Paleo-Immunology: Evidence Consistent with Insertion of a Primordial Herpes Virus-Like Element in the Origins of Acquired Immunity

Background

The RAG encoded proteins, RAG-1 and RAG-2 regulate site-specific recombination events in somatic immune B- and T-lymphocytes to generate the acquired immune repertoire. Catalytic activities of the RAG proteins are related to the recombinase functions of a pre-existing mobile DNA element in the DDE recombinase/RNAse H family, sometimes termed the “RAG transposon”.

Methodology/Principal Findings

Novel to this work is the suggestion that the DDE recombinase responsible for the origins of acquired immunity was encoded by a primordial herpes virus, rather than a “RAG transposon.” A subsequent “arms race” between immunity to herpes infection and the immune system obscured primary amino acid similarities between herpes and immune system proteins but preserved regulatory, structural and functional similarities between the respective recombinase proteins. In support of this hypothesis, evidence is reviewed from previous published data that a modern herpes virus protein family with properties of a viral recombinase is co-regulated with both RAG-1 and RAG-2 by closely linked cis-acting co-regulatory sequences. Structural and functional similarity is also reviewed between the putative herpes recombinase and both DDE site of the RAG-1 protein and another DDE/RNAse H family nuclease, the Argonaute protein component of RISC (RNA induced silencing complex).

Conclusions/Significance

A “co-regulatory” model of the origins of V(D)J recombination and the acquired immune system can account for the observed linked genomic structure of RAG-1 and RAG-2 in non-vertebrate organisms such as the sea urchin that lack an acquired immune system and V(D)J recombination. Initially the regulated expression of a viral recombinase in immune cells may have been positively selected by its ability to stimulate innate immunity to herpes virus infection rather than V(D)J recombination Unlike the “RAG-transposon” hypothesis, the proposed model can be readily tested by comparative functional analysis of herpes virus replication and V(D)J recombination.     

Compound Heterozygous Mutation of Rag1 Leading to Omenn Syndrome

Omenn syndrome is a primary immunodeficiency disorder, featuring susceptibility to infections and autoreactive T cells and resulting from defective genomic rearrangement of genes for the T cell and B cell receptors. The most frequent etiologies are hypomorphic mutations in “non-core” regions of the Rag1 or Rag2 genes, the protein products of which are critical members of the cellular apparatus for V(D)J recombination. In this report, we describe an infant with Omenn syndrome with a previously unreported termination mutation (p.R142*) in Rag1 on one allele and a partially characterized substitution mutation (p.V779M) in a “core” region of the other Rag1 allele. Using a cellular recombination assay, we found that while the p.R142* mutation completely abolished V(D)J recombination activity, the p.V779M mutation conferred a severe, but not total, loss of V(D)J recombination activity. The recombination defect of the V779 mutant was not due to overall misfolding of Rag1, however, as this mutant supported wild-type levels of V(D)J cleavage. These findings provide insight into the role of this poorly understood region of Rag1 and support the role of Rag1 in a post-cleavage stage of recombination.     

Reprint of “Functional overlaps between XLF and the ATM-dependent DNA double strand break response”

Developing B and T lymphocytes generate programmed DNA double strand breaks (DSBs) during the V(D)J recombination process that assembles exons that encode the antigen-binding variable regions of antibodies. In addition, mature B lymphocytes generate programmed DSBs during the immunoglobulin heavy chain (IgH) class switch recombination (CSR) process that allows expression of different antibody heavy chain constant regions that provide different effector functions. During both V(D)J recombination and CSR, DSB intermediates are sensed by the ATM-dependent DSB response (DSBR) pathway, which also contributes to their joining via classical non-homologous end-joining (C-NHEJ). The precise nature of the interplay between the DSBR and C-NHEJ pathways in the context of DSB repair via C-NHEJ remains under investigation. Recent studies have shown that the XLF C-NHEJ factor has functional redundancy with several members of the ATM-dependent DSBR pathway in C-NHEJ, highlighting unappreciated major roles for both XLF as well as the DSBR in V(D)J recombination, CSR and C-NHEJ in general. In this review, we discuss current knowledge of the mechanisms that contribute to the repair of DSBs generated during B lymphocyte development and activation with a focus on potential functionally redundant roles of XLF and ATM-dependent DSBR factors.     

