Joining Segments Research Articles

Genetic Characteristics of Non B Cell-Derived Immunoglobulin Genes.

It is widely acknowledged that immunoglobulins (Igs) are produced solely by B-lineage cells. The Ig gene is created by the rearrangement of a group of gene segments [variable (V), diversity (D), and joining (J) segments rearrangement, or V(D)J recombination], which results in the vast diversity of B cell-derived Ig responsible for recognising various antigens. Ig subsequently undergoes somatic hypermutation (SHM) and class switch recombination (CSR) after exposure to antigens, thus converting the low-affinity IgM to IgG, IgA, or IgE antibodies. IgM and IgD are primarily expressed in naïve B cells that have not been exposed to antigens, they do not undergo somatic hypermutation; hence, their variable region sequences remain the same as those in the germline. In contrast, IgG, IgA, and IgE are expressed in antigen-stimulated memory B cells or plasma cells, and thus, they often possess high-frequency mutations in their variable region sequences. Since the discovery that Ig can be produced by non-B cells, Qiu's group has investigated and compared the genetic characteristics of B cell-derived Ig and non-B cell-derived Ig. These findings demonstrated that non-B cell-derived Ig shares certain similarities with B cell-derived Ig in that the sequence of its constant region is identical to that of B cell-derived Ig, and its variable region is also strictly dependent on the rearrangement of V, D, and J gene segments. Moreover, akin to B cell-derived Ig, the V regions of IgM and IgD are rarely mutated, while IgG, IgA, and IgE produced by cancer cells are frequently mutated. However, the non-B cell-derived Ig V region sequence displays unique characteristics. (1) Unlike the vast diversity of B cell-derived Igs, non-B cell-derived Igs exhibit restricted diversity; cells from the same lineage always select the same V(D)J recombination patterns; (2) Both mRNA and proteins of RAG1/RAG2 recombinase have been detected in Ig positive cancer cell lines and normal tissues. But Ig recombination could also be found in RAG1-/- and RAG2-/- mice, suggesting that they are not necessary for the rearrangement of non-B cell-derived Igs. These features of non-B cell-derived Igs suggest a potentially undiscovered mechanism of V(D)J recombination, ligation, and SHM in non-B cells, which necessitates further investigation with advanced technology in molecular biology.

Insights into IGH clonal evolution in BCP-ALL: frequency, mechanisms, associations, and diagnostic implications.

The malignant transformation leading to a maturation arrest in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) occurs early in B-cell development, in a pro-B or pre-B cell, when somatic recombination of variable (V), diversity (D), and joining (J) segment immunoglobulin (IG) genes and the B-cell rescue mechanism of VH replacement might be ongoing or fully active, driving clonal evolution. In this study of newly diagnosed BCP-ALL, we sought to understand the mechanistic details of oligoclonal composition of the leukemia at diagnosis, clonal evolution during follow-up, and clonal distribution in different hematopoietic compartments. Utilizing high-throughput sequencing assays and bespoke bioinformatics we identified BCP-ALL-derived clonally-related IGH sequences by their shared 'DNJ-stem'. We introduce the concept of 'marker DNJ-stem' to cover the entirety of, even lowly abundant, clonally-related family members. In a cohort of 280 adult patients with BCP-ALL, IGH clonal evolution at diagnosis was identified in one-third of patients. The phenomenon was linked to contemporaneous recombinant and editing activity driven by aberrant ongoing DH/VH-DJH recombination and VH replacement, and we share insights and examples for both. Furthermore, in a subset of 167 patients with molecular subtype allocation, high prevalence and high degree of clonal evolution driven by ongoing DH/VH-DJH recombination were associated with the presence of KMT2A gene rearrangements, while VH replacements occurred more frequently in Ph-like and DUX4 BCP-ALL. Analysis of 46 matched diagnostic bone marrow and peripheral blood samples showed a comparable clonal and clonotypic distribution in both hematopoietic compartments, but the clonotypic composition markedly changed in longitudinal follow-up analysis in select cases. Thus, finally, we present cases where the specific dynamics of clonal evolution have implications for both the initial marker identification and the MRD monitoring in follow-up samples. Consequently, we suggest to follow the marker DNJ-stem (capturing all family members) rather than specific clonotypes as the MRD target, as well as to follow both VDJH and DJH family members since their respective kinetics are not always parallel. Our study further highlights the intricacy, importance, and present and future challenges of IGH clonal evolution in BCP-ALL.

