Genome-level rearrangements of Ig genes during B cell development are critical for generation of a diverse repertoire of BCRs that bind to a multitude of foreign Ags and some self Ags. Bone marrow B cell development involves a variety of cell–cell interactions, cell migration, and receptor signaling that likely benefit from the activity of membrane-cytoskeletal reorganizing proteins. However, the specific contribution of such proteins toward BCR repertoire diversification is poorly understood. Ezrin is a membrane-cytoskeletal linker protein that regulates mature B cell activation through spatial organization of the BCR. We employed next-generation sequencing to investigate whether Ezrin plays a role in IgH rearrangements and generation of BCR diversity in developing bone marrow B cells. BCR repertoire development occurred stochastically in B cell progenitors from both control and B cell conditional Ezrin-deficient mice. However, the loss of Ezrin resulted in fewer unique CDRs (CDR3s) in the BCRs and reduced Shannon entropy. Ezrin-deficient pre-B cells revealed similar utilization of joining (J) genes but significantly fewer variable (V) genes, thereby decreasing V-J combinatorial diversity. V-J junctional diversity, measured by CDR3 length and nucleotide additions and deletions, was not altered in Ezrin-deficient pre-B cells. Mechanistically, Ezrin-deficient cells showed a marked decrease in RAG1 gene expression, indicating a less efficient DNA recombination machinery. Overall, our results demonstrate that Ezrin shapes the BCR repertoire through combinatorial diversification.
B cell development is a highly complex and regulated process (1) starting with hematopoietic stem cell progenitors in the bone marrow that commit to the B cell fate as pro-B cells, followed by serial differentiation into pre-B and immature B cells (2). Ig H and L chain gene rearrangement during B cell development generates a diverse repertoire of BCRs to tackle a wide plethora of infectious agents (3). Pro-B cells undergo V(D)J recombination in the IgH variable (IGHV) genes of the BCR and progress to the pre-B cell stage, where IGLV gene recombination occurs. Each combination of the H and L chains further results in a new Ag specificity and contributes to the overall BCR repertoire (3). The specificity for Ag binding is determined by the IGHV CDRs 1, 2, and 3, and because the CDR3 region makes more contact with Ag than any other portion of the BCR, it exhibits the most variability and is the main determinant of Ag binding (4, 5). The primary source of diversity in the B cell repertoire is V(D)J recombination, triggered by expression of RAG1 and RAG2 recombinases (6) that introduce site-specific DNA double-strand breaks and recruit other proteins to enable joining (7). Junctional diversity results from nontemplated nucleotide additions (N-additions) and deletions at the blunt ends of junctions between V, D, and J genes (8).
Cell–cell interactions and receptor signaling in progenitor B cells orchestrate the optimal progression of bone marrow B cell development (9, 10) and likely involve the activity of membrane-cytoskeletal reorganizing proteins. The plasma membrane-cytoskeleton cross-linking protein Ezrin is involved in a variety of cellular processes such as migration, protein localization, receptor signaling, and cytokine secretion (11–15). We have previously reported that Ezrin regulates BCR organization and signaling, as well as Ag-specific Ab responses of mature B cells (11, 16). However, it is not known whether Ezrin regulates BCR repertoire diversification. In this study, we show that the genetic deletion of Ezrin in B cells reduces RAG1 gene expression, V gene usage, V-J combinations, and CDR3 diversity, revealing an important role for Ezrin in promoting BCR diversity during B cell development.
Materials and Methods
Ezrinfl/flMb1cre/+ (Ezrin B cell conditional knockout [Ez-def]) mice have been previously described (11, 15, 17). Age- and sex-matched Mb1cre/+ mice (18) were used as controls in all experiments. All animals were used in compliance with the guidelines approved by the Cleveland Clinic Institutional Animal Care and Use Committee.