Assigning and visualizing germline genes in antibody repertoires

Identifying the germline genes involved in immunoglobulin rearrangements is an essential first step in the analysis of antibody repertoires. Based on our prior work in analysing diverse recombinant viruses, we present IgSCUEAL (Immunoglobulin Subtype Classification Using Evolutionary ALgorithms), a phylogenetic approach to assign V and J regions of immunoglobulin sequences to their corresponding germline alleles, with D regions assigned using a simple pairwise alignment algorithm. We also develop an interactive web application for viewing the results, allowing the user to explore the frequency distribution of sequence assignments and CDR3 region length statistics, which is useful for summarizing repertoires, as well as a detailed viewer of rearrangements and region alignments for individual query sequences. We demonstrate the accuracy and utility of our method compared with sequence similarity-based approaches and other non-phylogenetic model-based approaches, using both simulated data and a set of evaluation datasets of human immunoglobulin heavy chain sequences. IgSCUEAL demonstrates the highest accuracy of V and J assignment amongst existing approaches, even when the reassorted sequence is highly mutated, and can successfully cluster sequences on the basis of shared V/J germline alleles.     

Generation of “LYmph Node Derived Antibody Libraries” (LYNDAL) for selecting fully human antibody fragments with therapeutic potential

The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro, the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)₂. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential.     

Selective expression of RAG-2 in chicken B cells undergoing immunoglobulin gene conversion

Chickens create their immunoglobulin (Ig) repertoires during B cell development in the bursa of Fabricius by intrachromosomal gene conversion. Recent evidence has suggested that Ig gene conversion may involve cis-acting DNA elements related to those involved in V(D)J recombination. Therefore, we have examined the potential role of the V(D)J recombination activating genes, RAG-1 and RAG-2, in regulating chicken Ig gene conversion. In contrast to the coexpression of RAG-1 and RAG-2 observed in mammalian B cells that undergo V(D)J recombination, chicken B cells isolated from the bursa of Fabricius express high levels of the RAG-2 mRNA but do not express RAG-1 mRNA. The developmental and phenotypic characteristics of the bursal lymphocytes and chicken B cell lines that express RAG-2 mRNA demonstrate that selective RAG-2 expression occurs specifically in B cells undergoing Ig diversification by gene conversion. These data suggest that RAG-2 plays a fundamental role in Ig-specific gene conversion.     

V(D)J recombinase-mediated processing of coding junctions at cryptic recombination signal sequences in peripheral T cells during human development

V(D)J recombinase mediates rearrangements at immune loci and cryptic recombination signal sequences (cRSS), resulting in a variety of genomic rearrangements in normal lymphocytes and leukemic cells from children and adults. The frequency at which these rearrangements occur and their potential pathologic consequences are developmentally dependent. To gain insight into V(D)J recombinase-mediated events during human development, we investigated 265 coding junctions associated with cRSS sites at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in peripheral T cells from 111 children during the late stages of fetal development through early adolescence. We observed a number of specific V(D)J recombinase processing features that were both age and gender dependent. In particular, TdT-mediated nucleotide insertions varied depending on age and gender, including percentage of coding junctions containing N-nucleotide inserts, predominance of GC nucleotides, and presence of inverted repeats (Pr-nucleotides) at processed coding ends. In addition, the extent of exonucleolytic processing of coding ends was inversely related to age. We also observed a coding-partner-dependent difference in exonucleolytic processing and an age-specific difference in the subtypes of V(D)J-mediated events. We investigated these age- and gender-specific differences with recombination signal information content analysis of the cRSS sites in the human HPRT locus to gain insight into the mechanisms mediating these developmentally specific V(D)J recombinase-mediated rearrangements in humans.     

IMonitor: A Robust Pipeline for TCR and BCR Repertoire Analysis

The advance of next generation sequencing (NGS) techniques provides an unprecedented opportunity to probe the enormous diversity of the immune repertoire by deep sequencing T-cell receptors (TCRs) and B-cell receptors (BCRs). However, an efficient and accurate analytical tool is still on demand to process the huge amount of data. We have developed a high-resolution analytical pipeline, Immune Monitor (“IMonitor”) to tackle this task. This method utilizes realignment to identify V(D)J genes and alleles after common local alignment. We compare IMonitor with other published tools by simulated and public rearranged sequences, and it demonstrates its superior performance in most aspects. Together with this, a methodology is developed to correct the PCR and sequencing errors and to minimize the PCR bias among various rearranged sequences with different V and J gene families. IMonitor provides general adaptation for sequences from all receptor chains of different species and outputs useful statistics and visualizations. In the final part of this article, we demonstrate its application on minimal residual disease detection in patients with B-cell acute lymphoblastic leukemia. In summary, this package would be of widespread usage for immune repertoire analysis.     