Abstract 615: Spatially resolved expression of T cell receptors elucidates spatial relationships between T cells, immune infiltration, and cancer-associated pathways

Abstract Spatial distribution of T cells is key in understanding the escape of tumors from immune surveillance via the adaptive immune response, including interactions between immune cells and the surrounding tumor microenvironment. T cells are critical to the adaptive immune response to pathogens and cancers, mediating an antigen-specific response through both specificity and diversity of T cell receptor (TCR) clonotypes. Many methods exist to determine specific clonotypes and overall TCR diversity present from bulk tissues or sorted cell populations; however, nearly all fail to capture spatial orientation and arrangement of T cells engaging with their microenvironment, and most require large amounts of starting material from precious samples. Here, we present a TCR expression profiling panel for the GeoMx® Digital Spatial Profiler that can be combined with the GeoMx Cancer Transcriptome Atlas (CTA) or Human Whole Transcriptome Atlas (WTA) on archival formalin-fixed paraffin embedded (FFPE) tissue specimens. This represents the first commercial spatial expression profiling assay for the simultaneous quantification of TCR constant, variable, and joining segments in situ. We show reliable sensitivity and specificity (>90%) with respect to orthogonal sequencing and robust detection of TCR chains with evidence of clonal expansion and CD8 infiltration across tumor regions in colorectal cancer tissue. These events also corresponded to increased signatures of exhaustion from the T cells and suggest that the T cells resident in or near the tumor are tumor-specific and poised for activation via checkpoint blockade. Signaling pathways and tumor-specific signatures were also evaluated to look for mechanisms through which tumor cells respond to T cell infiltration. We further validated the performance of the TCR probe pool in cell pellet arrays with orthogonal TCR sequencing, tonsil and colorectal cancer tissues. Together, the combination of our TCR add-on panel with the CTA or WTA illuminates T cell phenotypes, signaling pathways, population dynamics, and transcriptomic changes, yielding an unparalleled view of the T cell response in any context. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Katrina van Raay, Michelle Kriner, Jason Reeves, Erin Piazza, Hargita Kaplan, John Vivian, Francis Fernandez, Margaret Hoang, Joseph Beechem. Spatially resolved expression of T cell receptors elucidates spatial relationships between T cells, immune infiltration, and cancer-associated pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 615.

A Hands-on Activity for V(D)J Recombination in an Undergraduate Immunology Class.

This learning activity teaches the difficult concept of V(D)J recombination as it occurs in B cells. Following the traditional lecture, this hands-on activity uses pipe cleaners of various colors representing variable, joining, and diversity gene segments and recombination signal sequences. Students are provided with instructions for using the pipe cleaners to assemble specific light and heavy immunoglobulin chains. Students each assemble their own light and heavy chains and compare the products made by classmates. This activity uses materials that are easy and affordable to acquire and provides a tactile approach to reinforcing concepts that students often struggle to visualize and master from lecture and textbook material alone.

Clonotype pattern in T-cell lymphomas map the cell of origin to immature lymphoid precursors

AbstractMature T-cell lymphomas (TCLs) are rare, clinically heterogeneous hematologic cancers with high medical need. TCLs have an inferior prognosis which is attributed to poor understanding of their pathogenesis. On the basis of phenotypic similarities between normal and neoplastic lymphocytes, it has been assumed that TCLs develop in the periphery, directly from various subtypes of normal T cells. To address the debated question of the cell of origin in TCLs, we attempted to identify the highly variable complementarity-determining regions (CDRs) of T-cell receptors (TCRs) to trace the clonal history of the T cells. We have collected previously published whole-genome, whole-exome, and whole-transcriptome sequencing data from 574 patients with TCL. TCR clonotypes were identified by de novo assembly of CDR3 regions of TCRα, TCRβ, and TCRγ. We have found that the vast majority of TCLs are clonotypically oligoclonal, although the pattern of oligoclonality varied. Anaplastic large-cell lymphoma was the most diverse comprising multiple clonotypes of TCRα, TCRβ, and TCRγ, whereas adult TCL or leukemia and peripheral TCLs often showed monoclonality for TCRβ and TCRγ but had diverse TCRα clonotypes. These patterns of rearrangements indicated that TCLs are initiated at the level of the lymphoid precursor. In keeping with this hypothesis, TCR rearrangements in TCLs resembled the pattern seen in the human thymus, which showed biased usage of V (variable) and J (joining) segments of high combinatorial probability resulting in recurrent public CDR3 sequences shared across unrelated patients and different clinical TCL entities. Clonotypically diverse initiating cells may seed target tissues that are then responsible for disease relapses after therapy.

