Kras Is Critical for B Cell Lymphopoiesis Free

B cell development occurs through pro–, pre–, immature, and mature B cell stages (1). The pre-BCR instructs the transition from pro–B to pre–B cells whereas the BCR directs B cell maturation and subsequent immune responses (2, 3). Both the pre-BCR and BCR initiate signals via transmembrane molecules, Igα and Igβ, and activation of three distinct protein tyrosine kinases, Lyn, Syk, and Btk (3). Ultimately, these kinases activate several signaling pathways, including the Ras-Raf-MEK1/2-ERK1/2 cascade (3, 4). A dominant-negative Ras protein that inhibits this pathway blocks the pre-pro– to pro–B cell transition (5). In contrast, constitutively active Ras drives Rag1-deficient pro–B cells into pre–B-like cells and promotes maturation of BCR-low immature B cells (6). Additionally, ERK1/2 double deficiency blocks pre–BCR-mediated early B cell development (7). These findings demonstrate that the Ras-dependent pathway is critical for B cell development.

Ras protein is a 21-kDa membrane-associated small GTPase that cycles between an active GTP-bound state and an inactive GDP-bound state and functions as a molecular switch relaying signals from cell surface receptors to the Raf/MEK/ERK1/2 pathway (8). Guanine nucleotide exchange factors (GEFs) activate Ras by catalyzing the exchange of GDP for GTP whereas GTPase-activating proteins inactivate Ras via facilitating the hydrolysis of GTP to GDP (9). GTP-bound Ras specifically activates the serine/threonine kinase Raf through direct interaction (10–13). In turn, Raf phosphorylates and activates the dual-specificity threonine/tyrosine kinases MEK1/2, which phosphorylate and activate the serine/threonine kinases ERK1/2 (14, 15). Activation of ERK1/2 leads to upregulation of c-fos, a component of the transcription factor AP-1, and promotes a wide variety of cellular events (16, 17).

The family of highly conserved GTPases consists of the Ras, Rho, Rab, and Ran subfamilies (18). The mammalian Ras subfamily has three highly homologous members, Kras, Hras, and Nras, which are ubiquitously expressed (8). Studies of dominant-negative Ras proteins demonstrate a critical role of Ras activity in cell growth and embryogenesis (19). However, mice deficient in either or both Nras and Hras are viable and largely normal, demonstrating functional redundancy of these ras genes (20, 21). In contrast, Kras-deficient mice are embryonically lethal (22). Although all three Ras isoforms are activated by TCR or BCR engagement, disruption of a specific Ras isoform has distinct effects (22–26). Deficiency of Hras or Nras does not affect early T cell development, positive selection, or T cell activation, but it specifically impairs Th1 response of CD4 T cells (23). Nras deficiency also reduces CD8 thymocyte numbers and impairs CD8 T cell memory (25, 26). These findings demonstrate specific and distinct functions of the individual Ras isoforms.

Embryonic lethality of Kras-deficient mice precludes analysis of the role, if any, of Kras in lymphocyte development and function (22). In this study, we report on mice with hematopoietic deletion of Kras and bone marrow (BM) chimeric mice with B cell–specific targeted deletion of Kras. Our results demonstrate that Kras is important for B cell development.

VavCreKras^fl/fl mice were generated in J. Zhang’s laboratory (University of Wisconsin–Madison). Briefly, exon 1 of Kras was flanked with two LoxP sites (Supplemental Fig. 1). The generated Kras^fl/fl mice were crossed with VavCre transgenic mice, in which Cre expression mediates deletion of “floxed” gene throughout the entire hematopoietic compartment. The mouse line was maintained on C57BL/6 genetic background (>N10). Experimental VavCreKras^fl/fl and control VavCreKras^fl/+ or VavCreKras^+/+ mice were 8–12 wk old.

BM chimeric mice were generated. First, BM cells from VavCreKras^fl/fl or control mice were mixed 1:4 with BM cells from μMT mice and transplanted into sublethally irradiated (600 rad) Rag1-deficient or lethally irradiated (1000 rad) μMT mice by i.v. injection (5 × 10⁶ cells/recipient). Eight weeks after transplantation, the recipients were analyzed for B cell development and function.

Rag1-deficient and μMT mice from The Jackson Laboratory were maintained in the Biological Resource Center at the Medical College of Wisconsin. Animal protocols were approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee.

