Nonproductive Rearrangements Research Articles

78. Identification of a non-productive KMT2A rearrangement in B-ALL with apparent concurrent ETV6::RUNX1 and KMT2A fusions

78. Identification of a non-productive KMT2A rearrangement in B-ALL with apparent concurrent ETV6::RUNX1 and KMT2A fusions

Potential Unreliability of Uncommon ALK, ROS1, and RET Genomic Breakpoints in Predicting the Efficacy of Targeted Therapy in NSCLC.

Variable genomic breakpoints have been identified through the application of target-capture DNA next-generation sequencing (NGS) for ALK, ROS1, and RET fusion detection in NSCLC. We investigated whether ALK, ROS1, and RET genomic breakpoint location can predict matched targeted therapy efficacy. NSCLCs were analyzed by DNA NGS, target-specific RNA NGS, whole-transcriptome sequencing, and immunohistochemistry. In total, 3787 NSCLC samples were analyzed. DNA NGS detected ALK, ROS1, and RET fusions in 241, 59, and 76 cases, respectively. These fusions were divided into canonical (single EML4-ALK, CD74/EZR/TPM3/SDC4-ROS1, and KIF5B/CCDC6-RET fusions), noncanonical (single non-EML4-ALK, non-CD74/EZR/TPM3/SDC4-ROS1, and non-KIF5B/CCDC6-RET fusions), and primary/reciprocal (both primary and reciprocal rearrangements were detected) subtypes on the basis of genomic breakpoint position, and noncanonical and primary/reciprocal subtypes were defined as uncommon fusions. Further RNA sequencing and immunohistochemistry revealed that six of 47 (12.8%) uncommon fusions were actually nonproductive rearrangements that generated no aberrant transcripts or proteins. Moreover, genomic breakpoints of canonical ALK and RET, but not ROS1, fusions always predicted breakpoints at the transcript level, whereas 85.4% (35 of 41) of uncommon fusions actually produced canonical fusion transcripts. Patients with uncommon ALK fusion (n= 31) who received first-line crizotinib exhibited shorter median progression-free survival than those with canonical ALK fusion (n= 53, 8.4 mo versus 12.0 mo, p= 0.004). However, no difference in progression-free survival was observed when only ALK RNA or protein-positive cases were analyzed (p= 0.185). Uncommon ALK, ROS1, and RET genomic breakpoint is an unreliable predictor of matched targeted therapy efficacy. Functional validation by RNA or protein assay may add value for the accurate detection and interpretation of rare fusions.

Characteristics of Plasmablast Repertoire in Chronically HIV-Infected Individuals for Immunoglobulin H and L Chain Profiled by Single-Cell Analysis.

Characterization of the diversified immunoglobulin (Ig) repertoire may provide insight into pathways that shape an efficient antibody (Ab) repertoire for immune response against human immunodeficiency virus (HIV) infection. This study aimed to profile characteristics of the plasmablast repertoire during chronic HIV infection. Ig variable regions of plasmablasts from both chronically HIV-infected donors (HIVDs) previously treated with antiretroviral therapy (ART) and healthy donors (HDs) were amplified by single-cell PCR to establish the basis for further repertoire analysis. We compared the plasmablast repertoires expressed in multiple chronically HIVDs after ART treatment cessation and HDs. We also examined the non-productive repertoire to identify the indication of the immediate products of the rearrangement machinery without an impact of selection during HIV infection. We found multiple differences between the productive repertoires of HIVD and HD subjects, including biased usages of VH3-49, VH1-2, VH3-33, VH3-74, and VH5-51 in VH and D1-7, D1-14, D1-20, and D5-5/18 in D segments in the HIVD group, as well as shorter and preferential glycine usages in CDRH3 regions. Gene selections were also detected in light chains. Notably, differences between productive rearrangements of HIVDs and HDs outnumbered those between productive and non-productive rearrangements within HIVDs. HIV infection may exert a dominant impact on the development of the plasmablast repertoire. The impact of selection is of limited significance in shaping the plasmablast repertoire. Overall, the data indicate that the environment in which the plasmablasts live can affect the distribution of the VH and VL genes in the repertoire and the amino acid compositions of the expressed Abs.

