The Sµ-3'RR recombination (Sµ-3'RRrec) in B cells, a genomic rearrangement occurring within the immunoglobulin heavy chain (IGH) locus is believed to lead to B cell receptor (BCR) loss. Its increased frequency in patients with chronic lymphocytic leukemia (CLL), especially those with high MYC expression, suggests c-MYC contribute to genetic instability during oncogenesis To explore c-MYC's role in enhancing Sµ-3'RRrec, the study used a λ-c-MYC transgenic (Tg) mouse model overexpressing MYC specifically in B cells, along with wild-type (WT) and activation-induced cytidine deaminase knockout (AIDKO) mice. The results show that MYC overexpression leads to a higher proportion of BCR− B cells, which undergo Sµ-3'RRrec. These BCR− B cells are sensitive to apoptosis and represent activated mature B cells that likely originate outside the germinal center (GC). Further analysis demonstrated that Sµ-3'RRrec occurs more frequently in BCR− B cells than BCR+ B cells. These BCR− cells also display a polyclonal IGHV repertoire, indicating their diverse origins. Additionally, we observed changes in the class switch recombination (CSR) junctions in BCR− B cells, hinting at DNA repair differences.
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Activated mature B cells undergo enforced Sµ-3'RRrec in the λ-c-MYC mouse model
https://doi.org/10.21203/rs.3.rs-5331665/v1
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The recombination of the immunoglobulin heavy chain (IGH) locus between the switch µ (Sµ) region and the 3' regulatory regions (3'RR), known as Sµ-3'RRrec, was first identified over a decade ago (1). Since its discovery, its specific role in B cell physiology remains unclear, as no study to date has conclusively defined its function. Originally, this recombination event was termed 'locus suicide recombination' because, when it occurs on the productive IGH allele, it leads to the deletion of the entire constant region of the IGH locus. This deletion prevents the expression of a functional IGH, leading to the loss of the B cell receptor (BCR) and, ultimately, B cell death due to the absence of immunoglobulin production (2). This mechanism would explain why Sµ-3'RRrec might contribute to the regulation of B cell lifespan and homeostasis by eliminating defective or unwanted B cells. However, subsequent studies in both mice (3, 4) and humans (5–7) have revealed that Sµ-3'RRrec can also occur on the non-functional IGH allele, which does not affect B cell survival. This broader detection, including amplification of Sµ-3'RRrec junctions from peripheral blood mononuclear cells (PBMCs), has led us to reconsider the initial 'suicide' interpretation and adopt the more neutral term 'Sµ-3'RRrec' to reflect its potential role beyond just eliminating B cells. Furthermore, this recombination event has been detected at higher frequencies in patients with chronic lymphocytic leukemia (CLL) with unfavorable prognosis—CLL being a B-cell malignancy—compared to healthy individuals, suggesting that it could be a consequence or a biological artifact of disease progression (6). The increased frequency of Sµ-3'RRrec in CLL patients has been associated with elevated MYC expression, a hallmark of aggressive B cell cancers, raising important questions about its involvement in oncogenesis. It is possible that ongoing recombination at the IGH locus, as seen with Sµ-3'RRrec, reflects broader genomic instability in CLL, potentially affecting other loci as well. This observation highlights the role of Sµ-3'RRrec as a marker of an active genetic rearrangement process, raising questions about its molecular mechanism and the impact of c-MYC on it. In our previous study, we investigated the influence of c-MYC on IGH recombination, demonstrating in vitro that c-MYC significantly enhances both class switch recombination (CSR) and Sµ-3’RRrec in the murine B cell lymphoma CH12F3 cell line (6). Given these findings, we now seek to determine whether the overexpression of MYC in vivo can similarly elevate the levels of Sµ-3’RRrec. To