The Repertoire of Newly Developing Regulatory T Cells in the Type 1 Diabetes-Prone NOD Mouse Is Very Diverse.

Regulatory T lymphocytes expressing the forkhead/winged helix transcription factor Foxp3 (Treg) play a vital role in the protection of the organism from autoimmune disease and other immunopathologies. The antigen specificity of Treg plays an important role in their in vivo activity. We therefore assessed the diversity of the T-cell receptors (TCRs) for antigen expressed by Treg newly developed in the thymus of autoimmune type 1 diabetes-prone NOD mice and compared it to the control mouse strain C57BL/6. Our results demonstrate that use of the TCRα and TCRβ variable (V) and joining (J) segments, length of the complementarity determining region (CDR) 3, and the diversity of the TCRα and TCRβ chains are comparable between NOD and C57BL/6 mice. Genetic defects affecting the diversity of the TCR expressed by newly developed Treg therefore do not appear to be involved in the etiology of type 1 diabetes in the NOD mouse.

Serum IgM heavy chain sub-isotypes and light chain variants revealed in giant grouper (Epinephelus lanceolatus) via protein A affinity purification, mass spectrometry and genome sequencing

Two IgM heavy (H) chain sub-isotypes (80 and 40 kDa) and two light (L) chain variants (25 and 30 kDa) were detected in the serum of giant grouper (Epinephelus lanceolatus), purified by ammonium sulphate precipitation followed by protein A affinity chromatography. This method yielded 5.6 mg/mL high purity IgM from grouper serum, with efficiency estimated at 39.5% recovery from crude serum. The H and L chains were identified by SDS-PAGE and mass spectrometry (MS). Nanopore long-read sequencing was used to generate a genomic contig (MW768935), containing Cμ, Cδ loci, VH regions, and a H chain Joining segment. cDNA sequencing of Cμ transcripts (MW768933 and MW768934) were used to polish the genomic contig and determine the exons and introns of the corresponding locus. MS peptide mapping revealed that the 80 kDa H chain consisted of CH1-4 domains while peptides from the 40 kDa H chain only mapped to CH1-2 domains. Our genomic contig showed the Cμ locus has a Cμ1-Cμ2-Cμ3-Cμ4 arrangement on the same strand as the other Ig loci identified in this genomic sequence. Our study corrects the NCBI annotations of the opposing Cμ loci (LOC117268697 and LOC117268550) in chromosome 16 (NC_047006). Further, we identified both κ and λ L chain isotypes in serum IgM. The molecular weight differences observed may result from different combinations of CL and VL genes. Putative IgM sub-isotypes have also been reported in Epinephelus itajara and Epinephelus coioides. The presence of IgM sub-isotypes may be a conserved trait among Epinephelus species.

The Yin and Yang of RNA surveillance in B lymphocytes and antibody-secreting plasma cells

The random V(D)J recombination process ensures the diversity of the primary immunoglobulin (Ig) repertoire. In two thirds of cases, imprecise recombination between variable (V), diversity (D), and joining (J) segments induces a frameshift in the open reading frame that leads to the appearance of premature termination codons (PTCs). Thus, many B lineage cells harbour biallelic V(D)J-rearrangements of Ig heavy or light chain genes, with a productively-recombined allele encoding the functional Ig chain and a nonproductive allele potentially encoding truncated Ig polypeptides. Since the pattern of Ig gene expression is mostly biallelic, transcription initiated from nonproductive Ig alleles generates considerable amounts of primary transcripts with out-of-frame V(D)J junctions. How RNA surveillance pathways cooperate to control the noise from nonproductive Ig genes will be discussed in this review, focusing on the benefits of nonsense-mediated mRNA decay (NMD) activation during B-cell development and detrimental effects of nonsense-associated altered splicing (NAS) in terminally differentiated plasma cells.