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to FcεRII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.     

ATM is not required in somatic hypermutation of VH,but is involved in the introduction of mutations in the switch mu region

Class switch recombination (CSR) and somatic hypermutation (SHM) are mechanistically related processes that share common key factors such as activation-induced cytidine deaminase. We have previously shown a role for ATM (mutated in ataxia-telangiectasia) in CSR. In this paper we show that the frequency, distribution, and nature of base pair substitutions in the Ig variable (V) heavy chain genes in ataxia-telangiectasia patients are largely similar to those in normal donors, suggesting a normal SHM process. Characterization of the third complementarity-determining region in B cells from ataxia-telangiectasia patients also shows a normal V(D)J recombination process. SHM-like mutations could be identified in the switch (S) mu region (up to several hundred base pairs upstream of the S mu -S(alpha) breakpoints) in normal in vivo switched human B cells. In the absence of ATM, mutations can still be found in this region, but at less than half the frequency of that in normal donors. The latter mutations are mainly due to transitions (86% compared with 58% in controls) and are biased to A or T nucleotides. An ATM-dependent mechanism, different from that generating SHM in V genes, is therefore likely to be involved in introducing SHM-like mutations in the S region. ATM may thus be one of the factors that is not shared by the CSR and SHM processes.     

Immune fingerprinting through repertoire similarity

Immune repertoires provide a unique fingerprint reflecting the immune history of individuals, with potential applications in precision medicine. However, the question of how personal that information is and how it can be used to identify individuals has not been explored. Here, we show that individuals can be uniquely identified from repertoires of just a few thousands lymphocytes. We present “Immprint,” a classifier using an information-theoretic measure of repertoire similarity to distinguish pairs of repertoire samples coming from the same versus different individuals. Using published T-cell receptor repertoires and statistical modeling, we tested its ability to identify individuals with great accuracy, including identical twins, by computing false positive and false negative rates < 10⁻⁶ from samples composed of 10,000 T-cells. We verified through longitudinal datasets that the method is robust to acute infections and that the immune fingerprint is stable for at least three years. These results emphasize the private and personal nature of repertoire data.     

Defective signal joint recombination in fanconi anemia fibroblasts reveals a role for Rad50 in V(D)J recombination

V(D)J recombination of immunoglobulin loci is dependent on the immune cell-specific Rag1 and Rag2 proteins as well as a number of ubiquitously expressed cellular DNA repair proteins that catalyze non-homologous end-joining of DNA double-strand breaks. The evolutionarily conserved Rad50/Mre11/Nibrin protein complex has a role in DNA double-strand break-repair, suggesting that these proteins, too, may participate in V(D)J recombination. Recent findings demonstrating that Rad50 function is defective in cells from patients afflicted with Fanconi anemia provide a possible mechanistic explanation for previous findings that lymphoblasts derived from these patients exhibit subtle defects in V(D)J recombination of extrachromosomal plasmid molecules. Here, we describe a series of findings that provide convincing evidence for a role of the Rad50 protein complex in V(D)J recombination. We found that the fidelity of V(D)J signal joint recombination in fibroblasts from patients afflicted with Fanconi anemia was reduced by nearly tenfold, compared to that observed in fibroblasts from normal donors. Second, we observed that antibody-mediated inhibition of the Rad50, Mre11, or Nibrin proteins reduced the fidelity of signal joint recombination significantly in wild-type cells. The latter finding was somewhat unexpected, because signal joint rejoining in cells from patients with Nijmegen breakage syndrome, which results from mutations in the Nibrin gene, occurs with normal fidelity. However, introduction of anti-Nibrin antibodies into these cells reduced the fidelity of signal joint recombination dramatically. These data reveal for the first time a role for the Rad50 complex in V(D)J recombination, and demonstrate that the protein product of the disease-causing allele responsible for Nijmegen breakage syndrome encodes a protein with residual DNA double-strand break repair activity.     