explore this question, we used the λ-c-MYCTg mouse model, which is designed to overexpress the MYC gene specifically within the B cell lineage. In this model, the expression of the human MYC gene is driven by the immunoglobulin lambda (Igλ) promoter, allowing for targeted analysis of c-MYC's role in B cell physiology. Additionally, we used the activation-induced cytidine deaminase (AID) knock out (AIDKO) mouse model to assess the effects of MYC overexpression in the absence of AID, a key enzyme involved during CSR and somatic hypermutation (8, 9). This approach allowed us to establish an in vivo model that generates a greater number of BCR-negative (BCR−) B cells than in wild type mice, although we still face the limitation of B cell death induced by Sµ-3'RR recombination. This study makes an attempt to characterize the phenotype and cellular characteristics of the BCR− B cells that have undergone Sµ-3’RRrec. Ultimately, our data suggest that the BCR− B cells potentially have more numerous Sµ-3’RRrec junctions than in BCR positive (BCR+) B cells. In line with what is expected as a consequence of the Sµ-3’RRrec, BCR− B cells are sensitive to B cell apoptosis. BCR− B cells appear as activated mature B cells that have undergone a CSR process in which DNA repair is not similar as normally described (10, 11) and these cells could be activated B cells that do not participate in the germinal center reaction. Finally, these results are reminiscent of the proposition made in humans that CLL cells with recombinatory activity of the IGH locus associated with MYC overexpression originate from activated mature B lymphocytes outside the germinal center.
The overexpression of MYC results in an increase in BCR− B cells enriched in Sµ-3’RRrec junctions.
We quantified BCR− B cells because these cells potentially underwent Sµ-3’RR recombination. This quantification was performed using flow cytometry to determine the proportions of mature B cells expressing or lacking a BCR on their surface within splenic B cells. Our gating strategy specifically excluded B cell progenitors, as peripheral BCR− B cells may represent immature B cells at early developmental stages, which could bias the analysis. Notably, in the λ-c-MYCTg mice, it has been shown that tumoral B cell progenitors migrate from the bone marrow and are frequently present in the spleen (12). As shown in Fig. 1A, BCR− B cells were detected in all mice, and their percentages significantly increased with MYC overexpression, in both in AID wild-type (AIDWT) and AIDKO conditions. Indeed, we detected 2.4% ± 0.3 (mean ± standard error of the mean (SEM)) of BCR− B cells among splenic B cells from AIDWT mice (N = 21) compared to 2.9% ± 0.4 (mean ± SEM) in AIDKO samples (N = 13). This percentage increased with MYC overexpression, as we detected 18.1% ± 4.2 (mean ± SEM) BCR− B cells in AIDWT λ-c-MYCTg mice (N = 28) and 16.2% ± 5.0 (mean ± SEM) in AIDKO λ-c-MYCTg mice (N = 17). The mice used in our study were littermates, bred under the same conditions to generate the AIDWT / AIDWT λ-c-MYCTg and AIDKO / AIDKO λ-c-MYCTg lines. Thus, the percentage of BCR− B cells in spleen of mice are strongly influenced by c-MYC overexpression, but are not affected by the AID deficiency.
Among the analyzed λ-c-MYCTg mice, some had a normal-sized spleen, while others exhibited splenomegaly, and still others had tumors located typically in classic area of this model, such as the submandibular region or, less frequently, in the inguinal fossae. We then categorized the quantification of BCR− cells according to the mouse phenotype: normal-sized spleen (NSP), splenomegaly (S+), and tumor presence (S + T+) (Fig. 1B, Supplemental Fig. 1). Despite a tendency for the percentage of BCR− B cells to increase in mice with splenomegaly—regardless of their AID status—or in AID-proficient mice with tumors, we observed no significant effect due to the separation of phenotypes. Importantly, across all phenotypes, mice with MYC overexpression exhibited a significantly higher frequency of BCR− B cells.
