG3 = 474 ± 160; vav−/−, IgG3 = 48.5 ± 44. The differences in IgG3 production were statistically significant between the vav+/− and vav−/− groups on days 5 and 7 (Student’s t test, p < 0.05). One result representative of three experiments is shown.

Close modal

To further analyze the role of Vav in B cell responses to nonrepetitive T cell-dependent Ags, vav+/− and vav−/− mice were immunized with the T cell-dependent hapten NIP conjugated to OVA (NIP-OVA). Whereas vav+/− mice exhibit high titers of anti-NIP-specific IgG1 and IgG2a Abs, IgG1 and IgG2a Ab responses to NIP were absent in vav−/− mice (Fig. 4). In addition, germinal center formation was not observed in vav−/− mice following challenge with NIP-OVA (data not shown). These data show that vav−/− mice can mount biologically relevant responses against VSV and recombinant vaccinia VSV-G, and that Vav has no crucial role in B cell responses to the polyvalent hapten Ag TNP-Ficoll. However, Vav expression is required to generate functional B cell responses to nonrepetitive hapten Ags in vivo.

FIGURE 4.

Impaired responses to the T cell-dependent hapten NIP-OVA. A–D, vav−/− and vav+/− littermate mice were immunized with NIP-OVA, and serum IgG1 and IgG2a titers were determined 8 and 15 days later by ELISA on NIP-BSA-coated plates. Arbitrary units of OD of NIP-specific IgG1 and IgG2a titers are shown for individual mice. Titers represent 2-fold dilutions of sera starting from 1/80. One result representative of two experiments is shown.

FIGURE 4.

Impaired responses to the T cell-dependent hapten NIP-OVA. A–D, vav−/− and vav+/− littermate mice were immunized with NIP-OVA, and serum IgG1 and IgG2a titers were determined 8 and 15 days later by ELISA on NIP-BSA-coated plates. Arbitrary units of OD of NIP-specific IgG1 and IgG2a titers are shown for individual mice. Titers represent 2-fold dilutions of sera starting from 1/80. One result representative of two experiments is shown.

Close modal

Previously, it has been shown in vav−/−rag−/− blastocyst complementation studies that Vav has a role in the development of conventional B cells and peritoneal CD5+ B1 B lymphocytes (16, 17). Our results in viable vav−/− mice demonstrate that Vav is dispensable for B cell differentiation and pre-BCR-driven expansion. Stimulation of vav−/− B cells by LPS, CD40, or IL-4 was normal. However, peripheral B cells from vav−/− mice exhibited an impaired proliferative response to IgM ligation, indicating a critical role for Vav in Ag receptor signaling. In contrast to the normal development of conventional B cells, numbers of unconventional CD5+ B1 cells in the peritoneal cavity were reduced significantly in vav−/− mice. This reduction could be a direct consequence of reduced Ag receptor-mediated signaling in the absence of Vav, because the size of the B1 cell population is dependent on their capacity for self-renewal (35). A significant reduction in the B1 B cell population has also been observed in CD19-deficient (36, 37) and Btk-deficient (38, 39) mice. Both CD19 and Btk interact with Vav (21, 22), and it was reported that Vav has an important role in CD19-mediated activation of lipid and protein kinases (23).

Importantly, vav−/− B cells display impaired proliferation and up-regulation of surface MHC class II molecules following IgM stimulation in vitro and vav null mice do not respond to the nonrepetitive hapten NIP-OVA in vivo. However, vav−/− mice mount a protective immune response to viral infections, and vav−/− B cells respond to viral and haptenated Ags that have repetitive, polyvalent structures. We have reported previously that Vav has no apparent role in TCR-mediated signaling pathways such as overall tyrosine phosphorylation, mitogen-activated protein kinase, and stress-activated protein kinase/c-Jun N-terminal kinase activation (12, 13). However, Vav was found to associate with the cytoskeletal membrane anchors Talin and Vinculin and to coordinate recruitment of the actin cytoskeleton to the Ag receptor complex. Consistent with a role for Vav in transducing Ag receptor signals to the actin cytoskeleton, vav−/− mice T cells displayed impaired actin polymerization in response to Ag receptor activation and exhibit defective clustering (patching and capping) of the TCR (12, 13). Moreover, gene-targeted mice with a mutation in the Wiskott-Aldrich syndrome protein (WASP), a cytoskeletal protein that associates with the Vav target CDC42 and regulates cytoskeletal reorganization, display a T cell phenotype similar to vav−/− mice, i.e., wasp−/− T cells exhibit impaired TCR capping, proliferation, and IL-2 production following TCR stimulation (40). Based on these results, it has been suggested that TCR-mediated cytoskeletal reorganization and receptor clustering are crucial prerequisites for T cell maturation, IL-2 production, and cell cycle progression. Cytoskeletal rearrangements and formation of caps probably relocate the signaling machinery to the site of receptor engagement and thus organize compartmentalized, actin-scaffolded signaling highways (12).

