Introduction

Figure 1. Lack of IQGAP1 results in dysregulated early B cell development in the BM. A) Calponin homology (CH) domain regulates the cytoskeleton by binding to N-WASp and F-actin. Coiled-coil (CC) regions, comprised of conserved amino acid repeats, bind to Ezrin. WW domain is involved in recruiting Erk1/2 to facilitate a sequential phosphorylation cascade. Isoleucine/glutamine-containing (IQ) domains recruit Rap1a, Rap1b, B-Raf, C-Raf, Mek1, and Mek2 to promote phosphoinositide (PIPKIγ) and calcium signaling. The Ras-GAP domain (GRD) interacts with small GTPases Cdc42, Rac1, and TC10. The Ras-GAP C-terminus (RGCT) domain interacts with CLIP-170, Clasp2, β-catenin, E-cadherin, and APC. B) Sorted total B220⁺ resting B cells from the BM were analyzed for the expression levels of Iqgap1, Iqgap2, and Iqgap3 transcripts using RTqPCR. Relative expression of these transcripts is shown as percent of Rpl19 house-keeping gene. The data shown are the average of three independent experiments. C) Expression of IQGAP1 protein in the WT and Iqgap1^−/− mice were analyzed by western blot using an anti-IQGAP1 antibody. Level of β-actin was used as loading controls. Blots are a representative set from three different experiments. D, E) Viable cells within the lymphocyte gate were analyzed for the absolute number of total B cells. Staining with anti-B220 antibody indicated the presence of both B220^High and B220^Low B cells in both the WT and Iqgap1^−/− mice. F, G) Absolute numbers of B220⁺IgM⁻ pro/pre-B, B220⁺IgM⁺ immature B, and B220^Hi recirculating B cells were defined using anti-B220 and anti-IgM antibodies, and their numbers were calculated from live lymphocyte gates. H, I) The absolute numbers of pro-B cells were further identified with anti-B220 and anti-CD43 antibody staining. Pro-B cells are B220^MedCD43^+, and pre-B cells are B220⁺CD43⁻. Data shown in C, E, and G are representative panels. Open and filled circles in D, F, and H are values obtained from an individual mouse from four independent experiments. Data are shown with the mean ± SEM. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph.

Figure 1. Lack of IQGAP1 results in dysregulated early B cell development in the BM. A) Calponin homology (CH) domain regulates the cytoskeleton by binding to N-WASp and F-actin. Coiled-coil (CC) regions, comprised of conserved amino acid repeats, bind to Ezrin. WW domain is involved in recruiting Erk1/2 to facilitate a sequential phosphorylation cascade. Isoleucine/glutamine-containing (IQ) domains recruit Rap1a, Rap1b, B-Raf, C-Raf, Mek1, and Mek2 to promote phosphoinositide (PIPKIγ) and calcium signaling. The Ras-GAP domain (GRD) interacts with small GTPases Cdc42, Rac1, and TC10. The Ras-GAP C-terminus (RGCT) domain interacts with CLIP-170, Clasp2, β-catenin, E-cadherin, and APC. B) Sorted total B220⁺ resting B cells from the BM were analyzed for the expression levels of Iqgap1, Iqgap2, and Iqgap3 transcripts using RTqPCR. Relative expression of these transcripts is shown as percent of Rpl19 house-keeping gene. The data shown are the average of three independent experiments. C) Expression of IQGAP1 protein in the WT and Iqgap1^−/− mice were analyzed by western blot using an anti-IQGAP1 antibody. Level of β-actin was used as loading controls. Blots are a representative set from three different experiments. D, E) Viable cells within the lymphocyte gate were analyzed for the absolute number of total B cells. Staining with anti-B220 antibody indicated the presence of both B220^High and B220^Low B cells in both the WT and Iqgap1^−/− mice. F, G) Absolute numbers of B220⁺IgM⁻ pro/pre-B, B220⁺IgM⁺ immature B, and B220^Hi recirculating B cells were defined using anti-B220 and anti-IgM antibodies, and their numbers were calculated from live lymphocyte gates. H, I) The absolute numbers of pro-B cells were further identified with anti-B220 and anti-CD43 antibody staining. Pro-B cells are B220^MedCD43^+, and pre-B cells are B220⁺CD43⁻. Data shown in C, E, and G are representative panels. Open and filled circles in D, F, and H are values obtained from an individual mouse from four independent experiments. Data are shown with the mean ± SEM. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph.

Results

Lack of IQGAP1 forms a blockade at early B cell development, and it is cell-autonomous

However, B220^MedCD43⁻ pre-B cells were significantly higher in Iqgap1^−/− (Iqgap1^−/−: 7.6 ± 3.2×10⁶ and WT: 5.0 ± 1.7×10⁶; n=8, 8; p=0.041), which is consistent with the increased B220⁺IgM⁻ pro/pre-B cells populations. As pro-pre-B-I cells mature into pre-B-II (large and small), c-kit expression decreases and CD25 level increases. In this context, we also observed a significantly increased B220⁺CD25⁺ pre-B-II population (Iqgap1^−/−: 4.10 ± 2.16×10⁶ and WT: 2.35 ± 1.31×10⁴; n=9, 9; p=0.05) in Iqgap1^−/− mice (Figure 2A, B). BM-derived cells were also stained with anti-B220, anti-IgM, and anti-IgD Abs and analyzed. The absolute numbers of IgM⁺IgD⁺ (Iqgap1^−/−: 0.57 ± 0.25×10⁴and WT: 0.42 ± 0.19×10⁴; n=13, 13; p=NS) and IgM⁻IgD⁺ (Iqgap1^−/−: 0.48 ± 0.15×10⁴ and WT: 0.52 ± 0.25×10⁴; n=13, 13; p=NS) B cells were comparable between Iqgap1^−/− and WT mice (Figure 2C, D). However, both the immature IgM⁻IgD⁻ (Iqgap1^−/−: 6.8 ± 3.0×10⁴ and WT: 4.0 ± 1.3×10⁴; n=13, 13; p=0.008) and IgM⁺IgD⁻ (Iqgap1^−/−: 3.38 ± 1.68×10⁴ and WT: 1.71 ± 0.62×10⁴; n=13, 13; p=0.003) B cells were increased in Iqgap1^−/− mice compared to that of WT (Figure 2C, D), indicating a loss of developmental regulation during the early stages of B cell maturation.

