Stitchr: stitching coding TCR nucleotide sequences from V/J/CDR3 information.

The study and manipulation of T cell receptors (TCRs) is central to multiple fields across basic and translational immunology research. Produced by V(D)J recombination, TCRs are often only recorded in the literature and data repositories as a combination of their V and J gene symbols, plus their hypervariable CDR3 amino acid sequence. However, numerous applications require full-length coding nucleotide sequences. Here we present Stitchr, a software tool developed to specifically address this limitation. Given minimal V/J/CDR3 information, Stitchr produces complete coding sequences representing a fully spliced TCR cDNA. Due to its modular design, Stitchr can be used for TCR engineering using either published germline or novel/modified variable and constant region sequences. Sequences produced by Stitchr were validated by synthesizing and transducing TCR sequences into Jurkat cells, recapitulating the expected antigen specificity of the parental TCR. Using a companion script, Thimble, we demonstrate that Stitchr can process a million TCRs in under ten minutes using a standard desktop personal computer. By systematizing the production and modification of TCR sequences, we propose that Stitchr will increase the speed, repeatability, and reproducibility of TCR research. Stitchr is available on GitHub.

Decombinator V4: an improved AIRR compliant-software package for T-cell receptor sequence annotation?

MOTIVATION: Analysis of the T-cell receptor repertoire is rapidly entering the general toolbox used by researchers interested in cellular immunity. The annotation of T-cell receptors (TCRs) from raw sequence data poses specific challenges, which arise from the fact that TCRs are not germline encoded, and because of the stochastic nature of the generating process. RESULTS: In this study, we report the release of Decombinator V4, a tool for the accurate and fast annotation of large sets of TCR sequences. Decombinator was one of the early Python software packages released to analyse the rapidly increasing flow of T-cell receptor repertoire sequence data. The Decombinator package now provides Python 3 compatibility, incorporates improved sequencing error and PCR bias correction algorithms, and provides output which conforms to the international standards proposed by the Adaptive Immune Receptor Repertoire Community. AVAILABILITY AND IMPLEMENTATION: The entire Decombinator suite is freely available at: https://github.com/innate2adaptive/Decombinator. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

High-throughput sequencing of the T-cell receptor repertoire: pitfalls and opportunities.

T-cell specificity is determined by the T-cell receptor, a heterodimeric protein coded for by an extremely diverse set of genes produced by imprecise somatic gene recombination. Massively parallel high-throughput sequencing allows millions of different T-cell receptor genes to be characterized from a single sample of blood or tissue. However, the extraordinary heterogeneity of the immune repertoire poses significant challenges for subsequent analysis of the data. We outline the major steps in processing of repertoire data, considering low-level processing of raw sequence files and high-level algorithms, which seek to extract biological or pathological information. The latest generation of bioinformatics tools allows millions of DNA sequences to be accurately and rapidly assigned to their respective variable V and J gene segments, and to reconstruct an almost error-free representation of the non-templated additions and deletions that occur. High-level processing can measure the diversity of the repertoire in different samples, quantify V and J usage and identify private and public T-cell receptors. Finally, we discuss the major challenge of linking T-cell receptor sequence to function, and specifically to antigen recognition. Sophisticated machine learning algorithms are being developed that can combine the paradoxical degeneracy and cross-reactivity of individual T-cell receptors with the specificity of the overall T-cell immune response. Computational analysis will provide the key to unlock the potential of the T-cell receptor repertoire to give insight into the fundamental biology of the adaptive immune system and to provide powerful biomarkers of disease.

Feature selection using a one dimensional naïve Bayes' classifier increases the accuracy of support vector machine classification of CDR3 repertoires.