A pivotal question underlying our study is whether BCR− B cells are those that have undergone Sµ-3'RR recombination on the functional allele. To investigate this, we assessed whether there was an enrichment of Sµ-3'RRrec junctions in mature BCR− B cells compared to mature BCR+ B cells in the spleen. To achieve this objective, we performed flow cytometric sorting of AIDWT λ-c-MYCTg mature B cells with or without a BCR. The harvested cells were then divided for DNA and RNA extraction. The extracted DNA was used to amplify the Sµ-3'RRrec junctions. Murine embryonic splenic stem (ES) cells (data not shown, N = 1, 0 Sµ-3’RRrec junctions) from wild-type (WT) mice served as negative controls. Given the low number of cells recovered during cell sorting and the limited quantity and/or quality of DNA obtained, we were only able to test a small number of samples for IGH locus recombination Sµ-3’RRrec and CSR: BCR+ B cells from AIDWT λ-c-MYCTg, N = 1 and BCR+ B cells from AIDWT λ-c-MYCTg, N = 2. Nevertheless, for these three samples, we detected and analyzed both Sµ-3’RRrec and CSR recombination. As shown in Fig. 1C, we detected 11 and 14 or 15 junctions in the BCR+ and BCR− samples, respectively. The two BCR− samples show slightly higher rates of Sµ-3'RRec junctions compared to the BCR+ sample, suggesting a moderate enrichment of Sµ-3'RRec junctions in BCR− B cells. Although this conclusion should be interpreted cautiously due to the small sample size, it is supported by the sequencing read counts for Sµ-3'RRec junctions in each sample. We obtained 118 reads for BCR+ B cells and 610 or 8,347 reads for BCR− B cells containing Sµ-3'RRec junctions (Fig. 1C). These results indicate that BCR− B cells exhibit an increased number of Sµ-3'RRec junction sequences, with some unique junctions being more frequently detected, whereas in BCR+ B cells, Sµ-3'RRec junctions appeared less enriched.
Each Sµ-3'RRec junction arises from a unique recombination event, and its structure can be determined by alignment with reference sequences. Similar to CSR, the Sµ-3’RRrec junction sequences produced following recombination between two DNA double-strand breaks (DSBs) are shaped by the DSB repair machinery. These recombination junctions can be categorized as blunt, containing insertions between the ligated DNA ends, or exhibiting homology between the recombination acceptor and donor sequences. The lengths of these insertions and homologies vary in base pairs (bp). Short sequence homologies are referred to as microhomologies. Analysis of Sµ-3'RRec junction structures in both BCR+ and BCR− samples revealed that the joint structures were comparable, regardless of BCR status (Fig. 1D). We observed more junctions with insertions compared to those with microhomologies or blunt junctions, with long insertions (≥ 4 bp) being predominant (data not shown : BCR+ AIDWT λ-c-MYCTg, N = 1, 11 Sµ-3'RRec junctions, including 6 with insertions ≥ 4 bp; BCR− AIDWT λ-c-MYCTg, N = 2, 34 Sµ-3'RRec junctions, including 18 with insertions ≥ 4 bp). This Sµ-3'RRec structural profile is consistent with previous reports (3).
Finally, the data suggest, as expected, an enrichment of Sµ-3'RRec junctions in BCR− B cells. The detection of junctions in BCR+ samples is likely from non-functional IGH allele, as previously proposed (5), and the Sµ-3'RRec junctions exhibit a structurally consistent profile across all three tested samples.
BCR − B cells exhibiting Sµ-3’RRrec junctions are polyclonal mature B cells.