Intra-Vκ Cluster Recombination Shapes the Ig Kappa Locus Repertoire

During V(D)J recombination, RAG proteins introduce DNA double-strand breaks (DSBs) at recombination signal sequences (RSSs) that contain either 12- or 23-nt spacer regions. Coordinated 12/23 cleavage predicts that DSBs at variable (V) gene segments should equal the level of breakage at joining (J) segments. Contrary to this, here we report abundant RAG-dependent DSBs at multiple Vκ gene segments independent of V-J rearrangement. We find that a large fraction of Vκ gene segments are flanked not only by a bone-fide 12 spacer but also an overlapping, 23-spacer flipped RSS. These compatible pairs of RSSs mediate recombination and deletion inside the Vκ cluster even in the complete absence of Jκ gene segments and support a V(D)J recombination center (RC) independent of the conventional Jκ-centered RC. We propose an improved model of Vκ-Jκ repertoire formation by incorporating these surprisingly frequent, evolutionarily conserved intra-Vκ cluster recombination events.

RNA-Seq Based Immunoglobulin Repertoire Analysis of Normal Plasma Cells Generated in an in Vitro B Cell Differentiation System

Antibody secreting plasma cells (PCs) play an important role in effective humoral immune responses. The low frequency of bone marrow PCs in humans makes it challenging to obtain sufficient numbers of PCs for biologic studies. Previous studies have employed in vitro model systems to generate cells that morphologically, phenotypically, and functionally resemble normal polyclonal PCs. Gene expression profiles of in vitro generated PCs (IVPCs) mirror their normal counterparts, however to date extensive immunoglobulin (Ig) repertoire analysis of IVPCs is lacking. Here, we used a modified 3-step protocol to generate IVPCs and used RNA-seq to explore the transcriptome with emphasis on the Ig repertoire of plasmablasts and PCs. Total B cells were isolated from 3 normal donors and cultured with various cytokines and the B cell activators CpG ODN and CD40L. RNA was obtained from freshly isolated B cells (Day 0; D0) as well as from Day 4 (D4) plasmablasts, and Day 10 (D10) IVPCs. Morphologically, D10 cells exhibited typical PC morphology, including an eccentric nucleus and perinuclear hof. RNA-seq was performed on total RNA from all 3 donors and time points using the Standard TRuSeq v2 library prep and with paired end sequencing on the Illumina HiSeq 4000 platform. Principle component analysis of gene expression data showed that D0, D4 and D10 cells could be clearly segregated across all 3 normal donors. Of importance, transcripts previously described as distinguishing B cells from PCs were found to be differentially expressed including overexpression of CXCR5, CD19, EBF, CD83, PAX5, IRF8 in D0 B cells and overexpression of IRF4, Blimp-1, XBP1, BCMA, SLAMF7, Syndecan-1, CD38 and CD27 in IVPCs, thus validating our in vitro model for generating PCs. Furthermore, expression of cell cycle related transcripts such as CKS1, CDK1, and CCDN2 followed the pattern of low expression in resting B cells, increased expression in plasmablasts, and decreased expression in IVPCs confirming the cells are actively cycling in a manner comparable to cells in vivo. D10 IVPCs also overexpressed transcripts known to be upregulated during the unfolded protein response. As expected from Ig secreting cells, D10 IVPCs had an over-representation of Ig transcripts. At D0, resting B cells had high levels of IgD and IgM heavy chain (HC) transcripts. At D10, IgM transcripts modestly increased with Log2 fold change (FC) = 3 and as expected, IgD levels decreased significantly (Log2 FC = -2.2). IgA and IgG isotype transcripts significantly increased at D10 (Log2 FC > 6.0) with the IgG4 subtype having the greatest Log2 FC at 8.4. Next we focused on the Ig repertoire of D0, D4, and D10 cells. By aligning to known germline Ig sequences in IMGT/V-Quest (www.imgt.org) and then assembling the paired ends of D0, D4 and D10 Ig transcripts, we were able to analyze the Ig repertoire. Since the Ig HC variable (V) region is encoded by V, diversity (D) and joining (J) segments, only fragments that could be confidently determined were considered. All but 3 IGHV transcripts (IGHV3-35, IGHV3-47 and IGHV7-8) and 2 IGHD transcripts (IGHD4-4 and IGHD5-5) were found and all IGHJ segments were represented across the differentiation spectrum. In D0 cells, the number of unique VDJ combinations ranged from 643 to 863 across all 3 normal samples and increased to a range of 2524 to 2867 in D10 IVPCs. When looking at the differential expression of each VDJ combination from D0 to D10, a pairwise t-test for relative frequency showed that there was no significant change greater than 1%, suggesting the repertoire diversity was not skewed, thus proving the conditions for stimulation were not targeting any one starting B cell. Our data also allowed us to track clonal expansions during differentiation as defined by the increasing frequency of sequences with identical nucleotide sequence in the V region and CDR3 (including D and J regions). Hence, a single sequence could be tracked from D0 to D10. Of interest, in a small sampling of the total available sequences, only those B cells with a mutated IGHV region, characteristic of a memory B cell, went on to expand in this system whereas B cells with an unmutated IGHV did not. Our analysis of the Ig repertoire of IVPCs suggests this system provides a functional model to study Ig repertoire along the B cell differentiation process and further delineate the conditions that may result in a clonal expansion, a hallmark of many hematologic malignancies including multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Structure in mind, structure in vocal tract