Motivation: Somatic DNA recombination, the hallmark of vertebrate adaptive immunity, has the potential to generate a vast diversity of antigen receptor sequences. How this diversity captures antigen specificity remains incompletely understood. In this study we use high throughput sequencing to compare the global changes in T cell receptor ß chain complementarity determining region 3 (CDR3ß) sequences following immunization with ovalbumin administered with complete Freund's adjuvant (CFA) or CFA alone. Results: The CDR3ß sequences were deconstructed into short stretches of overlapping contiguous amino acids. The motifs were ranked according to a one-dimensional Bayesian classifier score comparing their frequency in the repertoires of the two immunization classes. The top ranking motifs were selected and used to create feature vectors which were used to train a support vector machine. The support vector machine achieved high classification scores in a leave-one-out validation test reaching >90% in some cases. Summary: The study describes a novel two-stage classification strategy combining a one-dimensional Bayesian classifier with a support vector machine. Using this approach we demonstrate that the frequency of a small number of linear motifs three amino acids in length can accurately identify a CD4 T cell response to ovalbumin against a background response to the complex mixture of antigens which characterize Complete Freund's Adjuvant. Availability and implementation: The sequence data is available at www.ncbi.nlm.nih.gov/sra/?term»SRP075893 . The Decombinator package is available at github.com/innate2adaptive/Decombinator . The R package e1071 is available at the CRAN repository https://cran.r-project.org/web/packages/e1071/index.html . Contact: b.chain@ucl.ac.uk. Supplementary information: Supplementary data are available at Bioinformatics online.

Accelerated Loss of TCR Repertoire Diversity in Common Variable Immunodeficiency.

Although common variable immunodeficiency (CVID) has long been considered as a group of primary Ab deficiencies, growing experimental data now suggest a global disruption of the entire adaptive immune response in a segment of patients. Oligoclonality of the TCR repertoire was previously demonstrated; however, the manner in which it relates to other B cell and T cell findings reported in CVID remains unclear. Using a combination approach of high-throughput TCRß sequencing and multiparametric flow cytometry, we compared the TCR repertoire diversity between various subgroups of CVID patients according to their B cell immunophenotypes. Our data suggest that the reduction in repertoire diversity is predominantly restricted to those patients with severely reduced class-switched memory B cells and an elevated level of CD21(lo) B cells (Freiburg 1a), and may be driven by a reduced number of naive T cells unmasking underlying memory clonality. Moreover, our data indicate that this loss in repertoire diversity progresses with advancing age far exceeding the expected physiological rate. Radiological evidence supports the loss in thymic volume, correlating with the decrease in repertoire diversity. Evidence now suggests that primary thymic failure along with other well-described B cell abnormalities play an important role in the pathophysiology in Freiburg group 1a patients. Clinically, our findings emphasize the integration of combined B and T cell testing to identify those patients at the greatest risk for infection. Future work should focus on investigating the link between thymic failure and the severe reduction in class-switched memory B cells, while gathering longitudinal laboratory data to examine the progressive nature of the disease.

Immune dysregulation in immunodeficiency disorders: The role of T-cell receptor sequencing.

Immune dysregulation is a prominent feature of primary immunodeficiency disorders, which commonly manifested as autoimmunity, cytopenias and inflammatory bowel disease. In partial T-cell immunodeficiency disorders, it has been proposed that the imbalance between effector and regulatory T-cells drives the breakdown of peripheral tolerance. While there is no robust test for immune dysregulation, the T-cell receptor repertoire is used as a surrogate marker, and has been shown to be perturbed in a number of immunodeficiency disorders featuring immune dysregulation including Omenn's Syndrome, Wiskott-Aldrich Syndrome, and common variable immunodeficiency. This review discusses how recent advances in TCR next-generation sequencing and bioinformatics have led to the in-depth characterization of CDR3 sequences and an exponential growth in examinable parameters. Specifically, we highlight the use of junctional diversity as a means to differentiate intrinsic T-cell defects from secondary causes of repertoire perturbation in primary immunodeficiency disorders. However, key questions, such as the identity of antigenic targets for large, expanded T-cell clonotypes, remain unanswered despite the fact that such clones are likely to play a pathogenic role in driving immune dysregulation and autoimmunity. Finally, we discuss a number of emerging technologies such as in silico reconstruction, high-throughput pairwise αß sequencing and single-cell RNAseq that offer the potential to define the antigenic epitope and function of a given T-cell, thereby enhancing our understanding in this field.

The sequence of sequencers: The history of sequencing DNA.

