Somatic Diversification Research Articles

Combining mutation and recombination statistics to infer clonal families in antibody repertoires.

B-cell repertoires are characterized by a diverse set of receptors of distinct specificities generated through two processes of somatic diversification: V(D)J recombination and somatic hypermutations. B-cell clonal families stem from the same V(D)J recombination event, but differ in their hypermutations. Clonal families identification is key to understanding B-cell repertoire function, evolution, and dynamics. We present HILARy (high-precision inference of lineages in antibody repertoires), an efficient, fast, and precise method to identify clonal families from single- or paired-chain repertoire sequencing datasets. HILARy combines probabilistic models that capture the receptor generation and selection statistics with adapted clustering methods to achieve consistently high inference accuracy. It automatically leverages the phylogenetic signal of shared mutations in difficult repertoire subsets. Exploiting the high sensitivity of the method, we find the statistics of evolutionary properties such as the site frequency spectrum and dN/dS ratio do not depend on the junction length. We also identify a broad range of selection pressures spanning two orders of magnitude.

Somatic epigenetic drift during shoot branching: a cell lineage-based model.

Plant architecture is shaped by the production of new organs, most of which emerge postembryonically. This process includes the formation of new lateral branches along existing shoots. Current evidence supports a detached-meristem model as the cellular basis of lateral shoot initiation. In this model, a small number of undifferentiated cells are sampled from the periphery of the shoot apical meristem (SAM) to act as precursors for axillary buds, which eventually develop into new shoots. Repeated branching thus creates cellular bottlenecks (i.e. somatic drift) that affect how de novo (epi)genetic mutations propagate through the plant body during development. Somatic drift could be particularly relevant for stochastic DNA methylation gains and losses (i.e. spontaneous epimutations), as they have been shown to arise rapidly with each cell division. Here, we formalize a special case of the detached-meristem model, where precursor cells are randomly sampled from the SAM periphery in a way that maximizes cell lineage independence. We show that somatic drift during repeated branching gives rise to a mixture of cellular phylogenies within the SAM over time. This process is dependent on the number of branch points, the strength of drift as well as the epimutation rate. Our model predicts that cell-to-cell DNA methylation heterogeneity in the SAM converges to nonzero states during development, suggesting that epigenetic variation is an inherent property of the SAM cell population. Our insights have direct implications for empirical studies of somatic (epi)genomic diversity in long-lived perennial and clonal species using bulk or single-cell sequencing approaches.

Lymphocyte pathway analysis using naturally lymphocyte-deficient fish.

Comparative phylogenetic analyses are of potential value to establish the essential components of genetic networks underlying physiological traits. For species that naturally lack particular lymphocyte lineages, we show here that this strategy readily distinguishes trait-specific actors from pleiotropic components of the genetic network governing lymphocyte differentiation. Previously, three of the four members of the DNA polymerase X family have been implicated in the junctional diversification process during the somatic assembly of antigen receptors. Our phylogenetic analysis indicates that the presence of terminal deoxynucleotidyl transferase is strictly associated with the facility of V(D)J recombination, whereas PolL and PolM genes are retained even in species lacking Rag-mediated somatic diversification of antigen receptor genes.

Origin and evolutionary malleability of T cell receptor α diversity

Lymphocytes of vertebrate adaptive immune systems acquired the capability to assemble, from split genes in the germline, billions of functional antigen receptors1–3. These receptors show specificity; unlike the broadly tuned receptors of the innate system, antibodies (Ig) expressed by B cells, for instance, can accurately distinguish between the two enantiomers of organic acids4, whereas T cell receptors (TCRs) reliably recognize single amino acid replacements in their peptide antigens5. In developing lymphocytes, antigen receptor genes are assembled from a comparatively small set of germline-encoded genetic elements in a process referred to as V(D)J recombination6,7. Potential self-reactivity of some antigen receptors arising from the quasi-random somatic diversification is suppressed by several robust control mechanisms8–12. For decades, scientists have puzzled over the evolutionary origin of somatically diversifying antigen receptors13–16. It has remained unclear how, at the inception of this mechanism, immunologically beneficial expanded receptor diversity was traded against the emerging risk of destructive self-recognition. Here we explore the hypothesis that in early vertebrates, sequence microhomologies marking the ends of recombining elements became the crucial targets of selection determining the outcome of non-homologous end joining-based repair of DNA double-strand breaks generated during RAG-mediated recombination. We find that, across the main clades of jawed vertebrates, TCRα repertoire diversity is best explained by species-specific extents of such sequence microhomologies. Thus, selection of germline sequence composition of rearranging elements emerges as a major factor determining the degree of diversity of somatically generated antigen receptors.

