Klhl6 belongs to the KLHL gene family, which is composed of an N-terminal BTB-POZ domain and four to six Kelch motifs in tandem. Several of these proteins function as adaptors of the Cullin3 E3 ubiquitin ligase complex. In this article, we report that Klhl6 deficiency induces, as previously described, a 2-fold reduction in mature B cells. However, we find that this deficit is centered on the inability of transitional type 1 B cells to survive and to progress toward the transitional type 2 B cell stage, whereas cells that have passed this step generate normal germinal centers (GCs) upon a T-dependent immune challenge. Klhl6-deficient type 1 B cells showed a 2-fold overexpression of genes linked with cell proliferation, including most targets of the anaphase-promoting complex/cyclosome complex, a set of genes whose expression is precisely downmodulated upon culture of splenic transitional B cells in the presence of BAFF. These results thus suggest a delay in the differentiation process of Klhl6-deficient B cells between the immature and transitional stage. We further show, in the BL2 Burkitt’s lymphoma cell line, that KLHL6 interacts with Cullin3, but also that it binds to HBXIP/Lamtor5, a protein involved in cell-cycle regulation and cytokinesis. Finally, we report that KLHL6, which is recurrently mutated in B cell lymphomas, is an off-target of the normal somatic hypermutation process taking place in GC B cells in both mice and humans, thus leaving open whether, despite the lack of impact of Klhl6 deficiency on GC B cell expansion, mutants could contribute to the oncogenic process.

B cell generation follows a stepwise process of differentiation, first in the bone marrow and later in the periphery, where fully mature B cells reside. After a productive rearrangement and expression of a functional BCR at the surface, B cells are still at an immature stage. Cross-linking of the BCR at this stage induces cell death, a negative selection process purging the B cell repertoire from autoreactive specificities (1, 2).

Immature B cells that survive past this checkpoint migrate to the spleen and mature into follicular (FO) and marginal zone (MZ) B cells (3). This maturation occurs in successive steps and implies the formation of two subsets of transitional B cells, transitional type 1 (T1) and transitional type 2 (T2) (4), which have been proposed to be located in different areas of the spleen, the periarteriolar lymphoid sheath and the B cell follicles, respectively (5). Several surface markers have been used to characterize T1 and T2 B cells, notably expression of CD93 and CD23, which distinguish T1 (CD93+CD23) from T2 B cells (CD93+CD23+) (68). It has been observed that immature B cells can also differentiate into T1-like and T2-like B cells within the bone marrow (9, 10). T2 B cells, which emerge in spleen with a parallel but slightly delayed timing, could thus originate from both in situ T1 cell differentiation and migration of T2-like cells from bone marrow (11, 12). Interaction between the B cell–activating cytokine BAFF and its specific receptor (BAFF-R) on B cells appears to play different roles in this process, depending on the B cell differentiation stage and the lymphoid organ concerned (1319). Whereas the immature to T1-like B cell progression in bone marrow seems to occur in a BAFF-independent mode, this is not the case in the spleen, where T1 cells require BAFF signals for differentiation and/or survival, as shown in mixed bone marrow chimeras with BAFF-R–deficient and BAFF-R–competent cells (20). Conversely, the differentiation of both splenic and bone marrow T1 into T2 B cells is strongly dependent on BAFF signaling, because B cell maturation in BAFF- and BAFF-R–deficient mice does not proceed beyond the T1 stage (13, 15, 19).

The KLHL gene family encodes 42 different proteins, defined as being composed of an N-terminal BTB-POZ domain followed by a BTB and C-terminal kelch (BACK) domain and four to six tandem Kelch motifs (21). The BTB domain, named by its homology with the Drosophila Bric-a-brac, Tramtrac, and Broad complex, is involved in protein–protein interaction and protein self-oligomerization (22). The BACK domain is very conserved, but its role is not unequivocally identified (23). The Kelch motif, which was discovered as a six-repeat element in the Drosophila kelch protein, is a segment of 44–56 aa that adopts a β-propeller structure and contains multiple potential protein contact sites (24). Despite their shared secondary structure, the primary sequence of KLHL family members has little homology, suggesting a large diversity of interacting partners and consequently multiple biological functions. Although data on the molecular function of KLHL proteins are still fragmentary, several of them have been identified as adaptors of Cullin 3 (Cul3)-based E3 ubiquitin ligases (25). These KLHL proteins integrate the function of ubiquitin ligase adaptor and substrate recognition by forming a complex with Cul3 through the BTB domain and binding the substrate through the Kelch modules, mediating through ubiquitylation the degradation (26, 27) or relocalization of their substrate within the cell (28). Whereas Cul3 is a ubiquitous protein, several KLHL genes show marked tissue-restricted expression, allowing their differential specificity (29, 30).

We previously reported the isolation of the Klhl6 gene through its overexpression in B cells from sheep ileal Peyer’s patches, an overexpression that is also observed in mouse and human germinal center (GC) B cells (31). More recently, recurrent mutations in KLHL6 have been described in B cell lymphomas of GC origin (32), as well as in chronic lymphocytic leukemias (CLLs) with mutated Ig V genes (33, 34). The group of T. Sato (35) generated Klhl6 knockout (KO) mice and reported a 2-fold reduction of mature B cells. The B cell deficit was linked to an impaired proliferative response and signal transduction after BCR cross-linking, and a marked reduction in GC B cell formation was observed in T-dependent immune responses (35). We report in this article an independent inactivation of the Klhl6 gene. Although we observed the same reduction in mature B cells, we identified the origin of this B cell deficit as being essentially due to an impaired survival and maturation of transitional T1 cells during B cell development. Klhl6-deficient B cells that matured beyond this stage behave similarly to normal B cells, and accordingly, no impact of Klhl6 deficiency was observed on GC responses.

The Comité National de Réflexion Ethique sur l'Expérimentation Animale and the ethics committee of French Ministry of Research and Higher Education approved all animal experiments (project 014033.03). Klhl6−/− mice were generated from E14.1 embryonic stem cells and backcrossed to C57BL/6 background for >10 generations. Klhl6−/− × activation-induced cytidine deaminase (AID)-Cre-EYFP mice were generated by backcrossing Klh6−/− and AID-Cre-EYFP knock-in mice (AID-Cre-ERT2 crossed with ROSA26-loxP-EYFP reporter mice) (36).

Chimeric mice were created by bone marrow transplantation. Bone marrow was collected from tibial and femoral bone marrow cavities of 15-d-old C57BL/6 Ly5.1 (CD45.1) control and C57BL/6 Ly5.2 (CD45.2) Klhl6−/− mice. RBCs were lysed using RBC lysis buffer. Nucleated cells (107 cells/mouse) were injected i.v. via marginal sinus to 8-wk-old RAG-2–deficient mice that were previously irradiated (3 Gy, 137Cs irradiator). Mice were analyzed 2–3 mo after reconstitution.

Tissue samples were collected from the appropriate mice as described in the text. After RBC lysis, cells were stained with a combination of fluorophore-conjugated Abs (Supplemental Table I). Surface markers were detected and analyzed using a FACSCanto II flow cytometry apparatus and the BD FACSDiva software (BD Biosciences).