We update our understanding of the view that grammar regulates inter-segmental temporal coordination and present an extension of that view to a new domain: we argue that inter-segmental coordination is basic to prosody. It is the glue joining segments together differently in different languages (here, illustrated with examples from Arabic and Spanish) and orchestrates their unfolding in ways corresponding to constructs posited in theoretical analysis. The correspondence is one between organization in mind-brain and organization in vocal tract. Moreover, for both mind-brain and vocal tract, the organization is phonological and abstract. It is so because it holds over segments of various identities: in Arabic, the first segment in // is not prosodified as part of the same unit as // and this holds true also for //, // and so on, regardless of sonority. In contrast, in English or Spanish, a different organization holds. Crucially, uniformity in organization (same organization presiding over sequences with varying segmental makeup) does not imply uniqueness of phonetic exponents: prosodic organization is pleiotropic, simultaneously expressed by more than one phonetic exponent. Finally, two properties of coordination relations are underscored: lawful flexibility and abstractness. The first is revealed in the degrees of freedom with which movements corresponding to any given effector begin; the second in invariances of task-relevant kinematic signatures regardless of the effectors implicated in any given segmental sequence. Once again, abstract phonological structure is mirrored in vocal tracts via coordination relations holding across physiology and the particular modes of its operation.

Immunoglobulin light chain (IGL) genes in torafugu: Genomic organization and identification of a third teleost IGL isotype

Here, we report a genome-wide survey of immunoglobulin light chain (IGL) genes of torafugu (Takifugu rubripes) revealing multi-clusters spanning three separate chromosomes (v5 assembly) and 45 scaffolds (v4 assembly). Conventional sequence similarity searches and motif scanning approaches based on recombination signal sequence (RSS) motifs were used. We found that three IGL isotypes (L1, L2, and L3) exist in torafugu and that several loci for each isotype are present. The transcriptional orientations of the variable IGL (VL) segments were found to be either the same (in the L2 isotype) or opposite (in the L1 and L3 isotypes) to the IGL joining (JL) and constant (CL) segments, suggesting they can undergo rearrangement by deletion or inversion when expressed. Alignments of expressed sequence tags (ESTs) to corresponding germline gene segments revealed expression of the three IGL isotypes in torafugu. Taken together, our findings provide a genomic framework for torafugu IGL genes and show that the IG diversity of this species could be attributed to at least three distinct chromosomal regions.

Characterization of T and B cell repertoire diversity in patients with RAG deficiency.