Somatic A-to-I RNA-edited RHOA isoform 2 specific-R176G mutation promotes tumor progression in lung adenocarcinoma.

Adenosine-to-inosine (A-to-I) RNA editing is the most common posttranscriptional editing to create somatic mutations and increase proteomic diversity. However, the functions of the edited mutations are largely underexplored. To identify novel targets in lung adenocarcinoma (LUAD), we conducted a genome-wide somatic A-to-I RNA editing analysis of 23 paired adjacent normal and LUAD transcriptomes and identified 26,280 events, including known nonsynonymous AZIN1-S367G and novel RHOAiso2 (RHOA isoform 2)-R176G, tubulin gamma complex associated protein 2(TUBGCP2)-N211S, and RBMXL1-I40 M mutations. We validated the edited mutations in silico in multiple databases and in newly collected LUAD tissue pairs with the SEQUENOM MassARRAY® and TaqMan PCR Systems. We selected RHOAiso2-R176G due to its significant level, isoform-specificity, and being the most common somatic edited nonsynonymous mutation of RHOAiso2 to investigate its roles in LUAD tumorigenesis. RHOAiso2 is a ubiquitous but low-expression alternative spliced isoform received a unique Alu-rich exon at the 3' RHOA mRNA to become an editing RNA target, leading to somatic hypermutation and protein diversity. Interestingly, LUAD patients harboring the RHOAiso2-R176G mutation were associated with aberrant RHOA functions, cancer cell proliferation and migration, and poor clinical outcomes in transcriptome analysis. Mechanistically, RHOAiso2-R176G mutation-expressing LUAD cells potentiate RHOA-guanosine triphosphate (GTP) activity to phosphorylate ROCK1/2 effectors and enhance cell proliferation and migration in vitro and increase tumor growth in xenograft and systemic metastasis models in vivo. Taken together, the RHOAiso2-R176G mutation is a common somatic A-to-I edited mutation of the hypermutated RHOA isoform 2. It is an oncogenic and isoform-specific theranostic target that activates RHOA-GTP/p-ROCK1/2 signaling to promote tumor progression.

Partial RAG deficiency in humans induces dysregulated peripheral lymphocyte development and humoral tolerance defect with accumulation of T-bet+ B cells

The recombination-activating genes (RAG) 1 and 2 are indispensable for diversifying the primary B cell receptor repertoire and pruning self-reactive clones via receptor editing in the bone marrow; however, the impact of RAG1/RAG2 on peripheral tolerance is unknown. Partial RAG deficiency (pRD) manifesting with late-onset immune dysregulation represents an ‘experiment of nature’ to explore this conundrum. By studying B cell development and subset-specific repertoires in pRD, we demonstrate that reduced RAG activity impinges on peripheral tolerance through the generation of a restricted primary B cell repertoire, persistent antigenic stimulation and an inflammatory milieu with elevated B cell-activating factor. This unique environment gradually provokes profound B cell dysregulation with widespread activation, remarkable extrafollicular maturation and persistence, expansion and somatic diversification of self-reactive clones. Through the model of pRD, we reveal a RAG-dependent ‘domino effect’ that impacts stringency of tolerance and B cell fate in the periphery.