Flow cytometry and cell sorting
Bone marrow cells were stained with FITC-, PE-, PE-Cy7-, allophycocyanin-, allophycocyanin-Cy7-, or PerCP-Cy5.5-conjugated Abs to B220, IgM, IgD, IL-7R, c-Kit, and BP-1 (Supplemental Table I) for analysis of developing B cell populations and sorting pre-B cells. Pro-B cells were identified as B220+IgM−IL-7R+c-Kit+BP-1−, pre-B cells as B220+IgM−IL-7R+c-Kit−BP-1+, and immature B cells as B220+IgM+IL-7R−IgD−. Bone marrow cells were sorted using FACSAria Fusion II, pelleted, and stored in RNAprotect (Qiagen). All samples were acquired on a BD LSRFortessa flow cytometer using BD FACSDiva software, and flow cytometry data were analyzed using FlowJo software (Tree Star).
RNA extraction and BCR sequencing
RNA extraction was performed by iRepertoire using the RNeasy mini column kit (Qiagen) for samples containing ≥106 cells and the RNeasy micro column kit (Qiagen) for samples containing <106 cells, and assessed for concentration and quality using NanoDrop (Thermo Scientific). The average mass input for each bone marrow B cell developmental stage was 20 ng. PCR1 was performed and amplicons were rescued using iRepertoire’s in-house Beckman Coulter SPRIselect bead protocol. The entire product was used as template for PCR2. The amplicon length from RNA mouse BCR H chain libraries is ∼400 bp. Amplicons were separated by gel extraction using a QIAquick gel purification kit and checked for concentration and quality using Thermo Scientific NanoDrop 8000. The cDNA libraries were pooled on the Illumina NextSeq and quality controlled using Mic quantitative PCR analysis. Sequenced data were put through iRepertoire’s software pipeline and processed to view on iRweb.
Quantitative real-time PCR
RNA was isolated from sorted bone marrow pre-B cells (Supplemental Table I). RNA from replicates (three mice per genotype) was pooled due to low cell counts, followed by cDNA synthesis, and quantitative real-time PCR (qRT-PCR) was performed using the PowerUp SYBR Green master mix (Applied Biosystems) on a QuantStudio 5 real-time PCR system (Applied Biosystems). Gene expression was analyzed using forward and reverse primers for GAPDH, RAG1, RAG2, XRCC4, Ligase IV, Artemis, Ku70, and DNApkcs genes (Supplemental Table I). Expression was calculated by normalizing target gene expression to the housekeeping gene GAPDH (ΔCt) and calculating the fold change in gene expression in Ez-def pre-B cells relative to Mb1cre/+ control cells (2−ΔΔCt).
Statistically significant differences between Mb1Cre/+ and Ez-def B cells were determined by calculating p values using an unpaired t test and an α level of 0.05. Mean ± SEM is shown and each symbol indicates an individual biological replicate. Data were plotted and analyzed using Prism 7 software (GraphPad).
Single-cell RNA sequencing of bone marrow cells (https://tabula-muris.ds.czbiohub.org) has revealed that Ezrin is expressed throughout B cell development (Fig. 1A), and expression increases as the B cells progress through the pro, pre, and immature stage (Fig. 1B). We have previously shown that the genetic deletion of Ezrin in B cells does not grossly impact the bone marrow proportions of proper-B cells and immature B cells (11). However, because Ezrin regulates BCR signaling (11, 16), B cell migration, and cell adhesion (13, 14), all of which are important elements of developing B cell behavior in the bone marrow, we hypothesized that its deficiency in developing B cells may alter the BCR repertoire. To investigate this, we employed a gating scheme to sort pro-B, pre-B, and immature B cells based on differential expression of IL-7R, c-Kit, BP-1, surface IgM, and surface IgD (Fig. 1C). No differences in the proportions of pro-B, pre-B, and immature B cells were observed between Mb1cre/+ and Ez-def mice (Fig. 1D). Because V(D)J recombination is complete by the time pro-B cells transition to the pre-B cell stage and RAG enzymes are expressed at both stages, we reasoned that IGHV sequencing in pre-B cells would yield useful information on the impact of Ezrin deficiency on V and J chain repertoire. Sorted pre-B cells were shipped to iRepertoire where RNA extraction, library generation, and murine IGHV (mIGHV) sequencing were performed (Fig. 2A) using 20 ng of RNA per replicate (n = 3) from each genotype. The outputs in iRweb provided various measures of repertoire complexity and diversity, including the number and frequency of CDR3s (19), that were further analyzed to examine the effect of Ezrin deficiency on BCR repertoire diversity.
