Impaired antibody response in Hoxc4^−/− mice. (a) Four pairs of littermate Hoxc4^+/+ and Hoxc4^−/− mice were immunized with NP₁₆-CGG and boost-injected 21 d later. Circulating IgM and IgG1, NP₃₀-binding IgM and IgG1, and (high-affinity) NP₃-binding IgM and IgG1 were measured 7 d after the boost-injection, and, their levels were expressed as 50% effective concentration (EC₅₀) units. These were defined as the number of dilutions needed to reach 50% of saturation binding. (b and c) Normal plasma cell and memory B cell development in Hoxc4^−/− mice 14 d after immunization with NP₁₆-CGG. (b) Surface CD138 and B220 expression in spleen cells from immunized littermate Hoxc4^+/+ and Hoxc4^−/− mice. Numbers in boxes are B220^lo CD138⁺ (plasma) cells, as percentage of total B220⁺ cells. (c) Spleen cells from immunized littermate Hoxc4^+/+ and Hoxc4^−/− mice were analyzed by flow cytometry after staining with FITC-PNA, APC-anti-mouse IgG1 mAb, PE-NP and PECy7-anti-mouse CD38 mAb. Insets in left panels show NP-binding surface IgG1⁺ B cells. Right panels show CD38 expression by the gated NP-binding IgG1⁺ B cells. Data are representative of 3 independent experiments (b and c).

Impaired CSR in Hoxc4^−/− B cells. (a) Spleen Hoxc4^+/+ and Hoxc4^−/− B cells were stimulated with LPS or CD154 in the presence of nil (for IgG2b and IgG3), IL-4 (for IgG1 and IgE), IFN-γ (for IgG2a), or TGF-β1/IL-4/IL-5/anti-δ mAb-dextran (for IgA). After 7 d, the supernatants from the cultures of Hoxc4^+/+ B cells (empty symbols) and Hoxc4^−/− B cell (full symbols) stimulated with LPS or LPS and cytokines were collected and analyzed for concentration of different Ig isotypes. Data are from 4 pairs of Hoxc4^+/+ and Hoxc4^−/− mice. (b) Hoxc4^+/+ and Hoxc4^−/− B cells were labeled with cell division-tracking fluorochrome CFSE and stimulated with LPS and IL-4 or CD154 and IL-4 to induce switching to IgG1. This showed that proliferation of Hoxc4^−/− B cells was normal. After 4 d of culture, Hoxc4^−/− and Hoxc4^+/+ B cells completed the same number of divisions, but the percentage of surface IgG1⁺ B cells was significantly lower among Hoxc4^−/− B cells. Data are from 2 independent experiments. (c) HoxC4 deficiency does not alter plasma cell differentiation. Hoxc4^+/+ and Hoxc4^−/− B cells were stimulated with LPS, LPS and IL-4 or CD154 and IL-4. After 4 d of culture, the cells were stained with PE-anti-mouse B220 mAb and FITC-anti-mouse CD138 mAb for flow cytometry analysis. Numbers in boxes indicate B220^loCD138⁺ (plasma) cells as percentage of total cells analyzed. Data are from one representative of 3 independent experiments.

Decreased somatic mutation frequency in the Ig H chain intronic J_H4-iEµ DNA of Peyer’s patch PNA^hiB220⁺ (GC) B cells from 3 12-week-old Hoxc4^−/− mice as compare to their Hoxc4^+/+ littermates. (a) Pie charts depict the proportions of sequences that carry 1, 2, 3, etc. mutations over the 720 bp J_H4-iE_µ DNA analyzed. The numbers of the sequences analyzed are at the center of the pies. (b) HoxC4 deficiency does not alter the level of V_J558DJ_H-Cµ transcripts. V_J558DJ_H-C_µ transcripts in Peyer’s patches B cells of these mice were measured by real-time qRT-PCR performed in triplicate samples using SYBR-green. In each sample, mRNA expression was normalized to CD79b expression. Data are means ± s.e. (bars) of triplicate samples from 3 independent pairs of Hoxc4^+/+ and Hoxc4^−/− mice. The analysis of the spectrum of mutations in Hoxc4^+/+ and Hoxc4^−/− mice is the subject of Supplementary Fig. 4.