Determining the order of nucleic acid residues in biological samples is an integral component of a wide variety of research applications. Over the last fifty years large numbers of researchers have applied themselves to the production of techniques and technologies to facilitate this feat, sequencing DNA and RNA molecules. This time-scale has witnessed tremendous changes, moving from sequencing short oligonucleotides to millions of bases, from struggling towards the deduction of the coding sequence of a single gene to rapid and widely available whole genome sequencing. This article traverses those years, iterating through the different generations of sequencing technology, highlighting some of the key discoveries, researchers, and sequences along the way.

Decombinator: a tool for fast, efficient gene assignment in T-cell receptor sequences using a finite state machine.

SUMMARY: High-throughput sequencing provides an opportunity to analyse the repertoire of antigen-specific receptors with an unprecedented breadth and depth. However, the quantity of raw data produced by this technology requires efficient ways to categorize and store the output for subsequent analysis. To this end, we have defined a simple five-item identifier that uniquely and unambiguously defines each TcR sequence. We then describe a novel application of finite-state automaton to map Illumina short-read sequence data for individual TcRs to their respective identifier. An extension of the standard algorithm is also described, which allows for the presence of single-base pair mismatches arising from sequencing error. The software package, named Decombinator, is tested first on a set of artificial in silico sequences and then on a set of published human TcR-ß sequences. Decombinator assigned sequences at a rate more than two orders of magnitude faster than that achieved by classical pairwise alignment algorithms, and with a high degree of accuracy (>88%), even after introducing up to 1% error rates in the in silico sequences. Analysis of the published sequence dataset highlighted the strong V and J usage bias observed in the human peripheral blood repertoire, which seems to be unconnected to antigen exposure. The analysis also highlighted the enormous size of the available repertoire and the challenge of obtaining a comprehensive description for it. The Decombinator package will be a valuable tool for further in-depth analysis of the T-cell repertoire. AVAILABILITY AND IMPLEMENTATION: The Decombinator package is implemented in Python (v2.6) and is freely available at https://github.com/uclinfectionimmunity/Decombinator along with full documentation and examples of typical usage.

Hypofractionated Radiotherapy-Related Lymphopenia Is Associated With Worse Survival in Unresectable Intrahepatic Cholangiocarcinoma.

OBJECTIVE: The aim of this study was to evaluate the incidence of radiotherapy (RT)-related lymphopenia, its predictors, and association with survival in unresectable intrahepatic cholangiocarcinoma (ICC) treated with hypofractionated-RT (HF-RT). METHODS: Retrospective analysis of 96 patients with unresectable ICC who underwent HF-RT (median 58.05 Gy in 15 fractions) between 2009 and 2022 was performed. Absolute lymphocyte count (ALC) nadir within 12 weeks of RT was analyzed. Primary variable of interest was severe lymphopenia, defined as Grade 3+ (ALC <0.5 k/µL) per CTCAE v5.0. Primary outcome of interest was overall survival (OS) from RT. RESULTS: Median follow-up was 16 months. Fifty-two percent of patients had chemotherapy pre-RT, 23% during RT, and 40% post-RT. Pre-RT, median ALC was 1.1 k/µL and 5% had severe lymphopenia. Post-RT, 68% developed RT-related severe lymphopenia. Patients who developed severe lymphopenia had a significantly lower pre-RT ALC (median 1.1 vs. 1.5 k/µL, P =0.01) and larger target tumor volume (median 125 vs. 62 cm 3 , P =0.02). In our multivariable Cox model, severe lymphopenia was associated with a 1.7-fold increased risk of death ( P =0.04); 1-year OS rates were 63% vs 77% ( P =0.03). Receipt of photon versus proton-based RT (OR=3.50, P =0.02), higher mean liver dose (OR=1.19, P <0.01), and longer RT duration (OR=1.49, P =0.02) predicted severe lymphopenia. CONCLUSIONS: HF-RT-related lymphopenia is an independent prognostic factor for survival in patients with unresectable ICC. Patients with lower baseline ALC and larger tumor volume may be at increased risk, and use of proton therapy, minimizing mean liver dose, and avoiding treatment breaks may reduce RT-related lymphopenia.

AIRR-C IG Reference Sets: curated sets of immunoglobulin heavy and light chain germline genes.