Splenic B cells, after purification (B cell isolation kit, MACS separation), were cultured in complete RPMI 1640 medium supplemented with 10% HyClone FetalClone I serum (Thermo Scientific), 100 U/ml penicillin and 100 U/ml streptomycin, 1 mM sodium pyruvate, MEM nonessential amino acids (1×), 25 mM HEPES (Invitrogen), 0.5 μM 2-ME (Sigma), and the indicated activating factors. All reagents, including anti-IgM (Jackson ImmunoResearch), LPS, CpG (Sigma), and recombinant BAFF protein (RD Biosystem), were prepared as per manufacturer’s instructions and used at the concentration indicated in Supplemental Fig. 2A. To monitor lymphocyte proliferation, we labeled cells with CFSE (2.5 μM; Miltenyi Biotec). Dye dilution was analyzed at the indicated time by flow cytometry after dead cell exclusion.

Cells were washed in PBS and resuspended in Bio-Rad sample buffer or in lysis buffer (50 mM Tris-HCl [pH 8], 0.1 mM EDTA, 200 mM NaCl, 10% glycerol, Nonidet P-40 0.5%, 0.4 mM PMSF, 3 μg/ml aprotinin, 1 μg/ml leupeptin, 0.5 μg/ml pepstatin). Whole cell extracts were fractionated by SDS-PAGE and transferred to a nitrocellulose membrane according to the manufacturer’s protocols (Bio-Rad). After incubation with 5% nonfat milk in TBST (10 mM Tris-HCl [pH 8], 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was washed once with TBST and incubated with Abs against NF-κB2 p100/p52 (1:1000), Survivin (1:1000), HBXIP (1:2000), GAPDH (1:2000), or β-actin (1:10,000) at 4°C for 12 h. Membranes were washed three times for 10 min and incubated with a 1:10,000 dilution of HRP-conjugated anti-rabbit Abs for 2 h. Blots were washed with TBST three times and visualized with the ECL system according to the provided protocol (Bio-Rad). Reagents are listed in Supplemental Table I. Signals were detected with a charge-coupled device camera (Fujilas 1000 Plus) and quantified with Multi Gauge software.

The DNA-binding capacity of p52 was evaluated by TransAM NF-κB Family Transcription Factor Assay kit (Active Motif), following manufacturer’s instructions. Nuclear cell extracts were prepared using the Nuclear Extract kit (Active Motif).

Transitional B cells from spleen of 17-d-old mice were sorted on a FACSAria II (Becton Dickinson) after staining with fluorescent Abs for CD19 and CD93. Total RNA was harvested from these cells using the RNeasy RNA preparation Kit (Qiagen). RNA quality and concentration were assessed using RNA 6000 Pico LabChips with a 2100 Bioanalyzer (Agilent Technologies). In brief, 100 ng of total RNA was reverse transcribed, and second-strand DNA was produced and amplified by in vitro transcription in the presence of biotinylated ribonucleotides using the IVT Express kit (Affymetrix). Gene expression analysis was performed using GeneChip Mouse Genome 430 2.0 arrays (Affymetrix), as recommended by the manufacturer. Fluorescence data were imported into two analysis software packages: Affymetrix Expression Console and R Bioconductor. Gene expression levels were normalized using the GC-RMA algorithm, and flags were computed using MAS5. The group comparisons were done using Student t test. To estimate the false discovery rate, we filtered the resulting p values at 5%. Enriched functional annotations for genes differentially expressed were analyzed with the computational method GSEA (gene set enrichment analysis). Microarray data are available at the ArrayExpress website, accession number E-MTAB 5928.

The BTZ cell line, which is a BL2 clone harboring pTet-tTAk-zeo (37), was cultured in RPMI 1640 supplemented with 10% HyClone FetalClone I serum, 100 U/ml penicillin, and 100 U/ml streptomycin. These cells were transfected using the Amaxa technique with a pBI vector in which KLHL6-EGFP fusion cDNA was cloned. Positive clones were selected and cultured with blasticidin (5 μg/ml; Invitrogen) and tetracycline (1 μg/ml; Sigma). Removing tetracycline from the medium induced the expression of KLHL6 fusion protein.

Induced and not induced KLHL6-expressing BL2 cells (105) were left attached for 1 h at 4°C on a microscope slide (SuperFrost Ultra Plus; Thermo Scientific) and then fixed with 4% (w/v) paraformaldehyde (Sigma) in PBS for 15 min at room temperature. PLA was performed with Duolink II Red Starter kit (Supplemental Table I) according to the manufacturer’s instructions (Sigma). The primary Abs, anti-HBXIP (1:100) and anti-EGFP (1:500) (Supplemental Table I), were incubated at room temperature for 2 h in a humidity chamber. Slides were mounted using Vectashield mounting medium with DAPI (Vector Laboratories). The samples were imaged through a Plan Apochromat 63/1.4 numeric aperture oil-immersion lens on a Zeiss LSM 710 confocal microscope (Zeiss), and Z-stacks were acquired using Zen 2009 software (Zeiss). High-resolution images were obtained with the Imaris software. The mean number of signals per cell was obtained by dividing the total number of PLA signals by the number of nuclei (counted manually).

The Y2H system was performed as previously described (38). The bait, human KLHL6 cDNA, was cloned into pDBa (Leu) using the Gateway technology (Invitrogen). The bait plasmid and the human spleen cDNA library [cloned into pEXP502-AD (Trp) ProQuest libraries; Invitrogen] were transformed in MAV03 yeast strain. The cDNA of 14 selected clones was amplified by PCR (oligo YL1 5′-CGCGTTTGGAATCACTACAGGG-3′, YL2 5′-GGAGACTTGACCAAACCTCTGGCG-3′). DNA was sequenced using the ABI Prism 3130xl Genetic Analyzer.

Mouse GC B cells (B220+ GL7+ PNA+) were sorted from Peyer’s patches of two 7- to 9-mo-old Ung−/−/Msh2−/− mice and two littermate controls. Human memory B cells (IgDCD27+) were sorted from blood of two individuals. Amplification of a region flanking exon 1 was achieved with Phusion polymerase from genomic DNA with the following oligonucleotides: for the mouse gene, MoKlhl6F 5′-CCTGATCTACGCACCCACTC-3′, MoKlhl6R 5′-GTTAGCTTGACTCTAAGAGATG-3′; for the human gene, HuKLHL6F 5′-CCCCTCCCACACACCTATTC-3′, HuKLHL6R 5′-GTCTCCTGACTTCAGGCCTTC-3′. Sequence of the PCR products was performed after Zero-blunt cloning (Invitrogen) using the BigDye Terminator sequencing kit and analyzed on an ABI PRISM 3130x Genetic Analyzer.

All statistical analyses were done with GraphPad Prism software using Welch t test. Data were reported as mean ± SD. Statistically significant differences are indicated on the figures: *p < 0.05, **p < 0.01, ***p < 0.001.

Klhl6 was inactivated in embryonic stem cells by replacement of exon 1 with a NeoR cassette, and mutant mice were generated (Supplemental Fig. 1A). Homozygous KO animals were viable, fertile, and were obtained with normal Mendelian segregation.

The B cell compartment was analyzed in Klhl6−/− and age-matched littermates. Early stages of bone marrow B cell development (pro-B and pre-B cells, i.e., from fraction A to fraction D, according to Hardy’s nomenclature) were not affected (Supplemental Fig. 1B). However, the frequency of immature (fraction E, B220+ CD93+IgM+) and mature recirculating (fraction F, B220+CD93IgM+) B cells differed between Klhl6−/− and control mice (Fig. 1A), with an increase in the absolute numbers of immature B cells and a 2-fold decrease in recirculating B cells in mutant mice (Table I).

FIGURE 1.