Figure 2. Lack of IQGAP1 leads to a potential blockade at pre-B cell stages in the BM and the defects in the transition of pro-B to pre-B cells in the BM of Iqgap1^−/− mice is cell-intrinsic. A, B) Absolute numbers of B220⁺CD25⁺ large and small pre-B-II stage. C, D) Absolute numbers of B220⁺IgM⁻IgD⁻ or B220⁺IgM⁺IgD⁻ immature B and B220⁺IgM⁺IgD⁺ or B220⁺IgM⁻IgD⁺ mature B cells. BM cells were stained with anti-B220, anti-IgM, and anti-IgD antibodies, and the absolute numbers were calculated per million total gated lymphocytes. Data shown in A, C, and E are one representative panel from a minimum of three independent experiments. Open and filled circles in B and D are values obtained from an individual mouse from four independent experiments. Data are shown with the mean ± SEM. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph. E) Indicated host mice were sub-lethally irradiated and reconstituted with donor-derived 5 × 10⁵ BM cells. Eight weeks later, cells from the host BM were harvested and analyzed. The absolute numbers of reconstituted pro-B cells were identified with anti-B220 and anti-CD43 antibody staining. Pro-B cells (B220^MedCD43⁺) and pre-B cells (B220⁺CD43⁻) were quantified from six host mice, and the average absolute numbers are presented as bar graphs. F) Absolute numbers of reconstituted B220⁺IgM⁻IgD⁻ or B220⁺IgM⁺IgD⁻ immature B and B220⁺IgM⁺IgD⁺ or B220⁺IgM⁻IgD⁺ mature B cells. BM cells were stained with anti-B220, anti-IgM, and anti-IgD antibodies, and the absolute numbers were calculated per million total gated lymphocytes. Data shown on the left are representative panels. The average absolute numbers are presented as bar graphs. Data shown are from two independent experiments with three mice each. Data are shown with the mean ± SEM. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph.

Figure 2. Lack of IQGAP1 leads to a potential blockade at pre-B cell stages in the BM and the defects in the transition of pro-B to pre-B cells in the BM of Iqgap1^−/− mice is cell-intrinsic. A, B) Absolute numbers of B220⁺CD25⁺ large and small pre-B-II stage. C, D) Absolute numbers of B220⁺IgM⁻IgD⁻ or B220⁺IgM⁺IgD⁻ immature B and B220⁺IgM⁺IgD⁺ or B220⁺IgM⁻IgD⁺ mature B cells. BM cells were stained with anti-B220, anti-IgM, and anti-IgD antibodies, and the absolute numbers were calculated per million total gated lymphocytes. Data shown in A, C, and E are one representative panel from a minimum of three independent experiments. Open and filled circles in B and D are values obtained from an individual mouse from four independent experiments. Data are shown with the mean ± SEM. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph. E) Indicated host mice were sub-lethally irradiated and reconstituted with donor-derived 5 × 10⁵ BM cells. Eight weeks later, cells from the host BM were harvested and analyzed. The absolute numbers of reconstituted pro-B cells were identified with anti-B220 and anti-CD43 antibody staining. Pro-B cells (B220^MedCD43⁺) and pre-B cells (B220⁺CD43⁻) were quantified from six host mice, and the average absolute numbers are presented as bar graphs. F) Absolute numbers of reconstituted B220⁺IgM⁻IgD⁻ or B220⁺IgM⁺IgD⁻ immature B and B220⁺IgM⁺IgD⁺ or B220⁺IgM⁻IgD⁺ mature B cells. BM cells were stained with anti-B220, anti-IgM, and anti-IgD antibodies, and the absolute numbers were calculated per million total gated lymphocytes. Data shown on the left are representative panels. The average absolute numbers are presented as bar graphs. Data shown are from two independent experiments with three mice each. Data are shown with the mean ± SEM. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph.

IQGAP1 is expressed in most of the hematopoietic cell lineages ²¹. Since IQGAP1 has been globally deleted ²⁰ in the mice we used, the defects we observed in absolute B cell numbers can be cell-intrinsic or due to an abnormality in the stromal microenvironment. To distinguish between these two possibilities, we performed bone marrow (BM) transplantation (WT→WT; Iqgap1^−/−→WT; WT→Iqgap1^−/−; Iqgap1^−/−→Iqgap1^−/−). After eight weeks, B cell development was analyzed in the reconstituted host mice. Lymphocyte reconstitution was successfully observed in all hosts. Compared to WT, the transfer of Iqgap1^−/− BM cells into WT or Iqgap1^−/− hosts resulted in an increased absolute number of lymphocytes in the BM (Supplementary Figure S1A). Absolute numbers of B220⁺ B, B220^High, and B220^Low cell numbers were significantly increased in host mice that received IQGAP1^−/− BM cells (Supplementary Figure S1B). Also, although B220⁺IgM^Neg cells did not show any considerable change, B220⁺IgM^High and B220⁺IgM^Low cells were significantly increased in the recipient mice that were transplanted with Iqgap1^−/− BM cells compared to that of WT (Supplementary Figure S1C). Analyzing Pro/Pre-B cells (B220⁺CD43⁺), Pro-B cells (B220^MedCD43⁻), and Pre-B cells (B220^HighCD43⁻) in reconstituted mice confirmed accumulation of Pro-B cells (Figure 2E). Staining for immature (IgM⁺IgD⁻) and mature (IgM⁺IgD⁺ and IgM⁻IgD⁺) B cell populations in the reconstituted mice indicated a significant increase in the immature but not the mature B cells (Figure 2F). In the spleens from the BM reconstituted mice, we did not observe an increase in the absolute number of lymphocytes or B220⁺ B cells (Supplementary Figure S2A). However, the transfer of Iqgap1^−/− BM cells into the WT or Iqgap1^−/− mice lead to a significant increase in the absolute numbers of immature (IgM⁺IgD⁻) but not mature (IgM⁺IgD⁺ and IgM⁻IgD⁺) B cell populations (Supplementary Figure S2B). These results demonstrate that the lack of IQGAP1 in the hematopoietic compartment but not the stromal environment is primarily responsible for the B cell developmental defects.