Introduction: Analysis of an individual's immunoglobulin (IG) gene repertoire requires the use of high-quality germline gene reference sets. When sets only contain alleles supported by strong evidence, AIRR sequencing (AIRR-seq) data analysis is more accurate and studies of the evolution of IG genes, their allelic variants and the expressed immune repertoire is therefore facilitated. Methods: The Adaptive Immune Receptor Repertoire Community (AIRR-C) IG Reference Sets have been developed by including only human IG heavy and light chain alleles that have been confirmed by evidence from multiple high-quality sources. To further improve AIRR-seq analysis, some alleles have been extended to deal with short 3' or 5' truncations that can lead them to be overlooked by alignment utilities. To avoid other challenges for analysis programs, exact paralogs (e.g. IGHV1-69*01 and IGHV1-69D*01) are only represented once in each set, though alternative sequence names are noted in accompanying metadata. Results and discussion: The Reference Sets include less than half the previously recognised IG alleles (e.g. just 198 IGHV sequences), and also include a number of novel alleles: 8 IGHV alleles, 2 IGKV alleles and 5 IGLV alleles. Despite their smaller sizes, erroneous calls were eliminated, and excellent coverage was achieved when a set of repertoires comprising over 4 million V(D)J rearrangements from 99 individuals were analyzed using the Sets. The version-tracked AIRR-C IG Reference Sets are freely available at the OGRDB website (https://ogrdb.airr-community.org/germline_sets/Human) and will be regularly updated to include newly observed and previously reported sequences that can be confirmed by new high-quality data.

Antibody-Peptide Epitope Conjugates for Personalized Cancer Therapy.

Antibody-peptide epitope conjugates (APEC) are a new class of modified antibody-drug conjugates that redirect T-cell viral immunity against tumor cells. APECs contain a tumor-specific protease cleavage site linked to a patient-specific viral epitope, resulting in presentation of viral epitopes on cancer cells and subsequent recruitment and killing by CD8+ T cells. Here we developed an experimental pipeline to create patient-specific APECs and identified new preclinical therapies for ovarian carcinoma. Using functional assessment of viral peptide antigen responses to common viruses like cytomegalovirus (CMV) in patients with ovarian cancer, a library of 192 APECs with distinct protease cleavage sequences was created using the anti-epithelial cell adhesion molecule (EpCAM) antibody. Each APEC was tested for in vitro cancer cell killing, and top candidates were screened for killing xenograft tumors grown in zebrafish and mice. These preclinical modeling studies identified EpCAM-MMP7-CMV APEC (EpCAM-MC) as a potential new immunotherapy for ovarian carcinoma. Importantly, EpCAM-MC also demonstrated robust T-cell responses in primary ovarian carcinoma patient ascites samples. This work highlights a robust, customizable platform to rapidly develop patient-specific APECs. SIGNIFICANCE: This study develops a high-throughput preclinical platform to identify patient-specific antibody-peptide epitope conjugates that target cancer cells and demonstrates the potential of this immunotherapy approach for treating ovarian carcinoma.

Persistent T Cell Repertoire Perturbation and T Cell Activation in HIV After Long Term Treatment.

Objective: In people living with HIV (PLHIV), we sought to test the hypothesis that long term anti-retroviral therapy restores the normal T cell repertoire, and investigate the functional relationship of residual repertoire abnormalities to persistent immune system dysregulation. Methods: We conducted a case-control study in PLHIV and HIV-negative volunteers, of circulating T cell receptor repertoires and whole blood transcriptomes by RNA sequencing, complemented by metadata from routinely collected health care records. Results: T cell receptor sequencing revealed persistent abnormalities in the clonal T cell repertoire of PLHIV, characterized by reduced repertoire diversity and oligoclonal T cell expansion correlated with elevated CD8 T cell counts. We found no evidence that these expansions were driven by cytomegalovirus or another common antigen. Increased frequency of long CDR3 sequences and reduced frequency of public sequences among the expanded clones implicated abnormal thymic selection as a contributing factor. These abnormalities in the repertoire correlated with systems level evidence of persistent T cell activation in genome-wide blood transcriptomes. Conclusions: The diversity of T cell receptor repertoires in PLHIV on long term anti-retroviral therapy remains significantly depleted, and skewed by idiosyncratic clones, partly attributable to altered thymic output and associated with T cell mediated chronic immune activation. Further investigation of thymic function and the antigenic drivers of T cell clonal selection in PLHIV are critical to efforts to fully re-establish normal immune function.

