IgH Allelic Exclusion Research Articles

Long-read sequencing unveils IGH-DUX4 translocation into the silenced IGH allele in B-cell acute lymphoblastic leukemia

IGH@ proto-oncogene translocation is a common oncogenic event in lymphoid lineage cancers such as B-ALL, lymphoma and multiple myeloma. Here, to investigate the interplay between IGH@ proto-oncogene translocation and IGH allelic exclusion, we perform long-read whole-genome and transcriptome sequencing along with epigenetic and 3D genome profiling of Nalm6, an IGH-DUX4 positive B-ALL cell line. We detect significant allelic imbalance on the wild-type over the IGH-DUX4 haplotype in expression and epigenetic data, showing IGH-DUX4 translocation occurs on the silenced IGH allele. In vitro, this reduces the oncogenic stress of DUX4 high-level expression. Moreover, patient samples of IGH-DUX4 B-ALL have similar expression profile and IGH breakpoints as Nalm6, suggesting a common mechanism to allow optimal dosage of non-toxic DUX4 expression.

The Ataxia Telangiectasia Mutated and Cyclin D3 Proteins Cooperate To Help Enforce TCRβ and IgH Allelic Exclusion

Coordination of V rearrangements between loci on homologous chromosomes is critical for Ig and TCR allelic exclusion. The Ataxia Telangietasia mutated (ATM) protein kinase promotes DNA repair and activates checkpoints to suppress aberrant Ig and TCR rearrangements. In response to RAG cleavage of Igκ loci, ATM inhibits RAG expression and suppresses further Vκ-to-Jκ rearrangements to enforce Igκ allelic exclusion. Because V recombination between alleles is more strictly regulated for TCRβ and IgH loci, we evaluated the ability of ATM to restrict biallelic expression and V-to-DJ recombination of TCRβ and IgH genes. We detected greater frequencies of lymphocytes with biallelic expression or aberrant V-to-DJ rearrangement of TCRβ or IgH loci in mice lacking ATM. A preassembled DJβ complex that decreases the number of TCRβ rearrangements needed for a productive TCRβ gene further increased frequencies of ATM-deficient cells with biallelic TCRβ expression. IgH and TCRβ proteins drive proliferation of prolymphocytes through cyclin D3 (Ccnd3), which also inhibits VH transcription. We show that inactivation of Ccnd3 leads to increased frequencies of lymphocytes with biallelic expression of IgH or TCRβ genes. We also show that Ccnd3 inactivation cooperates with ATM deficiency to increase the frequencies of cells with biallelic TCRβ or IgH expression while decreasing the frequency of ATM-deficient lymphocytes with aberrant V-to-DJ recombination. Our data demonstrate that core components of the DNA damage response and cell cycle machinery cooperate to help enforce IgH and TCRβ allelic exclusion and indicate that control of V-to-DJ rearrangements between alleles is important to maintain genomic stability.

Clonal Evolution of Malignant Populations In Potentially Biclonal Chronic Lymphocytic Leukemia Patients

AbstractAbstract 2412 Background:A majority of CLL cases represent monoclonal lymphoproliferation. On the other hand, simultaneous biclonal/biphenotypic disease has been sporadically recorded (Gonzales-Campos et al., 1997). Moreover, initially minor, aggressive subclone harboring p53 inactivation in monoclonal CLL can be selected after therapy (Malcikova el al., 2009). Here we present an analysis of patients expressing two or three functional IGH rearrangements. We also closely describe one case of a patient who developed another CLL population after therapy. In this case, the original malignant clone was forced out by the more aggressive CLL population that led to therapy resistance. Aims:We characterized clonal evolution in cases expressing two functional Ig rearrangements (potentially biclonal CLL) and described in detail the alteration of CLL populations in one case. Methods:B lymphocytes were separated from peripheral blood samples using Ficoll-Paque PLUS (GE Healthcare)/RosetteSep Kits (StemCell). IGHV status was assessed according to the ERIC recommendations (cDNA as an input material). A clonal rearrangement of IGH was confirmed by BIOMED-2 protocol, and Ig light chain utilization was assessed. Chromosomal alterations were analyzed by FISH (deletions 13q14, 17p13, 11q22 and trisomy 12) and metaphase cytogenetics with CpG/IL-2 stimulation. Results:From 1065 patients screened, 31 cases were identified as expressing two (28/31) or three (3/31) functional IGH in their disease history. 11 of them (35.5%) were analyzed repeatedly during the course of the disease with median follow-up between the first and the last IGHV examination 19.9 months (range 7.4 – 48.1). According to a number of light chains expressed and the presence of multiple IGH, all patients were divided as having a biclonal CLL (17/31) or a lack of IGH allelic exclusion (14/31). Patients lacking IGH allelic exclusion were omitted from further analysis. Interestingly, in five repeatedly analyzed cases, a mutated CLL clone vanished during a course of the disease and only unmutated clone VH1-69 (4/5) or VH4-34 (1/5) was still present; in one of these cases, therapy accelerated unmut VH1-69 clone selection. In another two patients, unmut CLL clone VH1-69 or VH3-30, respectively, was later selected beside the original one resulting to biclonal CLL. Importantly, we found a patient with an original monoclonal population who developed the second CLL clone after therapy. The original clone was, afterwards, totally overcome by the second one. In this case, Ig analysis of the sample prior to the first therapy showed only unmut VH3-21 rearrangement combined with VL1-51. Two distinct malignant populations were detected by flow-cytometry nine months after the therapy. Ig analysis assessed the original clone and a second unmut VH1-69/VK1-39 clone. IGH and IGL sequences were compared between both populations, and BCR editing was excluded as unlikely. The selection of distinct CLL was supported by cytogenetics which detected the second clone with complex karyotype including loss of chromosome 17 beside the original one with sole deletion in region 5q14.1-q21.3. Because of disease progression, second-line therapy was administered without therapeutic effect. Subsequent analysis of CLL cells revealed a total eradication of the original VH3-21 clone and persistence of the second aggressive clone with p53 inactivation. Conclusion:Frequency of biclonal cases in our cohort is 1.6% (17/1065). Our results show for the first time that aggressive CLL clones, especially VH1-69, can grow up beside and even displace more indolent mutated populations. Therapy can accelerate the clonal selection; this is documented by two cases. We also analyzed in detail one of these patients who developed the second aggressive CLL after therapy. We conclude that subsequent monitoring of CLL patients is necessary for understanding of malignant disease evolution and the role of therapy in the clone selection process. Supported by IGA MZ CR NS10439-3, NS9858-3, MSM0021622430, 204/09/H058 and MPO FR-TI2/254. Disclosures:No relevant conflicts of interest to declare.