Single-cell suspensions from BM and spleen were treated with Gey’s solution to remove RBCs and resuspended in PBS supplemented with 2% BSA. The cells were then stained with a combination of fluorescence-conjugated Abs. Allophycocyanin-conjugated anti-B220, anti-IgM, anti-CD4, and anti-CD44, and PE-Cy7–conjugated anti-B220, anti–IFN-γ, anti-CD25, and anti-CD23 were purchased from eBioscience. PE-conjugated anti-CD43, anti-CD21, anti-Thy1.2, anti-CD5, anti-CD8, and anti-IgD were purchased from BD Biosciences. Samples were applied to a flow cytometer (LSR II, Becton Dickinson). Data were collected and analyzed using FACSDiva software (Becton Dickinson) or FlowJo software (Tree Star).

Isolated mature B cells were resuspended in RPMI 1640 medium with 1% BSA. The B cells (10 × 10⁶/ml) were stimulated with anti-IgM (20 μg/ml, Jackson ImmunoResearch Laboratories) at 37°C for the indicated times. B220⁺CD25⁺ pre–B cells were sorted from the mouse BM and incubated with biotin-conjugated anti-Igβ (30 μg/ml, anti-CD79B, HM-79-12, Novus Biologicals), followed by adding streptavidin (30 μg/ml, Thermo Fisher Scientific) at 37°C for the indicated times. Cell lysates were subjected to Western blot analysis with the indicated Abs or Ras-GTP pull-down assay with the Ras activation kit (Millipore). Rabbit polyclonal anti-ERK1/2 (sc-093) and mouse monoclonal anti–phospho-ERK1/2 (pThr²⁰²/pTyr²⁰⁴, sc-7383), anti-Nras (sc-31), anti-Kras (sc-30), anti-Akt (sc-8312), anti-JNK (sc-571), anti-MEK1/2 (sc-436), anti-Raf-1 (sc-133), anti-p38 and anti–phospholipase C (PLC)γ2 (sc-407) Abs were purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti–phospho-Akt (phospho-Thr³⁰⁸, no. 9275), anti–phospho-JNK (pThr¹⁸³/pTyr¹⁸⁵, no. 4668), anti–phospho-MEK1/2 (pThr¹⁸⁰/pTyr¹⁸², no. 2338), and mouse monoclonal anti–phospho-p38 (pThr¹⁸⁰/pTyr¹⁸², no. 9216), as well as anti-IκBα (no. 9242) Abs, were purchased from Cell Signaling Technology. Mouse monoclonal anti–phospho-Raf1 (Ser³³⁸, no. 05-338) and anti-Ras (no. 05-516) Abs were purchased from EMD Millipore. Anti-Hras (no. 610001) Ab was purchased from BD Biosciences.

Purified mature B cells (2.5 × 10⁴) were stimulated with anti-IgM (10 μg/ml) or anti-IgM (10 μg/ml) plus IL-4 (10 ng/ml) for 48 h in a 96-well plate. The cells were then stained with propidium iodide for cell cycle analysis or pulsed for 16 h with [³H]thymidine (1 μCi/well). Thymidine-pulsed samples were collected with the use of a MACH III harvester (Tomtec, Hamden, CT), and the incorporation of [³H]thymidine was determined with a Wallac MicroBeta TriLux scintillation system (PerkinElmer, Waltham, MA).

Sorted pre–B cells were stimulated with biotin-conjugated anti-Igβ (30 μg/ml) plus streptavidin (30 μg/ml) at 37°C for the indicated times. The cells (1 × 10⁶) were lysed in the lysis buffer (20 mM HEPES [pH 7.9], 350 mM NaCl, 1 mM MgCl₂, 0.5 mM EDTA, 0.5 mM DTT, 20% glycerol, 1% Nonidet P-40). Cell lysates were incubated with [³²P]-labeled NF-κB or AP-1 probe (Santa Cruz Biotechnology) for 15 min at room temperature and then resolved on a 4% polyacrylamide gel at 4°C.