Total RNA obtained from AIDWT λ-c-MYCTg cell sorting was used to assess the expression of B cell lineage-specific markers that are developmentally stage-specific (Fig. 2). The identification of CD19+ BCR+ cells as part of the B lymphoid lineage is unequivocal, but CD19+ BCR− cells may represent plasmablasts or plasma cells that retain low CD19 expression and display little to no BCR on their surface (15). To further characterize BCR− B cells in terms of B cell specific gene expression, we first examined the expression of PAX5, the transcription factor recognized as the 'guardian' of B cell identity (15). PAX5 is specifically expressed in B lymphocytes from early developmental stages through to the differentiation of activated B cells into plasma cells. Since PAX5 expression is well-established in BCR+ B cells, additional testing in these cells was deemed unnecessary. Detection of PAX5 transcripts in BCR− B cells confirmed their B lymphocyte identity (Fig. 2A). In a previous study on the transgenic mouse model, BCR-deficient B cells were identified as immature B cells due to the impact of MYC overexpression on B progenitor development in the bone marrow (12). Taking these findings into account, we adapted our flow cytometry strategy to specifically study BCR− B cells, excluding B progenitor cells. To rule out any contamination, we compared the expression of early-stage markers (16, 17) between BCR+ B cells from the spleen of AIDWT λ-c-MYCTg, representing the mature B cell compartment, B cells isolated from the bone marrow of WT mice as positive controls, and the BCR− B cells of interest isolated from the spleen of AIDWT λ-c-MYCTg (Fig. 2B). The AIDWT λ-c-MYCTg BCR− B cells lacked expression of c-kit (CD117) and CD25. The CD43 expression levels were comparable between BCR− B cells and BCR+ B cells, lower than those found in bone marrow samples from WT mice. Furthermore, we characterized these mature B cells as PRDM1-negative (Fig. 2C), excluding that they are plasmablasts or plasma cells (18). Finally, we tested the expression of AICDA, which encodes the AID enzyme, in BCR+ B cells and BCR− B cells to determine whether they are potentially activated. We observed a slight increase in AICDA expression in BCR− samples compared to BCR + samples, but this difference was not statistically significant (Fig. 2D). Thus, the low level of AICDA expression in BCR− B cells suggests that these cells are unlikely to be undergoing activation. Collectively, our data suggest that BCR− B cells, enriched for Sµ-3'RRrec junctions, are indeed mature B cells. Given the observed reduction in BCR− cells due to apoptosis, we hypothesize that the BCR− B cells identified in our study likely represent only a subset of the total BCR− population, potentially leading to an underestimation of their overall prevalence.
Although BCR− B cells are rare, their increased frequency in the λ-c-MYCTg mouse models raises questions about the clonality of this population. To investigate this, we employed a method that measures IGHV rearrangement lengths after PCR amplification using consensus primers for V and J segments. This approach allowed us to detect and semi-quantify length-homogeneous IGHV clonotypes from DNA samples derived from B cells, using splenic B cells as controls for 'polyclonal' IGHV rearrangements. We found that the absolute number of IGHV rearrangements with homogeneous length was generally lower in BCR− B cells compared to total splenic B cells (Fig. 3A). This restriction in IGHV rearrangements within BCR− B cells may be explained by the low percentage and low number of these cells. In all analyzed samples, predominant clonotypes with homogeneous length were observed (Fig. 3B). While all samples displayed a polyclonal rearrangement profile, the presence of one or a few dominant IGHV rearrangements with homogeneous length was detected in both total B cells and BCR− B cells from transgenic AIDWT λ-c-MYCTg mice. To assess whether this observation is different between to the cell populations, we compared the diversity between total splenic B cells from WT mice, total splenic B cells from AIDWT λ-c-MYCTg mice, and sorted BCR− B cells from AIDWT λ-c-MYCTg mice. We evaluated IGHV diversity using the Shannon index, calculated based on the number and frequency of different IGHV rearrangement lengths. We observed that the diversity of IGHV junctions in AIDWT λ-c-MYCTg B cells was significantly reduced compared to WT B cells. This observation is directly linked to the effect of the MYC transgene, which promotes B cell transformation, leading to a situation where the polyclonal B cell population, typically represented by equivalent clones, becomes imbalanced due to the emergence of one or more dominant clones. However, we found that the diversity between sorted BCR− B cells and total B cells from AIDWT λ-c-MYCTg mice was similar (Fig. 3C). Consequently, the diverse IGHV rearrangements in BCR− B cells indicate a polyclonal population. Notably, the dominant length-homogeneous clonotypes identified in unsorted B cells were present in BCR− B cells at higher ranks (Fig. 3D). Together, our results suggest that BCR− B cells constitute a polyclonal population that displays a range of diverse IGHV rearrangements and representative of the overall diversity within the B cell population.
