A novel unconventional T cell population enriched in Crohn's disease.

OBJECTIVE: One of the current hypotheses to explain the proinflammatory immune response in IBD is a dysregulated T cell reaction to yet unknown intestinal antigens. As such, it may be possible to identify disease-associated T cell clonotypes by analysing the peripheral and intestinal T-cell receptor (TCR) repertoire of patients with IBD and controls. DESIGN: We performed bulk TCR repertoire profiling of both the TCR alpha and beta chains using high-throughput sequencing in peripheral blood samples of a total of 244 patients with IBD and healthy controls as well as from matched blood and intestinal tissue of 59 patients with IBD and disease controls. We further characterised specific T cell clonotypes via single-cell RNAseq. RESULTS: We identified a group of clonotypes, characterised by semi-invariant TCR alpha chains, to be significantly enriched in the blood of patients with Crohn's disease (CD) and particularly expanded in the CD8+ T cell population. Single-cell RNAseq data showed an innate-like phenotype of these cells, with a comparable gene expression to unconventional T cells such as mucosal associated invariant T and natural killer T (NKT) cells, but with distinct TCRs. CONCLUSIONS: We identified and characterised a subpopulation of unconventional Crohn-associated invariant T (CAIT) cells. Multiple evidence suggests these cells to be part of the NKT type II population. The potential implications of this population for CD or a subset thereof remain to be elucidated, and the immunophenotype and antigen reactivity of CAIT cells need further investigations in future studies.

The energy sensor AMPK orchestrates metabolic and translational adaptation in expanding T helper cells.

The importance of cellular metabolic adaptation in inducing robust T cell responses is well established. However, the mechanism by which T cells link information regarding nutrient supply to clonal expansion and effector function is still enigmatic. Herein, we report that the metabolic sensor adenosine monophosphate-activated protein kinase (AMPK) is a critical link between cellular energy demand and translational activity and, thus, orchestrates optimal expansion of T cells in vivo. AMPK deficiency did not affect T cell fate decision, activation, or T effector cell generation; however, the magnitude of T cell responses in murine in vivo models of T cell activation was markedly reduced. This impairment was global, as all T helper cell subsets were similarly sensitive to loss of AMPK which resulted in reduced T cell accumulation in peripheral organs and reduced disease severity in pathophysiologically as diverse models as T cell transfer colitis and allergic airway inflammation. T cell receptor repertoire analysis confirmed similar clonotype frequencies in different lymphoid organs, thereby supporting the concept of a quantitative impairment in clonal expansion rather than a skewed qualitative immune response. In line with these findings, in-depth metabolic analysis revealed a decrease in T cell oxidative metabolism, and gene set enrichment analysis indicated a major reduction in ribosomal biogenesis and mRNA translation in AMPK-deficient T cells. We, thus, provide evidence that through its interference with these delicate processes, AMPK orchestrates the quantitative, but not the qualitative, manifestation of primary T cell responses in vivo.

SeqURE - a new copy-capture based method for sequencing of unknown Retroposition events.

BACKGROUND: Retroelements (REs) occupy a significant part of all eukaryotic genomes including humans. The majority of retroelements in the human genome are inactive and unable to retrotranspose. Dozens of active copies are repressed in most normal tissues by various cellular mechanisms. These copies can become active in normal germline and brain tissues or in cancer, leading to new retroposition events. The consequences of such events and their role in normal cell functioning and carcinogenesis are not yet fully understood. If new insertions occur in a small portion of cells they can be found only with the use of specific methods based on RE enrichment and high-throughput sequencing. The downside of the high sensitivity of such methods is the presence of various artifacts imitating real insertions, which in many cases cannot be validated due to lack of the initial template DNA. For this reason, adequate assessment of rare (< 1%) subclonal cancer specific RE insertions is complicated. RESULTS: Here we describe a new copy-capture technique which we implemented in a method called SeqURE for Sequencing Unknown of Retroposition Events that allows for efficient and reliable identification of new genomic RE insertions. The method is based on the capture of copies of target molecules (copy-capture), selective amplification and sequencing of genomic regions adjacent to active RE insertions from both sides. Importantly, the template genomic DNA remains intact and can be used for validation experiments. In addition, we applied a novel system for testing method sensitivity and precisely showed the ability of the developed method to reliably detect insertions present in 1 out of 100 cells and a substantial portion of insertions present in 1 out of 1000 cells. Using advantages of the method we showed the absence of somatic Alu insertions in colorectal cancer samples bearing tumor-specific L1HS insertions. CONCLUSIONS: This study presents the first description and implementation of the copy-capture technique and provides the first methodological basis for the quantitative assessment of RE insertions present in a small portion of cells.