Distinct Signaling Requirements for Dμ Selection, IgH Allelic Exclusion, Pre-B Cell Transition, and Tumor Suppression in B Cell Progenitors

The pre-B cell receptor triggers expansion and differentiation of pre-B cells (the pre-B cell transition), as well as inhibition of V H to DJ H recombination (allelic exclusion). The latter also accounts for counter-selection of pro-B cells expressing Dμ protein (Dμ selection). However, the signaling pathways responsible for these events remain poorly defined. Here we show complete arrest of B cell development at the pre-B cell transition in BASH/CD19 double mutant mice, indicating partial redundancy of the two B cell-specific adaptors. Allelic exclusion remained intact in the double mutant mice, whereas Dμ selection was abolished in BASH mutant mice. Thus, distinct signals are required for these events. In addition, both mutant mice succumbed to pre-B cell leukemia, indicating that BASH and CD19 contribute to tumor suppression.

The pre-B cell receptor and its role in proliferation and Ig heavy chain allelic exclusion

The pre-B cell receptor (pre-BCR) is composed of the immunoglobulin (Ig) heavy ( μH) chain and the surrogate light chain encoded by VpreB and λ5. The pre-BCR has been implicated in precursor B cell proliferation, differentiation and IgH chain allelic exclusion. B cell development in mice lacking the transmembrane form of μH chain is blocked at the precursor B cell stage: the cells cannot proliferate or differentiate further and the IgH locus is allelically included. In mice lacking λ5, the precursor B cells, although unable to proliferate, can nonetheless differentiate, whereas the IgH locus is allelically excluded. It was, therefore, postulated that μH chain together with VpreB could form a pre-BCR-like receptor that would allow IgH allelic exclusion but not proliferation. In mice lacking both VpreB genes, precursor B cells do not proliferate but are able to differentiate. Surprisingly, the IgH locus is allelically excluded. This suggests that μH chains find other partner proteins to signal allelic exclusion.

VpreB1/VpreB2/λ5 Triple-Deficient Mice Show Impaired B Cell Development but Functional Allelic Exclusion of the IgH Locus

At the precursor B cell stage during bone marrow B cell development, Ig muH chain associates with surrogate L (SL) chain, which is encoded by the three genes VpreB1, VpreB2, and lambda 5, to form the pre-B cell receptor (pre-BCR). Surface expression of the pre-BCR is believed to signal both proliferation and allelic exclusion of the IgH locus. Mice which lack either VpreB1/VpreB2 or lambda 5 show a lack of precursor B cell expansion but normal IgH allelic exclusion. This would suggest that one of either lambda 5 or VpreB can make a pre-BCR-like complex which is still able to signal allelic exclusion but not proliferation. To investigate this, we established mice lacking all components of the SL chain. These mice showed severely impaired B cell development which was similar to that previously found in mice lacking either lambda 5 or VpreB1/VpreB2. Surprisingly, the IgH locus was still allelically excluded and thus the SL chain appears not to be involved in allelic exclusion.

Mice triallelic for the Ig heavy chain locus: implications for VHDJH recombination.