Recombination-activating genes 1 and 2 (RAG1 and RAG2) play a critical role in T and B cell development by initiating the recombination process that controls the expression of T cell receptor (TCR) and immunoglobulin genes. Mutations in the RAG1 and RAG2 genes in humans cause a broad spectrum of phenotypes, including severe combined immunodeficiency (SCID) with lack of T and B cells, Omenn syndrome, leaky SCID, and combined immunodeficiency with granulomas or autoimmunity (CID-G/AI). Using next-generation sequencing, we analyzed the TCR and B cell receptor (BCR) repertoire in 12 patients with RAG mutations presenting with Omenn syndrome (n = 5), leaky SCID (n = 3), or CID-G/AI (n = 4). Restriction of repertoire diversity skewed usage of variable (V), diversity (D), and joining (J) segment genes, and abnormalities of CDR3 length distribution were progressively more prominent in patients with a more severe phenotype. Skewed usage of V, D, and J segment genes was present also within unique sequences, indicating a primary restriction of repertoire. Patients with Omenn syndrome had a high proportion of class-switched immunoglobulin heavy chain transcripts and increased somatic hypermutation rate, suggesting in vivo activation of these B cells. These data provide a framework to better understand the phenotypic heterogeneity of RAG deficiency.

Development of a Bioinformatics Framework for the Detection of Gene Conversion and the Analysis of Combinatorial Diversity in Immunoglobulin Heavy Chains in Four Cattle Breeds.

We have developed a new bioinformatics framework for the analysis of rearranged bovine heavy chain immunoglobulin (Ig) variable regions by combining and refining widely used alignment algorithms. This bioinformatics framework allowed us to investigate alignments of heavy chain framework regions (FRHs) and the separate alignments of FRHs and heavy chain complementarity determining regions (CDRHs) to determine their germline origin in the four cattle breeds Aubrac, German Black Pied, German Simmental, and Holstein Friesian. Now it is also possible to specifically analyze Ig heavy chains possessing exceptionally long CDR3Hs. In order to gain more insight into breed specific differences in Ig combinatorial diversity, somatic hypermutations and putative gene conversions of IgG, we compared the dominantly transcribed variable (IGHV), diversity (IGHD), and joining (IGHJ) segments and their recombination in the four cattle breeds. The analysis revealed the use of 15 different IGHV segments, 21 IGHD segments, and two IGHJ segments with significant different transcription levels within the breeds. Furthermore, there are preferred rearrangements within the three groups of CDR3H lengths. In the sequences of group 2 (CDR3H lengths (L) of 11–47 amino acid residues (aa)) a higher number of recombination was observed than in sequences of group 1 (L≤10 aa) and 3 (L≥48 aa). The combinatorial diversity of germline IGHV, IGHD, and IGHJ-segments revealed 162 rearrangements that were significantly different. The few preferably rearranged gene segments within group 3 CDR3H regions may indicate specialized antibodies because this length is unique in cattle. The most important finding of this study, which was enabled by using the bioinformatics framework, is the discovery of strong evidence for gene conversion as a rare event using pseudogenes fulfilling all definitions for this particular diversification mechanism.

High Throughput Sequencing of T Cell Antigen Receptors Reveals a Conserved TCR Repertoire.

The T-cell receptor (TCR) repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next-generation sequencing has become a powerful tool for deep TCR profiling. Herein, we used this technology to study the repertoire features of TCR beta chain in the blood of healthy individuals.Peripheral blood samples were collected from 10 healthy donors. T cells were isolated with anti-human CD3 magnetic beads according to the manufacturer's protocol. We then combined multiplex-PCR, Illumina sequencing, and IMGT/High V-QUEST to analyze the characteristics and polymorphisms of the TCR.Most of the individual T cell clones were present at very low frequencies, suggesting that they had not undergone clonal expansion. The usage frequencies of the TCR beta variable, beta joining, and beta diversity gene segments were similar among T cells from different individuals. Notably, the usage frequency of individual nucleotides and amino acids within complementarity-determining region (CDR3) intervals was remarkably consistent between individuals. Moreover, our data show that terminal deoxynucleotidyl transferase activity was biased toward the insertion of G (31.92%) and C (27.14%) over A (21.82%) and T (19.12%) nucleotides.Some conserved features could be observed in the composition of CDR3, which may inform future studies of human TCR gene recombination.