Lack of IQGAP1 significantly increases IgM^HighCD23⁻ T1 B cells in the spleens

Next, we analyzed the B cell maturation in the spleen. Lack of IQGAP1 did not alter the total lymphocyte numbers in the Iqgap1^−/− mice (Iqgap1^−/−: 39.0 ± 8.2×10⁶ and WT: 40.1 ± 8.4×10⁶; n=16, 16; p=NS) (Figure 3A). Moreover, similar to the BM, the absolute numbers of B220⁺ B cells in the spleen were moderately but significantly increased in Iqgap1^−/− compared to that of the WT mice (Iqgap1^−/−: 16.58 ± 6.26×10⁶ and WT: 13.17 ± 5.16×10⁶; n=31, 31; p=0.022) (Figure 3B). To further analyze the role of IQGAP1 in splenic B cell development, we stained them with anti-IgM and anti-IgD Abs. Although there were moderate increases in immature IgM⁺IgD⁻ B (Iqgap1^−/−: 2.91 ± 2.17×10⁶ and WT: 1.85 ± 1.37×10⁶; n=12, 12; p=NS) and mature IgM⁺IgD⁺ (Iqgap1^−/−: 5.3 ± 2. 95×10⁶ and WT: 3.46 ± 2.01×10⁶; n=12, 12; p=NS) or IgM⁻IgD⁺ (Iqgap1^−/−: 5.08 ± 2.49×10⁶ and WT: 3.88 ± 1.82×10⁶; n=12, 12; p=NS) B cells in the spleen of Iqgap1^−/− mice, they did not differ significantly (Figure 3C).

Figure 3. Lack of IQGAP1 leads to increased T1 B cells in the spleen. A) Absolute number of total lymphocytes within the spleens of WT and Iqgap1^−/− mice were comparable. Absolute numbers were calculated from live gating of the lymphocyte population. Data shown represent the total lymphocytes per spleen from both strains. B) Absolute total splenic B cells were comparable between the WT and Iqgap1^−/− mice. Viable cells within the lymphocyte gate were analyzed for the absolute number of total B cells using anti-B220 antibody. Data shown represent the B220⁺ B cells per spleen from both strains. C) Absolute numbers of immature and mature B cells do not differ between the WT and Iqgap1^−/− mice. Total splenic B cells were stained with anti-B220, anti-IgM, and anti-IgD antibodies. Data shown represent the B220⁺IgM⁺ immature B and B220⁺IgM⁺IgD⁺ or B220⁺IgM⁻IgD⁺ mature B cells. Data represent absolute numbers of immature or mature B cells per spleen from both strains. D) Absolute numbers of T1-B but not T2-B or T3-B are altered in Iqgap1^−/− mice. Total splenocytes were stained with anti-B220, and anti-CD93 antibodies and the double-positive cells were gated and quantified. E) B220⁺CD93⁺ B cells were further sub-divided into B220⁺CD93⁺IgM⁺ T1-B, B220⁺CD93⁺IgM⁺CD23⁺ T2-B, and B220⁺CD93⁺IgM⁻CD24⁺ T3-B cells. Open and filled circles in A, B, C, and E are values obtained from an individual mouse from four independent experiments. Data are shown with the mean ± SEM. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph.

Figure 3. Lack of IQGAP1 leads to increased T1 B cells in the spleen. A) Absolute number of total lymphocytes within the spleens of WT and Iqgap1^−/− mice were comparable. Absolute numbers were calculated from live gating of the lymphocyte population. Data shown represent the total lymphocytes per spleen from both strains. B) Absolute total splenic B cells were comparable between the WT and Iqgap1^−/− mice. Viable cells within the lymphocyte gate were analyzed for the absolute number of total B cells using anti-B220 antibody. Data shown represent the B220⁺ B cells per spleen from both strains. C) Absolute numbers of immature and mature B cells do not differ between the WT and Iqgap1^−/− mice. Total splenic B cells were stained with anti-B220, anti-IgM, and anti-IgD antibodies. Data shown represent the B220⁺IgM⁺ immature B and B220⁺IgM⁺IgD⁺ or B220⁺IgM⁻IgD⁺ mature B cells. Data represent absolute numbers of immature or mature B cells per spleen from both strains. D) Absolute numbers of T1-B but not T2-B or T3-B are altered in Iqgap1^−/− mice. Total splenocytes were stained with anti-B220, and anti-CD93 antibodies and the double-positive cells were gated and quantified. E) B220⁺CD93⁺ B cells were further sub-divided into B220⁺CD93⁺IgM⁺ T1-B, B220⁺CD93⁺IgM⁺CD23⁺ T2-B, and B220⁺CD93⁺IgM⁻CD24⁺ T3-B cells. Open and filled circles in A, B, C, and E are values obtained from an individual mouse from four independent experiments. Data are shown with the mean ± SEM. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph.

Next, we identified the transitional B cells among the total B220⁺ splenocytes based on their CD93 positivity. Transitional T1, T2, and T3 B cells are defined as IgM^HighCD23⁻, IgM^HighCD23⁺, and IgM^LowCD23⁺, respectively. We gated the B220⁺ splenocytes into CD93⁺ and CD93⁻ B cells (Figure 3D). We selected the CD93⁺ B cells and subdivided them based on IgM and CD23 positivity. Total percentages of B220⁺CD93⁺ B cells did not vary between the WT and the Iqgap1^−/− splenocytes. However, the absolute number of IgM^HighCD23⁻ T1 B cells was significantly increased among the splenocytes from Iqgap1^−/− mice (Figure 3E). Absolute numbers of T2 and T3 B cells were comparable between the WT and the Iqgap1^−/− mice.