BCR − B cells with Sµ-3’RRrec have undergone CSR due to activation.
We analyzed counts of CSR junctions in DNA samples. Junction counts were compared using data from sorted BCR+ B cells (N = 1) and BCR− B cells (N = 2) from two AIDWT λ-c-MYCTg mice. Murine ES stem cells (data not shown, N = 1, 3 CSR junctions) from WT mice served as negative controls. CSR junctions were detected in BCR+ as expected and were also detected in BCR− B cell samples (Fig. 4A). While CSR junction levels appeared to be lower in BCR− B cells compared to BCR+ samples (78 and 72 junctions compared to 95 junctions). The detection of CSR junctions in sorted BCR− B cells, even if slightly lower in count, was unexpected. Based on our initial hypothesis, in the absence of a surface BCR, we would expect the functional and productive IGH allele to carry the Sµ-3'RRrec junction. As CSR can occur on both alleles (13–15), CSR rearrangement amplification likely originate from the non-functional IGH allele. Analysis of CSR junction structures revealed differences in profiles among the samples (Fig. 4B). Specifically, as shown in Fig. 4B, junctions in sorted BCR+ B cell were enriched in junctions with blunt breakpoints and/or small microhomologies, at the expense of junctions with insertions and/or longer microhomologies. The repair junction structural profile of CSR in this case is similar to what is expected. In contrast, the structure of CSR junctions in sorted BCR− B cells differed significantly. BCR− samples exhibited fewer junctions with small microhomologies (1–2 bp) and blunt junctions, while longer microhomologies (≥ 4 bp) were predominant. Strikingly, CSR joint structures in BCR− B cells seem to differ from those in BCR+ B cells, which was not the case for Sµ-3’RRrec (Fig. 1D). Notably, the CSR structural profile in BCR− samples is reminiscent of CSR profiles observed in condition of defect in the non-homologous end joining (NHEJ) repair system, which is the major repair pathway involved in CSR (10, 11).
The presence of CSR junctions, even on the non-functional allele, suggests that BCR− B cells have undergone prior activation. To determine whether BCR− B cells derived from activated B cells and germinal center (GC) B cells, we characterized the expression of CD38, CD95 and GL7 markers on splenocytes from AIDWT λ-c-MYCTg mice, specifically focusing on BCR− B cells. We did not observe difference in CD38Low CD95+ GC B cell population proportion in the CD19+ cells in the different mouse genotypes (Fig. 5A). Nevertheless, this was the case when analysis focused on BCR− B cells and the decrease of GC BCR− B cell population in the AIDKO cells (Fig. 5B). These results indicate that BCR− GC B cells emergence depends on AID. Also, the mature GL7+ splenic BCR− or BCR+ B cell populations in AIDKO and AIDKO λ-c-MYCTg mice were significantly reduced relative to WT mice (Fig. 5C and 5D), indicating that the GL7 B cell population depends on AID. Of note, while MYC overexpression induced an increase in GL7+ BCR− cells in the AID-deficient condition, this was not found with normal AIDWT λ-c-MYCTg BCR− B cells where we observed a decrease in GL7+ B cells. Thus, mature GL7+ cells, as BCR− GC B cells, appeared to depend on AID and deregulated MYC expression induces an increase of the GL7 + BCR− B cells in case of AID deficiency. Finally, we compared on GL7 mean fluorescence intensity (MFI) between GL7+ BCR+ and GL7+ BCR− B cells. Indeed, GL7high and GL7int phenotypes allow to distinguish GC B cells from activated B cells not participating in GC (16). The GL7 MFI ratio between BCR+ and BCR− B mature GL7 + B cells revealed that BCR− B cells expressed lower level of GL7 relative to BCR+ B cells (Fig. 5E).
