Long-Range Control of Class Switch Recombination by Transcriptional Regulatory Elements

Immunoglobulin class switch recombination (CSR) plays a crucial role in adaptive immune responses through a change of the effector functions of antibodies and is triggered by T-cell-dependent as well as T-cell-independent antigens. Signals generated following encounter with each type of antigen direct CSR to different isotypes. At the genomic level, CSR occurs between highly repetitive switch sequences located upstream of the constant gene exons of the immunoglobulin heavy chain locus. Transcription of switch sequences is mandatory for CSR and is induced in a stimulation-dependent manner. Switch transcription takes place within dynamic chromatin domains and is regulated by long-range regulatory elements which promote alignment of partner switch regions in CSR centers. Here, we review recent work and models that account for the function of long-range transcriptional regulatory elements and the chromatin-based mechanisms involved in the control of CSR.

IGKC, the immunoglobulin kappa constant gene segment, is differentially expressed in brain metastatic breast cancer.

Metastasis to the brain is a clinical problem in patients with breast cancer (1-3). We mined published microarray data (4-6) to compare primary and metastatic tumor transcriptomes to discover genes associated with brain metastasis in patients with metastatic breast cancer. We found that the immunoglobulin light chain kappa constant locus encoded by IGKC was among the genes whose expression was most different in the brain metastases of patients with metastatic breast cancer as compared to primary tumors of the breast. We observed a significant correlation between IGKC expression in primary tumors of the breast and distant-metastasis free survival, as well as overall survival. Down-regulation of IGKC may be an important event for metastasis of primary tumor-derived cancer cells to the brain in humans with metastatic breast cancer.

Recombinant T cell receptors specific for HLA-A*02:01-restricted neoepitopes containing KRAS codon 12 hotspot mutations

KRAS codon 12 mutations are among the most common hotspot mutations in human cancer. Using a functional screening platform we set out to identify αβ T-cell receptors (TCRs) as potential targeting reagents for KRASG12D and/or KRASG12V neoepitopes presented by the prevalent HLA-A*02:01 allele. Here we describe isolation and characterization of three distinct CD8+ T cell clones from a pre-treated 76 year old patient with pancreatic ductal adenocarcinoma (PDAC). One clone was KRASG12V reactive and two clones were KRASG12D reactive. Tetramer staining showed high specificity of each T cell clone for its cognate HLA-A*02:01 restricted KRASG12V or KRASG12D neoepitope (>98% tetramer positive) without appreciable cross-reactivity to wild-type KRAS (<2% tetramer positive). We amplified and sequenced the full-length TCR alpha and beta chains from each of the three T cell clones and determined that these three TCRs comprised distinct combinations of two different TCR alpha chains and two distinct TCR beta chains. We resynthesized these TCR alpha and beta chain nucleotide sequences and reconstituted the original pairs in healthy donor CD8+ T cells by lentiviral transduction, substituting the human αβ TCR constant gene segments with murine αβ TCR constant gene segments to prevent mispairing with endogenous TCR subunits. Tetramer analysis and IFN-γ ELISpot analysis confirmed the specificity of each reconstituted TCR for its cognate HLA-A*02:01 restricted KRAS neoepitope. To test cytolytic activity TCR-transduced healthy donor CD8+ T cells were co-cultured with KRASG12V, KRASG12D or KRASwt peptide-pulsed K562-HLA-A*02:01 antigen presenting cells at an effector to target cell ratio of 4:1. Under these conditions we observed neoepitope-specific killing of 16.5% to 19.0% of target cell populations. To assess in vivo activity we developed a KRASG12V/A*02:01 patient-derived xenograft (PDX) mouse model. Over a 56-day period, PDX bearing mice infused with human TCR-transduced T cells had significantly reduced tumor growth and longer survival compared to mice infused with non-transduced control T cells. In conjunction with other therapeutic approaches, immune effector cell therapies expressing these TCRs may improve outcomes for HLA-A*02:01 patients with KRASG12V and/or KRASG12D positive tumors.