Lack of IQGAP1 differentially affects FO B and MZ B cells in the spleens

B220⁺ splenic B cells can also be separated into new-forming (NF B) (CD21^Low/−CD23^Low/−), follicular (FO B) (CD23^HighCD21^Low/−) and marginal zone (MZ B) (CD21^HighCD23^Low/−) cells. Therefore, we next analyzed the numbers FO, NF, and MZ B cells (Figure 4A). The absolute numbers of B220-gated CD21^Low/−CD23^High FO B (Iqgap1^−/−: 11.7 ± 3.5×10⁶ and WT: 8.92 ± 3.82×10⁶; n=14, 14; p=0.026) and the CD21^Low/−CD2 3^Low/− NF B cells (Iqgap1^−/−: 6.37 ± 2.71×10⁶ and WT: 4.33 ± 1.18×10⁶; n=14, 14; p=0.018) were significantly increased in Iqgap1^−/− mice. However, the absolute number of MZ B was significantly reduced in the spleens of IQGAP1^−/− mice (Iqgap1^−/−: 1.18 ± 0.6×10⁶ and WT: 1.59 ± 0.87×10⁶; n=14, 14; p=0.05). In addition to these changes, the B cell follicles in the spleens of Iqgap1^−/− mice were extended and multi-centered compared to that of WT. MZ B cells can also be defined by their exclusive high-level expression of IgM, CD21, and CD1d ²². Staining for IgM and CD21 (Iqgap1^−/−: 0.93 ± 0.44×10⁶ and WT: 1.56 ± 0.0.53×10⁶; n=11, 11; p=0.0049) or IgM and CD1d (Iqgap1^−/−: 0.75 ± 0.32×10⁶ and WT: 1.18 ± 0.31×10⁶; n=9, 9; p=0.0364) confirmed the significant decrease in the absolute number of MZ B cells (Figure 4B, C). We immune-stained spleen cryosections to establish further the exclusive reduction in MZ B cells. Iqgap1^−/− mice displayed normal B cell follicle architecture (Figure 4D), as revealed by the anti-CD3ε and anti-B220 mAb staining. Since the metallophilic macrophages form a boundary between the follicular and marginal zones, we used MOMA-1 mAb to determine the changes in the marginal zone. The spleen sections from the WT mice revealed a well-defined band of B220⁺ MZ area outside of the MOMA-1⁺ metallophilic macrophages. However, similar regions were greatly reduced and less contiguous in Iqgap1^−/− mice, which further confirmed a reduction in the number of MZ B cells (Figure 4E).

Figure 4. Lack of IQGAP1 leads to increased NF-B, FO-B but decreased MZ-B cells in the spleen and the dysregulated early B cell development in the spleen of Iqgap1^−/− mice is B cell-intrinsic. A) Absolute numbers of CD21^Low/−CD23^Low/− NF-B, CD21^LowCD23^High FO-B, and CD21^HighCD23^Low/− MZ-B cells are shown. Total splenic B cells were stained with anti-B220, anti-CD21, and anti-CD23 antibodies, and live cells were gated and compared between the WT and Iqgap1^−/− mice. Data shown represent absolute numbers per million live B220⁺ B cells. B) Reduction in MZ-B cells was validated using anti-IgM and anti-CD21 antibody staining of total B220⁺ splenic B cells. Data shown represent absolute numbers per million live B220⁺ B cells. C) Reduction in MZ-B cells was validated using anti-IgM and anti-CD1d antibody staining of total B220⁺ splenic B cells. Data shown represent absolute numbers per million live B220⁺ B cells. Open and filled circles in A, B, and C are values obtained from an individual mouse from four independent experiments. Data are shown with the mean ± SEM. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph. D, E) Immunohistochemical analyses of splenic sections from WT and Iqgap1^−/− mice. Spleens were isolated, embedded in paraffin, 7-micron thick sections were cut, and stained for total B cells (anti-B220), T cells (anti-CD3ε), and metallophilic macrophages (anti-MOMA antibody). Panels show the locations of T and B cells as part of the B cell follicles and metalophillic antigen-1⁺ macrophages surrounding the follicles. F) Indicated host mice were sub-lethally irradiated and reconstituted with donor-derived 5 × 10⁵ BM cells. Eight weeks later, cells from the host BM were harvested and analyzed. Absolute numbers of reconstituted CD21^Low/−CD23^Low/− NF-B, CD21^LowCD23^High FO-B, and CD21^HighCD23^Low/− MZ-B cells are shown. Total splenic B cells were stained with anti-B220, anti-CD21, and anti-CD23 antibodies, and live cells were gated and compared between the WT and Iqgap1^−/− mice. Data shown represent absolute numbers per million live B220⁺ B cells.

Figure 4. Lack of IQGAP1 leads to increased NF-B, FO-B but decreased MZ-B cells in the spleen and the dysregulated early B cell development in the spleen of Iqgap1^−/− mice is B cell-intrinsic. A) Absolute numbers of CD21^Low/−CD23^Low/− NF-B, CD21^LowCD23^High FO-B, and CD21^HighCD23^Low/− MZ-B cells are shown. Total splenic B cells were stained with anti-B220, anti-CD21, and anti-CD23 antibodies, and live cells were gated and compared between the WT and Iqgap1^−/− mice. Data shown represent absolute numbers per million live B220⁺ B cells. B) Reduction in MZ-B cells was validated using anti-IgM and anti-CD21 antibody staining of total B220⁺ splenic B cells. Data shown represent absolute numbers per million live B220⁺ B cells. C) Reduction in MZ-B cells was validated using anti-IgM and anti-CD1d antibody staining of total B220⁺ splenic B cells. Data shown represent absolute numbers per million live B220⁺ B cells. Open and filled circles in A, B, and C are values obtained from an individual mouse from four independent experiments. Data are shown with the mean ± SEM. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph. D, E) Immunohistochemical analyses of splenic sections from WT and Iqgap1^−/− mice. Spleens were isolated, embedded in paraffin, 7-micron thick sections were cut, and stained for total B cells (anti-B220), T cells (anti-CD3ε), and metallophilic macrophages (anti-MOMA antibody). Panels show the locations of T and B cells as part of the B cell follicles and metalophillic antigen-1⁺ macrophages surrounding the follicles. F) Indicated host mice were sub-lethally irradiated and reconstituted with donor-derived 5 × 10⁵ BM cells. Eight weeks later, cells from the host BM were harvested and analyzed. Absolute numbers of reconstituted CD21^Low/−CD23^Low/− NF-B, CD21^LowCD23^High FO-B, and CD21^HighCD23^Low/− MZ-B cells are shown. Total splenic B cells were stained with anti-B220, anti-CD21, and anti-CD23 antibodies, and live cells were gated and compared between the WT and Iqgap1^−/− mice. Data shown represent absolute numbers per million live B220⁺ B cells.