Similar to T cells, Ab-mediated cross-linking of the BCR on B lymphocytes induces the formation of cap structures localizing at one pole of the cell, and formation of the caps is partially dependent on Vav expression (13). Although the functional relevance of cap formation is equivocal in B cells, IgM-associated Igα together with Lyn and Syk translocate to the membrane skeleton following BCR cross-linking (41, 42) and p21ras has been shown to co-cap with surface Ig molecules in mouse splenic B lymphocytes (43). A potential role of BCR caps in the generation of B cell responses is in line with our findings that increased degrees of Ag receptor cross-linking can partially reverse the proliferative defect in the anti-IgM response of vav−/− B cells in vitro, and that vav−/− B cells can be activated in vivo with repetitive Ags that effectively cross-link BCR. However, vav−/− B cells do not respond to nonrepetitive hapten Ags and low doses of anti-IgM Ab stimulation. Strong antigenicity of repetitive Ags has been described previously, and B cell responses against these molecules normally do not require T cell help (31). By contrast, B cell responses to nonrepetitive weak Ags and Ig class switching are Th cell dependent.

The impaired responses of vav−/− mice to the hapten NIP-OVA could be due to a defect in T cell help and/or an intrinsic defect in BCR-mediated stimulation. We have shown previously that peripheral T cells from vav−/− mice have a defect in IL-2 production and cell cycle progression following TCR activation (12). Thus, the defective response of vav−/− B cells to the T cell-dependent hapten Ag NIP-OVA and reduced T cell-dependent Ig class switching following VSV and Vacc-G infections in vav−/− mice can be attributed to compromised T cell help. However, vav−/− mice can mount protective Th cell-dependent IgG responses to VSV and Vacc-G infections, indicating that T cell help must be, at least in part, functional in the absence of Vav. Moreover, vav−/− B cells have an impaired response to IgM cross-linking in vitro, indicating that Vav has a direct role in BCR-mediated activation. The relative importance of the Vav deficiency in B and T cells in vivo needs to be further examined using adoptive transfer experiments. Recently, it has been shown that Vav regulates CD19-mediated PIP5 kinase activation in B cells (23). Interestingly, in vivo immune responses of CD19−/− mice resemble immune responses in vav−/− mice, i.e., CD19−/− mice exhibit nearly normal Ig responses following infections with repetitive VSV, but impaired B cell responses to challenge with nonrepetitive LCMV (44). These results suggest that the positive regulatory B cell coreceptor CD19 and Vav mediate similar signaling pathways required for B cell activation in vivo.

We report that the splenic B cells lacking the guanine-nucleotide exchange factor Vav do not respond to IgM cross-linking and do not respond to challenge with the T cell-dependent hapten NIP-OVA. By contrast, vav−/− mice mounted protective antiviral IgM and IgG responses to infections with VSV and Vacc-G, which harbor repetitive surface epitopes that directly cross-link the Ag receptor and activate B cells in the absence of T cell help. vav−/− B cells also responded normally to the polyvalent, T cell-independent hapten Ag TNP-Ficoll in vivo. Increased degrees of Ag receptor cross-linking can partially reverse the proliferative defect in the anti-IgM response of vav−/− B cells. These results suggest that Vav has an important role in setting the threshold for Ag receptor-mediated stimulation of T and B lymphocytes depending on the nature of the Ag.