Characterization of human αβTCR repertoire and discovery of D-D fusion in TCRβ chains

The characterization of the human T-cell receptor (TCR) repertoire has made remarkable progress, with most of the work focusing on the TCRβ chains. Here, we analyzed the diversity and complexity of both the TCRα and TCRβ repertoires of three healthy donors. We found that the diversity of the TCRα repertoire is higher than that of the TCRβ repertoire, whereas the usages of the V and J genes tended to be preferential with similar TRAV and TRAJ patterns in all three donors. The V-J pairings, like the V and J gene usages, were slightly preferential. We also found that the TRDV1 gene rearranges with the majority of TRAJ genes, suggesting that TRDV1 is a shared TRAV/DV gene (TRAV42/DV1). Moreover, we uncovered the presence of tandem TRBD (TRB D gene) usage in ～2% of the productive human TCRβ CDR3 sequences.     

An evolutionary view of the mechanism for immune and genome diversity

An ortholog of activation-induced cytidine deaminase (AID) was, evolutionarily, the first enzyme to generate acquired immune diversity by catalyzing gene conversion and probably somatic hypermutation (SHM). AID began to mediate class switch recombination (CSR) only after the evolution of frogs. Recent studies revealed that the mechanisms for generating immune and genetic diversity share several critical features. Meiotic recombination, V(D)J recombination, CSR, and SHM all require H3K4 trimethyl histone modification to specify the target DNA. Genetic instability related to dinucleotide or triplet repeats depends on DNA cleavage by topoisomerase 1, which also initiates DNA cleavage in both SHM and CSR. These similarities suggest that AID hijacked the basic mechanism for genome instability when AID evolved in jawless fish. Thus, the risk of introducing genome instability into nonimmunoglobulin loci is unavoidable but tolerable compared with the advantage conferred on the host of being protected against pathogens by the enormous Ig diversification.     

Isolation of high affinity human antibodies directly from large synthetic repertoires.

Antibody fragments of moderate affinity (approximately microM) can be isolated from repertoires of approximately 10(8) immunoglobulin genes by phage display and rounds of selection with antigen, and the affinities improved by further rounds of mutation and selection. Here, as an alternative strategy, we attempted to isolate high affinity human antibodies directly from large repertoires. We first created highly diverse repertoires of heavy and light chains entirely in vitro from a bank of human V gene segments and then, by recombination of the repertoires in bacteria, generated a large (close to 6.5 x 10(10)) synthetic repertoire of Fab fragments displayed on filamentous phage. From this repertoire we isolated Fab fragments which bound to a range of different antigens and haptens, and with affinities comparable with those of antibodies from a secondary immune response in mice (up to 4 nM). Although the VH-26 (DP-47) segment was the most commonly used segment in both artificial and natural repertoires, there were also major differences in the pattern of segment usage. Such comparisons may help dissect the contributions of biological mechanisms and structural features governing V gene usage in vivo.     

Antibody repertoire analysis of mouse immunization protocols using microfluidics and molecular genomics

ABSTRACT

Immunization of mice followed by hybridoma or B-cell screening is one of the most common antibody discovery methods used to generate therapeutic monoclonal antibody (mAb) candidates. There are a multitude of different immunization protocols that can generate an immune response in animals. However, an extensive analysis of the antibody repertoires that these alternative immunization protocols can generate has not been performed. In this study, we immunized mice that transgenically express human antibodies with either programmed cell death 1 protein or cytotoxic T-lymphocyte associated protein 4 using four different immunization protocols, and then utilized a single cell microfluidic platform to generate tissue-specific, natively paired immunoglobulin (Ig) repertoires from each method and enriched for target-specific binders using yeast single-chain variable fragment (scFv) display. We deep sequenced the scFv repertoires from both the pre-sort and post-sort libraries. All methods and both targets yielded similar oligoclonality, variable (V) and joining (J) gene usage, and divergence from germline of enriched libraries. However, there were differences between targets and/or immunization protocols for overall clonal counts, complementarity-determining region 3 (CDR3) length, and antibody/CDR3 sequence diversity. Our data suggest that, although different immunization protocols may generate a response to an antigen, performing multiple immunization protocols in parallel can yield greater Ig diversity. We conclude that modern microfluidic methods, followed by an extensive molecular genomic analysis of antibody repertoires, can be used to quickly analyze new immunization protocols or mouse platforms.