Finally, as the expected consequence of the Sµ-3’RRrec is the death of B cells, we analyzed the percentage of annexinV+ cells on B cells and we compared the results between BCR− and BCR+ B cells. This was done on in vitro cultured splenic B cells with conditions that mimic the GC environment of activated B cells (17). We observed that BCR− B cells are more sensitive to apoptosis than BCR+ B cells (Fig. 6A), whatever the AID and c-MYC status (Fig. 6B) meaning that activated mature BCR− B cells appear to be predisposed to apoptosis.
In this study, we used a MYC-overexpressing mouse model to force the Sµ-3'RR recombination on the IGH allele in B cells and to access these cells by identifying them as negative for a BCR on their surface.
Our results indeed show that BCR− cells appear to carry a higher number of Sµ-3'RRrec junctions, confirming our initial hypothesis. However, these results should be interpreted with caution and expressed as a trend due to the small sample size. The subsequent study of BCR− cells showed that these cells, by not expressing B precursor markers, are mature B cells. An intriguing point is the detection of CSR junctions in these cells. The presence of CSR junctions supports prior activation of cells. Supporting the notion of prior cellular activation of BCR− cells, we noted that the expression levels of AID and PRDM1 in these cells tend to be higher than those observed in BCR+ cells, even though they are statistically similar. Finally, these cells appear to express GL7. Their classification into early B lymphoid developmental stages has been ruled out due to the absence of expression of c-KIT and CD25 markers or similar CD43 expression to that of BCR+ cells. Thus, the GL7 marker seems to be linked to the phenotype of activated B cells or germinal center B cells, suggesting that at least a fraction of BCR− cells may have been activated. Ultimately, the observation that the average fluorescence intensity of GL7 on BCR− cells is lower compared to that of BCR+ cells can categorize BCR− cells as GL7LOW cells, which are defined in the literature as activated B cells not participating in the germinal center reaction.
In light of these data, we propose that BCR− cells, or a subset of these cells, represent mature B cells that have been activated and undergone isotype switching. The dependency of GC BCR− and GL7 BCR− B cells on AID expression suggests that the BCR− B cell population emerges following AID activity. In this context, the detection of CSR junctions is plausible, provided they originate from a non-productive IGH allele. Notably, the structural profile of CSR junctions observed in BCR− B cells differs from what is expected under normal conditions and compared to junctions in BCR+ cells from the same animal as one of the BCR− samples. The pathways for DSB repair shape the structure of the repair junctions. CSR predominantly occurs through the coordinated action of NHEJ and, to a lesser extent, alternative end-joining (Alt-EJ)(10, 11, 18). In the case of BCR− samples, the structural characteristics of CSR junctions resemble those seen in NHEJ defect, leading to reduced CSR efficiency. Our results may be interpreted as evidence of CSR induction in activated mature B cells, wherein an aberrant CSR process during DSB repair results in the functional IGH allele being involved in Sµ-3'RR recombination. The loss of surface BCR expression sensitizes BCR− B cells to apoptosis, and since CSR events can occur before B cell entry into the germinal center (19), the low expression of GL7 may inhibit BCR− B cells from entering the germinal center.
In this model, Sµ-3'RRrec could facilitate the elimination of B cells that have not undergone productive CSR due to defects in DNA repair processes. The causes of non-productive CSR in normal B cells remain unknown, and data regarding the frequency of such occurrences are lacking. Furthermore, the mechanism by which non-productive CSR could lead to Sµ-3'RR recombination is unknown, although extensive DNA resection might play a role. Investigating these mechanisms is complicated by the disappearance of cells. Efforts to promote the survival of B cells that undergo apoptosis following BCR loss have been attempted by Denis-Lagache et al. (4), although this model may not be optimal. Ultimately, the data and the proposed model are consistent with the findings of Al Jamal (6), which suggest that CLL cells exhibiting IGH locus recombination activity and MYC overexpression originate from activated mature B lymphocytes outside of the germinal center.