Next-generation amplicon TRB locus sequencing can overcome limitations of flow-cytometric Vβ expression analysis and confirms clonality in all T-cell prolymphocytic leukemia cases.

T-cell receptor (TCR) β repertoire analysis can distinguish monoclonal from polyclonal T-cell proliferations and crucially aid in the diagnosis of T-cell malignancies. TCR repertoire can be assessed either by flow cytometry (FCM), or by molecular genetic techniques. We compared the results of parallel analyses of Vβ expression by FCM and TRB rearrangements by DNA-based next-generation sequencing (NGS) in 80 diagnostic peripheral blood samples of patients with T-cell prolymphocytic leukemia (T-PLL) for (1) the diagnosis of clonality and (2) the assessment of dominant Vβ usage. FCM-based analysis of the surface expression was performed using the IOTest Beta Mark kit. The NGS-based analysis employed the multiplex Biomed-2 VB-JB primers. In all the samples, one or two clonal TRB rearrangements were detected by NGS. Although a dominant Vβ domain usage was detected by FCM in only 41/80 (51%) samples, clonality was suspected in all of them. In a total of 12 cases, the FCM missed the clone detected by NGS, despite theoretical coverage by the antibodies, the functionality of the rearrangement, and the expression of TCRαβ on the cell surface. Partly overlapping with those cases, FCM discovered predominant Vβ usage in the T-PLL population that differed from the one detected by NGS in 10 cases. Overall, the concordant NGS and FCM results were obtained on 61/80 (76%) of samples. We conclude that NGS-based TRB analysis can overcome certain limitations of FCM-based analysis by the identification of both productive and nonproductive rearrangements and by covering the whole Vβ spectrum. Currently available FCM analysis of Vβ expression lacks this breadth but has advantages, such as parallel immunophenotyping and a more accurate quantification of the Vβ usage. © 2018 International Society for Advancement of Cytometry.

Evidence for Ig Light Chain Isotype Exclusion in Shark B Lymphocytes Suggests Ordered Mechanisms.

Unlike most vertebrates, the shark IgL gene organization precludes secondary rearrangements that delete self-reactive VJ rearranged genes. Nurse sharks express four L chain isotypes, κ, λ, σ, and σ-2, encoded by 35 functional minigenes or clusters. The sequence of gene activation/expression and receptor editing of these isotypes have not been studied. We therefore investigated the extent of isotypic exclusion in separated B cell subpopulations. Surface Ig (sIg)κ-expressing cells, isolated with mAb LK14 that recognizes Cκ, carry predominantly nonproductive rearrangements of other L chain isotypes. Conversely, after depletion with LK14, sIgM+ cells contained largely nonproductive κ and enrichment for in-frame VJ of the others. Because some isotypic inclusion was observed at the mRNA level, expression in the BCR was examined. Functional λ mRNA was obtained, as expected, from the LK14-depleted population, but was also in sIgκ+ splenocytes. Whereas λ somatic mutants from the depleted sample displayed evidence of positive selection, the λ genes in sIgκ+ cells accumulated bystander mutations indicating a failure to express their products at the cell surface in association with the BCR H chain. In conclusion, a shark B cell expresses one L chain isotype at the surface and other isotypes as nonproductive VJ, sterile transcripts, or in-frame VJ whose products may not associate with the H chain. Based on the mRNA content found in the B cell subpopulations, an order of L chain gene activation is suggested as: σ-2 followed by κ, then σ and λ.

Mechanisms That Shape Human Antibody Repertoire Development in Mice Transgenic for Human Ig H and L Chain Loci.