Splenocytes (2 × 10⁶) were incubated with indo-1^AM (10 μg/ml, Molecular Probes) and allophycocyanin-conjugated anti-IgM, PE-Cy7–conjugated anti-CD23, and PE-conjugated anti-CD21 Abs at room temperature for 30 min. Then cells were washed and stimulated with an anti-IgM Ab. Calcium concentrations were determined in mature B cells by flow cytometry.

All statistical analysis was performed with the two-tailed unpaired Student t test.

To determine whether Kras has a distinct role in the development and function of lymphocytes, we compared the expression of Kras with that of Nras and Hras during B and T cell development. Levels of expression of Kras and Hras were higher in pro–B cells and lower in B cells at later developmental stages whereas Nras was expressed at high levels throughout B cell development (Fig. 1A). In thymocytes, all Ras members were expressed at a higher level in double-negative (DN) T cells and at a lower level in double-positive (DP) cells and CD4 or CD8 single-positive (SP) T cells (Fig. 1B). Thus, Kras is expressed in both B and T lymphocytes.

To overcome the embryonic lethality associated with Kras deficiency and study the role of Kras in lymphocyte development, mice with “floxed” Kras allele were generated (Supplemental Fig. 1). Then, Kras floxed mice were crossed with VavCre transgenic mice, in which Cre expression mediates deletion of the floxed gene throughout the entire hematopoietic compartment (27). Lysates of BM cells from VavCreKras^fl/fl mice displayed a complete deletion of Kras (Fig. 2A).

BM cell–specific deletion of Kras did not affect total BM cell numbers in VavCreKras^fl/fl relative to control (VavCreKras^+/+ and VavCreKras^fl/+) mice (Fig. 2B). In contrast, the percentages and absolute numbers of BM B cells were markedly reduced in VavCreKras^fl/fl relative to control mice (Fig. 2C). Additionally, the percentages and numbers of pro–/pre– (B220⁺IgM⁻), immature (B220⁺IgM⁺), and mature (B220^hiIgM⁺) B cells were all reduced in the BM of VavCreKras^fl/fl relative to control mice (Fig. 2D, 2E). Within the pro–/pre–B cell (B220⁺IgM⁻) population, the percentage of IgM⁻CD43⁺ pro–B cells was increased in VavCreKras^fl/fl relative to control mice (Fig. 2F). In contrast, the percentage of B220⁺CD25⁺ pre–B cells was decreased in VavCreKras^fl/fl relative to control mice (Fig. 2G). The absolute number of pre–B, but not pro–B, cells was markedly reduced in VavCreKras^fl/fl mice (Fig. 2H). Within the population of B220⁺CD43⁺ pro–B cells, the percentages and numbers of pre–/pro–B (fraction A, BP-1⁻CD24⁻), early pro–B (fraction B, BP-1⁻CD24⁺), late pro– (fraction C, BP-1⁺CD24⁺), and large pre–B (fraction C′, BP-1⁺CD24^hi) cells were comparable between VavCreKras^fl/fl and control mice (Supplemental Fig. 2). Thus, Kras deficiency impairs early B cell development at the pro– to pre–B cell transition stage.

Early T cell development was also examined in VavCreKras^fl/fl mice. Thymic T cell development was normal in VavCreKras^fl/fl relative to control mice, based on the presence of comparable populations of total, DN, DP, and SP thymocytes (Fig. 2I–K). Additionally, among DN thymocytes, the populations of CD44⁺CD25⁻ (DN1), CD44⁺CD25⁺ (DN2), CD44⁻CD25⁺ (DN3), and CD44⁻CD25⁻ (DN4) were comparable between VavCreKras^fl/fl and control mice (data not shown). Thus, Kras deficiency has no effect on early T cell development.