Reduced CDR3 diversity in developing Ezrin-deficient B cells
To investigate the impact of Ezrin deficiency on BCR diversity, we focused on the hypervariable CDR3 regions of the BCR where most Ag interaction occurs. Bubble plots were used to assess the number and frequency of unique CDR3s in pre-B cells from each of the three biological replicates, wherein each bubble corresponds to a unique CDR3 and bubble size is indicative of corresponding CDR3 frequency. This visual representation revealed that Ez-def pre-B cells expressed fewer unique CDR3s, albeit at higher frequencies compared with Mb1cre/+ pre-B cells for all replicates (Fig. 2B). Quantitative analysis of the bubble plots revealed ∼2-fold fewer unique CDR3 pre-B cells in Ez-def than in Mb1cre/+ pre-B cells (Fig. 2C). The diversity of CDR3 sequences in Ez-def B cells was further quantified by Shannon entropy, a measure of diversity that increases as both richness and evenness of the repertoire increases (20). Shannon entropy was ∼20% lower in Ez-def pre-B cells compared with that in corresponding Mb1cre/+ pre-B cell counterparts (Fig. 2D). Analysis of the top 10 CDR3 sequences in pre-B cells showed many differences between replicates within each genotype, as well as between Ez-def and Mb1cre/+ cells (Fig. 3), consistent with the stochastic nature of BCR repertoire generation (3).
Absence of Ezrin decreases combinatorial diversity
Because combinatorial diversity of the CDR3s is a major contributor to the overall diversity of the BCR repertoire, we investigated whether these processes were altered in Ez-def B cells. As combinatorial diversity is generated primarily through V(D)J recombination, usage of V and J genes is often used to measure changes in combinatorial diversity. Two-dimensional heatmaps were used to represent V-J combinations, wherein overlap in the usage of a specific V gene and a specific J gene corresponds to a colored square (Fig. 4A). Black squares indicate absence of that V-J combination, whereas blue, white, and red squares denote the presence of a V-J combination in increasing order of frequency (Fig. 4A). Ez-def pre-B cells showed lower combinatorial V-J gene usage accompanied by higher frequencies than Mb1cre/+ pre-B cells (Fig. 4A). Arrows indicate the V-J gene combinations with the biggest differences in usage and frequency (Fig. 4A). These data suggest that altered V and J gene usage in Ez-def developing B cells may underlie the difference in overall BCR diversity in the absence of Ezrin. Therefore, we further investigated whether the altered V-J combinations were derived from differential V gene or J gene usage. iRepertoire sequencing data revealed 16 different V gene families, each containing a number of alleles that can be used to construct the CDR3 binding region (Fig. 4B). Families 1 and 14 showed significant differences in usage between Mb1cre/+ and Ez-def, with family 8 showing differences approaching significance (Fig. 4B). Among the 16 V gene families, a total of 216 possible alleles can be used. For ease of depiction, frequencies of only significantly different alleles and those approaching significance are shown (Fig. 4C). There was an overall trend for reduced usage of the V alleles in Ez-def pre-B cells compared with corresponding Mb1cre/+ B cells (Fig. 4C). The mIGHV1-55, mIGHV1-64, mIGHV1-85, mIGHV2-3, mIGHV5-2, mIGHV5-16, mIGHV8-12, mIGHV9-4, mIGHV14-1, and mIGHV14-4 genes were all underutilized by at least 2-fold in Ez-def pre-B cells, whereas mIGHV1-7 and mIGHV9-4 alleles had significantly higher expression (Fig. 4B). The altered V-J combinations described in (Fig. 4A corresponded to the significantly reduced alleles in (Fig. 4C. The frequency of J gene family usage was not different between Mb1cre/+ and Ez-def B cells at the pre-B cell stage (Fig. 4D), indicating that the decreased usage of V alleles contributes most to the reduced V-J combinatorial diversity in the absence of Ezrin.