Klhl6 deficiency impacts the mature B cell pool, but not the GC B cell response. (A) Analysis of bone marrow immature B cells (FrE) (B220+IgM+CD93+) and mature recirculating B cells (B220+IgM+CD93) from 8- to 14-wk-old Klhl6-deficient mice (red squares) and age-matched littermate controls (blue circles). (B) Analysis of total splenocytes (upper row) and splenic B cell subsets: T1 B cells (CD19+CD93+CD23), T2 B cells (CD19+CD93+CD23+), FO B cells (CD19+CD93CD23+CD21int), and MZ B cells (CD19+CD93CD23CD21high). (C) Analysis of Peyer’s patch B cells (B220+) and GC cells (B220+GL7+). (D) Analysis of the anti-SRBC response of AID-Cre-EYFPxKlhl6−/− mice and Klhl6 heterozygous littermates, after immunization with 5 × 109 SRBC and tamoxifen administration, performed according to the experimental protocol depicted in the upper left panel. The lower left panels represent EYFP+ splenic B cell subsets analyzed 5 d after the boost. From left to right, EYFP+ cells, further gated into B220 PCs and B220+GL7+ GC B cells, and EYFP+ GC B cells analyzed for IgM and IgG1 isotypes. Upper and lower middle panels display the percentage of GC B cells among EYFP+ cells and the distribution of IgM and IgG1 isotypes among EYFP+ GC B cells; upper right panel shows percentage of PCs among EYFP+ cells. Each symbol represents an individual mouse. Data (mean ± SD) are from two independent experiments. Statistical analysis was performed with unpaired, two-tailed t test with Welch correction. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 1.

Klhl6 deficiency impacts the mature B cell pool, but not the GC B cell response. (A) Analysis of bone marrow immature B cells (FrE) (B220+IgM+CD93+) and mature recirculating B cells (B220+IgM+CD93) from 8- to 14-wk-old Klhl6-deficient mice (red squares) and age-matched littermate controls (blue circles). (B) Analysis of total splenocytes (upper row) and splenic B cell subsets: T1 B cells (CD19+CD93+CD23), T2 B cells (CD19+CD93+CD23+), FO B cells (CD19+CD93CD23+CD21int), and MZ B cells (CD19+CD93CD23CD21high). (C) Analysis of Peyer’s patch B cells (B220+) and GC cells (B220+GL7+). (D) Analysis of the anti-SRBC response of AID-Cre-EYFPxKlhl6−/− mice and Klhl6 heterozygous littermates, after immunization with 5 × 109 SRBC and tamoxifen administration, performed according to the experimental protocol depicted in the upper left panel. The lower left panels represent EYFP+ splenic B cell subsets analyzed 5 d after the boost. From left to right, EYFP+ cells, further gated into B220 PCs and B220+GL7+ GC B cells, and EYFP+ GC B cells analyzed for IgM and IgG1 isotypes. Upper and lower middle panels display the percentage of GC B cells among EYFP+ cells and the distribution of IgM and IgG1 isotypes among EYFP+ GC B cells; upper right panel shows percentage of PCs among EYFP+ cells. Each symbol represents an individual mouse. Data (mean ± SD) are from two independent experiments. Statistical analysis was performed with unpaired, two-tailed t test with Welch correction. *p < 0.05, **p < 0.01, ***p < 0.001.

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Table I.
Total number of cells ×106 among B220+ cells in bone marrow (hind legs)
IgMFrEFrE CD23FrE CD23+FrF
Adult mice      
 Control (n = 4) 3.09 ± 1.10 1.46 ± 0.43 1.08 ± 0.35 0.379 ± 0.11 2.23 ± 0.96 
 Klhl6−/− (n = 4) 3.37 ± 0.69 1.98 ± 0.71 1.77 ± 0.68 0.198 ± 0.04 1.04 ± 0.23 
Young mice      
 Control (n = 4) 4.38 ± 2.19 3.18 ± 1.49 2.02 ± 1.01 0.912 ± 0.38  
 Klhl6−/− (n = 4) 4.91 ± 1.56 3.53 ± 1.06 2.98 ± 0.79 0.485 ± 0.27  
IgMFrEFrE CD23FrE CD23+FrF
Adult mice      
 Control (n = 4) 3.09 ± 1.10 1.46 ± 0.43 1.08 ± 0.35 0.379 ± 0.11 2.23 ± 0.96 
 Klhl6−/− (n = 4) 3.37 ± 0.69 1.98 ± 0.71 1.77 ± 0.68 0.198 ± 0.04 1.04 ± 0.23 
Young mice      
 Control (n = 4) 4.38 ± 2.19 3.18 ± 1.49 2.02 ± 1.01 0.912 ± 0.38  
 Klhl6−/− (n = 4) 4.91 ± 1.56 3.53 ± 1.06 2.98 ± 0.79 0.485 ± 0.27  

The absolute number of splenocytes in adult mice was reduced by ∼30% in Klhl6−/− compared with wild type (wt) mice, a difference entirely accounted for by a marked reduction in frequency and number of the mature B cell compartment (Fig. 1B), whereas splenic T cells appeared unaffected (data not shown). Total numbers of splenic T1, T2, FO mature, and MZ B cells were all decreased ∼2-fold by Klhl6 deficiency, whereas the distribution among the different B cell subsets was comparable in KO and control mice (Fig. 1B). Reduction of mature B cells was also observed in Peyer’s patches, inguinal lymph nodes, and mesenteric lymph nodes (Fig. 1C, data not shown).

We evaluated in vitro the proliferation capacity of Klhl6-deficient splenic FO B cells, after CFSE labeling and BCR (anti-IgM) or TLR stimulation (LPS, CpG). Klhl6 deficiency did not alter B cell division profiles including the number of divisions performed and the cell viability (Supplemental Fig. 2A). The survival capacity of Klhl6−/− mature B cells was also similar to controls during culture in the presence of BAFF, a major B cell survival factor (Supplemental Fig. 2B).

The B cell deficit observed is therefore similar to the one described by Kroll and coworkers (35), but our results did not reveal a proliferation or survival defect consecutive to BCR or TLR signaling in mature B cells. These data altogether suggest that Klhl6 is required for optimal differentiation, rather than for maintenance or activation of the mature B cell compartment.

Klhl6 expression is markedly increased in GC B cells, both in mice and in humans. We therefore investigated the impact of Klhl6 deficiency on GC formation in chronically stimulated B cells from Peyer’s patches and in spleen after a secondary antigenic challenge with SRBCs.

No significant difference was observed in the proportion of Peyer’s patch GC B cells in Klhl6-deficient and age-matched littermates (Fig. 1C). To study the SRBC recall response, we crossed Klhl6 null mice with AID-Cre-ERT2 × ROSA26-EYFP reporter mice (AID-Cre-EYFP mice), in which B cells engaged in an immune response and expressing AID can irreversibly acquire EYFP expression upon simultaneous tamoxifen feeding (36). The proportion of EYFP+ GC B cells was analyzed 5 d after secondary immunization with SRBC in AID-Cre-EYFP/Klhl6−/− mice and heterozygous littermates, with tamoxifen feeding at days 7 and 12 of the primary challenge and day 1 of the boost (Fig. 1D). The percentage of GL7+EYFP+ B cells was equivalent in Klhl6−/− and control mice, as well as the IgG1 and IgM isotype distribution (Fig. 1D). Plasma cell (PC) formation was also similar in Klhl6−/− mice and controls during this recall response (Fig. 1D).

These data indicate that Klhl6 is dispensable for GC formation and maintenance, and therefore that Klhl6-deficient B cells do not show impaired activation, neither in vitro nor in vivo.