The effect of Kras deficiency on lymphocyte maturation was also examined. The number of total splenocytes was comparable between VavCreKras^fl/fl and control mice (Fig. 3A). However, the percentage and number of B220⁺ B cells in the spleens of VavCreKras^fl/fl relative to control mice were reduced whereas the population of Thy1.2⁺ splenic T cells was comparable between the two types of mice (Fig. 3B, 3C). Additionally, the percentages of transitional 1 (T1) (IgM^hiIgD^lo), transitional 2 (T2) (IgM^hiIgD^hi), and follicular (FO) mature (IgM^loIgD^hi) B cells were reduced in VavCreKras^fl/fl relative to control mice (Fig. 3D). Based on the expression of IgM, CD21, and CD23, splenic B cells can also be separated into different subsets of immature and mature B cells. Among CD23⁺ B cells, the population of FO (CD23⁺CD21^intIgM^lo) but not T2 (CD23⁺CD21^hiIgM^hi) B cells was reduced in VavCreKras^fl/fl mice relative to control mice (Fig. 3E, 3F). In CD23⁻ B cells, the population of marginal zone (MZ; CD23⁺CD21^hiIgM^hi) but not T1 (CD23⁺CD21^loIgM^hi) B cells was dramatically reduced in VavCreKras^fl/fl mice (Fig. 3E, 3F). Of note, the populations of CD4⁺ and CD8⁺ T cells in the spleen were comparable between VavCreKras^fl/fl relative to control mice (Fig. 3G). The self-renewing mature B1 B cells reside mainly in the peritoneal and pleural cavities. Within peritoneal lymphocytes, the population of B1 B cells (CD5⁺IgM⁺) was largely reduced in VavCreKras^fl/fl relative to control mice whereas the population of B2 B cells (CD5⁻IgM⁺) was normal in VavCreKras^fl/fl mice (Fig. 3H). Therefore, Kras deficiency severely impairs B cell maturation, resulting in a marked reduction of FO, MZ, and B1 B cells, but it has no effect on T cell maturation.

We examined whether the defective B cell development in VavCreKras^fl/fl mice is the result of an intrinsic abnormality of the B cells. We generated BM chimeric mice with B cell–specific deficiency of Kras by mixing BM cells from VavCreKras^fl/fl or control mice with BM cells from B cell–deficient μMT mice, and transplanting the mixture into sublethally irradiated B and T cell–null Rag1-deficient or lethally irradiated μMT mice. Subsequently, B cell development in the recipients was examined. The populations of pre–, immature, and mature B cells in the BM were markedly reduced in the Rag1-deficient or μMT recipients of a mixture of VavCreKras^fl/fl and μMT BM cells relative to the recipients of a mixture of control and μMT BM cells (Fig. 4A, 4B, and data not shown). Moreover, the populations of splenic B cells in the recipients of a mixture of VavCreKras^fl/fl and μMT BM cells were dramatically reduced compared with the recipients that received control and μMT BM cells (Fig. 4C, 4D, and data not shown). Of note, the total number of splenocytes was markedly reduced in the Rag1-deficient but not μMT recipients of VavCreKras^fl/fl and μMT BM cells relative to the corresponding recipients of a mixture of control and μMT BM cells (data not shown). Thus, the total numbers of splenic T cells were reduced in the B cell–specific Kras-deficient BM chimeric Rag1-deficient but not μMT recipients, although the reduction was not significant (Fig. 4C, 4D). Additionally, BM cells from VavCreKras^fl/fl or control mice were transplanted into lethally irradiated CD45.1 congenic mice. The populations of splenic B but not T cells were dramatically reduced in the CD45.1 recipients of VavCreKras^fl/fl BM cells relative to the corresponding recipients of control BM cells (data not shown). Taken together, these data demonstrate that the defective B cell development in VavCreKras^fl/fl mice is B cell autonomous.

We studied the effect of Kras deficiency on B cell proliferation and survival. We examined BCR-induced proliferation of Kras-deficient B cells using [³H]thymidine incorporation. Mature splenic B cells were sorted from VavCreKras^fl/fl or control mice and cultured in the presence of anti-IgM or anti-IgM plus IL-4. The [³H]thymidine incorporation rate of Kras-deficient, relative to control, mature B cells in response to anti-IgM or anti-IgM plus IL-4 was markedly reduced (Fig. 5A). To determine whether the reduction of BCR-induced [³H]thymidine incorporation in Kras-deficient B cells was due to a decrease of cell proliferation and/or an increase of cell apoptosis, propidium iodide staining in combination with FACS analysis was used to detect the stimulation-induced cell proliferation and apoptosis of experimental B cells. Following anti-IgM or anti-IgM plus IL-4 stimulation, the entry into S and G₂/M phase of Kras-deficient B cells was markedly reduced compared with that of control B cells (Fig. 5B). Additionally, the population of apoptotic cells (sub-G₀) was obviously increased in Kras-deficient relative to control B cells in the absence or presence of stimulation (Fig. 5B). The marked increase of BCR-induced apoptosis in Kras-deficient B cells was further confirmed by a short period of anti-IgM stimulation (Fig. 5C). Thus, Kras deficiency impairs BCR-induced B cell proliferation and survival.