Effect of Ezrin deficiency on junctional diversity
Junctional diversity contributes to overall diversity and results from differences in CDR3 length and variability in N-additions and deletions/trimming (1). To investigate junctional diversity in developing Ez-def B cells, we first assessed the overall length of the CDR3s. The percentage of total CDR3s with specific nucleotide lengths were compared in Mb1cre/+ and Ez-def B cells at the pre-B cell stage of development. Mb1cre/+ and Ez-def pre-B cells did not show many differences in CDR3 length, except for CDR3s with 42 nt, which were significantly fewer in Ez-def pre-B cells (Fig. 5A). CDR3 nucleotide length is determined by N-additions during the D to J and the V to D-J joining events by the enzyme TdT, and nucleotide trimming at the junctions through exonuclease activity. The number of nucleotides added or trimmed can contribute to junctional diversity. Therefore, we next analyzed the percentage of CDR3s with specific numbers of N-additions ranging from 0 to 10 nt. No significant differences in N-additions were observed between Ez-def and Mb1cre/+ pre-B cells (Fig. 5B). Furthermore, H chain V and J region trimmings were calculated to assess differences in deletions. Ez-def pre-B cells showed a marginal but significantly higher percentage of CDR3 sequences with 1 nt trimming in the V regions compared with Mb1cre/+ (Fig. 5C). J trimmings analysis showed no significant differences in pre-B cells from the two genotypes (Fig. 5D).
RAG gene expression is reduced in Ezrin-deficient B cells
RAG proteins bind to heptamer and nonamer recombination signal sequences to initiate recombination of V, D, and J genes (3). In studies employing mutant mice with lower RAG1 gene expression it was observed that the RAG1 hypomorphic pro-B cells, pre-B cells, splenic B cells, and peritoneal B1 cells had fewer rearrangements of normally highly used V allele families (21). Therefore, we quantified expression of RAG1 and RAG2 genes in sorted pre-B cells by qRT-PCR. In two separate experiments, RAG1 gene expression was reduced 0.643- and 0.574-fold, respectively, in Ez-def pre-B cells compared with Mb1cre/+ cells (Fig. 6A). In contrast, there was a 1.055- and 1.10-fold increase in RAG2 gene expression in Ez-def pre-B cells (Fig. 6A). We additionally quantified gene expression of other key enzymes that facilitate productive V(D)J recombination: XRCC4, Ligase IV, Artemis, Ku70, and DNApkcs (22–26). Pre-B cell RNA extracted from three replicate Mb1cre/+ and Ez-def mice were pooled and used to perform gene expression analysis by qRT-PCR. XRCC4, Ku70, and DNApkcs showed a 0.64-, 0.73-, and 0.835-fold decrease, respectively (Supplemental Fig. 1). In contrast, Ligase IV was increased 1.63-fold increase and Artemis was largely unaltered at 1.03-fold (Supplemental Fig. 1). Overall, our data suggest that deletion of Ezrin reduces V gene usage and combinatorial diversity in developing Ez-def B cells, likely through reduced RAG1 gene expression.
In this study, we report that Ezrin promotes diversification of the BCR repertoire through greater utilization of mIGHV genes. Our published (11) and current data did not reveal changes in pro-, pre-, and immature B cell proportions in mice with a B cell–specific deletion of Ezrin. However, the diversity of the developing Ez-def BCR repertoire showed clear quantitative and qualitative alterations. The loss of Ezrin in developing B cells resulted in fewer unique CDR3s, decreased V gene usage across several V gene families, a lower number of V-J combinations, and reduced overall combinatorial diversity of the BCR repertoire. Junctional analysis did not reveal many additional differences between Mb1cre/+ and Ez-def cells, and this is likely not a contributing factor to lower CDR3 diversity observed in Ez-def pre-B cells.