To identify the differentiation stage affected by the absence of Klhl6, the bone marrow immature B cell subset was further resolved in two subpopulations (9, 10), newly formed/T1 (FrE, CD23) and late differentiated T2-like (FrE, CD23+) B cells. Klhl6−/− mice displayed a 2-fold reduced frequency for the more mature CD23+ B cell population, whereas the CD23 subset was slightly increased (Fig. 2A, Table I).

FIGURE 2.

Klhl6 deficiency impairs B cell maturation and survival in bone marrow and spleen. (A) Analysis of bone marrow B cell subpopulations from adult or young Klhl6-deficient mice (14–17 d old) and age-matched littermate controls. The surface marker CD23 was used to discriminate newly formed, T1-like B cells (B220+IgM+CD93+CD23) from T2-like B cells (B220+IgM+CD93+CD23+). (B) Analysis of splenocytes from young Klhl6-deficient and age-matched control mice (14–20 d old), with percentage and absolute numbers of T1 and T2 B cell subsets. Results (mean ± SD) represent two (A) and three (B) independent experiments. Each symbol (red squares for Klh6−/− and blue circles for controls) represents an individual mouse (n = 5–10 mice). Statistical analysis by unpaired, two-tailed t test with Welch correction. **p < 0.01, ***p < 0.001. (C) Competitive reconstitution of Rag2-deficient mice with bone marrow cells from 12- to 14-d-old Ly5.2 Klhl6−/− and Ly5.1 control mice at 1:1 ratio. The gating strategy for discriminating B cell subsets is described in Fig. 1 for spleen and blood and in (A) for bone marrow. Density plots depict the B cell subpopulations and contour plots the frequency of Klhl6−/− (red) and control cells (blue). The numbers adjacent to the outlined areas represent the percentage of cells within the gate. The bar graphs represent the ratio between Ly5.1 wt and Ly5.2 Klhl6−/− cells (mean ± SD, n = 3 mouse chimeras). Data shown correspond to one representative experiment out of two.

FIGURE 2.

Klhl6 deficiency impairs B cell maturation and survival in bone marrow and spleen. (A) Analysis of bone marrow B cell subpopulations from adult or young Klhl6-deficient mice (14–17 d old) and age-matched littermate controls. The surface marker CD23 was used to discriminate newly formed, T1-like B cells (B220+IgM+CD93+CD23) from T2-like B cells (B220+IgM+CD93+CD23+). (B) Analysis of splenocytes from young Klhl6-deficient and age-matched control mice (14–20 d old), with percentage and absolute numbers of T1 and T2 B cell subsets. Results (mean ± SD) represent two (A) and three (B) independent experiments. Each symbol (red squares for Klh6−/− and blue circles for controls) represents an individual mouse (n = 5–10 mice). Statistical analysis by unpaired, two-tailed t test with Welch correction. **p < 0.01, ***p < 0.001. (C) Competitive reconstitution of Rag2-deficient mice with bone marrow cells from 12- to 14-d-old Ly5.2 Klhl6−/− and Ly5.1 control mice at 1:1 ratio. The gating strategy for discriminating B cell subsets is described in Fig. 1 for spleen and blood and in (A) for bone marrow. Density plots depict the B cell subpopulations and contour plots the frequency of Klhl6−/− (red) and control cells (blue). The numbers adjacent to the outlined areas represent the percentage of cells within the gate. The bar graphs represent the ratio between Ly5.1 wt and Ly5.2 Klhl6−/− cells (mean ± SD, n = 3 mouse chimeras). Data shown correspond to one representative experiment out of two.

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These observations were confirmed for the bone marrow immature B cell compartment of young mice (14–17 d old), a stage at which recirculation of mature B cells is minimal. Like in adults, a 2-fold reduction in the proportion and absolute numbers of late differentiated T2-like cells was observed in Klhl6−/− mice compared with controls, together with a significant increase in the newly formed/T1 subpopulation (Fig. 2A, Table I). These results suggest an impaired B cell survival and/or differentiation at the late differentiated T2-like stage of B cell maturation in bone marrow, already present in newborn mice.

As mentioned earlier, splenic T1 and T2 B cells showed a 2-fold reduction in adult Klhl6-deficient mice compared with littermate controls, similar to the deficit observed for the mature FO and MZ subsets. In newborn mice (14–17 d old), in which the splenic B cell pool is mostly composed of transitional B cells, T1 and T2 cells were reduced to a similar extent in Klhl6−/− animals, to 50–60% of control values (Fig. 2B). This observation indicates that, whereas Klhl6 deficiency only affects B cell differentiation at the T2-like stage in bone marrow, it appears to be required for normal homeostasis of T1 cells in spleen.

To assess the behavior of Klhl6-deficient B cells in a competitive setting, we generated mixed bone marrow chimeras by reconstitution of irradiated Rag2−/− mice with equal number of allelically marked wt (CD45.1) and Klhl6-deficient (CD45.2) bone marrow cells (Fig. 2C). In bone marrow of restored mice, this competition revealed a selective advantage of Klhl6−/− cells (a 2-fold increase) in the immature B cell subset of newly formed CD23 cells, whereas the wt/Klhl6−/− cell ratio appeared reversed at the next step of cell differentiation, in CD23+ T2-like B cells (Fig. 2C). Interestingly, the proportion of control and mutant cells was unchanged in immature CD23 and CD23+ B cell fractions between bone marrow and blood (Fig. 2C), indicating that egress from bone marrow is not affected by the absence of Klhl6. In spleen, Klhl6-deficient B cells were increasingly disfavored along further B cell maturation steps compared with wt cells; this difference is already starting at the T1 differentiation stage, with stronger imbalance observed for naive and MZ B cells (Fig. 2C).

These results confirm the role of Klhl6 in normal T1 homeostasis and reveal an impact on MZ B cells, which is not observed outside such competition context.

BAFF is a key cytokine involved in the survival and maturation of T1 cells (39). To explore further the functional role of Klhl6 in T1 B cell maintenance, we purified and cultured transitional B cells from spleen of 14- to 17-d-old mice in vitro in the presence of BAFF. B cell survival was followed over 3 d. Klhl6-deficient B cells displayed a 40% reduction in viability compared with B cells isolated from spleen of littermate controls, whereas no difference in survival rate was observed in unstimulated cells (Fig. 3A).

FIGURE 3.

Impaired BAFF-induced differentiation of Klhl6-deficient transitional B cells. (A) Survival of MACS-purified transitional B cells from young Klhl6−/− mice and controls after BAFF stimulation at the concentration of 100 ng/ml (upper panel) and unstimulated (lower panel). Data are from three to nine experiments in which cells were pooled from two to four mice. Statistical analysis was performed with multiple t tests according to the Holm–Sidak method. (B) In vitro differentiation of T1 transitional B cells from young Klhl6−/− and control mice. CD19+CD93+CD23 cells were sorted and cultured in the presence of BAFF (100 ng/ml). Differentiation and viability were evaluated by staining with CD19, CD93, CD23, and SYTOX Blue at the indicated time points. Results are displayed as density plots and are representative of two independent experiments. The number of T1 cells is reported on the right upper panel and T2 cells on the right lower panel. (C) BAFF receptor expression of bone marrow newly formed and splenic transitional B cells from Klhl6−/− and control mice. Quantification of BAFF-R mean fluorescence intensity (MFI) from bone marrow and spleen subpopulations are shown, respectively, in the upper and lower panels (mean ± SD, n = 3–5). The p values with unpaired, two-tailed t test with Welch correction. (D) Activation of the noncanonical NF-κB pathway was monitored from purified Klhl6−/− and control transitional B cells. Cells were cultured for 22 h in the presence or absence of BAFF (150 ng/ml). The p52 transcription factor derived from the processing of p100 protein was detected by Western blot of total cell extracts (left panel). The middle and right panels show, respectively, the detection of p52 from transitional B cells ex vivo and from transitional B cells after 24-h activation in the presence of BAFF, using DNA-binding ELISA assay. The results represent two to four independent experiments in which purified B cells from two to three mice were pooled. **p < 0.01, ***p < 0.001.