These reductions were B cell-intrinsic as reconstitution with Iqgap1^−/− BM cells either into WT or Iqgap1^−/− mice confirmed these defects. In the spleen of the reconstituted mice, we did not observe an increase in the absolute number of lymphocytes or B220⁺ B cells. However, the transfer of Iqgap1^−/− BM cells into the WT or Iqgap1^−/− mice lead to a significant increase in the absolute numbers of immature (IgM⁺IgD⁻) but not mature (IgM⁺IgD⁺ and IgM⁻IgD⁺) B cell populations. Reconstitution with Iqgap1^−/− BM cells either into WT or Iqgap1^−/− mice showed an increase in the CD23^HiCD21^Low/− FO B and a decrease in CD21^HiCD23^Low/− MZ B cells (Figure 4F). A decrease in the MZ B cells was further validated by a reduction in IgM⁺CD21^High or IgM⁺CD1d^High B cells in the reconstituted mice (Figure 5A, B). Our results demonstrate that the B cell defects in the BM and spleen of Iqgap1^−/− mice were cell-intrinsic.

Figure 5. Dysregulated early B cell development in the spleen of Iqgap1^−/− mice is B cell-intrinsic. A) Reduction in reconstituted MZ-B cells was validated using anti-IgM and anti-CD1d antibody staining of total B220⁺ splenic B cells. Data shown represent absolute numbers per million live B220⁺ B cells. B) Reduction in reconstituted MZ-B cells was validated using anti-IgM and anti-CD21antibody staining of total B220⁺ splenic B cells. Data shown represent absolute numbers per million live B220⁺ B cells. Data are shown with the mean ± SEM. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph.

Figure 5. Dysregulated early B cell development in the spleen of Iqgap1^−/− mice is B cell-intrinsic. A) Reduction in reconstituted MZ-B cells was validated using anti-IgM and anti-CD1d antibody staining of total B220⁺ splenic B cells. Data shown represent absolute numbers per million live B220⁺ B cells. B) Reduction in reconstituted MZ-B cells was validated using anti-IgM and anti-CD21antibody staining of total B220⁺ splenic B cells. Data shown represent absolute numbers per million live B220⁺ B cells. Data are shown with the mean ± SEM. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph.

Lack of IQGAP1 impairs T-independent and T-dependent antibody production

The significant impairment in B cell response in Iqgap1^−/− mice could be due to either a developmental or a functional defect. To narrow it down, we first analyzed the basal immunoglobulin levels in the pre-immune sera of WT and Iqgap1^−/− mice. Then, we quantified each isotype in the pre-immune sera. Figure 6A shows that IgG1, IgG2a, IgG2b, and IgA levels were comparable between WT and Iqgap1^−/− mice. However, the levels of IgM were significantly reduced, and IgG3 was significantly increased in the sera of Iqgap1^−/− mice (Figure 6A). Secreted ‘natural’ IgM is central to neutralizing pathogens and self-antigens. Thus, the presence of multiple Ig isotypes in the sera of Iqgap1^−/− mice indicated that the lack of IQGAP1 did not result in a global hypo-responsiveness of B cells. IgG3 subtype possesses an extended hinge region, superior molecular flexibility, broad polymorphisms, and unique glycosylation sites compared to other IgG subclasses.

Figure 6. Lack of IQGAP1 results in significantly reduced IgM and IgG3 levels in the sera of Iqgap1^−/− mice and results in significantly reduced T-independent and T-dependent humoral responses. A) Pre-immune sera were collected from 20 WT or Iqgap1^−/− mice each and were analyzed for the levels of soluble IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 using isotype-specific capture and detection antibodies-based ELISA. Data presented were an average of duplicates for each isotype of each serum from multiple experiments. A total of 20 sera were used for each strain. Data are shown with the mean ± SEM. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph. B) T-independent B cell responses were measured following the TNP-AECM-Ficoll antigen challenge. Four WT or four Iqgap1^−/− mice were used to collect pre-immune or immune sera. ELISA was performed with titrated concentrations of sera (1:4, 1:8, and 1:16). IgM, IgG1, IgG2a, and IgG3 isotypes were tested and shown. C) T-dependent B cell responses were measured following the TNP-KLH antigen challenge. Four WT or four Iqgap1^−/− mice were used to collect pre-immune or immune sera. ELISA was performed with sera collected at different time points before (pre-immune) and after antigen challenge (post 7d, 21d, and following 7d after booster challenge). IgM, IgG1, IgG2a, and IgG2b isotypes were tested and shown. Open and filled circles in A and B are values obtained from an individual mouse. Black and grey bars represent, respectively. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph.

Figure 6. Lack of IQGAP1 results in significantly reduced IgM and IgG3 levels in the sera of Iqgap1^−/− mice and results in significantly reduced T-independent and T-dependent humoral responses. A) Pre-immune sera were collected from 20 WT or Iqgap1^−/− mice each and were analyzed for the levels of soluble IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 using isotype-specific capture and detection antibodies-based ELISA. Data presented were an average of duplicates for each isotype of each serum from multiple experiments. A total of 20 sera were used for each strain. Data are shown with the mean ± SEM. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph. B) T-independent B cell responses were measured following the TNP-AECM-Ficoll antigen challenge. Four WT or four Iqgap1^−/− mice were used to collect pre-immune or immune sera. ELISA was performed with titrated concentrations of sera (1:4, 1:8, and 1:16). IgM, IgG1, IgG2a, and IgG3 isotypes were tested and shown. C) T-dependent B cell responses were measured following the TNP-KLH antigen challenge. Four WT or four Iqgap1^−/− mice were used to collect pre-immune or immune sera. ELISA was performed with sera collected at different time points before (pre-immune) and after antigen challenge (post 7d, 21d, and following 7d after booster challenge). IgM, IgG1, IgG2a, and IgG2b isotypes were tested and shown. Open and filled circles in A and B are values obtained from an individual mouse. Black and grey bars represent, respectively. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph.

To test whether IQGAP1 plays a role in humoral immune responses, we challenged age and sex-matched WT and Iqgap1^−/− mice with T-independent (TI) antigen TNP-AECM-Ficoll (TNP (2,4,6-Trinitrophenyl), where the hapten is conjugated to Amino Ethyl Carboxy Methyl-Ficoll and collected sera to analyze the presence of antigen-specific IgM, IgG1, IgG2a, or IgG3. Our phenotypic analyses indicated an increased number of naïve B cells in Iqgap1^−/− mice expressed membrane-bound IgM and IgD in both the BM and spleen. Irrespective of this and an increase in the overall FO B cell number, Iqgap1^−/− mice could not produce similar amounts of TNP-specific antibodies during TI responses as WT mice (Figure 6B). Next, we examined the T-dependent (TD) immune responses by intraperitoneal injection of TNP-KLH. In the primary response, the generation of TNP-specific IgM, IgG1, IgG2a, or IgG2b was significantly reduced in Iqgap1^−/− mice when compared to that of WT mice (Figure 6C). Further, after the antigen boost, the KLH-specific IgG1 did not recover and remained significantly low in the Iqgap1^−/− mice compared to WT (Figure 6C). These results demonstrate that IQGAP1 plays a crucial role in the activation and functions of B cells.