We thank C. Paige and A. Rolink for reagents and M. Nghiem, K. Bachmaier, A. Hakem, and L. Zhang for critical comments. We also thank Christiane Ruedel and Manfred Kopf for critically reading the manuscript. The Basel Institute for Immunology was founded and is supported by F. Hoffmann-LaRoche (Basel, Switzerland).

1

J. M. P. is supported by the Medical Research Council (MRC) of Canada.

3

Abbreviations used in this paper: Btk, Bruton’s tyrosine kinase; BCR, B cell receptor; LCMV, lymphocytic choriomeningitis virus; NIP, (4-hydroxy-5-iodo-3-nitrophenyl)acetyl; sIg, soluble Ig; TNP, trinitrophenyl; VSV, vesicular stomatitis virus; Vacc-G, recombinant vaccinia virus expressing the VSV glycoprotein; VSV-G, VSV glycoprotein.

1
Fischer, K.-D., K. Tedford, J. M. Penninger.
1993
. Vav links antigen-receptor signaling to the actin cytoskeleton.
Semin. Immunol.
260
:
358
2
Collins, T. L., M. Deckert, A. Altman.
1997
. Views on Vav.
Immunol. Today
18
:
221
3
Katzav, S., Z.-D. Martin, M. Barbacid.
1989
.
vav, a novel human oncogene derived from a locus ubiquitously expressed in hematopoietic cells. EMBO J.
8
:
2283
4
Henning, S. W., D. A. Cantrell.
1998
. GTPases in antigen receptor signalling.
Curr. Opin. Immunol.
10
:
322
5
Margolis, B., P. Hu, S. Katzav, W. Li, J. M. Oliver, A. Ullrich, A. Weiss, J. Schlessinger.
1992
. Tyrosine phosphorylation of vav proto-oncogene product containing SH2 domain and transcription factor motifs.
Nature
356
:
71
6
Bustelo, X. R., J. A. Ledbetter, M. Barbacid.
1992
. Product of vav proto-oncogene defines a new class of tyrosine protein kinase substrates.
Nature
356
:
68
7
Knaus, U. G., S. Morris, H. J. Dong, J. Chernoff, G. M. Bokoch.
1995
. Regulation of human-leukocyte P21-activated kinases through G-protein-coupled receptors.
Science
269
:
221
8
Tapon, N., A. Hall.
1997
. Rho, Rac and Cdc42 GTPases regulate the organization of the actin cytoskeleton.
Curr. Opin. Cell Biol.
9
:
86
9
Crespo, P., K. E. Schuebel, A. A. Ostrom, J. S. Gutkind, X. R. Bustelo.
1997
. Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product.
Nature
385
:
169
10
Han, J. W., B. Das, W. Wei, L. Vanaelst, R. D. Mosteller, R. Khosravifar, J. K. Westwick, C. J. Der, D. Broek.
1997
. Lck regulates Vav activation of members of the Rho-family of GTPase.
Mol. Cell. Biol.
17
:
1346
11
Crespo, P., X. R. Bustelo, D. S. Aaronson, O. A. Coso, M. Lopezbarahona, M. Barbacid, J. S. Gutkind.
1996
. Rac-1 dependent stimulation of the JNK/SAPK signaling pathway by Vav.
Oncogene
13
:
455
12
Fischer, K. D., Y. Y. Kong, H. Nishina, K. Tedford, L. E. M. Marengere, I. Kozieradzki, T. Sasaki, M. Starr, G. Chan, S. Gardener, M. P. Nghiem, D. Bouchard, M. Barbacid, A. Bernstein, J. M. Penninger.
1998
. Vav is a regulator of cytoskeletal reorganization mediated by the T-cell receptor.
Curr. Biol.
8
:
554
13
Holsinger, L. J., I. A. Graef, W. Swat, T. Chi, D. M. Bautista, L. Davidson, R. S. Lewis, F. W. Alt, G. R. Crabtree.
1998
. Defects in actin-cap formation in Vav-deficient mice implicate an actin requirement for lymphocyte signal transduction.