To determine the impact of the milieu on the development of the human B cell repertoire, we carried out a comprehensive analysis of productive and nonproductive Ig gene rearrangements from transgenic mice engineered to express single copies of the unrearranged human H chain and L chain Ig gene loci. By examining the nonproductive repertoire as an indication of the immediate product of the rearrangement machinery without an impact of selection, we discovered that the distribution of human rearrangements arising in the mouse was generally comparable to that seen in humans. However, differences between the distribution of nonproductive and productive rearrangements that reflect the impact of selection suggested species-specific selection played a role in shaping the respective repertoires. Although expression of some VH genes was similar in mouse and human (IGHV3-23, IGHV3-30, and IGHV4-59), other genes behaved differently (IGHV3-33, IGHV3-48, IGHV4-31, IGHV4-34, and IGHV1-18). Gene selection differences were also noted in L chains. Notably, nonproductive human VH rearrangements in the transgenic mice expressed shorter CDRH3 with less N addition. Even the CDRH3s in the productive rearrangements were shorter in length than those of the normal human productive repertoire. Amino acids in the CDRH3s in both species showed positive selection of tyrosines and glycines, and negative selection of leucines. The data indicate that the environment in which B cells develop can affect the expressed Ig repertoire by exerting influences on the distribution of expressed VH and VL genes and by influencing the amino acid composition of the Ag binding site.

DJ Pairing during VDJ Recombination Shows Positional Biases That Vary among Individuals with Differing IGHD Locus Immunogenotypes.

Human IgH diversity is influenced by biases in the pairing of IGHD and IGHJ genes, but these biases have not been described in detail. We used high-throughput sequencing of VDJ rearrangements to explore DJ pairing biases in 29 individuals. It was possible to infer three contrasting IGHD-IGHJ haplotypes in nine of these individuals, and two of these haplotypes include deletion polymorphisms involving multiple contiguous IGHD genes. Therefore, we were able to explore how the underlying genetic makeup of the H chain locus influences the formation of particular DJ pairs. Analysis of nonproductive rearrangements demonstrates that 3' IGHD genes tend to pair preferentially with 5' IGHJ genes, whereas 5' IGHD genes pair preferentially with 3' IGHJ genes; the relationship between IGHD gene pairing frequencies and IGHD gene position is a near linear one for each IGHJ gene. However, striking differences are seen in individuals who carry deletion polymorphisms in the D locus. The absence of different blocks of IGHD genes leads to increases in the utilization frequencies of just a handful of genes, and these genes have no clear positional relationships to the deleted genes. This suggests that pairing frequencies may be influenced by additional complex positional relationships that perhaps arise from chromatin structure. In contrast to IGHD gene usage, IGHJ gene usage is unaffected by the IGHD gene-deletion polymorphisms. Such an outcome would be expected if the recombinase complex associates with an IGHJ gene before associating with an IGHD gene partner.

Biased Immunoglobulin Light Chain Gene Usage in the Shark.

This study of a large family of κ L chain clusters in nurse shark completes the characterization of its classical Ig gene content (two H chain isotypes, μ and ω, and four L chain isotypes, κ, λ, σ, and σ-2). The shark κ clusters are minigenes consisting of a simple VL-JL-CL array, where V to J recombination occurs over an ~500-bp interval, and functional clusters are widely separated by at least 100 kb. Six out of ~39 κ clusters are prerearranged in the germline (germline joined). Unlike the complex gene organization and multistep assembly process of Ig in mammals, each shark Ig rearrangement, somatic or in the germline, appears to be an independent event localized to the minigene. This study examined the expression of functional, nonproductive, and sterile transcripts of the κ clusters compared with the other three L chain isotypes. κ cluster usage was investigated in young sharks, and a skewed pattern of split gene expression was observed, one similar in functional and nonproductive rearrangements. These results show that the individual activation of the spatially distant κ clusters is nonrandom. Although both split and germline-joined κ genes are expressed, the latter are prominent in young animals and wane with age. We speculate that, in the shark, the differential activation of the multiple isotypes can be advantageously used in receptor editing.