Abstract 1883: Large scale viability screening with PRISM underscores non-inhibitory properties of small molecules

Abstract Oncology drug discovery has been dependent on phenotypic screening that lead to discovery of many drugs that remain essential cancer treatments to this day. Historically, logistical and technological limitations have hampered the scale of phenotypic small molecule screening across genomic diverse models. Until recently, large scale phenotypic screening across many cell lines, reflecting the somatic diversity in cancer, has not been possible. The PRISM technology does allow for a high-throughput approach to phenotypic viability screening by pooling cell lines and deconvoluting their response to small molecules using unique integrated DNA-barcodes. Here, we report on the results of a large phenotypic screening effort to identify novel small molecules and targets that can expand our knowledge on cancer vulnerabilities and can spur development of phenotypic drug discovery. A library of 18.000 compounds was assembled based on structural diversity and ability to induce gene-expression changes. Of these 18.000 compounds, around 3.500 were found to reduce cell viability of at least 5 cell lines in the PRISM assay. Of the cytotoxic compounds, we did not identify many small molecules that correlated strongly with CRISPR KO dependency data from project Achilles, and instead found that gene-expression correlations hold much more predictive value. By using additional approaches, we were able to identify the target of select novel cytotoxic small molecules and their respective mechanism-of-action. To our surprise, we have identified few compounds that act as canonical inhibitors of cancer drivers, and instead found compounds that act as molecular glues, prodrugs or inhibitors of more general cellular processes. These findings make a strong case for large scale phenotypic viability screening across genomically diverse models as a complementary approach to target identification next to target based drug discovery. Citation Format: Lucian de Waal, Kevin Larpenteur, Li Wang, Cong Zhu, Jordan Bryan, Mustafa Kocak, Corrie Aghia, Joshua Bittker, Victor Jones, Anita Vrcic, Edward McBride, Samantha Bender, Kalea Gore, Frank Li, Zhenghao Chen, Aviad Tsherniak, Aravind Subramanian, Daphne Koller, Jeffery Settleman, Chris C. Mader, David Stokoe, Ari Firestone, Jennifer A. Roth, Todd R. Golub. Large scale viability screening with PRISM underscores non-inhibitory properties of small molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1883.

Somatic Diversification of Rearranged Antibody Gene Segments by Intra- and Interchromosomal Templated Mutagenesis.

The ability of the humoral immune system to generate Abs capable of specifically binding a myriad of Ags is critically dependent on the somatic hypermutation program. This program induces both templated mutations (i.e., gene conversion) and untemplated mutations. In humans, somatic hypermutation is widely believed to result in untemplated point mutations. In this study, we demonstrate detection of large-scale templated events that occur in human memory B cells and circulating plasmablasts. We find that such mutations are templated intrachromosomally from IGHV genes and interchromosomally from IGHV pseudogenes as well as other homologous regions unrelated to IGHV genes. These same donor regions are used in multiple individuals, and they predominantly originate from chromosomes 14, 15, and 16. In addition, we find that exogenous sequences placed at the IgH locus, such as LAIR1, undergo templated mutagenesis and that homology appears to be the major determinant for donor choice. Furthermore, we find that donor tracts originate from areas in proximity with open chromatin, which are transcriptionally active, and are found in spatial proximity with the IgH locus during the germinal center reaction. These donor sequences are inserted into the Ig gene segment in association with overlapping activation-induced cytidine deaminase hotspots. Taken together, these studies suggest that diversity generated during the germinal center response is driven by untemplated point mutations as well as templated mutagenesis using local and distant regions of the genome.

Critical role of WASp in germinal center tolerance through regulation of B cell apoptosis and diversification

A main feature of Wiskott-Aldrich syndrome (WAS) is increased susceptibility to autoimmunity. A key contribution of B cells to development of these complications has been demonstrated through studies of samples from affected individuals and mouse models of the disease, but the role of the WAS protein (WASp) in controlling peripheral tolerance has not been specifically explored. Here we show that B cell responses remain Tcell dependent in constitutive WASp-deficient mice, whereas selective WASp deletion in germinal center B cells (GCBs) is sufficient to induce broad development of self-reactive antibodies and kidney pathology, pointing to loss of germinal center tolerance as a primary cause leading to autoimmunity. Mechanistically, we show that WASp is upregulated in GCBs and regulates apoptosis and plasma cell differentiation in the germinal center and that the somatic hypermutation-derived diversification is the basis of autoantibody development.