The conserved HoxC4-Oct- and Sp-NF-κB-binding sites are essential for full Aicda promoter activity; HoxC4, Oct1, Oct2, OcaB, Pax5, Sp1, Sp3 and NF-κB (p52 subunit) are recruited to the Aicda promoter in B cells expressing AICDA/Aicda and undergoing CSR or SHM. (a) pGL3-Enhancer luciferase gene reporter constructs containing WT or mutant Aicda promoter, in which the HoxC4-Oct- and/or Sp-NF-κB-binding sites were deleted or disrupted by site-directed mutagenesis, were used to transfect CH12F3-2A, 4B6 or Ramos B cells. Luciferase activity was measured after 24 h (CH12F3-2A B cells) or 16 h (Ramos and 4B6 B cells) of culture. Data are means ± s.e. (bars) of 3 independent experiments. (b) AICDA/Aicda expression in human spontaneously switching 4B6 and Ramos B cells, human inducible switching 2E2 B cells stimulated with nil or anti-CD40 mAb and IL-4, mouse CH12F2-2A B cells stimulated with nil or LPS, IL-4 and TGF-β1, WT C57BL/6 mouse spleen B cells stimulated nil, LPS and IL-4, or CD154 and IL-4 were analyzed by semi-quantitative RT-PCR using serially two-fold diluted cDNA as a template. Data are representative of 3 independent experiments. (c) Cross-linked chromatin was precipitated from the human or mouse B cells of panel b using a mouse mAb specific to HoxC4, rabbit Abs specific to Oct1, Oct2, OcaB, Pax5, Sp1, Sp3 or p52, or preimmune control mouse or rabbit IgG. The precipitated DNA was specified by PCR using AICDA or Aicda promoter primers. Data are representative of 3 independent experiments.

Hoxc4 expression correlates with Aicda expression. (a) Hoxc4 and Aicda transcripts in bone marrow, thymus, spleen, Peyer’s patches, liver or heart of C57BL/6 mice, as measured by real-time qRT-PCR performed in triplicate samples using SYBR-green. In each sample, mRNA expression was normalized to Gapdh expression. Data are means ± s.e. (bars) of fold mRNA in the indicated tissue compared to bone marrow of 3 independent experiments. (b) HoxC4, AID, PCNA and β-actin protein expression in PNA^hiB220⁺ (GC) B cells and PNA^loB220⁺ (non-GC) B cells from spleen of NP₁₆-CGG immunized mice, as detected by immunoblotting. Data are from 2 independent experiments. (c) Increased expression of Hoxc4 and Aicda mRNA in LPS and IL-4- or CD154 and IL-4-stimulated spleen B cells, as determined by real-time qRT-PCR. Spleen B cells from 3 C57BL/6 mice were cultured with nil, LPS plus IL-4 or CD154 plus IL-4, and harvested after 3 d to extract RNA and perform real-time qRT-PCR. mRNA expression was normalized to CD79b transcripts. Data are from 3 independent experiments. In each experiment, values are means ± s.e. (bars) of fold mRNA levels in B cells stimulated as indicated, as compared to unstimulated B cells (nil).

HoxC4 deficiency does not affect B and T cell numbers, CD4⁺ T cell numbers, B and T cell death in spleen and Peyer’s patches, B cell cycle or division, nor does it alter GC formation and in vivo B cell proliferation. (a–d) Flow cytometric profiles of cells from spleen and Peyer’s patches stained with (a) PE-anti-B220 mAb and FITC-anti-CD3 mAb, (b) FITC-anti-CD3 mAb and PerCP-anti-CD4 mAb, (c) 7-AAD and FITC-anti-CD3 mAb, and (d) 7-AAD and PE-anti-B220 mAb. (e) Cell cycle analysis of Hoxc4^+/+ and Hoxc4^−/− B cells. Spleen Hoxc4^+/+ and Hoxc4^−/− B cells were stimulated with LPS and IL-4 and harvested after 1 and 2 d for PI staining and flow cytometry analysis to measure DNA content and enumerate cells in G0/G1, S and G2/M phases. (f) Cell division in Hoxc4^+/+ and Hoxc4^−/− B cells. Spleen Hoxc4^+/+ and Hoxc4^−/− B cells were labeled with CFSE, cultured with LPS and IL-4, and harvested 2 and 3 d later for flow cytometry analysis. Cell division is indicated by progressive left shift of fluorescence histograms. Individual cell generations are enumerated above the graph. Data are from one representative of 3 experiments. (g) Staining of GCs in spleen sections prepared 10 d after immunization of Hoxc4^+/+ and Hoxc4^−/− mice. Scale bar: 200 µm. The original magnifications are indicated at the bottom of each set of panels. (h) Flow cytometric profiles of cells from Peyer’s patches from NP₁₆-CGG immunized Hoxc4^+/+ and Hoxc4^−/− mice stained with PE-labeled anti-B220 mAb and FITC-PNA. (i) In vivo B cell proliferation. Three 10-week-old Hoxc4^+/+ and Hoxc4^−/− mice were immunized with NP₁₆-CGG. After 10 d, the mice were injected with BrdU (1 mg) twice within 16 h and sacrificed 4 h after the last injection. Cells from spleen and Peyer’s patches were stained with PE-anti-mouse B220 mAb or this mAb together with FITC-PNA. Incorporated BrdU was detected with APC-anti-BrdU mAb and analyzed using by FACS. Data are from one representative of 3 pairs of Hoxc4^+/+ and Hoxc4^−/− mice.