Immunoglobulin kappa variable region gene selection during early human B cell development in health and systemic lupus erythematosus

The unique specificity of the B cell receptor is generated by an ordered sequence of gene rearrangement events. Once IGH genes have rearranged, rearrangement at the IGK locus is initiated followed by the IGL locus if functional IGK rearrangement is not achieved. Receptor specificity can subsequently be altered by secondary light chain editing based on the features of the heavy and light chain combination. The final profile of expressed genes is not random and biases in this profile are associated with several autoimmune diseases. However, how and when biases are created is not known.To increase our understanding of the processes of selection and editing of IGK rearrangements, we compared four groups of rearrangements of IGK acquired by next generation sequencing. First, expressed rearrangements of IGK from cDNA of IGK expressing B cells. Second, productive rearrangements of IGK from DNA of the same kappa expressing B cells. Third, non-productive rearrangements of IGK from DNA of IGK and IGL expressing B cells, and fourth productively rearranged IGK from DNA of IGL expressing B cells. The latter group would have been rejected during B cell development in favour of rearrangement at the IGL locus and are therefore selected against.We saw evidence that rearranged IGK segments can be selected at a checkpoint where the decision to rearrange the IGL locus is made. In addition, our data suggest that mechanisms regulating the expression or not of IGK rearrangements may also contribute to repertoire development and also that this latter component of the selection process is defective in SLE.

Sterile DJH rearrangements reveal that distance between gene segments on the human Ig H chain locus influences their ability to rearrange.

Rearrangement of the Ig locus occurs in two steps. First, a JH gene is rearranged to a D gene followed by a VH gene rearranging to the DJH rearrangement. By next generation sequencing, we analyzed 9969 unique DJH rearrangements and 5919 unique VHDJH rearrangements obtained from peripheral blood B cells from 110 healthy adult donors. We found that DJH rearrangements and nonproductive VHDJH rearrangements share many features but differ significantly in their use of D genes and propensity for somatic hypermutation. In D to JH gene rearrangements, the D genes proximal to the JH locus are used more frequently than JH locus distal D genes, whereas VH locus proximal D genes were observed more frequently in nonproductive VHDJH rearrangements. We further demonstrate that the distance between VH, D, and JH gene segments influence their ability to rearrange within the human Ig locus.

Evolution to Plasmablastic Lymphoma (PBL) after CAR-T Cell Therapy in a Case of SLL/CLL with Richter’s Transformation

