A revamped rat reference genome improves the discovery of genetic diversity in laboratory rats.

The seventh iteration of the reference genome assembly for Rattus norvegicus-mRatBN7.2-corrects numerous misplaced segments and reduces base-level errors by approximately 9-fold and increases contiguity by 290-fold compared with its predecessor. Gene annotations are now more complete, improving the mapping precision of genomic, transcriptomic, and proteomics datasets. We jointly analyzed 163 short-read whole-genome sequencing datasets representing 120 laboratory rat strains and substrains using mRatBN7.2. We defined ∼20.0 million sequence variations, of which 18,700 are predicted to potentially impact the function of 6,677 genes. We also generated a new rat genetic map from 1,893 heterogeneous stock rats and annotated transcription start sites and alternative polyadenylation sites. The mRatBN7.2 assembly, along with the extensive analysis of genomic variations among rat strains, enhances our understanding of the rat genome, providing researchers with an expanded resource for studies involving rats.

The Rat Genome Database: Genetic, Genomic, and Phenotypic Data Across Multiple Species.

The laboratory rat, Rattus norvegicus, is an important model of human health and disease, and experimental findings in the rat have relevance to human physiology and disease. The Rat Genome Database (RGD, https://rgd.mcw.edu) is a model organism database that provides access to a wide variety of curated rat data including disease associations, phenotypes, pathways, molecular functions, biological processes, cellular components, and chemical interactions for genes, quantitative trait loci, and strains. We present an overview of the database followed by specific examples that can be used to gain experience in employing RGD to explore the wealth of functional data available for the rat and other species. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Navigating the Rat Genome Database (RGD) home page Basic Protocol 2: Using the RGD search functions Basic Protocol 3: Searching for quantitative trait loci Basic Protocol 4: Using the RGD genome browser (JBrowse) to find phenotypic annotations Basic Protocol 5: Using OntoMate to find gene-disease data Basic Protocol 6: Using MOET to find gene-ontology enrichment Basic Protocol 7: Using OLGA to generate gene lists for analysis Basic Protocol 8: Using the GA tool to analyze ontology annotations for genes Basic Protocol 9: Using the RGD InterViewer tool to find protein interaction data Basic Protocol 10: Using the RGD Variant Visualizer tool to find genetic variant data Basic Protocol 11: Using the RGD Disease Portals to find disease, phenotype, and other information Basic Protocol 12: Using the RGD Phenotypes & Models Portal to find qualitative and quantitative phenotype data and other rat strain-related information Basic Protocol 13: Using the RGD Pathway Portal to find disease and phenotype data via molecular pathways.

A revamped rat reference genome improves the discovery of genetic diversity in laboratory rats.

The seventh iteration of the reference genome assembly for Rattus norvegicus-mRatBN7.2-corrects numerous misplaced segments and reduces base-level errors by approximately 9-fold and increases contiguity by 290-fold compared to its predecessor. Gene annotations are now more complete, significantly improving the mapping precision of genomic, transcriptomic, and proteomics data sets. We jointly analyzed 163 short-read whole genome sequencing datasets representing 120 laboratory rat strains and substrains using mRatBN7.2. We defined ~20.0 million sequence variations, of which 18.7 thousand are predicted to potentially impact the function of 6,677 genes. We also generated a new rat genetic map from 1,893 heterogeneous stock rats and annotated transcription start sites and alternative polyadenylation sites. The mRatBN7.2 assembly, along with the extensive analysis of genomic variations among rat strains, enhances our understanding of the rat genome, providing researchers with an expanded resource for studies involving rats.

Rare disease research resources at the Rat Genome Database.

Rare diseases individually affect relatively few people, but as a group they impact considerable numbers of people. The Rat Genome Database (https://rgd.mcw.edu) is a knowledgebase that offers resources for rare disease research. This includes disease definitions, genes, quantitative trail loci (QTLs), genetic variants, annotations to published literature, links to external resources, and more. One important resource is identifying relevant cell lines and rat strains that serve as models for disease research. Diseases, genes, and strains have report pages with consolidated data, and links to analysis tools. Utilizing these globally accessible resources for rare disease research, potentiating discovery of mechanisms and new treatments, can point researchers toward solutions to alleviate the suffering of those afflicted with these diseases.

2022 updates to the Rat Genome Database: a Findable, Accessible, Interoperable, and Reusable (FAIR) resource.