The naive T-cell receptor repertoire has an extremely broad distribution of clone sizes.

The clone size distribution of the human naive T-cell receptor (TCR) repertoire is an important determinant of adaptive immunity. We estimated the abundance of TCR sequences in samples of naive T cells from blood using an accurate quantitative sequencing protocol. We observe most TCR sequences only once, consistent with the enormous diversity of the repertoire. However, a substantial number of sequences were observed multiple times. We detect abundant TCR sequences even after exclusion of methodological confounders such as sort contamination, and multiple mRNA sampling from the same cell. By combining experimental data with predictions from models we describe two mechanisms contributing to TCR sequence abundance. TCRα abundant sequences can be primarily attributed to many identical recombination events in different cells, while abundant TCRß sequences are primarily derived from large clones, which make up a small percentage of the naive repertoire, and could be established early in the development of the T-cell repertoire.

Massively parallel interrogation and mining of natively paired human TCRαß repertoires.

T cells engineered to express antigen-specific T cell receptors (TCRs) are potent therapies for viral infections and cancer. However, efficient identification of clinical candidate TCRs is complicated by the size and complexity of T cell repertoires and the challenges of working with primary T cells. Here we present a high-throughput method to identify TCRs with high functional avidity from diverse human T cell repertoires. The approach used massively parallel microfluidics to generate libraries of natively paired, full-length TCRαß clones, from millions of primary T cells, which were then expressed in Jurkat cells. The TCRαß-Jurkat libraries enabled repeated screening and panning for antigen-reactive TCRs using peptide major histocompatibility complex binding and cellular activation. We captured more than 2.9 million natively paired TCRαß clonotypes from six healthy human donors and identified rare (<0.001% frequency) viral-antigen-reactive TCRs. We also mined a tumor-infiltrating lymphocyte sample from a patient with melanoma and identified several tumor-specific TCRs, which, after expression in primary T cells, led to tumor cell killing.

Antibody-mediated delivery of viral epitopes to tumors harnesses CMV-specific T cells for cancer therapy.

Several cancer immunotherapy approaches, such as immune checkpoint blockade and adoptive T-cell therapy, boost T-cell activity against the tumor, but these strategies are not effective in the absence of T cells specific for displayed tumor antigens. Here we outline an immunotherapy in which endogenous T cells specific for a noncancer antigen are retargeted to attack tumors. The approach relies on the use of antibody-peptide epitope conjugates (APECs) to deliver suitable antigens to the tumor surface for presention by HLA-I. To retarget cytomegalovirus (CMV)-specific CD8+ T cells against tumors, we used APECs containing CMV-derived epitopes conjugated to tumor-targeting antibodies via metalloprotease-sensitive linkers. These APECs redirect pre-existing CMV immunity against tumor cells in vitro and in mouse cancer models. In vitro, APECs activated specifically CMV-reactive effector T cells whereas a bispecific T-cell engager activated both effector and regulatory T cells. Our approach may provide an effective alternative in cancers that are not amenable to checkpoint inhibitors or other immunotherapies.

An Economical, Quantitative, and Robust Protocol for High-Throughput T Cell Receptor Sequencing from Tumor or Blood.

The T cell receptor repertoire provides a window to the cellular adaptive immune response within a tumor, and has the potential to identify specific and personalized biomarkers for tracking host responses during cancer therapy, including immunotherapy. We describe a protocol for amplifying, sequencing, and analyzing T cell receptors which is economical, robust, sensitive, and versatile. The key experimental step is the ligation of a single-stranded oligonucleotide to the 3' end of the T cell receptor cDNA, which allows easy amplification of all possible rearrangements using only a single set of primers per locus, while simultaneously introducing a unique molecular identifier to label each starting cDNA molecule. After sequencing, this molecular identifier can be used to correct both sequence errors and the effects of differential PCR amplification efficiency, thus producing a more accurate measure of the true T cell receptor frequency within the sample. This method has been applied to the analysis of unfractionated human tumor lysates, subpopulations of tumor-infiltrating lymphocytes, and peripheral blood samples from patients with a variety of solid tumors.