Chimeric antigen receptor (CAR)-modified T cells specific for CD19 have resulted in complete responses in B lymphoblastic leukemia (B-ALL) and in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). In B-ALL cases, clinical relapse with CD19-negative cells has been reported (Grupp et al NEJM 368; 1509-18). Here we report relapse in a CLL case treated with CAR-T cells specific for CD19 (CTL019) which demonstrated clonal evolution into plasmablastic lymphoma (PBL) lacking residual markers of B lymphoid differentiation.The patient was a 62 year old man diagnosed with CLL 4.5 years prior to the CTL019 therapy. At diagnosis his absolute lymphocyte count was 253K/uL, with 98% of lymphocytes being positive for CD19, CD20, CD5(dim), CD23, and lambda-restricted, but negative for CD10, CD38, and Zap-70. CT scans revealed splenomegaly (16 cm) and extensive lymphadenopathy (up to 6.9 cm). Standard CLL FISH panel was negative. Brief therapeutic responses were achieved but progression occurred on each of the following regimens: fludarabine, cyclophosphamide and rituximab for 6 cycles, bendamustine/rituximab for 2 cycles, ofatumumab for 5 infusions, and CHOP for 3 cycles. He was then enrolled on a series of clinical trials including flavopiridol and PCI 32765 (ibrutinib). Six months prior to CTL019 infusion, further node progression was demonstrated with trisomy 12, del 12p14, del 11q, and del 17p by FISH. Node and marrow biopsies were consistent with large B cell/Richter’s transformation. He received OFAR X 1 and was referred for the CTL019 CLL trial at the University of Pennsylvania where he had successful apheresis and expansion of T cells. Nodes were debulked with the XP01 inhibitor (KPT-330). He received radiation to a 9 cm right axillary mass without response. He received one cycle of fludarabine and cyclophosphamide followed by CTL019 cell infusion. There was marked expansion of CTL019 cells and he experienced cytokine release syndrome. He had a significant tumor response with 50% or more reduction in most nodal areas on CT scan and 80% reduction in tumor involvement of the marrow 1 month after infusion. He was lymphopenic and received monthly IVIG replacement.Approximately 6 months after the CTL019 infusion, he developed right jaw pain. A gingival biopsy revealed plasmablastic lymphoma (PBL). The cells were lambda-restricted by in-situ hybridization, and positive for CD45, CD138, MUM-1, CD56 (subset), and EMA, but negative for CD19, CD20, Pax-5, CD5, CD23, BCL-2, cyclin D1, CD30, ALK-1, HHV-8, and EBV (by EBER in situ hybridization). The Ki67 expression was >90%. At the time WBC was 3.7K/uL, HCT 44%, and platelets 78K/uL with ANC 2.2K/uL. About one month after relapse, neutropenia occurred, and atypical cells emerged which typed by flow cytometry as positive for CD45(dim), CD5, CD23, CD81, lambda-restricted, and negative for CD19. The patient developed sepsis with pancytopenia and died 7 months after his CTL019 cell infusion.Molecular analysis revealed the same size monoclonal gene rearrangements of the immunoglobulin heavy chain (IGH) and kappa light chain (IGK) genes in both the PBL, subsequent peripheral blood, and a prior SLL (2010) lymph node biopsy. Sequencing revealed identical productive and nonproductive IGH gene rearrangements in all three specimens, with the exception of a 2bp insertion in the CDR1 region which destroyed the IGH reading frame in the PBL. P53 sequencing revealed multiple mutations [p.Gly245Ser(heterozygous) and p.Val197Glu(heterozygous)] from the prior SLL lymph node and recent peripheral blood, but only a single mutation [p.Gly245Ser(homozygous)] present in the PBL, consistent with previously documented loss of 17p prior to CTL019 therapy.CAR-T cell therapy is an effective treatment modality for CLL and ALL. In this case, a plasmablastic lymphoma lacking well-differentiated B cell features, including CD19, emerged 6 months after CTL019 therapy, along with residual circulating CD19-negative/CD5-positive lambda-restricted B cells. This finding suggests that anti-CD19 selective pressure may divert lymphoid differentiation into other pathways or may foster proliferation of an alternatively differentiated stage of the original clonal tumor. This case demonstrates the ability of CD19-negative tumors to emerge after CD19 directed therapy in CLL, which also attests to the efficiency and continued activity of these directed T cells over time. DisclosuresPorter:Novartis: Patents & Royalties, Research Funding. Liesveld:Glycomimetics: Honoraria, Membership on an entity’s Board of Directors or advisory committees; GSK: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Detection of the Malignant B Cell Clone in Multiple Myeloma Via High Throughput Sequencing Is Robust to Significant Levels of Somatic Hypermutation