The Rat Genome Database (RGD, https://rgd.mcw.edu) has evolved from simply a resource for rat genetic markers, maps, and genes, by adding multiple genomic data types and extensive disease and phenotype annotations and developing tools to effectively mine, analyze, and visualize the available data, to empower investigators in their hypothesis-driven research. Leveraging its robust and flexible infrastructure, RGD has added data for human and eight other model organisms (mouse, 13-lined ground squirrel, chinchilla, naked mole-rat, dog, pig, African green monkey/vervet, and bonobo) besides rat to enhance its translational aspect. This article presents an overview of the database with the most recent additions to RGD's genome, variant, and quantitative phenotype data. We also briefly introduce Virtual Comparative Map (VCMap), an updated tool that explores synteny between species as an improvement to RGD's suite of tools, followed by a discussion regarding the refinements to the existing PhenoMiner tool that assists researchers in finding and comparing quantitative data across rat strains. Collectively, RGD focuses on providing a continuously improving, consistent, and high-quality data resource for researchers while advancing data reproducibility and fulfilling Findable, Accessible, Interoperable, and Reusable (FAIR) data principles.

Ontological Analysis of Coronavirus Associated Human Genes at the COVID-19 Disease Portal.

The COVID-19 pandemic stemmed a parallel upsurge in the scientific literature about SARS-CoV-2 infection and its health burden. The Rat Genome Database (RGD) created a COVID-19 Disease Portal to leverage information from the scientific literature. In the COVID-19 Portal, gene-disease associations are established by manual curation of PubMed literature. The portal contains data for nine ontologies related to COVID-19, an embedded enrichment analysis tool, as well as links to a toolkit. Using these information and tools, we performed analyses on the curated COVID-19 disease genes. As expected, Disease Ontology enrichment analysis showed that the COVID-19 gene set is highly enriched with coronavirus infectious disease and related diseases. However, other less related diseases were also highly enriched, such as liver and rheumatic diseases. Using the comparison heatmap tool, we found nearly 60 percent of the COVID-19 genes were associated with nervous system disease and 40 percent were associated with gastrointestinal disease. Our analysis confirms the role of the immune system in COVID-19 pathogenesis as shown by substantial enrichment of immune system related Gene Ontology terms. The information in RGD's COVID-19 disease portal can generate new hypotheses to potentiate novel therapies and prevention of acute and long-term complications of COVID-19.

MOET: a web-based gene set enrichment tool at the Rat Genome Database for multiontology and multispecies analyses.

Biological interpretation of a large amount of gene or protein data is complex. Ontology analysis tools are imperative in finding functional similarities through overrepresentation or enrichment of terms associated with the input gene or protein lists. However, most tools are limited by their ability to do ontology-specific and species-limited analyses. Furthermore, some enrichment tools are not updated frequently with recent information from databases, thus giving users inaccurate, outdated or uninformative data. Here, we present MOET or the Multi-Ontology Enrichment Tool (v.1 released in April 2019 and v.2 released in May 2021), an ontology analysis tool leveraging data that the Rat Genome Database (RGD) integrated from in-house expert curation and external databases including the National Center for Biotechnology Information (NCBI), Mouse Genome Informatics (MGI), The Kyoto Encyclopedia of Genes and Genomes (KEGG), The Gene Ontology Resource, UniProt-GOA, and others. Given a gene or protein list, MOET analysis identifies significantly overrepresented ontology terms using a hypergeometric test and provides nominal and Bonferroni corrected P-values and odds ratios for the overrepresented terms. The results are shown as a downloadable list of terms with and without Bonferroni correction, and a graph of the P-values and number of annotated genes for each term in the list. MOET can be accessed freely from https://rgd.mcw.edu/rgdweb/enrichment/start.html.

Interpreting Sequence Variation in PDAC-Predisposing Genes Using a Multi-Tier Annotation Approach Performed at the Gene, Patient, and Cohort Level.