Receptor editing at κ the locus is moderately defective in B cells lacking IQGAP1

The diversity in the length of hypervariable complementarity-determining region 3 (CDR3) of the heavy chain (CDRH3) indicates a repertoire of rearranged V_H genes and polyclonality ²³. Using PCR-based spectrotyping, we analyzed the fragment size and peak height for IgHV3, IgHV5, IgHV6, and IgHV7 in B cells from the BM. Our results show that the length of the variable regions and the relative number of sequence-occurrences of the heavy chains in B cells from the WT and Iqgap1^−/− mice followed an expected normal Gaussian distribution indicative of diverse and comparable repertoire (Figure 7A). Thus, the lack of IQGAP1 did not affect the B cell repertoire diversity at the heavy chain rearrangement. Furthermore, the preserved heavy chain rearrangement in B cells from Iqgap1^−/− mice indicated that lack of IQGAP1 did not affect the B cell repertoire diversity at the heavy chain rearrangement.

Figure 7. Light chain but not heavy recombination is affected by the lack of IQGAP1. A) PCR-based spectrotyping was employed to quantify the diversity in the length of hypervariable CDR3 of the heavy chain (CDRH3) in B cells from the BM of WT and Iqgap1^−/− mice. The fragment size and peak height for IgHV3, IgHV5, IgHV6, and IgHV7 indicated that the repertoire of rearrangement V_H genes and polyclonality between these two strains are comparable and followed the expected normal Gaussian distribution indicative of diverse and comparable repertoire. B) Igκ and Igλ light chains in the sera are reduced in Iqgap1^−/− mice. C) RS rearrangement at the k-locus is reduced in the absence of IQGAP1. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph.

Figure 7. Light chain but not heavy recombination is affected by the lack of IQGAP1. A) PCR-based spectrotyping was employed to quantify the diversity in the length of hypervariable CDR3 of the heavy chain (CDRH3) in B cells from the BM of WT and Iqgap1^−/− mice. The fragment size and peak height for IgHV3, IgHV5, IgHV6, and IgHV7 indicated that the repertoire of rearrangement V_H genes and polyclonality between these two strains are comparable and followed the expected normal Gaussian distribution indicative of diverse and comparable repertoire. B) Igκ and Igλ light chains in the sera are reduced in Iqgap1^−/− mice. C) RS rearrangement at the k-locus is reduced in the absence of IQGAP1. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph.

CD43⁺ B cell progenitors in mice rearrange immunoglobulin Igκ genes before the assembly of a productive V_HD_HJ_H joint. Thus, μ chain rearrangement and expression are not prerequisites to Igκ light chain gene rearrangements in normal development ²⁴. Therefore, we next analyzed the quantity of κ and λ light chains in the sera of non-challenged healthy WT and Iqgap1^−/− mice using an ELISA-based assay. We found a significant reduction in both the κ and λ light chains in Iqgap1^−/− mice compared to the WT (Figure 7B). This demonstrates that the defect we see in the antibody production in challenged mice could be related to the production of the light but not the heavy chains. Indeed small GTPase, Ras, plays an essential role via Mek1/2 and Erk1/2 in silencing Ccnd3 transcription, exiting from the cell cycle, and initiating Igκ recombination and light chain editing ²⁵. Receptor editing in the light chain loci forms a critical basis for high-affinity maturation that is a continuous process in an ongoing immune response ^{26, 27}, and impairment in κ chain rearrangement could account for the functional defects in T-dependent and independent antibody responses. Therefore, we next analyzed the light chain rearrangement. The orderly recombination of genes at the κ locus is the first step in the light chain rearrangement. The successful recombination of genes at the κ locus is proceeded by rearranging non-coding gene segments defined as ‘recombining sequence’ (RS) ²⁸. RS rearrangements occur higher in the most highly edited B cells. Thus, quantifying RS provides a readout for successful κ chain rearrangement ²⁹. Our results show that the levels of amplified RS products were moderately lower in the B cells derived from Iqgap1^−/− mice (Figure 7C), indicating that the early pre-B cell development is largely intact in Iqgap1^−/− mice. Thus, the functional defects observed in the B cells from Iqgap1^−/− mice are due to an inability to rearrange the light chains appropriately.

B cell activation requires IQGAP1-Mek1/2-Erk1/2 signalosome

IQGAP1 is an essential scaffold in organizing Erk1/2 activation and phosphorylation ³⁰. Four tandem Isoleucine-Glutamine (IQ) domains are located between 745-864 amino acids. These IQ domains primarily mediate interactions with Rap1 ³¹, B-Raf or C-Raf ³², and Mek1/2 ³⁰. Therefore, we hypothesized that IQGAP1 regulates the sequential activation of the Raf/Mek1/2/Erk1/2 cascade. To test this, we activated purified total splenic B cells with soluble anti-IgM mAb. Rac1/Cdc42 activates p21-activated kinase (Pak) that connects the upstream signaling to Raf by phosphorylating S445 in B-Raf and S338 in C-Raf ³³. We found comparable amounts of phosphorylated B-Raf (Serine 445), C-Raf (Serine 338), and total B-Raf and C-Raf were present in Iqgap1^−/− and WT B cells (Figure 8A). B-Raf or C-Raf catalyzes the phosphorylation of Mek1/2, which in turn phosphorylates Erk1/2 ³⁴. Since IQ domains recruit Mek1/2, we analyzed their activation status. and found the phosphorylation of Mek1/2 were considerably reduced in B cells lacking IQGAP1. The WW domain of IQGAP1 (685-710 amino acids) contains two conserved tryptophan, positioned about 20-22 amino acids apart (Wx_20-22W) that function as an interaction module for proline-rich ligands ³⁵. This domain is also responsible for recruiting Erk1/2 via a polyproline (APPPxxPY) motif ³⁶. Therefore, we next analyzed the phosphorylation status of Erk1/2 along with the two other MAPKs, Jnk1/2, and p38. All three kinases were active in the WT B cells. p38 phosphorylation was not affected in any of the Iqgap1^−/− B cells. However, the levels of Erk1/2 and Jnk1/2 phosphorylation were considerably reduced in Iqgap1^−/− B cells (Figure 8A).