FIGURE 3.

Impaired BAFF-induced differentiation of Klhl6-deficient transitional B cells. (A) Survival of MACS-purified transitional B cells from young Klhl6−/− mice and controls after BAFF stimulation at the concentration of 100 ng/ml (upper panel) and unstimulated (lower panel). Data are from three to nine experiments in which cells were pooled from two to four mice. Statistical analysis was performed with multiple t tests according to the Holm–Sidak method. (B) In vitro differentiation of T1 transitional B cells from young Klhl6−/− and control mice. CD19+CD93+CD23 cells were sorted and cultured in the presence of BAFF (100 ng/ml). Differentiation and viability were evaluated by staining with CD19, CD93, CD23, and SYTOX Blue at the indicated time points. Results are displayed as density plots and are representative of two independent experiments. The number of T1 cells is reported on the right upper panel and T2 cells on the right lower panel. (C) BAFF receptor expression of bone marrow newly formed and splenic transitional B cells from Klhl6−/− and control mice. Quantification of BAFF-R mean fluorescence intensity (MFI) from bone marrow and spleen subpopulations are shown, respectively, in the upper and lower panels (mean ± SD, n = 3–5). The p values with unpaired, two-tailed t test with Welch correction. (D) Activation of the noncanonical NF-κB pathway was monitored from purified Klhl6−/− and control transitional B cells. Cells were cultured for 22 h in the presence or absence of BAFF (150 ng/ml). The p52 transcription factor derived from the processing of p100 protein was detected by Western blot of total cell extracts (left panel). The middle and right panels show, respectively, the detection of p52 from transitional B cells ex vivo and from transitional B cells after 24-h activation in the presence of BAFF, using DNA-binding ELISA assay. The results represent two to four independent experiments in which purified B cells from two to three mice were pooled. **p < 0.01, ***p < 0.001.

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The differentiation capacity of transitional T1 B cells was further evaluated after purification of CD93+CD23 B cells from newborn spleens and culture in the presence of BAFF, and differentiation into T2 B cells was assessed by CD23 surface marker acquisition (Fig. 3B). The frequency of Klhl6-deficient T2 cells was reduced during the in vitro culture compared with controls, and the impaired survival of T1 transitional B cells contributed by Klhl6 deficiency resulted in a 5-fold decrease in absolute T2 cell numbers (Fig. 3B).

Expression of the BAFF-R increases as B cells mature, and we therefore analyzed BAFF-R expression by flow cytometry on bone marrow and spleen B cell subsets in young Klhl6-deficient mice and age-matched littermates, as a marker of their differentiation status. BAFF-R expression, which is first measurable on a fraction of bone marrow T1-like CD23 B cells (40), was barely detectable in Klhl6-deficient mice compared with littermate controls. An increase in BAFF-R expression was observed along B cell maturation in bone marrow, with lower levels observed for Klhl6-deficient T2-like cells compared with controls. Similar reduction of BAFF-R expression was observed in T1 mutant spleen cells, a difference that was still present but less pronounced at the T2 stage (Fig. 3C). In contrast, no significant difference in BAFF-R expression was observed at the mature B cell stage, in agreement with the normal phenotype observed for Klhl6-deficient cells that survived along this differentiation pathway (Supplemental Fig. 2B, right panel).

Signaling through the BAFF-R activates the noncanonical NF-κB pathway (41). Activation of this pathway was therefore evaluated on purified transitional B cells from young Klhl6−/− and control mice ex vivo and after 24 h BAFF stimulation by quantification of the activated transcription factor p52 derived from p100 processing. Western blot analysis on total protein extracts did not show a significant difference in p52 amounts between Klhl6-deficient and control transitional B cells (Fig. 3D). A second approach, based on ELISA assays of nuclear extracts, designed to detect p52 binding to its DNA target, gave similar results (Fig. 3D).

These data thus provide evidence for a role of Klhl6 in BAFF-mediated differentiation and survival of splenic transitional T1 cells, but did not reveal a functional defect in the activation of the alternate NF-κB pathway.

To understand the molecular processes underlying the impaired BAFF-induced survival observed in Klhl6-deficient mice, we performed gene expression profiling of splenic transitional B cells isolated from 17-d-old Klhl6−/− and littermate controls, ex vivo (T0) and after 24 h of in vitro culture in the presence of BAFF (T24).

At T0, 460 probe sets showed a 1.5 difference between KO and control cells, with 258 underexpressed genes in Klhl6-deficient cells and 202 overexpressed (Fig. 4A). Among underexpressed genes are genes related with immune cell differentiation, like CD40, CTIIA, FCGR2B, CD274 (PDL1), and CD22; with cell migration, like CCR6 and CXCR5; or with cytokine signaling, like LTA, EBI3, IL27RA, IRF7, and IFIT1-3 (Fig. 4A). Among overexpressed genes, GSEA highlighted genes linked with cell proliferation, with a remarkable set of genes comprising most substrates of the anaphase-promoting complex/cyclosome (APC/C), showing a difference in the 2-fold range (Fig. 4A, 4C) (42). This large ubiquitin ligase complex has an essential role in the control of the cell cycle, through ubiquitylation of various protein substrates, marking them for degradation at specific cell-cycle checkpoints (43).

FIGURE 4.

Gene expression profile reveals a maturation delay of Klhl6−/− transitional B cells. (A) GSEA comparison of upregulated and downregulated genes in Klhl6−/− and control transitional B cells identified by microarray analysis. (B) GSEA comparison of upregulated and downregulated genes in Klhl6−/− (left panel) and control (middle panel) transitional B cells after 24-h BAFF stimulation. Venn diagrams (right panel) show the number of downregulated or upregulated genes shared by Klhl6-deficient and control mice during BAFF activation. (C) Heat map representation (normalized log2 expression) of selected genes representing APC/C substrates or cofactors, as well as genes involved in proliferation control, whose expression differed by >1.5-fold between Klhl6−/− and control cells. (D) GSEA plots showing enrichment of upregulated cell-cycle genes in Klhl6−/− transitional B cells compared with controls. The normalized enrichment score (NES), nominal p value, and q (false discovery rate) are given for each plot. (E) Similar GSEA plots for both control (middle panel) and Klhl6−/− cells (right panel) after BAFF stimulation, showing an inverse profile compared with the one shown in (D) for the same gene set. (F) Increased expression of survivin (encoded by Birc5) in Klhl6−/− T1 B cells. Upper left panel displays immunoblot analysis of transitional B cell extracts (106 cells) obtained by MACS enrichment, from young Klhl6−/− and littermate control mice. Quantification of relative fold change for survivin expression, after β-actin normalization, is reported in lower left panel. Data are mean ± SD from 11 experiments. Right panel shows Western blot analysis of total cell extracts from FACS-sorted T1 and T2 B cells. The values 1 and 1/2 indicate that protein extracts from 5 × 105 and 2.5 × 105 cells were loaded on the gel. Blot was probed with anti-survivin and anti-GAPDH. Results are representative of one of two independent experiments.

FIGURE 4.