HoxC4 deficiency does not alter germline I_H-C_H transcripts but reduces the expression of post-recombination Iµ-C_H transcripts. Spleen Hoxc4^+/+ and Hoxc4^−/− B cells were cultured with LPS or LPS and cytokines for 3 d and then harvested for RNA extraction. This was used as template in real-time qRT-PCR to measure the levels of germline I_µ-C_µ, I_γ3-C_γ3, I_γ1-C_γ1, I_γ2b-C_γ2b, I_γ2a -C_γ2a, I_ε-C_εand I_α-C_α transcripts, and post-recombination I_µ-C_γ3, I_µ-C_γ1, I_µ-C_γ2b, I_µ-C_γ2a, I_µ-C_ε and I_µ-C_α transcripts, as normalized to CD79b expression. Values are means ± s.e. (bars) of triplicate samples. Data are from one representative of 3 pairs of Hoxc4^+/+ and Hoxc4^−/− mice. The data from the remaining 2 pairs are depicted in Supplementary Fig. 3.

(a,b) Decreased Aicda expression in Hoxc4^−/− B cells. Spleen Hoxc4^+/+ and Hoxc4^−/− B cells were cultured in the presence of nil, LPS and IL-4, or CD154 and IL-4. After 0, 12, 24, 48 and 72 h of culture, cells were harvested for RNA or protein extraction. (a) RNA (2 µg) was reverse-transcribed to cDNA and used as template in real-time qRT-PCR, in which Aicda expression was normalized to CD79b expression. Data are means ± s.e. (bars) of 3 independent experiments. (b) AID and β-actin proteins were detected by immunoblotting. Data are representative of 3 independent experiments. (c) Depicted, not to scale, are portion of the human AICDA and mouse Aicda promoter region (AID-Pro), flanking 5’ region (5’R), cr1 and cr2 in the first intron, and five exons, within which the coding region is depicted in light blue. (d) The −349 to −1 (AID-Pro) region, which we tentatively defined as Aicda promoter based on its high conservation, but not the immediately adjacent 5’ region, nor the downstream cr1 or cr2 region promotes transcription. Human Ramos (undergoing spontaneous SHM) B cells and mouse CH12F3-2A (CSR inducible) B cells were transfected with the indicated pGL3-reporter gene vector to assess Aicda promoter activity. After transfection, CH12F3-2A cells were treated with LPS, IL-4 and TGF-β1 to induce CSR. Luciferase activity was measured after 16 h (Ramos) or 24 h (CH12F3-2A) of culture. Data are the means ± s.e. (bars) of 3 independent experiments.

Enforced expression of AID rescues CSR in Hoxc4^−/− B cells. Hoxc4^+/+ and Hoxc4^−/− B cells activated with LPS were transduced with the TAC control or AID-expression TAC-Aicda retrovirus and cultured in the presence of LPS and IL-4. Three or 4 d after transduction, B cells were harvested for analysis of surface expression of B220 and IgG1 (a) and expression of Aicda by real-time qRT-PCR and semi-quantitative RT-PCR, germline I_µ-C_µ and I_γ1-C_γ1 transcripts by real-time qRT-PCR, circle I_γ1-C_µ transcripts by semi-quantitative RT-PCR and post-recombination I_µ-C_γ1 transcripts by real-time qRT-PCR (b). Expression of these transcripts was normalized in each case to CD79b transcripts; expression of transcripts in Hoxc4^−/− B cells were depicted as ratios to those in Hoxc4^+/+ B cells. FACS data are from one representative of 5 independent experiments. Real-time qRT-PCR and semi-quantitative RT-PCR data are means ± s.e. (bars) of 3 independent experiments.