Figure 8. IQGAP1 orchestrates ERK1/2 activation in B cells. A) Sorted splenic B cells were activated with soluble anti-IgM antibody for indicated time points and lysed, protein quantities were estimated and analyzed by western blots. Lysates were analyzed for the total and phosphorylated forms of Raf1, B-Raf, Mek1, Erk1/2, JNK1/2, and p38. Numbers below each blot represent fold change calculated following normalized values against their respective total and based on the phosphorylated protein levels of the WT. B) PCR array results. C) PCR array results. Data shown are one representative panel of three independent experiments. Data shown are obtained by repeated probing of the same membrane for indicated proteins.

Figure 8. IQGAP1 orchestrates ERK1/2 activation in B cells. A) Sorted splenic B cells were activated with soluble anti-IgM antibody for indicated time points and lysed, protein quantities were estimated and analyzed by western blots. Lysates were analyzed for the total and phosphorylated forms of Raf1, B-Raf, Mek1, Erk1/2, JNK1/2, and p38. Numbers below each blot represent fold change calculated following normalized values against their respective total and based on the phosphorylated protein levels of the WT. B) PCR array results. C) PCR array results. Data shown are one representative panel of three independent experiments. Data shown are obtained by repeated probing of the same membrane for indicated proteins.

To further define the status of specific transcription factors altered in the early B cells from Iqgap1^−/− mice, we performed a PCR array analysis. We sorted total B cells from the BM or spleen of three mice each and pooled the mRNA for each strain. cDNA preparations were analyzed in triplicates, and average expression was presented as fold change (Fc) between Iqgap1^−/− B cells over WT (Figure 8B). Total naïve BM B cells were analyzed for the expression levels of transcription factors that play an essential role in early B cell commitment and maturation. Expressions of Foxo3, Foxo4, MafK, Stat5a, and Tcf4 were increased two-fold in Iqgap1^−/− B cells compared to WT. Id2, Ikzf3, Lef, Nfil3, Rel, Sox4, and Tbx21 were reduced in the Iqgap1^−/− B cells. It is important to note that the expression of Id2 is regulated by Erk/Jnk/Cdk2 pathway. Ikzf3 (Ailos) is essential for the heterochromatic sequestration of the Igκ gene. Next, we stimulated the sorted splenic B cells with anti-IgM mAb and compared the expression of transcription factors in between unstimulated and stimulated conditions from both strains. Averages from triplicates were used to calculate the fold change (Fc) between Iqgap1^−/− B cells over WT under both the experimental conditions (Figure 8C). Similar to the total B cells from the BM, the levels of Foxo3, Foxo4, MafK, Stat5a, and Tcf4 have increased two or more folds in non-stimulated Iqgap1^−/− B cells compared to WT. The expression levels of all these factors were considerably reduced in Iqgap1^−/− B cells following anti-IgM mAb-mediated activation. However, the levels of c-Fos, Jun, Sox4, and Tbx21 were reduced in both unstimulated and stimulated conditions. Interestingly, levels of Id2, c-Kit, Myc, Nfatc1, and Rel were increased in Iqgap1^−/− B cells following anti-IgM mAb-mediated activation (Figure 8C). Expression of c-Kit is limited only to the pre-lymphoid progenitors, pro-B, and pre-B-I cells. Therefore, a high level of c-Kit expression in Iqgap1^−/− B cells at both unstimulated and stimulated conditions strongly validates our phenotypic characterization and supports our notion that lack of IQGAP1 results in a defect in the transition of pre-B-I into large pre-B-II cells.

IQGAP1 is essential for suppressing Stat5a and Stat5b

Earlier studies have shown that pre-BCR-mediated activation counters the IL-7R pathway ²⁵. Signaling via IL-7R sustains the survival and proliferation of pro-B cells ³⁷. This is primarily mediated by cell-cycle-related genes, including Ccnd3. In addition, phosphorylated Stat5A and Stat5B, downstream of IL-7R, translocated into the nucleus and form a complex with PRC2/EZH2 to bind to 5′ intronic enhancer of the κ gene (iEκ) to actively suppress its transcription by limiting the access to E2A, which is induced by the pre-BCR signaling. In addition, the pre-BCR-mediated Ras-Mek1/2-Erk1/2 pathway silences Ccnd3 to orchestrate the exit from the cell cycle and to initiate κ chain recombination ^{25, 38}. First, we investigated the status of Ccnd3 transcripts. For this, we purified the relatively immature T1 B cells from the spleens of WT and Iqgap1^−/− mice. Expression of cell cycle-related genes such as β-catenin, Bcl-xl, Ccnd3, Ccnd1, and Bim were analyzed. We found that the levels of β-catenin and Ccnd3 transcripts were significantly and moderately reduced, respectively (Supplementary Figure S3). Next, we investigated the status of E2A, Ezh2, Stat3, Stat5A, and Stat5B in WT and Iqgap1^−/− B cells. First, we analyzed the protein levels of these transcription factors in the total B cells derived from the BM of both strains (Figure 9A). The limited numbers of pro-and pre-B cells excluded our ability to use them in these assays. Compared to the WT, Iqgap1^−/− B cells possessed moderately higher levels of E2A, Stat5A, Stat5B, and Ezh2.