On the organization of human T-cell receptor loci: log-periodic distribution of T-cell receptor gene segments.

The human T-cell repertoire is complex and is generated by the rearrangement of variable (V), diversity (D) and joining (J) segments on the T-cell receptor (TCR) loci. The T-cell repertoire demonstrates self-similarity in terms clonal frequencies when defined by V, D and J gene segment usage; therefore to determine whether the structural ordering of these gene segments on the TCR loci contributes to the observed clonal frequencies, the TCR loci were examined for self-similarity and periodicity in terms of gene segment organization. Logarithmic transformation of numeric sequence order demonstrated that the V and J gene segments for both T-cell receptor α (TRA) and β (TRB) loci are arranged in a self-similar manner when the spacing between the adjacent segments was considered as a function of the size of the neighbouring gene segment, with an average fractal dimension of approximately 1.5. Accounting for the gene segments occurring on helical DNA molecules with a logarithmic distribution, sine and cosine functions of the log-transformed angular coordinates of the start and stop nucleotides of successive TCR gene segments showed an ordered progression from the 5' to the 3' end of the locus, supporting a log-periodic organization. T-cell clonal frequency estimates, based on V and J segment usage, from normal stem cell donors were plotted against the V and J segment on TRB locus and demonstrated a periodic distribution. We hypothesize that this quasi-periodic variation in gene-segment representation in the T-cell clonal repertoire may be influenced by the location of the gene segments on the periodic-logarithmically scaled TCR loci. Interactions between the two strands of DNA in the double helix may influence the probability of gene segment usage by means of either constructive or destructive interference resulting from the superposition of the two helices.

A discrete chromatin loop in the mouse Tcra-Tcrd locus shapes the TCRδ and TCRα repertoires.

The Tcra-Tcrd locus undergoes V(D)J recombination in CD4−CD8−double-negative thymocytes and CD4+CD8+ double-positive thymocytes to generate diverse TCRδ and TCRα repertoires, respectively. Here we reveal a Tcra-Tcrd locus chromatin interaction network in double-negative thymocytes that was formed by interactions between CTCF-binding elements. Disruption of a discrete chromatin loop encompassing Tcrd diversity, joining and constant gene segments allowed a single variable gene segment to frequently contact and rearrange to diversity and joining gene segments and dominate the adult TCRδ repertoire. Disruption of this loop also narrowed the TCRα repertoire, which, we believe, follows as a consequence of the restricted TCRδ repertoire. Hence, a single CTCF-mediated chromatin loop directly regulates TCRδ diversity and indirectly regulates TCRα diversity.

Abstract 4737: Comprehensive profiling of immunoglobulin sequences using hybrid capture-based next generation sequencing in B-cell hematologic malignancies