Aberrant Methylation of LINE-1 Transposable Elements: A Search for Cancer Biomarkers.

Cancer remains one of the main causes of human mortality despite significant progress in its diagnostics and therapy achieved in the past decade. Massive hypomethylation of retrotransposons, in particular LINE-1, is considered a hallmark of most malignant transformations as it results in the reactivation of retroelements and subsequent genomic instability. Accumulating data on LINE-1 aberrant methylation in different tumor types indicates its significant role in cancer initiation and progression. However, direct evidence that LINE-1 activation can be used as a cancer biomarker is still limited. The objective of this review was to critically evaluate the published results regarding the diagnostic/prognostic potential of the LINE-1 methylation status in cancer. Our analysis indicates that LINE-1 hypomethylation is a promising candidate biomarker of cancer development, which, however, needs validation in both clinical and laboratory studies to confirm its applicability to different cancer types and/or stages. As LINE-1 is present in multiple cell-free copies in blood, it has advantages over single-copy genes regarding perspectives of using its methylation status as an epigenetic cancer biomarker for cell-free DNA liquid biopsy.

Identification of Disease-associated Traits and Clonotypes in the T Cell Receptor Repertoire of Monozygotic Twins Affected by Inflammatory Bowel Diseases.

BACKGROUND AND AIMS: Intestinal inflammation in inflammatory bowel diseases [IBD] is thought to be T cell mediated and therefore dependent on the interaction between the T cell receptor [TCR] and human leukocyte antigen [HLA] proteins expressed on antigen presenting cells. The collection of all TCRs in one individual, known as the TCR repertoire, is characterised by enormous diversity and inter-individual variability. It was shown that healthy monozygotic [MZ] twins are more similar in their TCR repertoire than unrelated individuals. Therefore MZ twins, concordant or discordant for IBD, may be useful to identify disease-related and non-genetic factors in the TCR repertoire which could potentially be used as disease biomarkers. METHODS: Employing unique molecular barcoding that can distinguish between polymerase chain reaction [PCR] artefacts and true sequence variation, we performed deep TCRα and TCRß repertoire profiling of the peripheral blood of 28 MZ twin pairs from Denmark and Germany, 24 of whom were discordant and four concordant for IBD. RESULTS: We observed disease- and smoking-associated traits such as sharing, diversity and abundance of specific clonotypes in the TCR repertoire of IBD patients, and particularly in patients with active disease, compared with their healthy twins. CONCLUSIONS: Our findings identified TCR repertoire features specific for smokers and IBD patients, particularly when signs of disease activity were present. These findings are a first step towards the application of TCR repertoire analyses as a valuable tool to characterise inflammatory bowel diseases and to identify potential biomarkers and true disease causes.

An advanced enrichment method for rare somatic retroelement insertions sequencing.

BACKGROUND: There is increasing evidence that the transpositional activity of retroelements (REs) is not limited to germ line cells, but often occurs in tumor and normal somatic cells. Somatic transpositions were found in several human tissues and are especially typical for the brain. Several computational and experimental approaches for detection of somatic retroelement insertions was developed in the past few years. These approaches were successfully applied to detect somatic insertions in clonally expanded tumor cells. At the same time, identification of somatic insertions presented in small proportion of cells, such as neurons, remains a considerable challenge. RESULTS: In this study, we developed a normalization procedure for library enrichment by DNA sequences corresponding to rare somatic RE insertions. Two rounds of normalization increased the number of fragments adjacent to somatic REs in the sequenced sample by more than 26-fold, and the number of identified somatic REs was increased by 8-fold. CONCLUSIONS: The developed technique can be used in combination with vast majority of modern RE identification approaches and can dramatically increase their capacity to detect rare somatic RE insertions in different types of cells.

Quantitative profiling reveals minor changes of T cell receptor repertoire in response to subunit inactivated influenza vaccine.