Figure 9. IQGAP1 is required to suppress Stat5a and Stat5b and to initiate the transcription of IRF4 and IRF8. A) Total sorted BM B cells were used to analyze the protein levels of E2A, Stat3, Stat5A, Stat5B, IRF4, and β-actin. Data shown are one representative of three experiments. B) Total sorted BM B cells were activated with anti-IgM mAb, and the cytoplasmic and nuclear protein fractions were separated and analyzed for E2A, Stat3, Stat5A, Stat5B, and β-actin. Data shown are one representative of three experiments. C) mRNA from sorted BM B cells was used to quantify the expression of Irf4 and Irf8 transcripts. Data shown are the average relative expression of these transcripts from four mice. D) mRNA from sorted splenic B cells was used to quantify the expression of Irf4 and Irf8 transcripts. Data shown are the average relative expression of these transcripts from four mice. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph. E) Cartoon summarizing the role of IQGAP1 during early B cell development. IL-7R transduces its signal via Stat3, Stat5A, and Stat5B. Signals via pre-BCR initiate two distinct pathways, One via SLP65-mediated IRF4 and IRF8 activation and the second via the Ras-Raf-Mek-Erk/Jnk pathway mediated by IQGAP1. Four tandemly organized IQ domains can recruit C-Raf or B-Raf. IQ domains also have the specificity to Mek1/2. Rafs mediate the activation of Mek1/2 on the IQGAP1 scaffold. The WW domain of IQGAP1 recruits Erk1/2, which is phosphorylated by Mek1/2. Phosphorylated Erk1/2 activates several transcription factors, including E2A, Elk1, and CREB. E2A competes with Stat5 dimers for the 5′ intronic Eκ enhancer accessibility. Successful displacement of Stat5 dimers initiates the recombination process of Igκ gene. E2A also relieves the Stat5-mediated repression of Rag1 and Rag2 genes. Stat5-mediated transcription of Ccnd3 is relieved by the phosphorylated Ras-Mek1/2-Erk1/2 pathway. The potential candidates that repress the transcription of Ccnd3 are Cdkn2a or p21. The mechanism by which IQGAP1 regulates the expression of Irf4 and Irf8 genes are not known. IRF4 binds to the 3′ Eκ in further promoting light chain recombination. IRF4 also binds to the 5′ Eλ to initiate the transcription of Igλ gene. IRF4, along with Ikaros3 (Ailos) represses the surrogate light chain component λ5-encoding gene. IRF4 also plays an important role in initiating the transcription of Cxcr4, which is essential for the pre-B cells to traffic from their IL-7-producing stromal niche to IL-7-deficient niche to transition into immature B cells. Repressing Id2 and Id3 is also needed for the pre-B cells that is regulated by Ebf1, a potential target downstream of Erk1/2.

Figure 9. IQGAP1 is required to suppress Stat5a and Stat5b and to initiate the transcription of IRF4 and IRF8. A) Total sorted BM B cells were used to analyze the protein levels of E2A, Stat3, Stat5A, Stat5B, IRF4, and β-actin. Data shown are one representative of three experiments. B) Total sorted BM B cells were activated with anti-IgM mAb, and the cytoplasmic and nuclear protein fractions were separated and analyzed for E2A, Stat3, Stat5A, Stat5B, and β-actin. Data shown are one representative of three experiments. C) mRNA from sorted BM B cells was used to quantify the expression of Irf4 and Irf8 transcripts. Data shown are the average relative expression of these transcripts from four mice. D) mRNA from sorted splenic B cells was used to quantify the expression of Irf4 and Irf8 transcripts. Data shown are the average relative expression of these transcripts from four mice. Statistical significance was calculated using Student’s t-test, and p-values are shown below each graph. E) Cartoon summarizing the role of IQGAP1 during early B cell development. IL-7R transduces its signal via Stat3, Stat5A, and Stat5B. Signals via pre-BCR initiate two distinct pathways, One via SLP65-mediated IRF4 and IRF8 activation and the second via the Ras-Raf-Mek-Erk/Jnk pathway mediated by IQGAP1. Four tandemly organized IQ domains can recruit C-Raf or B-Raf. IQ domains also have the specificity to Mek1/2. Rafs mediate the activation of Mek1/2 on the IQGAP1 scaffold. The WW domain of IQGAP1 recruits Erk1/2, which is phosphorylated by Mek1/2. Phosphorylated Erk1/2 activates several transcription factors, including E2A, Elk1, and CREB. E2A competes with Stat5 dimers for the 5′ intronic Eκ enhancer accessibility. Successful displacement of Stat5 dimers initiates the recombination process of Igκ gene. E2A also relieves the Stat5-mediated repression of Rag1 and Rag2 genes. Stat5-mediated transcription of Ccnd3 is relieved by the phosphorylated Ras-Mek1/2-Erk1/2 pathway. The potential candidates that repress the transcription of Ccnd3 are Cdkn2a or p21. The mechanism by which IQGAP1 regulates the expression of Irf4 and Irf8 genes are not known. IRF4 binds to the 3′ Eκ in further promoting light chain recombination. IRF4 also binds to the 5′ Eλ to initiate the transcription of Igλ gene. IRF4, along with Ikaros3 (Ailos) represses the surrogate light chain component λ5-encoding gene. IRF4 also plays an important role in initiating the transcription of Cxcr4, which is essential for the pre-B cells to traffic from their IL-7-producing stromal niche to IL-7-deficient niche to transition into immature B cells. Repressing Id2 and Id3 is also needed for the pre-B cells that is regulated by Ebf1, a potential target downstream of Erk1/2.

Interestingly, no protein band was found for IRF4 in Iqgap1^−/− B cells (Figure 9A). Next, we evaluated the levels of these transcription factors translocated into the nucleus following anti-IgM mAb-mediated activation. The B cells activated by this method are not pro- or pre-B cells; but immature and mature B cells. However, it served as an indicator of the status of these transcription factors. We isolated total BM B cells and activated them with anti-IgM mAb for two hours. Nuclear and cytoplasmic fractions were isolated and analyzed for total E2A, Stat3, Stat5A, Stat5B, and β-actin (Figure 9B). Stat3, Stat5A, and Stat5B were present at comparable levels in the cytoplasmic fractions of B cells in both the WT and Iqgap1^−/− mice. However, following anti-IgM mAb-mediated activation, the levels of these transcription factors were higher in Iqgap1^−/− B cells compared to that of the WT. These results indicate a sustained presence of E2A, Stat3, Stat5A, and Stat5B in the nucleus of Iqgap1^−/− B cells. In addition to these transcription factors, IRF4 and IRF8 are obligatory for the cycling pre-B to initiate light chain rearrangement and transition into immature B cells ^{39, 40}. Therefore, next, we quantified the levels of IRF4 and IRF8 in total BM- and spleen-derived B cells. Both IRF4 and IRF8 were significantly reduced in B cells from Iqgap1^−/− compared to the WT mice (Figure 9C, D). These results demonstrate that the lack of IQGAP1 scaffold significantly impairs early B cell development due to the reduced levels of IRF4 and IRF8 transcription factors.

Discussion

Materials and Methods

References

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