We investigated germline variation in pancreatic ductal adenocarcinoma (PDAC) predisposition genes in 535 patients, using a custom-built panel and a new complementary bioinformatic approach. Our panel assessed genes belonging to DNA repair, cell cycle checkpoints, migration, and preneoplastic pancreatic conditions. Our bioinformatics approach integrated annotations of variants by using data derived from both germline and somatic references. This integrated approach with expanded evidence enabled us to consider patterns even among private mutations, supporting a functional role for certain alleles, which we believe enhances individualized medicine beyond classic gene-centric approaches. Concurrent evaluation of three levels of evidence, at the gene, sample, and cohort level, has not been previously done. Overall, we identified in PDAC patient germline samples, 12% with mutations previously observed in pancreatic cancers, 23% with mutations previously discovered by sequencing other human tumors, and 46% with mutations with germline associations to cancer. Non-polymorphic protein-coding pathogenic variants were found in 18.4% of patient samples. Moreover, among patients with metastatic PDAC, 16% carried at least one pathogenic variant, and this subgroup was found to have an improved overall survival (22.0 months versus 9.8; p=0.008) despite a higher pre-treatment CA19-9 level (p=0.02). Genetic alterations in DNA damage repair genes were associated with longer overall survival among patients who underwent resection surgery (92 months vs. 46; p=0.06). ATM alterations were associated with more frequent metastatic stage (p = 0.04) while patients with BRCA1 or BRCA2 alterations had improved overall survival (79 months vs. 39; p=0.05). We found that mutations in genes associated with chronic pancreatitis were more common in non-white patients (p<0.001) and associated with longer overall survival (52 months vs. 26; p=0.004), indicating the need for greater study of the relationship among these factors. More than 90% of patients were found to have variants of uncertain significance, which is higher than previously reported. Furthermore, we generated 3D models for selected mutant proteins, which suggested distinct mechanisms underlying their dysfunction, likely caused by genetic alterations. Notably, this type of information is not predictable from sequence alone, underscoring the value of structural bioinformatics to improve genomic interpretation. In conclusion, the variation in PDAC predisposition genes appears to be more extensive than anticipated. This information adds to the growing body of literature on the genomic landscape of PDAC and brings us closer to a more widespread use of precision medicine for this challenging disease.

RNA Sequencing Reveals Cancer-Associated Changes in Laryngeal Cells Exposed to Non-Acid Pepsin.

OBJECTIVE: Laryngopharyngeal reflux (LPR) is a common affliction that contributes to laryngeal inflammation, symptoms that impact quality of life, and life-threatening illnesses such as cancer. Effective treatment strategies for LPR are lacking. Pepsin is a proinflammatory and carcinogenic element of refluxate. Investigation of molecular pathways involved in pepsin-mediated damage may lead to identification of novel biomarkers and therapeutic targets for LPR. In this study, RNA sequencing was used to examine changes in human laryngeal epithelial cells following brief pepsin insult. Cells were immortalized to generate a model to aid future study of laryngeal injury and therapeutics. STUDY DESIGN: In vitro translational. METHODS: Laryngeal epithelial cells were cultured from a patient without signs or symptoms of LPR or laryngeal cancer. Cells were treated with 0.1 mg/ml pepsin for 1 hour or normal growth media (control) prior to RNA sequencing. Cells were immortalized via HPV E6/7 and characterized by microscopy, immunohistochemistry, G-banding, and soft agar assay. RESULTS: Three hundred ninety-seven genes exhibited differences in expression with pepsin treatment (P < .05). Pathway analysis revealed association with cancer and related signaling processes including dysregulation of cancer-associated molecules, Metastasis-Associated Lung Adenocarcinoma Transcript 1 and KRT82, and the long-noncoding RNA, lipoprotein receptor-related protein 1 (LRP1)-AS, which regulates the putative pepsin receptor LRP1. CONCLUSIONS: A single, brief exposure to pepsin activated cancer-associated signaling pathways in laryngeal cells in vitro, revealing novel mechanisms by which chronic reflux may contribute to carcinogenesis. The cell line developed herein represents a novel tool in which to investigate pepsin-dysregulated pathways identified by RNA sequencing and disparities of tumor proneness of laryngeal subsites. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:121-129, 2021.