Interestingly, decreased combinatorial diversity induced by B cell–specific loss of Ezrin was associated with lower RAG1 gene expression but unchanged RAG2 expression. Because an active synaptic complex is a heterotetramer containing two RAG1 and two RAG2 molecules (27, 28), and RAG2 recombinase cannot bind to DNA without RAG1 (29), the decrease in RAG1 gene expression observed in Ez-def cells is likely to be meaningful. Lower RAG1 gene expression in Ez-def B cells suggests that fewer recombination signal sequences will be recognized within the developing B cell genome, and thereby fewer Ig V genes used for recombination. RAG gene expression is regulated by a number of factors (25, 26, 30, 31) and a mechanistic dissection of this process in developing Ez-def B cells in the future will elucidate the molecular and functional intersection between Ezrin and RAG1 expression and V(D)J recombination. Additional V(D)J key recombination factors (25, 26, 32) were explored as potential actors in the relationship between V gene usage and overall BCR repertoire diversity in Ez-def pre-B cells. Ligase IV has an important role in ligating signal and coding ends of the V, D, and J genes, and XRCC4 stabilizes Ligase IV activity. Whereas XRCC4 expression was decreased, the expression of Ligase IV was increased in Ez-def pre-B cells. The opposite nature of gene expression changes in these two enzymes may indicate compensatory mechanisms. Regardless of the cause, reduced XRCC4 expression may decrease the stability and, hence, the activity of Ligase IV, and thereby disrupt the V(D)J recombination machinery. Artemis was chosen for analysis due to its function in opening hairpin-sealed coding ends for subsequent joining but did not reveal any difference in expression between developing Mb1cre/+ and Ez-def B cells, suggesting that the observed differences in V gene usage are independent of Artemis activity by itself. Interestingly, DNApkcs is required to form the coding joint, and promotes the activity of Artemis. The decrease in expression of DNApkcs in Ez-def cells by ∼20% may affect the activity of Artemis and ultimately make the joining of coding ends less productive. Finally, Ku70 has a role in repairing both signal and coding ends of V, D, and J genes, and its expression decreased by ∼30%. All of the gene expression changes in DNA recombination factors in this study are intriguing and consistent with the potential disruption of V(D)J recombination and reduction in V gene usage and V-J combinations. Future studies will examine the interactions between these proteins and the V genes to form the recombination complexes on a molecular level.
Another possible explanation for reduced expression of specific IGHV genes in Ez-def B cells in the bone marrow is altered transcriptional activity. Interestingly, Ezrin associates with ARID3A, a transcription factor that was reported to regulate BCR diversity by binding to certain IGHV promoter-proximal sites and inducing Ig gene transcription (33–35). ARID3A interacts with Ezrin within lipid rafts and shuttles between the nucleus and the cytoplasm (36). Therefore, it is conceivable that the deletion of Ezrin affects the nuclear translocation of ARID3A and associated IGHV transcription in developing B cells. Future studies will elucidate the effect of Ezrin deficiency on expression and localization of ARID3A in bone marrow B cells, as well as that of ARID3A deletion on V, D, and J gene usage in pre-B cells.
There are at least two main implications of our findings for the ability of Ez-def mice to recognize Ags. If the lower BCR diversity observed in Ez-def mice during bone marrow development is maintained in the mature B cells in secondary lymphoid tissues, it is likely that fewer infectious agents would be recognized. Along those lines, lower combinatorial diversity in the absence of Ezrin may also produce fewer self-reactive BCR specificities and protect from autoimmunity. Consistent with this idea, we have previously reported that B cell–specific deletion of Ezrin in the Lyn-deficient mouse model of systemic lupus erythematosus reduces self-reactive Abs and glomerulonephritis (17). Selection, maintenance, and Ag response of mature B cells in peripheral tissues depend on various factors, including survival signals from cytokines and the BCR (37, 38), which ultimately shape the available Ag recognition capacity. In addition to regulating BCR signaling (11), Ezrin may modulate survival signals from cytokines, and thereby further influence diversity of the mature B cell repertoire.
Taken together, our findings reveal, to our knowledge, a novel role for Ezrin in shaping the developing BCR repertoire.
The authors acknowledge assistance from the Lerner Research Institute Flow Cytometry Core Facility.
This work was supported by the National Institutes of Health Grant AR067705 to N.G.
The online version of this article contains supplemental material.
The authors have no financial conflicts of interest.