Abstract Background: Sequencing the genes encoding immunoglobulins is critical in detecting clonal cell populations as well as determining prognosis and therapeutic decisions in patients with lymphoid malignancies including B-cell leukemias, lymphomas and multiple myeloma. Current assays for identifying the rearranged immunoglobulin sequence in B-cell malignancies rely on sequence specific PCR based amplification of conserved immunoglobulin (IG) regions. We have developed a novel, hybrid capture-based approach to sequencing the immunoglobulin chains that enables identification of the heavy and light chain variable domains, complementarity-determining region (CDR) sequences and somatic hypermutation (SHM) status. Method: RNA and DNA were successfully extracted from 60 specimens, including 7 mantle cell lymphoma cell (MCL) lines and 53 clinical chronic lymphocytic leukemia (CLL) bone marrow aspirates. Adaptor-ligated DNA and cDNA sequencing libraries were captured by solution hybridization using custom bait-sets targeting the immunoglobulin variable, joining and class segments. All captured libraries were sequenced to high depth (Illumina HiSeq) in a CLIA-certified laboratory (Foundation Medicine). Results: The capture-based approach was validated using 7 MCL cell lines and 53 CLL samples profiled using a CLIA-certified commercial PCR-based assay (Invivoscribe). The immunoglobulin sequences derived from the 7 MCL cell lines were 100% concordant with identifying the published heavy and light chain variable domain and percent SHM. Comparison to 53 previously clinically tested samples showed 98% (52/53) concordance for identifying the presence of a clonal population, and 100% (39/39) concordance for identifying the IGHV domain. Comparison to 50 CLL samples previously tested for SHM resulted in 96% (48/50) overall concordance with 94% (31/33) concordant for no SHM and 100% (17/17) concordant for the presence of SHM. Additionally, secondary clones were identified in 11 samples. Conclusions: We have demonstrated that hybrid capture-based targeted DNA and RNA sequencing can be used to comprehensively characterize the immunoglobulin sequence of clonal tumor B cell populations. This capability enables quantification of SHM and identification of the variable domain, CDR3 sequence and class restriction. Integration of this methodology with comprehensive genomic profiling approaches will expand the clinical utility of such assays in patients with hematological malignancies and may provide important insights in immune oncology and response of patients to immunotherapies, including patients with solid tumors. Citation Format: Michelle K. Nahas, Lauren E. Young, Jeff Gardner, Omar Abdel-Wahab, Jie He, Amy L. Donahue, Kristina M. Knapp, Geoff A. Otto, Doron Lipson, Vincent A. Miller, Ross L. Levine, Philip J. Stephens. Comprehensive profiling of immunoglobulin sequences using hybrid capture-based next generation sequencing in B-cell hematologic malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4737. doi:10.1158/1538-7445.AM2015-4737

HTJoinSolver: Human immunoglobulin VDJ partitioning using approximate dynamic programming constrained by conserved motifs.

BackgroundPartitioning the human immunoglobulin variable region into variable (V), diversity (D), and joining (J) segments is a common sequence analysis step. We introduce a novel approximate dynamic programming method that uses conserved immunoglobulin gene motifs to improve performance of aligning V-segments of rearranged immunoglobulin (Ig) genes. Our new algorithm enhances the former JOINSOLVER algorithm by processing sequences with insertions and/or deletions (indels) and improves the efficiency for large datasets provided by high throughput sequencing.ResultsIn our simulations, which include rearrangements with indels, the V-matching success rate improved from 61% for partial alignments of sequences with indels in the original algorithm to over 99% in the approximate algorithm. An improvement in the alignment of human VDJ rearrangements over the initial JOINSOLVER algorithm was also seen when compared to the Stanford.S22 human Ig dataset with an online VDJ partitioning software evaluation tool.ConclusionsHTJoinSolver can rapidly identify V- and J-segments with indels to high accuracy for mutated sequences when the mutation probability is around 30% and 20% respectively. The D-segment is much harder to fit even at 20% mutation probability. For all segments, the probability of correctly matching V, D, and J increases with our alignment score.

Insertion of an imprinted insulator into the IgH locus reveals developmentally regulated, transcription-dependent control of V(D)J recombination.

The assembly of antigen receptor loci requires a developmentally regulated and lineage-specific recombination between variable (V), diversity (D), and joining (J) segments through V(D)J recombination. The process is regulated by accessibility control elements, including promoters, insulators, and enhancers. The IgH locus undergoes two recombination steps, D-J(H) and then V(H)-DJ(H), but it is unclear how the availability of the DJ(H) substrate could influence the subsequent V(H)-DJ(H) recombination step. The Eμ enhancer plays a critical role in V(D)J recombination and controls a set of sense and antisense transcripts. We epigenetically perturbed the early events at the IgH locus by inserting the imprinting control region (ICR) of the Igf2/H19 locus or a transcriptional insulator devoid of the imprinting function upstream of the Eμ enhancer. The insertions recapitulated the main epigenetic features of their endogenous counterparts, including differential DNA methylation and binding of CTCF/cohesins. Whereas the D-J(H) recombination step was unaffected, both the insulator insertions led to a severe impairment of V(H)-DJ(H) recombination. Strikingly, the inhibition of V(H)-DJ(H) recombination correlated consistently with a strong reduction of DJ(H) transcription and incomplete demethylation. Thus, developmentally regulated transcription following D-J(H) recombination emerges as an important mechanism through which the Eμ enhancer controls V(H)-DJ(H) recombination.