V(H)DJ(H) recombination has been extensively studied in mice carrying an Ig heavy chain rearranged transgene. In most models, inhibition of endogenous Ig rearrangement occurs, consistently with the feedback model of IgH recombination. Nonetheless, an incomplete IgH allelic exclusion is a recurrent observation in these animals. Furthermore, transgene expression in ontogeny is likely to start before somatic recombination, thus limiting the use of Ig-transgenic mice to access the dynamics of V(H)DJ(H) recombination. As an alternative approach, we challenged the regulation of somatic recombination with the introduction of an extra IgH locus in germline configuration. This was achieved by reconstitution of RAG2(-/-) mice with fetal liver cells trisomic for chromosome 12 (Ts12). We found that all three alleles can recombine and that the ratio of Ig allotype-expressing B cells follows the allotypic ratio in trisomic cells. Although these cells are able to rearrange the three alleles, the levels of Ig phenotypic allelic exclusion are not altered when compared with euploid cells. Likewise, we find that most VDJ rearrangements of the silenced allele are unable to encode a functional mu-chain, indicating that the majority of these cells are also genetically excluded. These results provide additional support for the feedback model of allelic exclusion.

Review article: role of the surrogate light chain and the pre-B-cell receptor in mouse B-cell development.

The generation of B lymphocytes from committed progenitor cells is a complex process involving the transit of cells through several critical stages of development. Throughout their transit, developing B cells are subject to choices between survival, proliferation or death; fates that are dictated by combinations of intrinsic and extrinsic signals. Thus, failure to express certain signalling molecules on their surface at particular stages of development results in the cell failing to receive a viability and/or proliferative signal. Likewise, failure to link surface receptor expression to survival machinery will result in cell death. During the early stages of B-cell development, one such receptor molecule is the pre-B-cell receptor (pre-BCR). The pre-BCR is a heterodimer composed of an immunoglobulin (Ig) heavy chain molecule (IgH) covalently associated with an immunoglobulin light chain-like molecule called the surrogate light chain (SL).1–3 The SL itself is made up of two non-covalently associated proteins called lambda-5 (λ5) and VpreB, which together form a molecule having structural homology with conventional light (IgL) chains.4,5 Thus, in the SL, λ5 replaces a light chain constant region and VpreB the variable part. It is likely that the membrane pre-BCR complex is composed of two IgH molecules associated with two SLs.1 However, SL can reach the surface in the absence of IgH but in association with other molecules, for example with a glycoprotein of approximately 130 000 MW, so-called gp130.6,7 In this case, the surface SL-containing complex is referred to as a pro-BCR.8 The detailed description of SL structure must await crystallographic analysis, but recently Melchers has proposed a model of SL chain assembly.9 In this review we will discuss the molecular structure and gene organization of pre-BCR components and attempt to outline how surface expression of the pre-BCR impacts on cell survival, differentiation, proliferation and repertoire selection of developing B cells. General scheme of B lymphocyte development Different stages of B-cell development can be defined by the use of a combination of cell-surface markers and the status of immunglobulin gene rearrangements10,11 (Fig. 1, B-lymphocyte development in adult bone marrow). Following commitment to the B-cell lineage, an event which is still poorly understood, the first stage of the B-cell lineage is called a pro-B cell and is defined phenotypically as a B220+CD19− c-kit+CD43+CD25−IgM−IgD− cell in which there does not have to be any immunoglobulin gene rearrangement.12,13 It should be noted though that some pro-B cells have DJH rearrangements but only on one allele.13 Thus pro-B cells are found in mice in which the recombinase-activating genes (RAG) have been deleted,14,15 and in such mice, they are CD19+. However, pro-B cells do contain RNA transcripts for components of the SL but it is unclear at the present time whether they contain SL protein either in their cytoplasm or at the cell surface.13,16 It is possible that SL might reach the cell surface in association with molecules other than IgH, for example gp130.6,7 Open in a separate window Figure 1 B-lymphocyte development in the adult mouse bone marrow.

Frequency and characterization of phenotypic Ig heavy chain allelically included IgM-expressing B cells in mice.

Ig H chain (IgH) allelic exclusion remains a puzzling topic. Here, we address the following question: Do phenotypic IgH allelically included cells exist in normal mice and, if so, at what frequency? Sorted cells from heterozygous mice were evaluated for the expression of both IgM allotypes by double intracytoplasmic stainings. Dual expressors were found at a frequency of 1 in 104 splenic B cells. These data were confirmed by direct sequencing of IgH-rearranged alleles obtained after single cell (or clone) PCR on dual expressors. Typically, these cells have one rearranged J558 VH whereas, in the other allele, a D-proximal VH gene is used. Interestingly, dual expressors have rearranged IgH alleles with similar CDR3 lengths. These results show that, in contrast to the kappa L chain and the TCR beta-chain, IgH allelic exclusion is the result of an extremely stringent mechanism. We discuss two non-mutually exclusive scenarios for the origin of IgH dual expressors: 1) IgH allelically included cells arise when the first allele to rearrange productively is unable to form a pre-BCR; dual expressors could be a subset of this population in which, upon conventional L chain rearrangement, both IgH are expressed at the surface; and 2) synchronous rearrangement of the IgH alleles.