Background: High-throughput sequencing (HTS) of antibody gene rearrangements is an emerging tool for minimal residual disease (MRD) monitoring in B cell malignancies in which the malignant clone harbors a monoclonal Ig heavy chain (IgH) and/or light chain (κ or λ) rearrangement. This approach has shown promise in B-ALL and CLL, but experience with this technique applied to samples from multiple myeloma patients is limited.Approach: We conducted HTS of PCR-amplified IgH (VDJ and DJ) rearrangements from bone marrow aspirates of 21 patients with various plasma cell dyscrasias (MM, MGUS, LPL) and peripheral blood of a patient with plasma cell leukemia. In 17/21 samples, an aliquot was enriched for CD138+ cells by immunomagnetic separation and analyzed separately. Dominant clones from enriched and un-enriched aliquots were compared to verify the malignant clonotype sequence(s). Disease burden in un-enriched samples was also evaluated by microscopy of the bone marrow aspirate smear and ranged from 0 (hemodilute) to 37% plasma cells.Results: In 19 out of 21 samples, a clearly dominant IgH gene rearrangement (>2.5% of total sequences, range 2.9-99.9%) was identified with clear separation from background frequency (at least 2.7-fold higher frequency than next most common clone). In 17/17 cases with paired CD138-enriched samples, the dominant sequences in the enriched and un-enriched samples were identical, indicating successful identification of the malignant clonal Ig rearrangements in the un-enriched sample. More than one IgH rearrangement suitable for longitudinally tracking the malignant clone was identified in 8 of 21 cases. The two cases without an expected, productive IgH rearrangement were IgG-κ and IgG-λ. This suggested that somatic hypermutation (SHM) in the primer binding sites might interfere with some clonal amplifications, so we investigated the degree of SHM in the VH segment of the 19 cases with at least one detected dominant Ig rearrangement. A total of 18 productive VDJ rearrangements were identified, and had SHM frequencies ranging from 2% to 19% in the sequenced portions of the rearranged VH gene. 8 myeloma clones harbored an identifiable DJ rearrangement, none of which showed evidence of SHM. Finally, 3 myeloma clones harbored nonproductive VDJ rearrangements, two with no SHM, and one with 2 SHM in 84 bp of the sequenced VH gene.Conclusion: HTS of Ig heavy and light chain rearrangements can successfully identify the malignant plasma cell clone in clinical specimens, including those with low disease burden and significant SHM. Application of this technique to MRD evaluation in multiple myeloma warrants further development. DisclosuresCarlson:Adaptive Biotechnologies: Consultancy, Equity Ownership. Vogl:Celgene Corporation: Consultancy; Amgen: Consultancy; Millennium/Takeda: Research Funding; GSK: Research Funding; Acetylon: Research Funding. Stadtmauer:Janssen: Consultancy.

Annotation of pseudogenous gene segments by high-throughput sequencing of rearranged lymphocyte receptor loci (TECH1P.844)

Abstract The adaptive immune system generates a huge breadth of antigen-specific T (TCRs) and B cell receptors (BCRs) by combinatoric shuffling of gene segments. Lymphocytes undergo somatic recombination of variable (V), diversity (D), and joining (J) region gene segments, with deletions and non-templated insertions at the junctions, collectively encoding the CDR3 region. Many of the gene segments are annotated as non-functional; however, full annotation of genes as functional, pseudogene or ORF has proven elusive. We used high-throughput sequencing to identify 100K’s of rearranged TCRB and IGH receptors from a large cohort of healthy adults. We then annotated V and J gene segments as functional or pseudogenes based on selection. Random chance predicts that less than one-third of somatic rearrangements will lead to in-frame transcripts, but only lymphocytes with a productive receptor are selected. Non-functional rearrangements (out-of-frame or pseudogene) persist only as a second allele in mature T and B cells. Based on these data we annotated the functional status of each gene segment based on the realized ratio of productive to non-productive rearrangements. Some gene segments are currently mis-annotated: in TCRB 2 presumed-functional gene segments are pseudogenes, in IgH 3 presumed-functional genes are pseudogenes and 1 annotated pseudogene is functional. Additionally, in both loci we identified cases of gene segment functional/pseudogene status segregating among healthy subjects.

Development of the expressed immunoglobulin &Mgr; chain repertoire during maturation of mice B cells

In the bone marrow and spleen, the developing B cell populations undergo both negative and positive selections to shape their B cell receptor repertoire. To gain insight into the shift of the immunoglobulin heavy (IgH) chain repertoire during B cell development, we undertook large scale Ig μ chain repertoire analysis of pre-B, immature B and spleen B cell populations. We found that the majority of VH gene segments, VH families, JH and D gene segments, were observed to have significantly different usage frequencies when three B cell populations were compared, but the usage profile of the VH, D, and JH genes between different B cell populations showed high correlations. In both productive and nonproductive rearrangements, the length of CDRH3 shortened signifi- cantly on average when B cells entered the periphery. However, the CDRH3 length distribution of nonproductive rearrangements did not follow a Gaussian distribution, but decreased successively in the order 3n-2, 3n-1 and 3n, suggesting a direct correlation between mRNA stability and CDRH3 length patterns of nonproductive rearrange- ments. Further analysis of the individual components comprising CDRH3 of productive rearrangements indi- cated that the decrease in CDRH3 length was largely due to the reduction of N addition at the 5' and 3' junctions. Moreover, with development, the amino acid content of CDRH3 progressed toward fewer positively charged and nonpolar residues but more polar residues. All these data indicated that the expressed Ig μ chain repertoire, especially the repertoire of CDRH3, was fine-tuned when B cells passed through several checkpoints of selection during the process of maturation.