Curr. Biol.
8
:
563
14
Fischer, K. D., A. Zmuldzinas, S. Gardner, M. Barbacid, A. Bernstein, C. Guidos.
1995
. Defective T-cell receptor signalling and positive selection of Vav-deficient CD4+CD8+ thymocytes.
Nature
374
:
474
15
Turner, M., P. J. Mee, A. E. Walters, M. E. Quinn, A. L. Mellor, R. Zamoyska, V. L. J. Tybulewicz.
1997
. A requirement for the Rho-family GTP exchange factor Vav in positive and negative selection of thymocytes.
Immunity
7
:
451
16
Zhang, R., F. W. Alt, L. Davidson, S. H. Orkin, W. Swat.
1995
. Defective signalling through the T- and B-cell antigen receptors in lymphoid cells lacking the vav proto-oncogene.
Nature
374
:
470
17
Tarakhovsky, A., M. Turner, S. Schaal, P. J. Mee, L. P. Duddy, K. Rajewsky, V. L. Tybulewicz.
1995
. Defective antigen receptor-mediated proliferation of B and T cells in the absence of Vav.
Nature
374
:
467
18
Wu, J., D. G. Motto, G. A. Koretzky, A. Weiss.
1996
. Vav and Slp-76 interact and functionally cooperate in IL-2 gene activation.
Immunity
4
:
593
19
Raab, M., A. J. da Silva, P. R. Findell, C. E. Rudd.
1997
. Regulation of Vav-SLP-76 binding by ZAP-70 and its relevance to TCR ζ/CD3 induction of interleukin-2.
Immunity
6
:
155
20
Tuosto, L., F. Michel, O. Acuto.
1996
. p95vav associates with tyrosine-phosphorylated SLP-76 in antigen-stimulated T cells.
J. Exp. Med.
184
:
1161
21
Guinamard, R., M. Fougereau, P. Seckinger.
1997
. The SH3 domain of Bruton’s tyrosine kinase interacts with Vav, Sam68 and EWS.
Scand. J. Immunol.
45
:
587
22
Weng, W. K., L. Jarvis, T. W. LeBien.
1994
. Signaling through CD19 activates Vav/mitogen-activated protein kinase pathway and induces formation of a CD19/Vav/phosphatidylinositol 3-kinase complex in human B cell precursors.
J. Biol. Chem.
269
:
32514
23
O’Rourke, L. M., R. Tooze, M. Turner, D. M. Sandoval, R. H. Carter, V. L. J. Tybulewicz, D. T. Fearon.
1998
. CD19 as a membrane-anchored adaptor protein of B lymphocytes: costimulation of lipid and protein kinases by recruitment of Vav.
Immunity
8
:
635
24
Sato, S., P. J. Jansen, T. F. Tedder.
1997
. CD19 and CD22 expression reciprocally regulates tyrosine phosphorylation of Vav protein during B lymphocyte signaling.
Proc. Natl. Acad. Sci. USA
94
:
13158
25
Nishina, H., M. Bachmann, A. J. Oliveiradossantos, I. Kozieradzki, K. D. Fischer, B. Odermatt, A. Wakeham, A. Shahinian, H. Takimoto, A. Bernstein, T. W. Mak, J. R. Woodgett, P. S. Ohashi, J. M. Penninger.
1997
. Impaired CD28-mediated interleukin-2 production and proliferation in stress kinase SAPK/ERK1 kinase (SEK1) mitogen-activated protein-kinase kinase-4 (MKK4)-deficient T-lymphocytes.
J. Exp. Med.
186
:
941
26
Cheng, G., A. M. Cleary, Z. S. Ye, D. I. Hong, S. Lederman, D. Baltimore.
1995
. Involvement of CRAF1, a relative of TRAF, in CD40 signaling.
Science
267
:
1494
27
Roost, H., S. Charan, R. Gobet, E. Rueedi, H. Hengartner, A. Althage, R. M. Zinkernagel.
1988
. An acquired immune suppression in mice caused by infection with lymphocytic choriomeningitis virus.
Eur. J. Immunol.
18
:
511
28