Biases in demographic modelling affect our understanding of recent divergence

Estimating patterns of gene flow during the early stages of divergence is central to understanding whether reproductive isolation arises through gradual erosion of gene flow or via successive stages of strict isolation and secondary contact. Such scenarios can be tested by comparing the joint allele frequency spectrum (jAFS) of a set of populations to jAFS simulated under scenarios of isolation with migration (IM) and secondary contact (SC). However, the potential effect of unaccounted demographic events (such as population expansions and bottlenecks) on model choice and parameter estimation remains largely unexplored. Using simulations, we demonstrate that under realistic divergence scenarios with constant gene flow, failure to account for population size (Ne) changes in daughter and ancestral populations leads to biases in divergence time estimates as well as model choice. On the other hand, when the simulations included long periods of strict isolation the correct gene flow scenario was usually retrieved. We illustrate these issues reconstructing the recent demographic history of North Sea and Baltic Sea turbots (Schopthalmus maximus) by testing 16 IM and 16 SC scenarios, modelling changes in Ne as well as the effects of linked selection and barrier loci. Failure to account for changes in Ne resulted in selecting SC models with long periods of isolation and divergence times preceding the formation of the Baltic Sea. In contrast, models accounting for Ne changes suggest that the Baltic Sea turbot population originated from a recent (<6 kya) invasion and has diverged with constant gene flow from the North Sea. We further show how interpreting genomic landscape of differentiation can help discerning among competing models. For example, turbots show no signature of increased absolute divergence (dxy) with respect to genetic diversity in the North Sea. Two islands of high differentiation show genomic signature characteristic of recent selective sweeps, rather than old divergence resisting secondary introgression. The results have broad implications for the study of recent population divergence by high-lighting the potential effects of ancestral size changes and bottlenecks followed by growth on demographic inference. In general, extreme caution should be exercised when interpreting results of demographic mode.

Long-Amplicon Sequencing of Rearranged IGH Chains Enables Simultaneous Measurement of Somatic Hypermutation and Isotype in Chronic Lymphocytic Leukemia

Background Chronic lymphocytic leukemia (CLL) is a common form of leukemia characterized by clonal expansion of neoplastic mature B cells and a heterogenous disease course ranging from aggressive clonal expansion requiring early intervention to indolent disease that does not require treatment. Accumulating evidence suggests that the somatic hypermutation (SHM) status of the IGHV gene of the malignant clone may serve as a prognostic marker of clinical outcome, where a SHM frequency of >2% indicates a favorable outcome (SHM-M) while < 2% SHM (SHM-U) indicates comparatively poor prognosis. Current next-generation sequencing (NGS) approaches for analyzing SHM commonly rely on multiplex primers targeting the framework 1 (FR1) or leader region of the IGH variable gene in combination with joining gene primers to amplify rearranged IGH chains from gDNA template. Limitations include the potential for joining gene mutations to interfere with primer binding and an inability to evaluate isotype, which could potentially serve as an additional prognostic marker given the mechanistic link between somatic hypermutation and class-switch recombination. Here we present a novel method for translational research investigations of IGH chain SHM employing multiplex FR1 and isotype (constant gene) specific primers to amplify IGH chains from RNA template. We demonstrate performance of the assay via sequencing of synthesized CLL IGH chains derived from literature. Methods Multiplex PCR primers were designed to target all IGH variable genes and constant genes and alleles in the IMGT database (Oncomine IGH-LR assay). Primers were used to amplify IGH chains in samples consisting of 25ng healthy donor peripheral blood leukocyte (PBL) total RNA spiked with one of 7 synthesized monoclonal CLL rearrangements including three germline rearrangements (0% SHM), and four mutated rearrangements (SHM ranging from 3-9%), each expressed with a different isotype. Resultant libraries were sequenced on the Gene Studio S5 and analyzed via Ion Reporter to identify clonotypes, quantify SHM, and identify B cell clonal lineages. Automated downsampling analysis was used to confirm that libraries had been sequenced to an appropriate depth. Results 7 of 7 synthesized rearrangements were correctly classified as SHM-M or SHM-U, and in each instance the isotype was correctly identified by the software. Clonal lineage analysis indicated that each spike in rearrangement was monoclonal, consistent with expectation. In silico analysis revealed a high correlation between SHM estimates obtained using the entire v-gene sequence and those obtained from FR1-targeting primers (Pearson's correlation >.99). Conclusions These results support the robustness and reliability of multiplex FR1 and constant gene based IGH chain amplification, combined with clonotyping and lineage analysis, for the translational research characterization of somatic hypermutation in CLL and other B cell neoplasms, including efforts to understand the potential prognostic value of isotype. Disclosures Looney: Thermo Fisher Scientific: Employment. Lowman:Thermo Fisher Scientific: Employment. Pickle:Thermo Fisher Scientific: Employment. Chang:Thermo Fisher Scientific: Employment. Topacio-Hall:Thermo Fisher Scientific: Employment. Toro:Thermo Fisher Scientific: Employment. Hyland:Thermo Fisher Scientific: Employment.