We examined the effect of Kras deficiency on B cell humoral immune response. The basal levels of serum Igs in VavCreKras^fl/fl mice were measured. The levels of IgM, IgG1, IgG2b, IgG2c, and IgG3 in the sera were comparable between naive VavCreKras^fl/fl and control mice (Fig. 6A). Additionally, Ag-specific Ab responses to T cell–dependent (TD) or T cell–independent (TI) Ags were examined in the B cell–specific Kras-deficient BM chimeric mice. Following TD Ag nitrophenol (NP)–chicken γ globulin or TI Ag trinitrophenol (TNP)-Ficoll immunization, the levels of NP-specific IgG1 and TNP-specific IgM were slightly but not significantly less in the chimeric mice that received a mixture of BM cells from VavCreKras^fl/fl and μMT mice relative to those that received a mixture of BM cells from control and μMT mice (Fig. 6B, 6C). Thus, Kras deficiency slightly, but not significantly, impairs B cell immune responses.

Kras deficiency impaired BCR-mediated functions. We further examined the role of Kras in BCR signaling. Consistent with the previous studies with a B cell line (24), Kras was activated by BCR ligation in primary B cells (Fig. 7A). Importantly, the level of total BCR-activated Ras (Ras-GTP) was largely reduced in Kras-deficient B cells (Fig. 7B). These data indicate a potential role of Kras in BCR signaling in B cells. Ras is able to regulate multiple signaling pathways, including the PI3K/AKT, PLC/Ca²⁺/protein kinase C, Raf/MEK/Erk, and others, in many types of cells. We further investigated the downstream pathways that are controlled by Kras during BCR signaling. BCR-induced Ca²⁺ flux was comparable between Kras-deficient and control B cells (Fig. 7C). BCR-induced phosphorylation of Akt and IκBα was normal in mutant relative to control B cells (Fig. 7D). However, BCR-induced activation of ERK1/2 but not JNK or p38 was impaired in Kras-deficient relative to control B cells (Fig. 7D). Consistently, BCR-induced activation of Raf-1 and MEK1/2, the upstream activators of ERK1/2, was markedly reduced in mutant B cells (Fig. 7E). Of note, TCR-induced AKT and Raf/MEK/ERK activation was normal in Kras-deficient CD4 T cells (data not shown). Thus, Kras plays an important role in BCR-mediated activation of the ERK pathway in B cells.

Signals from the pre–BCR control the expansion of pre–B cells. Kras deficiency resulted in a marked reduction of pre–B cells (Fig. 2G). We investigated whether Kras is the Ras member that controls pre–BCR-mediated activation of the Raf-1/MEK/ERK pathway. Pre–B cells FACS sorted from VavCreKras^fl/fl mice were stimulated with anti-Igβ. Pre–BCR-induced ERK1/2 activation was clearly impaired in Kras-deficient relative to control pre–B cells (Fig. 7F). Consistently, pre–BCR-induced activation of AP-1, the transcription factors downstream of ERK1/2, but not NF-κB was decreased in mutant relative to control pre–B cells (Fig. 7G). Thus, Kras critically controls pre–BCR-mediated activation of the ERK pathway in pre–B cells.

Signals emanating from the pre-BCR direct the expansion and differentiation of pre–B cells, and a lack of a functional pre-BCR arrests early B cell development at the pre–B cell stage (2). The pre-BCR initiates several signaling pathways, including the ERK pathway. ERK1/2 double deficiency or dominant-negative Ras can block pre–BCR-mediated B cell development (7, 28). Constitutively active Ras compensates for pre-BCR deficiency to drive Rag1-deficient pro–B cells to differentiate into pre–B-like cells (6). Thus, the Ras-dependent ERK pathway is critical for pre–BCR-mediated pre–B cell development. The ERK pathway is activated by Kras, Nras, or Hras through the Raf/MEK1/2 signaling cascade (10–15). Nras and Hras single- or Nras/Hras double-deficient mice are largely normal, whereas Kras-deficient mice are embryonic lethal (20–22). Although Nras and Hras, similar to Kras, are highly expressed in B cell progenitors as we find in this study, no defective B cell development is reported in mice deficient in either or both of Nras and Hras (20, 21). However, overexpression of a dominant-negative form of Hras in early B cells completely arrests B cell development at the pre–/pro–B to pro–B cell transition prior to pre-BCR signaling, suggesting an important role of the Ras pathway in the function of other receptors, such as the IL-7 receptor, during early B cell development (5). Mice with overexpression of the dominant-negative Hras in late B cell precursors display a more severe reduction in the numbers of late pre–B cells than do Kras-deficient mice, strongly indicating the involvement of other Ras members in pre–BCR-mediated early B cell development (29). Our present study demonstrates that Kras functions uniquely among Ras family members as its ability to contribute to pre–BCR-mediated activation of Raf/MEK/ERK pathway to control pre–B cell development.