The Restricted DH Gene Reading Frame Usage in the Expressed Human Antibody Repertoire Is Selected Based upon its Amino Acid Content

The Ab repertoire is not uniform. Some variable, diversity, and joining genes are used more frequently than others. Nonuniform usage can result from the rearrangement process, or from selection. To study how the Ab repertoire is selected, we analyzed one part of diversity generation that cannot be driven by the rearrangement mechanism: the reading frame usage of DH genes. We have used two high-throughput sequencing methodologies, multiple subjects and advanced algorithms to measure the DH reading frame usage in the human Ab repertoire. In most DH genes, a single reading frame is used predominantly, and inverted reading frames are practically never observed. The choice of a single DH reading frame is not limited to a single position of the DH gene. Rather, each DH gene participates in rearrangements of differing CDR3 lengths, restricted to multiples of three. In nonproductive rearrangements, there is practically no reading frame bias, but there is still a striking absence of inversions. Biases in DH reading frame usage are more pronounced, but also exhibit greater interindividual variation, in IgG(+) and IgA(+) than in IgM(+) B cells. These results suggest that there are two developmental checkpoints of DH reading frame selection. The first occurs during VDJ recombination, when inverted DH genes are usually avoided. The second checkpoint occurs after rearrangement, once the BCR is expressed. The second checkpoint implies that DH reading frames are subjected to differential selection. Following these checkpoints, clonal selection induces a host-specific DH reading frame usage bias.

Perturbation of thymocyte development in nonsense-mediated decay (NMD)-deficient mice

The random nature of T-cell receptor-β (TCR-β) recombination needed to generate immunological diversity dictates that two-thirds of alleles will be out-of-frame. Transcripts derived from nonproductive rearrangements are cleared by the nonsense-mediated mRNA decay (NMD) pathway, the process by which cells selectively degrade transcripts harboring premature termination codons. Here, we demonstrate that the fetal thymus in transgenic mice that ubiquitously express a dominant-negative form of Rent1/hUpf1, an essential trans-effector of NMD, shows decreased cell number, reduced CD4CD8 double-positive thymocytes, diminished expression of TCR-β, and increased expression of CD25, suggesting a defect in pre-TCR signaling. Transgenic fetal thymocytes also demonstrated diminished endogenous Vβ-to-DβJβ rearrangements, whereas Dβ-to-Jβ rearrangements were unperturbed, suggesting that inhibition of NMD induces premature shut-off of TCR-β rearrangement. Developmental arrest of thymocytes is prevented by the introduction of a fully rearranged TCR-β transgene that precludes generation of out-of-frame transcripts, suggesting direct mRNA-mediated trans-dominant effects. These data document that NMD has been functionally incorporated into developmental programs during eukaryotic evolution.

Pro-B cells sense productive immunoglobulin heavy chain rearrangement irrespective of polypeptide production

B-lymphocyte development is dictated by the protein products of functionally rearranged Ig heavy (H) and light (L) chain genes. Ig rearrangement begins in pro-B cells at the IgH locus. If pro-B cells generate a productive allele, they assemble a pre-B cell receptor complex, which signals their differentiation into pre-B cells and their clonal expansion. Pre-B cell receptor signals are also thought to contribute to allelic exclusion by preventing further IgH rearrangements. Here we show in two independent mouse models that the accumulation of a stabilized μH mRNA that does not encode μH chain protein specifically impairs pro-B cell differentiation and reduces the frequency of rearranged IgH genes in a dose-dependent manner. Because noncoding IgH mRNA is usually rapidly degraded by the nonsense-mediated mRNA decay machinery, we propose that the difference in mRNA stability allows pro-B cells to distinguish between productive and nonproductive Ig gene rearrangements and that μH mRNA may thus contribute to efficient H chain allelic exclusion.