Gene expression profile reveals a maturation delay of Klhl6−/− transitional B cells. (A) GSEA comparison of upregulated and downregulated genes in Klhl6−/− and control transitional B cells identified by microarray analysis. (B) GSEA comparison of upregulated and downregulated genes in Klhl6−/− (left panel) and control (middle panel) transitional B cells after 24-h BAFF stimulation. Venn diagrams (right panel) show the number of downregulated or upregulated genes shared by Klhl6-deficient and control mice during BAFF activation. (C) Heat map representation (normalized log2 expression) of selected genes representing APC/C substrates or cofactors, as well as genes involved in proliferation control, whose expression differed by >1.5-fold between Klhl6−/− and control cells. (D) GSEA plots showing enrichment of upregulated cell-cycle genes in Klhl6−/− transitional B cells compared with controls. The normalized enrichment score (NES), nominal p value, and q (false discovery rate) are given for each plot. (E) Similar GSEA plots for both control (middle panel) and Klhl6−/− cells (right panel) after BAFF stimulation, showing an inverse profile compared with the one shown in (D) for the same gene set. (F) Increased expression of survivin (encoded by Birc5) in Klhl6−/− T1 B cells. Upper left panel displays immunoblot analysis of transitional B cell extracts (106 cells) obtained by MACS enrichment, from young Klhl6−/− and littermate control mice. Quantification of relative fold change for survivin expression, after β-actin normalization, is reported in lower left panel. Data are mean ± SD from 11 experiments. Right panel shows Western blot analysis of total cell extracts from FACS-sorted T1 and T2 B cells. The values 1 and 1/2 indicate that protein extracts from 5 × 105 and 2.5 × 105 cells were loaded on the gel. Blot was probed with anti-survivin and anti-GAPDH. Results are representative of one of two independent experiments.

Close modal

Very strikingly, incubation in the presence of BAFF induced the downregulation of a similar set of proliferative genes (Fig. 4B, 4C), which were reduced ∼2-fold in both KO and control transitional B cells. This is illustrated by the reverse shape of the GSEA cell-cycle diagram of the KO/control comparison relative to the one of the BAFF stimulation (compare Fig. 4D, 4E). This indicated that Klhl6 deficiency resulted, in transitional B cells, in a higher expression of genes involved in proliferation that are precise targets for BAFF-induced downmodulation. Among genes induced upon culture in the presence of BAFF are genes linked to cholesterol, lipid, and lipoprotein metabolism, involved notably in the remodeling of membranes through protein–lipid linkage (farnesyl transferase and farnesyl synthase, squalene epoxidase) (44). Klhl6−/− and control cells presented a similar GSEA profile for both overexpressed and underexpressed genes during in vitro culture with BAFF, with a large number of shared genes evidenced in the Venn diagrams (Fig. 4B), suggesting that Klhl6 deficiency did not induce a general impairment of the response to BAFF signaling. Splenic mature B cells showed minimal differences in gene expression between Klhl6−/− and controls, including upon in vitro BAFF stimulation (data not shown), in agreement with the normal activation profile observed for Klhl6-deficient cells after the transitional B cell stage.

The differential expression of cell-cycle genes was confirmed at the protein level for survivin (encoded by the Birc5 gene), a protein involved in cell division and antiapoptotic processes (45). Survivin expression was increased ∼2-fold in transitional Klhl6-deficient B cells compared with controls, a differential regulation of expression that was restricted to T1 Klhl6−/− B cells, whereas T2 cells did not display such change (Fig. 4F).

Interestingly, downregulation of this set of proliferative genes is similarly observed during the transition from bone marrow immature to splenic T1 B cells (ImmGen database), further documenting that progressive transcriptional extinction of such proliferation markers, which are otherwise strictly controlled through ubiquitination/phosphorylation at the protein level, parallels maturation of cells that are already nondividing (Supplemental Fig. 3).

These results indicate that BAFF-induced maturation of transitional B cells involves notably the coordinated shutdown of proliferative genes, including most APC/C substrates, and that Klhl6 deficiency induces a delayed maturation along this gene regulation pathway that controls exit from the cell cycle.

Several members of the Kelch protein family behave as adaptor of the Cul3 E3 ubiquitin ligase, by binding Cul3 through the BTB domain and different substrates through the Kelch domain (25). To study KLHL6 interactions, we established a Burkitt’s lymphoma cell line (BL2, a GC B cell–derived lymphoma), transfected with an expression vector encoding human KLHL6 fused with EGFP at its C terminus, under the control of a tetracycline-regulated promoter, inducible by tetracycline removal. KLHL6-EGFP was localized in the cytoplasm, and part of the overexpressed protein was distributed in structures that were identified as aggresomes (Fig. 5).

FIGURE 5.

Klhl6 interacts with Culin-3 and HBXIP. (A) BL2 cells, with inducible KLHL6-EGFP expression, were lysed and subjected to immunoprecipitation with anti-EGFP Ab. Immunoprecipitated proteins were analyzed with an anti-Cul3 Ab. (B) Three-dimensional confocal images of Cul3-KLHL6 interactions detected by PLA in the inducible KLHL6-EGFP BL2 cell line. From left to right, KLHL6-EGFP protein (green), PLA signals (red), overlay, and representative section of two cells from the outlined area, showing cytoplasmic localization of signals. Nuclei are stained in blue. Graph shows the percentage of cells harboring the indicated number of interactions. Data correspond to two independent experiments in which 1398 PLA signals were detected in 115 cells. (C) Three-dimensional confocal images of KLHL6-HBXIP/LAMTOR5 interactions analyzed by PLA. From left to right, KLHL6-EGFP protein (green), PLA signals (red), overlay, and single section of two cells from outlined area, showing cytoplasmic localization of signals. The bar graph depicts the percentage of cells harboring the indicated number of interactions. PLA-positive signals were detected with two different anti-HBXIP Abs: black bar (Ab1) represents goat anti-HBXIP (Santa Cruz), and gray bar (Ab2) represents rabbit anti-HBXIP (Sigma). Results assemble two independent experiments for both anti-HBXIP Abs. 157 cells were scored detecting 3396 interactions. (D) Western blot analysis of HBXIP in the KLH6-EGFP–inducible BL2 cell line and Klhl6−/− and control mouse B cell subsets, with β-actin as loading control. Left panel, Protein extracts from 105 wt BL2 cells, EGFP-sorted cells induced for KHL6 expression (+), and repressed cells (−). Right panel, Protein extracts from mouse T1, T2, and mature (M) B cells (1.5 × 105) from Klhl6-deficient and control mice. (E) HBXIP–survivin interactions in the BL2 cell line. Left panel, Representative three-dimensional confocal image shows PLA-positive signals (red dots) that were identified with rabbit anti-HBXIP and mouse anti-survivin. Right panel, Single section from outlined area showing cytoplasmic localization of signals. Data are from two independent experiments. Scale bars, 5 μm.

FIGURE 5.