Motor Neuron Generation from iPSCs from Identical Twins Discordant for Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder characterized by the loss of the upper and lower motor neurons. Approximately 10% of cases are caused by specific mutations in known genes, with the remaining cases having no known genetic link. As such, sporadic cases have been more difficult to model experimentally. Here, we describe the generation and differentiation of ALS induced pluripotent stem cells reprogrammed from discordant identical twins. Whole genome sequencing revealed no relevant mutations in known ALS-causing genes that differ between the twins. As protein aggregation is found in all ALS patients and is thought to contribute to motor neuron death, we sought to characterize the aggregation phenotype of the sporadic ALS induced pluripotent stem cells (iPSCs). Motor neurons from both twins had high levels of insoluble proteins that commonly aggregate in ALS that did not robustly change in response to exogenous glutamate. In contrast, established genetic ALS iPSC lines demonstrated insolubility in a protein- and genotype-dependent manner. Moreover, whereas the genetic ALS lines failed to induce autophagy after glutamate stress, motor neurons from both twins and independent controls did activate this protective pathway. Together, these data indicate that our unique model of sporadic ALS may provide key insights into disease pathology and highlight potential differences between sporadic and familial ALS.

Mitochondrial Metabolic Reprogramming by CD36 Signaling Drives Macrophage Inflammatory Responses.

RATIONALE: A hallmark of chronic inflammatory disorders is persistence of proinflammatory macrophages in diseased tissues. In atherosclerosis, this is associated with dyslipidemia and oxidative stress, but mechanisms linking these phenomena to macrophage activation remain incompletely understood. OBJECTIVE: To investigate mechanisms linking dyslipidemia, oxidative stress, and macrophage activation through modulation of immunometabolism and to explore therapeutic potential targeting specific metabolic pathways. METHODS AND RESULTS: Using a combination of biochemical, immunologic, and ex vivo cell metabolic studies, we report that CD36 mediates a mitochondrial metabolic switch from oxidative phosphorylation to superoxide production in response to its ligand, oxidized LDL (low-density lipoprotein). Mitochondrial-specific inhibition of superoxide inhibited oxidized LDL-induced NF-κB (nuclear factor-κB) activation and inflammatory cytokine generation. RNA sequencing, flow cytometry, 3H-labeled palmitic acid uptake, lipidomic analysis, confocal and electron microscopy imaging, and functional energetics revealed that oxidized LDL upregulated effectors of long-chain fatty acid uptake and mitochondrial import, while downregulating fatty acid oxidation and inhibiting ATP5A (ATP synthase F1 subunit alpha)-an electron transport chain component. The combined effect is long-chain fatty acid accumulation, alteration of mitochondrial structure and function, repurposing of the electron transport chain to superoxide production, and NF-κB activation. Apoe null mice challenged with high-fat diet showed similar metabolic changes in circulating Ly6C+ monocytes and peritoneal macrophages, along with increased CD36 expression. Moreover, mitochondrial reactive oxygen species were positively correlated with CD36 expression in aortic lesional macrophages. CONCLUSIONS: These findings reveal that oxidized LDL/CD36 signaling in macrophages links dysregulated fatty acid metabolism to oxidative stress from the mitochondria, which drives chronic inflammation. Thus, targeting to CD36 and its downstream effectors may serve as potential new strategies against chronic inflammatory diseases such as atherosclerosis.

Age-Based Dynamics of a Stable Circulating Cd8 T Cell Repertoire Component.

T-cell memory to pathogens can be envisioned as a receptor-based imprint of the pathogenic environment on the naïve repertoire of clonotypes. Recurrent exposures to a pathogen inform and reinforce memory, leading to a mature state. The complexity and temporal stability of this system in man is only beginning to be adequately described. We have been using a rank-frequency approach for quantitative analysis of CD8 T cell repertoires. Rank acts as a proxy for previous expansion, and rank-frequency, the number of clonotypes at a particular rank, as a proxy for abundance, with the relation of the two estimating the diversity of the system. Previous analyses of circulating antigen-experienced cytotoxic CD8 T-cell repertoires from adults have shown a complex two-component clonotype distribution. Here we show this is also the case for circulating CD8 T cells expressing the BV19 receptor chain from five adult subjects. When the repertoire characteristic of clonotype stability is added to the analysis, an inverse correlation between clonotype rank frequency and stability is observed. Clonotypes making up the second distributional component are stable; indicating that the circulation can be a depot of selected clonotypes. Temporal repertoire dynamics was further examined for influenza-specific T cells from children, middle-aged, and older adults. Taken together, these analyses describe a dynamic process of system development and aging, with increasing distributional complexity, leading to a stable circulating component, followed by loss of both complexity and stability.

Clinical heterogeneity of mitochondrial NAD kinase deficiency caused by a NADK2 start loss variant.