CDR3 binding chemistry controls TCR V-domain rotational probability and germline CDR2 scanning of polymorphic MHC

The mechanism which adapts the T-cell antigen receptor (TCR) within a given major histocompatibility complex (MHC/HLA) genotype is essential for protection against pathogens. Historically attributed to relative affinity, genetically vast TCRs are surprisingly focused towards a micromolar affinity for their respective peptide (p) plus MHC (pMHC) ligands. Thus, the somatic diversity of the TCR with respect to MHC-restriction, and (ultimately) to pathogens, remains enigmatic. Here, we derive a triple integral solution (from fixed geometry) for any given V domain in TCR bound to pMHC. Solved complexes involving HLA-DR and HLA-DQ, where genetic linkage to the TCR is most profound, were examined in detail. Certain V domains displayed rare geometry within this panel—specifying a restricted rotational probability/volumetric density (dV). Remarkably, hydrogen (H) bond charge-relays distinguished these structures from the others; suggesting that CDR3 binding chemistry dictates CDR2 contacts on the opposite MHC-II alpha helix. Together, these data suggest that TCR recapitulate dV and specialise target pMHC recognition. As such, there are implications for the design of TCR-based therapeutics.

Germinal Centers.

Germinal centers (GCs) are microanatomical sites of B cell clonal expansion and antibody affinity maturation. Therein, B cells undergo the Darwinian process of somatic diversification and affinity-driven selection of immunoglobulins that produces the high-affinity antibodies essential for effective humoral immunity. Here, we review recent developments in the field of GC biology, primarily as it pertains to GCs induced by infection or immunization. First, we summarize the phenotype and function of the different cell types that compose the GC, focusing on GC B cells. Then, we review the cellular and molecular bases of affinity-dependent selection within the GC and the export of memory and plasma cells. Finally, we present an overview of the emerging field of GC clonal dynamics, focusing on how GC and post-GC selection shapes the diversity of antibodies secreted into serum.

Immunological Diversity Is a Cornerstone of Organismal Defense and Allorecognition across Metazoa

Abstract The ongoing arms race between hosts and microbes has fueled the evolution of novel strategies for diversifying the molecules involved in immune responses. Characterization of immune systems from an ever-broadening phylogenetic range of organisms reveals that there are many mechanisms by which this diversity can be generated and maintained. Diversification strategies operate at the level of populations, genomes, genes, and even individual transcripts. Lineage-specific innovations have been cataloged within the immune systems of both invertebrates and vertebrates. Furthermore, somatic diversification of immune receptor genes has now been described in jawless vertebrates and some invertebrate species. In addition to pathogen detection, immunological diversity plays important roles in several distinct allorecognition systems. In this Brief Review, we highlight some of the evolutionary innovations employed by a variety of metazoan species to generate the molecular diversity required to detect a vast array of molecules in the context of both immune response and self/nonself-recognition.

Use of ruxolitinib to control graft-versus-host–like disease in Omenn syndrome and successfully bridging to HSCT

Omenn syndrome (OS) is a rare variant of severe combined immunodeficiency characterized by autologous graft-versus-host–like disease (GVHLD) manifestations (erythroderma, hepatomegaly, diarrhea, alopecia, failure to thrive) and susceptibility to severe opportunistic infections. The typical form of the disease is caused by mutations of the recombination-activating genes (RAG1 and RAG2) that lead to an impairment of the V (D) J recombination process, the somatic diversification of T-cell receptors (TCR) and immunoglobulins.

Genomic Stress Responses Drive Lymphocyte Evolvability: An Ancient and Ubiquitous Mechanism.

Somatic diversification of antigen receptor genes depends on the activity of enzymes whose homologs participate in a mutagenic DNA repair in unicellular species. Indeed, by engaging error-prone polymerases, gap filling molecules and altered mismatch repair pathways, lymphocytes utilize conserved components of genomic stress response systems, which can already be found in bacteria and archaea. These ancient systems of mutagenesis and repair act to increase phenotypic diversity of microbial cell populations and operate to enhance their ability to produce fit variants during stress. Coopted by lymphocytes, the ancient mutagenic processing systems retained their diversification functions instilling the adaptive immune cells with enhanced evolvability and defensive capacity to resist infection and damage. As reviewed here, the ubiquity and conserved character of specialized variation-generating mechanisms from bacteria to lymphocytes highlight the importance of these mechanisms for evolution of life in general.