Mice
Experiments were conducted under an approved protocol according to European Directive 2010/63/EU on animals used for scientific purposes (French national authorization number: 870850 and French Ethics Committee registration numbers APAFIS#26105-2020061810023698 and APAFIS#49715-202405301232424). AIDWT λ-c-MYCTg mice and AIDKO mice (9), both with C57BL/6 background, were crossed to generate AIDKO λ-c-MYCTg mice. Lineage maintenance crosses were: AIDWT female with AIDWT λ-c-MYCTg male or AIDKO female with AIDKO λ-c-MYCTg male of the same litter. Mice analyzed were at least 8 weeks old in all experiments. All mice were housed in conventional facility. Three phenotypes were considered: mice with a normal spleen phenotype (NSP) or with a spleen weight < 140mg; pre-tumoral mice (S+) or mice with a spleen weight greater than the maximum NSP spleen weight (splenomegaly); and tumoral mice (S + T+) presenting splenomegaly (spleen > 140mg) and a submandibullary tumor (Supplemental Fig. 1).
Cell cytometry and cell sorting
Femurs, lymph nodes or spleen were extracted from mice. Cell suspensions of bone marrow, tumors, splenocytes (after erythrocytes lysis with Versalyse™ #IM3648, Beckman Coulter) or isolated B cells from spleen (with EasySep™ mouse B cell Isolation Kit #19854, STEMCELL Technologies) were stained (analyses are summarized in supplemental table 1 and antibodies used in supplemental table 2). Data acquisition was performed with CytoFLEX LX (Beckman Coulter) and results were analyzed using the analysis version 2.1 of Kaluza software (Beckman Coulter). BCR+ and BCR− B cells were sorted using M1bis mix (supplemental table 2) with FACSAria™ III instrument (BD Biosciences).
CSR amplification and sequencing
CSR junctions (between Sµ and Sγ1 regions of IGH locus) were amplified by nested PCR using specific primers (Supplemental table 3) and analyzed with CSReport as described (20). The amplicons were used to prepare 300bp sequencing libraries with Ion Xpress™ Plus Fragment Library Kit (Life technologies, Thermofisher, 447269) and sequenced using ion S5 chip (Life Technologies). Murine embryonic stem (ES) cells (N = 1) served as negative controls for CSR junction detection (3 CSR junctions were detected, data not shown).
Sµ-3’RRrec amplification and sequencing
Sµ-3’RRrec junctions (between Sµ and like switch 10 region (1) were amplified by nested PCR using specific primers (PCR code C and C’ in supplemental table 3) from 100ng of genomic DNA in triplicates. Nested-PCR protocol of Sµ-3’RRrec junctions amplification is indicated in supplemental table 3. The amplicons were used to prepare sequencing libraries from Illumina protocol and sequenced using 300bp paired-ended sequencing on a MiSeq (Illumina). Forward and reverse reads were merged and were analyzed using CSReport (20). Murine (ES) cells (N = 1) served as negative controls for Sµ-3’RRrec junction detection (0 junctions were detected, data not shown)
Sequence deposit
CSR and Sµ-3’RRrec sequencing data produced in this study have been deposited in the National Center for Biotechnology Information’s BioProject (PRJNA830327).
Real time quantitative PCR
Total RNA was extracted using RNeasy micro kit (#74004, Qiagen) or TRIzol™ Reagent (#15596018, Invitrogen). TRIzol-extracted samples were treated with DNase I (#18068015, Invitrogen) prior to retro-transcription, cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (#4368814, Applied Biosystems). Each real-time reaction was performed in duplicate on 50ng of RNA equivalent using Taqman assay (SensiFast Probe Hi-Rox kit #BIO820025, Ozyme) on QuantStudio 3 (Applied Biosystems). Thermal cycling conditions according to Applied protocol was: 1 cycle (95°C − 2 min); 40 cycles (95°C–10sec, 60°C – 30sec). All probe used are listed in supplemental table 4. Relative mRNA levels were normalized to CD19 transcripts.