Multiple RNA Surveillance Mechanisms Cooperate to Reduce the Amount of Nonfunctional Igκ Transcripts

Random V(D)J junctions ensure that the diversity of the Ig primary repertoire is adapted to the vast heterogeneity of Ags. In two-thirds of cases, recombination between variable segments induces a frameshift in the open reading frame and generates a premature termination codon. In B cells harboring biallelic V(D)J rearrangement of Ig genes, transcription is known to occur on both the functional and nonfunctional alleles, generating considerable amounts of primary transcripts with out-of-frame V regions. In this study, we analyzed in cell lines and primary B cells the RNA surveillance of nonfunctional Igkappa transcripts arising from nonproductive rearrangement. We demonstrated that splicing inhibition, nonsense-mediated decay and nonsense-altered splicing each have an individual partial effect that together associate into an efficient surveillance machinery, downregulating nonfunctional Igkappa mRNA. Moreover, we provide evidence that the RNA surveillance efficiency increases throughout B cell development. Whereas splicing inhibition remains constant in most cell lines, differences in nonsense-mediated decay and nonsense-altered splicing are responsible for the higher RNA surveillance observed in plasma cells. Altogether, these data show that nonfunctionally rearranged alleles are subjected to active transcription but that multiple RNA surveillance mechanisms eradicate up to 90% of out-of-frame Igkappa mRNA.

Analysis of somatic hypermutation in X-linked hyper-IgM syndrome shows specific deficiencies in mutational targeting

AbstractSubjects with X-linked hyper-IgM syndrome (X-HIgM) have a markedly reduced frequency of CD27+ memory B cells, and their Ig genes have a low level of somatic hypermutation (SHM). To analyze the nature of SHM in X-HIgM, we sequenced 209 nonproductive and 926 productive Ig heavy chain genes. In nonproductive rearrangements that were not subjected to selection, as well as productive rearrangements, most of the mutations were within targeted RGYW, WRCY, WA, or TW motifs (R = purine, Y = pyrimidine, and W = A or T). However, there was significantly decreased targeting of the hypermutable G in RGYW motifs. Moreover, the ratio of transitions to transversions was markedly increased compared with normal. Microarray analysis documented that specific genes involved in SHM, including activation-induced cytidine deaminase (AICDA) and uracil-DNA glycosylase (UNG2), were up-regulated in normal germinal center (GC) B cells, but not induced by CD40 ligation. Similar results were obtained from light chain rearrangements. These results indicate that in the absence of CD40-CD154 interactions, there is a marked reduction in SHM and, specifically, mutations of AICDA-targeted G residues in RGYW motifs along with a decrease in transversions normally related to UNG2 activity.

Lack of Nonfunctional B-cell Receptor Rearrangements in a Patient with Normal B Cell Numbers Despite Partial RAG1 Deficiency and Atypical SCID/Omenn Syndrome

A 2.5-month old boy presented with recurrent wheezing, protracted diarrhea, erythrodermia, and failure to thrive. Laboratory analysis showed lymphocytopenia with severely reduced T-cell numbers but normal numbers of B and NK cells. Serum IgE was increased and the patient had eosinophilia. These presentations are consistent with atypical severe combined immunodeficiency (SCID)/Omenn Syndrome and the diagnosis was confirmed by demonstration of homozygosity for the R841W mutation in the catalytic core of RAG1. Comparison of the patient's immunoglobulin heavy chain rearrangements to those of age-matched controls, cord blood, and adults revealed an almost total lack of nonproductive rearrangements (2.7% versus 14.7%, 27.6%, and 19.8% in the controls, respectively) indicating failure to correct out-of-frame rearrangements by a second rearrangement on the homologous chromosome 14. We hypothesize that the R841W mutation causes a malfunction of RAG1 that has differential outcome on V(D)J recombination in B and T cells, as the patient had normal B cell numbers but suffered severe alpha-beta T-cell immunodeficiency.