Mond, J. J., A. Lees, C. M. Snapper.
1995
. T cell-independent antigens type 2.
Annu. Rev. Immunol.
13
:
655
29
Bachmann, M. F., U. Kalinke, A. Althage, G. Freer, C. Burkhart, H. Roost, M. Aguet, H. Hengartner, R. M. Zinkernagel.
1997
. The role of antibody concentration and avidity in antiviral protection.
Science
276
:
2024
30
Bachmann, M. F., R. M. Zinkernagel.
1997
. Neutralizing antiviral B cell responses.
Annu. Rev. Immunol.
15
:
235
31
Bachmann, M. F., R. M. Zinkernagel.
1996
. The influence of virus structure on antibody responses and virus serotype formation.
Immunol. Today
17
:
553
32
Leist, T. P., S. P. Cobbold, H. Waldmann, M. Aguet, R. M. Zinkernagel.
1987
. Functional analysis of T lymphocyte subsets in antiviral host defense.
J. Immunol.
138
:
2278
33
Bachmann, M. F., T. M. Kuendig.
1994
. In vivo versus in vitro assays for assessment of T- and B-cell function.
Curr. Opin. Immunol.
6
:
320
34
Shahinian, A., K. Pfeffer, K. P. Lee, T. M. Kuendig, K. Kishihara, A. Wakeham, K. Kawai, P. S. Ohashi, C. B. Thompson, T. W. Mak.
1993
. Differential T cell costimulatory requirements in CD28-deficient mice.
Science
261
:
609
35
Tarakhovsky, A..
1997
. Bar Mitzvah for B-1 cells: how will they grow up?.
J. Exp. Med.
185
:
981
36
Rickert, R. C., K. Rajewsky, J. Roes.
1995
. Impairment of T-cell-dependent B-cell responses and B-1 cell development in CD19-deficient mice.
Nature
376
:
352
37
Engel, P., L. J. Zhou, D. C. Ord, S. Sato, B. Koller, T. F. Tedder.
1995
. Abnormal B lymphocyte development, activation, and differentiation in mice that lack or overexpress the CD19 signal transduction molecule.
Immunity
3
:
39
38
Satterthwaite, A. B., H. Cheroutre, W. N. Khan, P. Sideras, O. N. Witte.
1997
. Btk dosage determines sensitivity to B cell antigen receptor cross-linking.
Proc. Natl. Acad. Sci. USA
9
:
13152
39
Khan, W. N., F. W. Alt, R. M. Gerstein, B. A. Malynn, I. Larsson, G. Rathbun, L. Davidson, S. Mueller, A. B. Kantor, L. A. Herzenberg, et al
1995
. Defective B cell development and function in Btk-deficient mice.
Immunity
3
:
283
40
Snapper, S. B., F. S. Rosen, E. Mizoguchi, P. Cohen, W. Khan, C. H. Liu, T. L. Hagemann, S. P. Kwan, R. Ferrini, L. Davidson, A. K. Bhan, F. W. Alt.
1998
. Wiskott-Aldrich syndrome protein-deficient mice reveal a role for WASP in T but not B cell activation.
Immunity
9
:
81
41
Defranco, A. L..
1995
. Transmembrane signaling by antigen receptors of B-lymphocytes and T-lymphocytes.
Curr. Opin. Cell Biol.
7
:
163
42
Deckert, M., S. Tartaredeckert, C. Couture, T. Mustelin, A. Altman.
1996
. Functional and physical interactions of Syk family kinases with the Vav protooncogene product.
Immunity
5
:
591
43
Graziadei, L., K. Riabowol, S.-D. Bar.
1990
. Co-capping of ras proteins with surface immunoglobulins in B lymphocytes.
Nature
347
:
396
44
Binder, D., M. F. vandenBroek, D. Kagi, H. Bluethmann, J. Fehr, H. Hengartner, R. M. Zinkernagel.
1998
. Aplastic anemia rescued by exhaustion of cytokine-secreting CD8+ T cells in persistent infection with lymphocytic choriomeningitis virus.
J. Exp. Med.
187
:
1903