Klhl6 interacts with Culin-3 and HBXIP. (A) BL2 cells, with inducible KLHL6-EGFP expression, were lysed and subjected to immunoprecipitation with anti-EGFP Ab. Immunoprecipitated proteins were analyzed with an anti-Cul3 Ab. (B) Three-dimensional confocal images of Cul3-KLHL6 interactions detected by PLA in the inducible KLHL6-EGFP BL2 cell line. From left to right, KLHL6-EGFP protein (green), PLA signals (red), overlay, and representative section of two cells from the outlined area, showing cytoplasmic localization of signals. Nuclei are stained in blue. Graph shows the percentage of cells harboring the indicated number of interactions. Data correspond to two independent experiments in which 1398 PLA signals were detected in 115 cells. (C) Three-dimensional confocal images of KLHL6-HBXIP/LAMTOR5 interactions analyzed by PLA. From left to right, KLHL6-EGFP protein (green), PLA signals (red), overlay, and single section of two cells from outlined area, showing cytoplasmic localization of signals. The bar graph depicts the percentage of cells harboring the indicated number of interactions. PLA-positive signals were detected with two different anti-HBXIP Abs: black bar (Ab1) represents goat anti-HBXIP (Santa Cruz), and gray bar (Ab2) represents rabbit anti-HBXIP (Sigma). Results assemble two independent experiments for both anti-HBXIP Abs. 157 cells were scored detecting 3396 interactions. (D) Western blot analysis of HBXIP in the KLH6-EGFP–inducible BL2 cell line and Klhl6−/− and control mouse B cell subsets, with β-actin as loading control. Left panel, Protein extracts from 105 wt BL2 cells, EGFP-sorted cells induced for KHL6 expression (+), and repressed cells (−). Right panel, Protein extracts from mouse T1, T2, and mature (M) B cells (1.5 × 105) from Klhl6-deficient and control mice. (E) HBXIP–survivin interactions in the BL2 cell line. Left panel, Representative three-dimensional confocal image shows PLA-positive signals (red dots) that were identified with rabbit anti-HBXIP and mouse anti-survivin. Right panel, Single section from outlined area showing cytoplasmic localization of signals. Data are from two independent experiments. Scale bars, 5 μm.

Close modal

Pull-down experiment showed that the EGFP-tagged KLHL6 protein coimmunoprecipitated endogenous Cul3 (Fig. 5A). KLHL6 and Cul3 interactions were also detected in the BL2-KLHL6-EGFP clone using the proximity ligation assay (PLA). Positive PLA signals (red dots, located mostly outside aggresome structures) were present in the cytoplasm of BL2 cells at an average frequency of 12 interactions per cell, with a variation between 1 and >30 interactions per cell (Fig. 5B). The number of interactions observed with an unrelated Ab in place of the anti-Cul3 Ab was at background level (0.03 interaction per cell), similar to the level observed, based on anti-EGFP/anti-Cul3 Ab-mediated signals, in the BL2-KLHL6-EGFP clone in the presence of tetracycline. These results showed that KLHL6 is an interactor of the Cul3 ubiquitin ligase.

We then searched for KLHL6 substrates by a yeast two-hybrid system using the full-length human KLHL6 cDNA as bait. Screening of a cDNA library derived from human spleen yielded 14 clones that tested positive in two-hybrid assays. Nucleotide sequence analysis of these clones revealed a recurrent cDNA that encoded a protein of 91 aa identified as HBXIP. HBXIP is a ubiquitous protein, showing complete conservation between mice and humans, and initially identified through its interaction with the hepatitis B virus HBx protein (46). HBXIP is endowed with multiple functions, including control of cell proliferation, cytokinesis, cell survival, and mTORC1 activation (47, 48). Interestingly, HBXIP has also been reported to interact with survivin (49).

Accordingly, association of KLHL6 with endogenous HBXIP was detected in the BL2-KLHL6-EGFP cell line by PLA. Similar numbers of KLHL6-HBXIP interactions were found using two different Abs made in two different host species with an average of 25 positive PLA signals per cell, with a similar distribution frequency (Fig. 5C). We next evaluated the abundance of HBXIP in this cell line, with or without tetracycline induction. Western blot analysis of HBXIP did not show any difference in protein levels in BL2 induced or not for KLHL6 overexpression (Fig. 5D). We also confirmed HBXIP–survivin interactions in the BL2-KLHL6-EGFP cell line (Fig. 5E), but did not detect direct survivin–KLHL6 interactions by PLA (data not shown). To study HBXIP expression in a physiologically more relevant context for Klhl6 function, we analyzed HBXIP protein expression in splenic T1, T2, and mature B cells from Klhl6−/− and wt mice (Fig. 5D). HBXIP expression was altogether low, as expected from nonproliferating cells, but did not differ between Klhl6-deficient and -proficient cells, suggesting that binding of Klhl6 to HBXIP does not lead to its degradation.

Although HBXIP does not appear as a degradation target for Klhl6, its identification as a binding partner further links Klhl6 to cell cycle and survival controls.

Several mutations affecting the KLHL6 protein have been reported in B cell lymphomas, notably CLL with mutated VH genes, as well as some other lymphomas of GC origin (3234). Most of these mutations target the first half of the BTB domain contained in exon 1 and are therefore located within 1 kb domain downstream of the KLHL6 promoter, that is, within a domain compatible with an AID-driven process (Fig. 6A). However, KLHL6 has not been reported so far as an AID off-target (50).

FIGURE 6.

Somatic hypermutation targets Klhl6 in mouse and human GC B cells. Mutation analysis was performed on genomic DNA from B220+PNA+GL7+ cells isolated from wt or Ung−/−Msh2−/− mice (two mice), and for human samples, from IgDCD27+ memory B cells (two individuals) and naive B cells as controls. (A) Representation of the Klhl6 locus and the region flanking exon 1 that was sequenced. (B) Klhl6 mutation frequency in GC B cells from wt and Msh2−/−Ung−/− mice (left) and human naive and memory cells (right), with summary of mutation data.

FIGURE 6.

Somatic hypermutation targets Klhl6 in mouse and human GC B cells. Mutation analysis was performed on genomic DNA from B220+PNA+GL7+ cells isolated from wt or Ung−/−Msh2−/− mice (two mice), and for human samples, from IgDCD27+ memory B cells (two individuals) and naive B cells as controls. (A) Representation of the Klhl6 locus and the region flanking exon 1 that was sequenced. (B) Klhl6 mutation frequency in GC B cells from wt and Msh2−/−Ung−/− mice (left) and human naive and memory cells (right), with summary of mutation data.

Close modal

AID off-targets can be easily revealed in UngxMsh2 double-deficient mice in which AID-induced mutations are not corrected by error-free repair pathways (51). We therefore analyzed Klhl6 mutations in mouse Peyer’s patch GC B cells from wt and Ung−/−Msh2−/− mice. Klhl6 mutations were observed at a frequency of 29 × 10−5/bp in Ung−/−Msh2−/− mice, that is, a 4-fold lower level than the Bcl6 gene, which displays the highest mutation load at non-Ig loci (51), whereas mutation frequency at the Klhl6 locus was close to background values in wt animals (7.2 × 10−5/bp) (Fig. 6B). We also observed mutations within the same domain of the KLHL6 gene in human IgG memory B cells at a frequency of 17.5 × 10−5/bp, thus making KLHL6 a new example of a gene targeted by hypermutation in normal human B cells, together with BCL6 and FAS (5254).

Although it is possible that KLHL6 may be a contributing factor to cell-cycle deregulation in lymphomas, as observed during B cell differentiation in the mouse, the frequency of mutagenesis observed could also suggest that it only represents a bystander effect of hypermutation mistargeting.

Klhl6 is a member of the KLHL family that shows a preferential expression in hematopoietic and endothelial cells, and notably in the B cell lineage. It is still a poorly characterized gene, and its description in Gene Ontology databases links it with GC formation. We report in this article, as described previously (35), that Klhl6 deficiency results in a 2-fold reduction in the pool of mature B cells in the mouse. However, whereas the highest expression of Klhl6 during B cell development occurs at the pre-B cell stage (ImmGen database), we identified a marked impairment in the differentiation step occurring between immature and transitional T1 and T2 cells as the major cause of this deficiency.