Mitochondrial NAD kinase deficiency (NADK2D, OMIM #615787) is a rare autosomal recessive disorder of NADPH biosynthesis that can cause hyperlysinemia and dienoyl-CoA reductase deficiency (DECRD, OMIM #616034). NADK2 deficiency has been reported in only three unrelated patients. Two had severe, unremitting disease; one died at 4 months and the other at 5 years of age. The third was a 10 year old female with CNS anomalies, ataxia, and incoordination. In two cases mutations in NADK2 have been demonstrated. Here, we report the fourth known case, a 15 year old female with normal intelligence and a mild clinical and biochemical phenotype presumably without DECRD. Her clinical symptoms, which are now stable, became evident at the age of 9 with the onset of decreased visual acuity, bilateral optic atrophy, nystagmus, episodic lower extremity weakness, peripheral neuropathy, and gait abnormalities. Plasma amino acid levels were within normal limits except for mean lysine and proline levels that were 3.7 and 2.5 times the upper limits of normal. Whole exome sequencing (WES) revealed homozygosity for a g.36241900 A>G p. Met1Val start loss mutation in the primary NADK2 transcript (NM_001085411.1) encoding the 442 amino acid isoform. This presumed hypomorphic mutation has not been previously reported and is absent from the v1000GP, EVS, and ExAC databases. Our patient's normal intelligence and stable disease expands the clinical heterogeneity and the prognosis associated with NADK2 deficiency. Our findings also clarify the mechanism underlying NADK2 deficiency and suggest that this disease should be ruled out in cases of hyperlysinemia, especially those with visual loss, and neurological phenotypes.

RNF213 variants in a child with PHACE syndrome and moyamoya vasculopathy.

Segmental infantile hemangiomas (IH) can be associated with congenital anomalies in a regional distribution. PHACE refers to large cervicofacial segmental IH in association with congenital anomalies of the aortic arch and medium-sized arteries of the head and neck, as well as structural anomalies of the posterior fossa and eye. A subset of PHACE patients have arterial anomalies that progress to moyamoya vasculopathy (MMV). MMV is defined as stenosis of the supraclinoid segment of the internal carotid arteries and/or their major branches, with subsequent development of a compensatory collateral vessel network. We describe a patient with MMV and segmental IH on the back and lower body who meets diagnostic criteria for PHACE based on a posterior segment eye anomaly and cerebral arterial anomalies. Whole exome sequencing demonstrated two inherited heterozygous variants in RNF213. Variants in RNF213 are associated with increased susceptibility to MMV. Our findings suggest that RNF213 variants may play a role in the development of MMV in patients with hemangioma syndromes associated with congenital cerebral arterial anomalies.

Structural and Mechanistic Implications of Rearrangement Frequencies within Human TCRBV Genes.

The T cell repertoire is a function of thymic V(D)J rearrangement and of peripheral selection. The mature repertoire embodies TCR sequences that are important for survival and can identify important structural aspects of the TCR. Analysis of the circulating TCRBV19 CD8 T cell repertoire showed that a majority of NDN-encoded CDR3 amino acid motifs start at CDR3 position four, well within the V region. Rearrangement at this position indicates that the DNA hairpin loop is not opened at the position adjacent to the recombination signal sequence, but rather is trimmed back three or more bases. In this article, we show that the rearrangement frequency distribution within the V region reveals selection on CDR3 position four. The selection is already established in single-positive CD8 thymocytes. Crystal structures reveal a possible basis for this selection due to the location of this residue in a bend that positions the remaining portion of CDR3 to interact with the peptide and MHC. Examination of other TCRBV families also shows selection for rearrangement within the V region of a number of genes and for CD8 and CD4 cells. The exact profile of rearrangement within the V region appears to be V gene specific. The frequent observation of side chains associated with turn motifs at CDR3 positions three and four fits with the structural need for a bend. The data are discussed in terms of the generation of a structural turn motif, the rearrangement mechanism, and selection of the repertoire on the peptide and MHC.

CDR3 motif generation and selection in the BV19-utilizing subset of the human CD8 T cell repertoire.