Extranuclear DNA accumulates in aged cells and contributes to senescence and inflammation.

Systemic inflammation is central to aging-related conditions. However, the intrinsic factors that induce inflammation are not well understood. We previously identified a cell-autonomous pathway through which damaged nuclear DNA is trafficked to the cytosol where it activates innate cytosolic DNA sensors that trigger inflammation. These results led us to hypothesize that DNA released after cumulative damage contributes to persistent inflammation in aging cells through a similar mechanism. Consistent with this notion, we found that older cells harbored higher levels of extranuclear DNA compared to younger cells. Extranuclear DNA was exported by a leptomycin B-sensitive process, degraded through the autophagosome-lysosomal pathway and triggered innate immune responses through the DNA-sensing cGAS-STING pathway. Patient cells from the aging diseases ataxia and progeria also displayed extranuclear DNA accumulation, increased pIRF3 and pTBK1, and STING-dependent p16 expression. Removing extranuclear DNA in old cells using DNASE2A reduced innate immune responses and senescence-associated (SA) ß-gal enzyme activity. Cells and tissues of Dnase2a-/- mice with defective DNA degradation exhibited slower growth, higher activity of ß-gal, or increased expression of HP-1ß and p16 proteins, while Dnase2a-/- ;Sting-/- cells and tissues were rescued from these phenotypes, supporting a role for extranuclear DNA in senescence. We hypothesize a direct role for excess DNA in aging-related inflammation and in replicative senescence, and propose DNA degradation as a therapeutic approach to remove intrinsic DNA and revert inflammation associated with aging.

Aberrant X chromosome skewing and acquired clonal hematopoiesis in adult-onset common variable immunodeficiency.

Advances in genomic medicine have elucidated an increasing number of genetic etiologies for patients with common variable immunodeficiency (CVID). However, there is heterogeneity in clinical and immunophenotypic presentations and a limited understanding of the underlying pathophysiology of many cases. The primary defects in CVID may extend beyond the adaptive immune system, and the combined defect in both the myeloid and lymphoid compartments suggests the mechanism may involve bone marrow output and earlier progenitors. Using the methylation profile of the human androgen receptor (AR) gene as a surrogate epigenetic marker for bone marrow clonality, we examined the hematopoietic compartments of patients with CVID. Our data show that clonal hematopoiesis is common among patients with adult-onset CVID who do not have associated noninfectious complications. Nonblood tissues did not show a skewed AR methylation status, supporting a model of an acquired clonal hematopoietic event. Attenuation of memory B cell differentiation into long-lived plasma cells (CD20-CD27+CD38+CD138+) was associated with marked changes in the postdifferentiation methylation profile, demonstrating the functional consequence of clonal hematopoiesis on humoral immunity in these patients. This study sheds light on a potential etiology of a subset of patients with CVID, and the findings suggest that it is a stage of an acquired lymphocyte maturation disorder.

Murine xenograft bioreactors for human immunopeptidome discovery.

The study of peptides presented by MHC class I and class II molecules is limited by the need for relatively large cell numbers, especially when studying post-translationally modified or otherwise rare peptide species. To overcome this problem, we pose the hypothesis that human cells grown as xenografts in immunodeficient mice should produce equivalent immunopeptidomes as cultured cells. Comparing human cell lines grown either in vitro or as murine xenografts, we show that the immunopeptidome is substantially preserved. Numerous features are shared across both sample types, including peptides and proteins featured, length distributions, and HLA-binding motifs. Peptides well-represented in both groups were from more abundant proteins, or those with stronger predicted HLA binding affinities. Samples grown in vivo also recapitulated a similar phospho-immunopeptidome, with common sequences being those found at high copy number on the cell surface. These data indicate that xenografts are indeed a viable methodology for the production of cells for immunopeptidomic discovery.