DETECTION OF SELECTION SIGNALS IN CATTLE POPULATIONS BY PCA

The presented study provides a genome-wide scan of selection signals in cattle by principal component analysis (PCA). The aim was to identify SNP affected by intensive selection based on package PCAdapt implemented under software R. This analysis provided insight into the association between the SNP frequencies related to population differentiation. The four cattle populations were involved in the analysis (Slovak Spotted cattle, Ayrshire, Swiss Simmental and Holstein) with overall 272 of genotyped individuals. After applying quality control, the final dataset consisted of 35 675 SNPs, with an overall length of 2496.14 Mb and average space between adjacent SNP 70.03 ± 76.1 kb. After performing PCA analysis, the uniqueness of the breeds was revealed. On the other hand, a close genetic relationship and eleven SNPs affected by selection were found, with a position close to 162 genes involved in the various biological processes. The majority of genes were involved in the positive regulation of adenylate cyclase activity, embryo development and somatic diversification of immune receptors via somatic mutation. Several candidate genes for genetic control of the immune system (DNAJB9), muscle development (SEPT7, TRIM32, ROCK1, NRAP, PZDZ8, HSPA12A and FGFR2), milk production (SOCS5, CD46), reproduction (LHCGR, EEPD1, FSHR) and coat colour (KIT) were identified. Our results provide insights into the regions of the genome affected by the intensive selection of analysed cattle populations.

Genomic and Expression Analyses Define MUC17 and PCNX1 as Predictors of Chemotherapy Response in Breast Cancer.

Poor-prognosis breast cancers are treated with cytotoxic chemotherapy, but often without any guidance from therapy predictive markers because universally accepted markers are not currently available. Treatment failure, in the form of recurrences, is relatively common. We aimed to identify chemotherapy predictive markers and resistance pathways in breast cancer. Our hypothesis was that tumor cells remaining after neoadjuvant chemotherapy (NAC) contain somatic variants causing therapy resistance, while variants present pre-NAC but lost post-NAC cause sensitivity. Whole-exome sequencing was performed on matched pre- and post-NAC cancer cells, which were isolated by laser microdissection, from 6 cancer cases, and somatic variants selected for or against by NAC were identified. Somatic variant diversity was significantly reduced after therapy (P < 0.05). MUC17 variants were identified in 3 tumors and were selected against by NAC in each case, while PCNX1 variants were identified in 2 tumors and were selected for in both cases, implicating the function of these genes in defining chemoresponse. In vitro knockdown of MUC17 or PCNX1 was associated with significantly increased or decreased chemotherapy sensitivity, respectively (P < 0.05), further supporting their roles in chemotherapy response. Expression was tested for predictive value in two independent cohorts of chemotherapy-treated breast cancers (n = 53, n = 303). Kaplan-Meier analyses revealed that low MUC17 expression was significantly associated with longer survival after chemotherapy, whereas low PCNX1 was significantly associated with reduced survival. We concluded that therapy-driven selection of somatic variants allows identification of chemotherapy response genes. With respect to MUC17 and PCNX1, therapy-driven selection acting on somatic variants, in vitro knockdown data concerning drug sensitivity, and survival analysis of expression levels in patient cohorts all define the genes as mediators of and predictive markers for chemotherapy response in breast cancer.

Recent human genetic errors of innate immunity leading to increased susceptibility to infection.

The germline encoded innate immunity governs eukaryotic host defense through both hematopoietic and non-hematopoietic cells, whereas adaptive immunity actions mainly via T cells and B cells characterized by their somatic genetic diversification of antigen-specific responses. Human inborn errors of innate immunity typically underlie infectious diseases. Disturbed innate immunity can additionally result in auto-inflammation. Here, we review inborn errors of innate immunity that have been recently discovered as well as new insights into previously described inborn errors of innate immunity.

CELSR3 mRNA expression is increased in hepatocellular carcinoma and indicates poor prognosis.