The amino acids at the V-J rearrangement junction of TCR are encoded by the D region and by N or P nucleotides. Together they comprise the NDN region, the specific pMHC selection surface of the TCR ß-chain. As an extension of our earlier work on the recall response to influenza M158-66 in HLA-A2 individuals, we have been analyzing the circulating BV19 CD8 T cell repertoires. We observed that NDN regions of the CDR3 often start at positions that are V-region encoded. Here we examine NDN encoded amino acid motifs of BV19 rearrangements in circulating CD8 T cells based on the CDR3 length, the CDR3 start position of the NDN, and the motif length. Motifs that start at V region-encoded positions could be expected to be CDR3 length independent as indeed is the case. Motifs that included sequential proline and glycine showed a CDR3 length independent distribution and examining codon usage indicates that a large proportion of these can be explained by P-nucleotide addition from the 5' end of the D region. Other examples of skewed codon usage were observed indicating possible additional rearrangement mechanisms. Another pattern of motif distributions was a shift of position along the CDR3 as a function of the CDR3 length. As these data were collected from an older healthy individual they can be used to model successful repertoire selection and to further define characteristics associated with a positive history of responses to pathogen exposures.

CDR3 clonotype and amino acid motif diversity of BV19 expressing circulating human CD8 T cells.

Generating a detailed description of human T cell repertoire diversity is an important goal in the study of human immunology. The circulation is the source of most T cells used for studies in humans. Here we use high throughput sequencing of TCR BV19 transcripts from CD8 T cells derived from unmanipulated PBMC from an older HLA-A2 individual to provide a quantitative and qualitative description of the clonotypic CDR3 nucleotide and amino acid composition of the TCR ß-chain from this subset of circulating CD8 T cells. Aggregated samples from six time points spanning ∼1.5 years were analyzed to smooth possible temporal fluctuation. BV19 encompasses the well studied RS-encoding clonotypes involved in recognition of the M1(58-66) epitope from influenza A in HLA-A2 individuals. The clonotype distribution was diverse, complex and self-similar. The amino acid composition was generally skewed in favor of glycines and there were specific amino acids observed at higher frequency at the NDN start position. The motif repertoire distribution was also diverse, complex and self-similar with respect to CDR3 length, NDN start and length.

The functional CD8 T cell memory recall repertoire responding to the influenza A M1(58-66) epitope is polyclonal and shows a complex clonotype distribution.

The CD8 memory T cell repertoire to the influenza A derived M1(58-66) epitope shows a restricted V genes and CDR3 sequences usage. The repertoire is highly polyclonal and the clonotype distribution has been described as consisting of two components, one showing a power law-like distribution and the other composed of a few clonotypes with a very high relative frequency. The question is whether the complex repertoire defined by its ability to flourish in a short term recall culture corresponded to functional cells. Here we show that there is a relation between expression of the degranulation marker CD107 and cytotoxicity or IFN-γ production in CD8 T cell lines and clones. We then examine recently degranulated CD8 cells from recall cultures from four middle aged HLA-A2 subjects and show that these functional cells are polyclonal. The clonotype distributions of the CD8(+)CD107(+) repertoires are complex in the same manner as previously reported. The clonotype composition of CD8(+)CD107(+) repertoires is also very similar to CD8 only repertoires, and to CD8(+)HLA-A2-M1(58-66) pentamer positive repertoires. We postulate that multiple exposures during childhood to this conserved influenza A epitope has generated a complex functional repertoire in HLA-A2 individuals.

Naive T cell repertoire skewing in HLA-A2 individuals by a specialized rearrangement mechanism results in public memory clonotypes.

How the naive T cell repertoire arises and forms the memory repertoire is still poorly understood. This relationship was analyzed by taking advantage of the focused TCR usage in HLA-A2-restricted CD8 memory T cell responses to influenza M1(58-66). We analyzed rearranged BV19 genes from CD8 single-positive thymocytes, a surrogate for the naive repertoire, from 10 HLA-A2 individuals. CDR3 amino acid sequences associated with response to influenza were observed at higher frequencies than expected by chance, an indicator of preselection. We propose that a rearrangement mechanism involving long P-nucleotide addition from the J2.7 region explains part of this increase. Special rearrangement mechanisms can result in identical T cells in different individuals, referred to as public responses. Indeed, the rearrangements utilizing long P nucleotide additions were commonly observed in the response to the M1(58-66) epitope in 30 HLA-A2 middle-aged adults. Thus, in addition to negative and positive selection, special rearrangement mechanisms may influence the composition of the naive repertoire, resulting in more robust responses to a pathogen in some individuals.