No evidence for a loss of genetic diversity despite a strong decline in size of a European population of the Corncrake Crex crex

SummaryThe preservation of genetic diversity is an important aspect of conservation biology. Low genetic diversity within a population can lead to inbreeding depression and a reduction in adaptive potential, which may increase extinction risk. Here we report changes in genetic diversity over 12 years in a declining population of the Corncrake Crex crex, a grassland bird species of high conservation concern throughout Europe. Despite a twofold demographic decline during the same period, we found no evidence for a reduction of genetic diversity. The gradual genetic differentiation observed among populations of Corncrake across Europe suggests that genetic diversity is maintained in western populations by constant gene flow from the larger and more productive populations in eastern Europe and Asia. The maintenance of genetic diversity in this species is an opportunity that may help the implementation of effective conservation actions across the Corncrake’s European range.

Two modes of cis-activation of switch transcription by the IgH superenhancer

B cell isotype switching plays an important role in modulating adaptive immune responses. It occurs in response to specific signals that often induce different isotype (I) promoters driving transcription of switch regions, located upstream of the Ig heavy chain (IgH) constant genes. The transcribed switch regions can recombine, leading to a change of the constant gene and, consequently, of antibody isotype. Switch transcription is controlled by the superenhancer 3′ regulatory region (3′RR) that establishes long-range chromatin cis-interactions with I promoters. Most stimuli induce more than one I promoter, and switch transcription can occur on both chromosomes. Therefore, it is presently unknown whether induced I promoters compete for the 3′RR on the same chromosome. Here we performed single-chromosome RT-qPCR assays to examine switch transcription monoallelically in the endogenous context. We show that there are two modes of 3′RR-mediated activation of I promoters: coactivation and competition. The nature of the inducing signal plays a pivotal role in determining the mode of activation. Furthermore, we provide evidence that, in its endogenous setting, the 3′RR has a bidirectional activity. We propose that the coactivation and competition modes mediated by the 3′RR may have evolved to cope with the different kinetics of primary immune responses.

Long-amplicon TCRβ repertoire sequencing to reveal human T-cell receptor variable gene polymorphism: Implications for the prediction and interpretation of immunotherapy outcome.

129 Background: Human T cell antigen receptors play a critical role in protective immune responses but are also implicated in autoimmune disease and immune-mediated adverse events during immunotherapy. The antigen specificity of the T cell receptor is determined in part by the sequence of the CDR and Framework regions encoded by the TCRB variable gene. Previous studies of population sequencing data indicate that current antigen receptor allele databases, such as IMGT, fail to capture a significant portion of human variation. Here we use long-amplicon multiplex sequencing of rearranged TCRB receptors to validate putative novel human variable gene alleles previously recovered from 1000 genomes data. Methods: TCRB rearrangements were amplified from cDNA from 85 Caucasians undergoing treatment for melanoma using AmpliSeq-based multiplex Framework 1 and Constant gene primers to produce ~330bp amplicons. Samples were sequenced using the Ion Torrent S5 530 chip to produce ~1.5M raw reads per sample. Ion Reporter was used for clonotyping and identification of variable gene sequences absent from the IMGT database. Putatively novel sequences were compared to those reported in the Lym1k database of alleles recovered from 1000 genomes data. Results: We identified 15 novel variable gene alleles that are absent from the IMGT database and result in amino acid changes to the CDR or Framework regions of the TCR. Typically, a single individual was found to be heterozygous for a novel variant, though we note two instances where multiple individuals possessed a novel variant. We also identified novel variable gene alleles that are absent from the Lym1k database, potentially due to challenges in inferring receptor alleles from short-read population sequencing studies. Conclusions: We find evidence for significant human diversity in TCRB variable gene alleles beyond what is currently represented in the IMGT database. TCRB sequencing using multiplex Framework 1 and Constant gene targeting primers is ideally suited for studying the role of TCRB polymorphism in autoimmune disease and immune-mediated adverse events during immunotherapy.