Competitive restoration with bone marrow cells from wt and Klhl6−/− mice indicated that B cell maturation up to the T1-like stage in bone marrow was not compromised by Klhl6 deficiency, with T1-like cells (IgM+CD93+CD23) being even 2-fold increased compared with control cells. The proportion was re-equilibrated at the T1 stage in spleen, implying that their differentiation is compromised in this organ. Klhl6−/− cells were clearly also disfavored at the T2-like stage in bone marrow (IgM+CD93+CD23+), as well as at the T2 and FO stages in spleen. In vitro incubation with the BAFF cytokine revealed an impaired survival of Klhl6-deficient splenic transitional B cells, both in terms of T1 and T2 cell survival and of differentiation of T1 cells into T2. BAFF-R expression was markedly reduced in splenic T1 cells, a difference already observed in bone marrow T1- and T2-like cells. In contrast, naive Klhl6−/− B cells that passed this differentiation bottleneck displayed normal BAFF-R expression levels at their surface and showed comparable survival in vitro in the presence of BAFF and normal proliferative responses to various activation signals.

The reduction in BAFF-R expression and the specific stages at which Klhl6−/− B cell differentiation appears to be impaired strikingly mirrors the phenotype of BAFF/BAFF-R–deficient animals (1315, 19). One hypothesis could thus be a direct or indirect impact of the absence of Klhl6 on BAFF-R signaling or transcription. We failed, however, to detect any changes in the activation of the alternate NF-κB pathway upon in vitro culture of splenic transitional B cells in the presence of BAFF, a pathway whose activation is jointly promoted by BAFF and BCR signals (55, 56). Overexpression of KLHL6 in the BL2 Burkitt’s lymphoma cell line also did not induce changes in the level of BAFF-R expression at the surface of these cells (data not shown).

We therefore favor another hypothesis, namely that the reduction in BAFF-R expression may reflect, and not cause, a delayed maturation process of Klhl6−/− transitional B cells, leading to their subsequent apoptosis. Gene profiling performed on wt transitional B cells before and after 24-h culture in the presence of BAFF revealed a striking set of 1.5- to 2-fold downregulated genes linked with cell proliferation. Among them were many targets of the APC/C complex, thus revealing a coordinated transcriptional regulation of proteins controlled by this large E3 ubiquitin ligase complex that regulates entry into the different phases of the cell cycle through its sequential binding with two distinct stage-specific interactors, Cdc20 or Fzr1 (43). Such downmodulation of genes linked with proliferation control suggests that, in cells that are already nondividing, further transcriptional reduction may drive mitotic exit and enforce the G0 status of naive B cells. Very strikingly, the transcriptional profile of Klhl6−/− splenic T1 cells displayed an upregulation (around 1.5- to 2-fold) of the same set of cell-cycle genes, including those controlled by the APC/C complex, as compared with wt cells. The upregulation of a cell proliferation signature is reminiscent of the difference in expression profile observed between normal immature bone marrow B cells and splenic T1 cells, implying clearly that Klhl6-deficient transitional B cells lag behind wt cells for their maturation. Klhl6 could therefore target a protein controlling this differentiation process. Moreover, similar transcriptional changes, and of similar magnitude, were observed in transitional Klhl6-deficient and wt transitional B cells upon 24 h of in vitro culture in the presence of BAFF, suggesting again that absence of Klhl6 does not induce a specific defect in the BAFF-R signaling pathway.

We showed that Klhl6 is, like several other KLHL family members, a cofactor of the Cul3 ubiquitin complex and identified HBXIP as its binding partner. Interestingly, HBXIP is involved in multiple regulatory processes, controlling cell proliferation, cytokinesis, cell survival, and as described more recently, regulation of the mTOR nutrient-sensing pathway (47, 48, 57). We, however, failed to identify any modulation of the HBXIP protein performed by Klhl6, either upon KLHL6 overexpression in a BL2 cell line or in Klhl6−/− transitional B cells. Ubiquitinylation can also induce protein relocalization, as observed for AuroraB that, after ubiquitinylation by Klhl9 and Klhl13, is removed from mitotic chromosomes in prometaphase and relocalized at the spindle zone during anaphase (58). Further studies should address whether relocalization of HBXIP could take place and interfere with the maturation process of immature B cells during B cell development. It is worth mentioning that, in differentiation pathways like myogenesis, in which the role of multiple Klhl genetic mutants have been implicated, like Klhl19, Klhl40, and Klhl41, their Cul3 adaptor function has been well established, with interactors described for some of them, but so far no ubiquitinylation targets have been identified (59).

Klhl6 has been initially isolated through its marked overexpression in GC B cells (31), and Sato and colleagues (35) have reported an impaired GC response in Klhl6-deficient mice. We did not observe any reduction in GC B cells in the absence of Klhl6, either in chronically stimulated Peyer’s patches or during an anti-SRBC response, but we cannot exclude that differences may exist in other immune conditions, like viral infections. We nevertheless observed that active Klhl6 expression in GC B cells allows the targeting by AID of part of its BTB domain that lies within a promoter-proximal region, whereas it had not been listed so far as an AID off-target gene (50). These mutations at the Klhl6 locus were repaired in normal mouse GC B cells and could only be observed in Msh2−/−Ung−/− repair–deficient animals. On the contrary, we observed that KLHL6 was mutated in normal human memory B cells, which adds it to the short list of mutated genes in normal human GC B cells, together with BCL-6 and FAS/CD95 (5254). Recurrent mutations observed in B cell lymphomas, and notably CLL with mutated Ig V genes, may thus be explained by such a physiological process (3234). KLHL6 mutations observed in lymphomas could therefore be simple bystander products of AID off-target activity, because they were not identified as driver elements of the oncogenic process in a recent longitudinal study of CLL progression (60). In contrast, the cell division signature of Klhl6-deficient transitional B cells leaves open the possible contribution of KLHL6 mutants to deregulation of proliferation in CLL.

We thank our colleagues Robert Weil and Jürgen Wienands for expertise in the study of NF-κB and B cell signaling. We thank Sandra Weller for providing human B cell samples. We acknowledge the contributions of Jérome Megret (Flow Cytometry Core, Structure Fédérative de Recherche Necker) in performing cell sorting and Nicolas Goudin (Cell Imaging Core, Structure Fédérative de Recherche Necker) in assisting with confocal microscopy analysis. We thank Claire Boudet and the personnel of Laboratoire d'Expérimentation Animale et Transgénèse (Structure Fédérative de Recherche Necker) for mouse breeding and handling.

This work was supported by the Fondation Princesse Grace (to Team Développement du Système Immunitaire) and the Ligue contre le Cancer (Équipe Labellisée).

The microarray data presented in this article have been submitted to the ArrayExpress database (https://www.ebi.ac.uk/arrayexpress/browse.html) under accession number E-MTAB-5928.

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • AID

    activation-induced cytidine deaminase

  •  
  • APC/C

    anaphase-promoting complex/cyclosome

  •  
  • CLL

    chronic lymphocytic leukemia

  •  
  • Cul3

    Cullin 3

  •  
  • FO

    follicular

  •  
  • GC

    germinal center

  •  
  • GSEA

    gene set enrichment analysis

  •  
  • KO

    knockout

  •  
  • MZ

    marginal zone

  •  
  • PC

    plasma cell

  •  
  • PLA

    proximity ligation assay

  •  
  • T1

    transitional type 1

  •  
  • T2

    transitional type 2

  •  
  • wt

    wild type.

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The authors have no financial conflicts of interest.

Supplementary data