IGHV repertoire diversity evaluation
Splenocytes genomic DNA was used to amplify IGH VDJ. We performed four independent PCR on 250ng of DNA, using 12 degenerated forward primers previously designed on V leaders of the murine IGH locus (21), and a reverse primer with a 5’ fluorescence marker (FAM) for each of the 4 Js of the murine IGH locus (Supplemental table 5). Phusion DNA polymerase (New England Biolabs) was used according to the manufacturer’s instructions. Amplicons (ranged from 380 to 610bp) were analyzed by capillary electrophoresis (GeneScan 3130xl Genetic Analyzers, Applied Biosystems) and results were exported using GeneMapper® Software 6 (Applied Biosystems). This method allowed to separate IGHV rearrangements depending on their length. Using this approach, several IGHV rearrangements can have the same length and be indistinguishable. For this reason, we refer to length-homogeneous IGHV clonotype. Shannon diversity index or H (formula below) was used to estimate intra-clonal IGHV diversities and was calculated considering the area for each peak for each length-homogeneous IGHV clonotype (ni) and the area of all length-homogeneous IGHV clonotype peaks (N).
H range from 0 (when Sµ-3’RRrec junction diversity shrinks due to clonal dominance) to high values for samples with higher diversity.
Splenic cell cultures
0.5 × 106 isolated B cells from mice spleen (with EasySep™ mouse B cell Isolation Kit #19854, STEMCELL Technologies) were cultured (37°C incubator with 5% of CO2) in a 10-cm tissue culture dish (Nunc™ cell culture/Petri dishes #150318, Thermo Scientific™) previously coated with 3 × 106 40LB cells (17) cultured in complete DMEM-Glutamax medium. 24h before primary culture, 40LB growth has been chemically arrested (at confluence) with 10 µg/mL mitomycin C (#M4287, Sigma-Aldrich) for 4h. Primary culture was done in 20 mL complete RPMI-1640 medium with 1ng/mL recombinant murine IL4 (#214 − 14, ThermoScientific from Preprotech).
Statistical analysis
Graphs, histograms, curves, and standard statistical analyses were designed using GraphPad Prism 6x software. All data are shown as the mean ± SEM. Student t test or Mann-Whitney test was used to compare means. Chi-square (Χ2) was used to compare categorical data when appropriate. The differences were considered significant at ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. All experiments were repeated at least three times with four to five mice in each group.
Disclosure of conflicts of interest:
The authors declare no competing interests.
Fundings:
This work was supported by grants to SP from la Ligue Contre le Cancer (Comité de la Haute-Vienne, CD87, and Comité de la Creuse, CD23) and the Fondation ARC pour la recherche sur le cancer (ARCPJA2022060005138). KG is supported by Région Nouvelle Aquitaine and Délégation INSERM Nouvelle Aquitaine. MP is supported by Région Nouvelle Aquitaine and Université de Limoges. IAJ is supported by Fondation pour la Recherche Medicale (FRM) and Lebanese associations (AZM and Saade, LASeR). SP is a National Institute of Health and Medical Research (INSERM) investigator.
Author Contributions:
KG performed experiments and participated in writing of the original draft. MP, IAJ, CO, OT, MR and MB participated in the experiments. CVF, SA, DR, JC, JF and NG participated in data curation. NF and TF provided mouse models. KG, MP, CO, MR, TF, DR, JC, MR, JF and NG participated in writing the original draft. JF, NG and SP led the conceptualization and data curation. SP led funding acquisition and manuscript writing.
Acknowledgements:
The authors would like to thank Dr. Kitamura for providing the 40LB cell line. We thank the BISCEM facility for sequencing, flow cytometry, cell sorting and animal care.
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