The BCR also activates the ERK pathway, and constitutively active Ras rescues the differentiation of immature B cells with low levels of BCR expression (30). As we report in the present study, B cells at the later developmental stages, relative to pro–B cells, express lower levels of Kras. Nonetheless, Kras largely accounts for BCR-induced Ras activities, which might be due to its upregulation by BCR stimulation. Kras deficiency leads to a reduction of FO, MZ, and B1, but not transitional T1 or T2 B cells, indicating that the reduction of mutant mature B cells is a result of impaired BCR function but not decreased pre-B cells. Indeed, BCR-induced B cell proliferation and survival are impaired in Kras-deficient B cells. Of note, Kras deficiency impairs but does not completely block ERK activation, indicating potential roles of Nras and Hras in BCR signaling. Indeed, overexpression of the dominant-negative Hras in B cells markedly reduces the populations of peripheral mature B cells, including B1 B cells (5). Nonetheless, no defective B cell maturation is reported in mice with deletion of either or both of Nras and Hras (20, 21). Thus, studies of mice with compound deficiencies of Kras, Nras, and Hras are required to reveal any contributions of Nras and/or Hras to BCR-mediated ERK activation and B cell maturation.

B cell development in the BM chimeric recipients of a mixture of VavCreKras^fl/fl and μMT BM cells is more severely impaired than that in hematopoietic-specific Kras-deficient (VavCreKras^fl/fl) mice. This is likely due to the inability of Kras-deficient B cell progenitors to outcompete μMT B cell progenitors in the transplanted recipients. In fact, defective B cell development in the CD45.1 congenic recipients that receive Kras-deficient BM cells without BM cells from μMT mice is similar to that in the nontransplanted Kras-deficient mice (VavCreKras^fl/fl). Additionally, owing to unclear reasons, the total number of splenocytes in Rag1-deficient BM chimeric mice with B cell–specific Kras deficiency is markedly reduced compared with control Rag1-deficient BM chimeric mice. Consequently, the total number of splenic T cells in the experimental relative to control Rag1-deficient BM chimeric mice was reduced, although the reduction was not statistically significant. However, μMT BM chimeric mice with B cell–specific deficiency of Kras display a marked reduction of splenic B cells but a normal population of splenic T cells compared with control μMT BM chimeric mice. Moreover, Kras deficiency results in a marked reduction of all three subsets of mature B cells and an impairment of BCR-induced cell proliferation and survival, but only slightly affects the B cell humoral immune response in vivo. Kras-deficient mature B cells with the remaining ERK activities might be enough to drive Ab production following Ag challenge in vivo.

The molecular mechanism underlying the distinct functions of the closely related Ras family members is not clear. The posttranslational modification differences of the C termini of Ras lead to their differential localization. The palmitoylated C terminus of H-Ras directs its localization to lipid rafts in the plasma membrane whereas a stretch of basic residues in the C terminus of Kras guides its localization outside rafts (31, 32). The differential cellular localization of different Ras members could result in their differential activation and engagement of diverse downstream effector pathways. There are two types of RasGEFs, mSOS and RasGRP (33, 34). The BCR can activate Ras through mSos that is regulated by the Grb2/Shc complex or through RasGRP that is controlled by DAG, a product of PLCγ (4, 35). However, the contribution of differential localization of Ras members and differential activation of RasGEFs by the BCR or pre-BCR to distinct Ras activation in B cells requires further investigation.

The authors have no financial conflicts of interest.