Vaccination against influenza is widely used to protect against seasonal flu epidemic although its effectiveness is debated. Here we performed deep quantitative T cell receptor repertoire profiling in peripheral blood of a healthy volunteer in response to trivalent subunit influenza vaccine. We did not observe significant rebuilding of peripheral blood T cell receptors composition in response to vaccination. However, we found several clonotypes in memory T cell fraction that were undetectable before the vaccination and had a maximum concentration at day 45 after vaccine administration. These cells were found in lower concentration in the course of repertoire monitoring for two years period. Our observation suggests a potential for recruitment of only a limited number of new T cells after each seasonal influenza vaccination.

Dynamics of Individual T Cell Repertoires: From Cord Blood to Centenarians.

The diversity, architecture, and dynamics of the TCR repertoire largely determine our ability to effectively withstand infections and malignancies with minimal mistargeting of immune responses. In this study, we have employed deep TCRß repertoire sequencing with normalization based on unique molecular identifiers to explore the long-term dynamics of T cell immunity. We demonstrate remarkable stability of repertoire, where approximately half of all T cells in peripheral blood are represented by clones that persist and generally preserve their frequencies for 3 y. We further characterize the extremes of lifelong TCR repertoire evolution, analyzing samples ranging from umbilical cord blood to centenarian peripheral blood. We show that the fetal TCR repertoire, albeit structurally maintained within regulated borders due to the lower numbers of randomly added nucleotides, is not limited with respect to observed functional diversity. We reveal decreased efficiency of nonsense-mediated mRNA decay in umbilical cord blood, which may reflect specific regulatory mechanisms in development. Furthermore, we demonstrate that human TCR repertoires are functionally more similar at birth but diverge during life, and we track the lifelong behavior of CMV- and EBV-specific T cell clonotypes. Finally, we reveal gender differences in dynamics of TCR diversity constriction, which come to naught in the oldest age. Based on our data, we propose a more general explanation for the previous observations on the relationships between longevity and immunity.

High-throughput identification of antigen-specific TCRs by TCR gene capture.

The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.

Mother and child T cell receptor repertoires: deep profiling study.

The relationship between maternal and child immunity has been actively studied in the context of complications during pregnancy, autoimmune diseases, and haploidentical transplantation of hematopoietic stem cells and solid organs. Here, we have for the first time used high-throughput Illumina HiSeq sequencing to perform deep quantitative profiling of T cell receptor (TCR) repertoires for peripheral blood samples of three mothers and their six children. Advanced technology allowed accurate identification of 5 × 10(5) to 2 × 10(6) TCR beta clonotypes per individual. We performed comparative analysis of these TCR repertoires with the aim of revealing characteristic features that distinguish related mother-child pairs, such as relative TCR beta variable segment usage frequency and relative overlap of TCR beta complementarity-determining region 3 (CDR3) repertoires. We show that thymic selection essentially and similarly shapes the initial output of the TCR recombination machinery in both related and unrelated pairs, with minor effect from inherited differences. The achieved depth of TCR profiling also allowed us to test the hypothesis that mature T cells transferred across the placenta during pregnancy can expand and persist as functional microchimeric clones in their new host, using characteristic TCR beta CDR3 variants as clonal identifiers.

Next generation sequencing for TCR repertoire profiling: platform-specific features and correction algorithms.

The TCR repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next generation sequencing (NGS) is becoming a powerful tool for deep TCR profiling; yet, questions abound regarding the methodological approaches for sample preparation and correct data interpretation. Accumulated PCR and sequencing errors along with library preparation bottlenecks and uneven PCR efficiencies lead to information loss, biased quantification, and generation of huge artificial TCR diversity. Here, we compare Illumina, 454, and Ion Torrent platforms for individual TCR profiling, evaluate the rate and character of errors, and propose advanced platform-specific algorithms to correct massive sequencing data. These developments are applicable to a wide variety of next generation sequencing applications. We demonstrate that advanced correction allows the removal of the majority of artificial TCR diversity with concomitant rescue of most of the sequencing information. Thus, this correction enhances the accuracy of clonotype identification and quantification as well as overall TCR diversity measurements.

Quantitative tracking of T cell clones after haematopoietic stem cell transplantation.

Autologous haematopoietic stem cell transplantation is highly efficient for the treatment of systemic autoimmune diseases, but its consequences for the immune system remain poorly understood. Here, we describe an optimized RNA-based technology for unbiased amplification of T cell receptor beta-chain libraries and use it to perform the first detailed, quantitative tracking of T cell clones during 10 months after transplantation. We show that multiple clones survive the procedure, contribute to the immune response to activated infections, and form a new skewed and stable T cell receptor repertoire.