ObjectiveHepatocellular carcinoma (HCC) is a disease that is associated with high mortality; currently, there is no curative and reliable treatment. Cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3) is the key signaling molecule in the wingless and INT-1/planar cell polarity (WNT/PCP) pathway. This study aimed to elucidate the prognostic significance of CELSR3 in HCC patients.MethodsThe Cancer Genome Atlas (TCGA) database, the Cancer Cell Line Encyclopedia (CCLE) database and the Gene Expression Omnibus (GEO) database were used to analyze the expression of CELSR3 mRNA in HCC samples and cells. The relationship between CELSR3 mRNA and clinical features was assessed by the chi-square test. the diagnostic and predictive value of CELSR3 mRNA expression were analyzed using the receiver operating characteristic (ROC) curve. Kaplan–Meier curve and Cox regression analyses were performed to assess the prognostic value of CELSR3 mRNA in HCC patients. Finally, all three cohorts database was used for gene set enrichment analysis(GSEA) and the identification of CELSR3-related signal transduction pathways.ResultsThe expression of CELSR3 mRNA was upregulated in HCC, and its expression was correlated with age (P = 0.025), tumor status (P = 0.022), clinical stage (P = 0.003), T classification (P = 0.010), vital status (P = 0.001), and relapse (P = 0.005). The ROC curve assessment indicated that CELSR3 mRNA expression has high diagnostic value in HCC and in the subgroup analysis of stage. In addition, the Kaplan-Meier curve and Cox analyses suggested that patients with high CELSR3 mRNA expression have a poor prognosis, indicating that CELSR3 mRNA is an independent prognostic factor for the overall survival of HCC patients. GSEA showed that GO somatic diversification of immune receptors, GO endonuclease activity, GO DNA repair complex and GO somatic cell DNA recombination, were differentially enriched in the meta-GEO cohort, the HCC cell line cohort and the TCGA cohort of the high CELSR3 mRNA expression phenotype.ConclusionOur results indicate that CELSR3 mRNA is involved in the progression of cancer and can be used as a biomarker for the prognosis of HCC patients.

Clustered Mutations at the Murine and Human IgH Locus Exhibit Significant Linkage Consistent with Templated Mutagenesis.

Somatic hypermutation generates a myriad of Ab mutants in Ag-specific B cells, from which high-affinity mutants are selected. Chickens, sheep, and rabbits use nontemplated point mutations and templated mutations via gene conversion to diversify their expressed Ig loci, whereas mice and humans rely solely on untemplated somatic point mutations. In this study, we demonstrate that, in addition to untemplated point mutations, templated mutagenesis readily occurs at the murine and human Ig loci. We provide two distinct lines of evidence that are not explained by the Neuberger model of somatic hypermutation: 1) across multiple data sets there is significant linkage disequilibrium between individual mutations, especially among close mutations, and 2) among those mutations, those <8 bp apart are significantly more likely to match microhomologous regions in the IgHV repertoire than predicted by the mutation profiles of somatic hypermutation. Together, this supports the role of templated mutagenesis during somatic diversification of Ag-activated B cells.

Individual Sea Urchin Coelomocytes Undergo Somatic Immune Gene Diversification.

The adaptive immune response in jawed vertebrates is marked by the ability to diversify somatically specific immune receptor genes. Somatic recombination and hypermutation of gene segments are used to generate extensive repertoires of T and B cell receptors. In contrast, jawless vertebrates utilize a distinct diversification system based on copy choice to assemble their variable lymphocyte receptors. To date, very little evidence for somatic immune gene diversification has been reported in invertebrate species. Here we show that the SpTransformer (SpTrf ; formerly Sp185/333) immune effector gene family members from individual coelomocytes from purple sea urchins undergo somatic diversification by means of gene deletions, duplications, and acquisitions of single nucleotide polymorphisms. While sperm cells from an individual sea urchin have identical SpTrf gene repertoires, single cells from two distinct coelomocyte subpopulations from the same sea urchin exhibit significant variation in the SpTrf gene repertoires. Moreover, the highly diverse gene sequences derived from single coelomocytes are all in-frame, suggesting that an unknown mechanism(s) driving these somatic changes involve stringent selection or correction processes for expression of productive SpTrf transcripts. Together, our findings infer somatic immune gene diversification strategy in an invertebrate.