Different classes of genomic inserts contribute to human antibody diversity.

Recombination of antibody genes in B cells can involve distant genomic loci and contribute a foreign antigen-binding element to form hybrid antibodies with broad reactivity for Plasmodium falciparum. So far, antibodies containing the extracellular domain of the LAIR1 and LILRB1 receptors represent unique examples of cross-chromosomal antibody diversification. Here, we devise a technique to profile non-VDJ elements from distant genes in antibody transcripts. Independent of the preexposure of donors to malaria parasites, non-VDJ inserts were detected in 80% of individuals at frequencies of 1 in 104 to 105 B cells. We detected insertions in heavy, but not in light chain or T cell receptor transcripts. We classify the insertions into four types depending on the insert origin and destination: 1) mitochondrial and 2) nuclear DNA inserts integrated at VDJ junctions; 3) inserts originating from telomere proximal genes; and 4) fragile sites incorporated between J-to-constant junctions. The latter class of inserts was exclusively found in memory and in in vitro activated B cells, while all other classes were already detected in naïve B cells. More than 10% of inserts preserved the reading frame, including transcripts with signs of antigen-driven affinity maturation. Collectively, our study unravels a mechanism of antibody diversification that is layered on the classical V(D)J and switch recombination.

Reconstructing Antibody Repertoires from Error-Prone Immunosequencing Reads.

Transforming error-prone immunosequencing datasets into Ab repertoires is a fundamental problem in immunogenomics, and a prerequisite for studies of immune responses. Although various repertoire reconstruction algorithms were released in the last 3 y, it remains unclear how to benchmark them and how to assess the accuracy of the reconstructed repertoires. We describe an accurate IgReC algorithm for constructing Ab repertoires from high-throughput immunosequencing datasets and a new framework for assessing the quality of reconstructed repertoires. Surprisingly, Ab repertoires constructed by IgReC from barcoded immunosequencing datasets in the blind mode (without using information about unique molecular identifiers) improved upon the repertoires constructed by the state-of-the-art tools that use barcoding. This finding suggests that IgReC may alleviate the need to generate repertoires using the barcoding technology (the workhorse of current immunogenomics efforts) because our computational approach to error correction of immunosequencing data is nearly as powerful as the experimental approach based on barcoding.

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.

Application of nonsense-mediated primer exclusion (NOPE) for preparation of unique molecular barcoded libraries.

BACKGROUND: Recently we proposed efficient method to exclude undesirable primers at any stage of amplification reaction, here termed NOPE (NOnsense-mediated Primer Exclusion). According to this method, added oligonucleotide overlapping with the 3'-end of unwanted amplification primer (NOPE oligo) simultaneously provides a template for its elongation. This elongation disrupts specificity of unwanted primer, preventing its further participation in PCR. The suggested approach allows to rationally manage the course of PCR reactions in order to facilitate analysis of complex DNA mixtures as well as to perform multistage PCR bypassing intermediate purification steps. RESULTS: Here we apply NOPE method to DNA library preparation for the high-throughput sequencing (HTS) with the PCR-based introduction of unique molecular identifiers (UMI). We show that NOPE oligo efficiently neutralizes UMI-containing oligonucleotides after introduction of UMI into sample DNA molecules, thus allowing to proceed with further amplification steps without purification and associated loss of starting material. At the same time, NOPE oligo does not affect the efficiency of target PCR amplification. CONCLUSION: We describe a simple, robust and cheap modification of UMI-labeled HTS libraries preparation procedure, that allows to bypass purification step and thus to preserve starting material which may be limited, e.g. circulating tumor DNA, circulating fetal DNA, or small amounts of isolated cells of interest. Furthermore, demonstrated simplicity and robustness of NOPE method should make it popular in various PCR protocols.

Towards error-free profiling of immune repertoires.

Deep profiling of antibody and T cell-receptor repertoires by means of high-throughput sequencing has become an attractive approach for adaptive immunity studies, but its power is substantially compromised by the accumulation of PCR and sequencing errors. Here we report MIGEC (molecular identifier groups-based error correction), a strategy for high-throughput sequencing data analysis. MIGEC allows for nearly absolute error correction while fully preserving the natural diversity of complex immune repertoires.

VDJtools: Unifying Post-analysis of T Cell Receptor Repertoires.

Despite the growing number of immune repertoire sequencing studies, the field still lacks software for analysis and comprehension of this high-dimensional data. Here we report VDJtools, a complementary software suite that solves a wide range of T cell receptor (TCR) repertoires post-analysis tasks, provides a detailed tabular output and publication-ready graphics, and is built on top of a flexible API. Using TCR datasets for a large cohort of unrelated healthy donors, twins, and multiple sclerosis patients we demonstrate that VDJtools greatly facilitates the analysis and leads to sound biological conclusions. VDJtools software and documentation are available at https://github.com/mikessh/vdjtools.

Age-related decrease in TCR repertoire diversity measured with deep and normalized sequence profiling.

The decrease of TCR diversity with aging has never been studied by direct methods. In this study, we combined high-throughput Illumina sequencing with unique cDNA molecular identifier technology to achieve deep and precisely normalized profiling of TCR ß repertoires in 39 healthy donors aged 6-90 y. We demonstrate that TCR ß diversity per 10(6) T cells decreases roughly linearly with age, with significant reduction already apparent by age 40. The percentage of naive T cells showed a strong correlation with measured TCR diversity and decreased linearly up to age 70. Remarkably, the oldest group (average age 82 y) was characterized by a higher percentage of naive CD4(+) T cells, lower abundance of expanded clones, and increased TCR diversity compared with the previous age group (average age 62 y), suggesting the influence of age selection and association of these three related parameters with longevity. Interestingly, cross-analysis of individual TCR ß repertoires revealed a set >10,000 of the most representative public TCR ß clonotypes, whose abundance among the top 100,000 clones correlated with TCR diversity and decreased with aging.

Pairing of T-cell receptor chains via emulsion PCR.

Our ability to analyze adaptive immunity and engineer its activity has long been constrained by our limited ability to identify native pairs of heavy-light antibody chains and alpha-beta T-cell receptor (TCR) chains--both of which comprise coupled "halves of a key", collectively capable of recognizing specific antigens. Here, we report a cell-based emulsion RT-PCR approach that allows the selective fusion of the native pairs of amplified TCR alpha and beta chain genes for complex samples. A new type of PCR suppression technique was developed that makes it possible to amplify the fused library with minimal noise for subsequent analysis by high-throughput paired-end Illumina sequencing. With this technique, single analysis of a complex blood sample allows identification of multiple native TCR chain pairs. This approach may be extended to identify native antibody chain pairs and, more generally, pairs of mRNA molecules that are coexpressed in the same living cells.

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.

Accounting for B-cell Behavior and Sampling Bias Predicts Anti-PD-L1 Response in Bladder Cancer.

Cancer immunotherapy is predominantly based on T cell-centric approaches. At the same time, the adaptive immune response in the tumor environment also includes clonally produced immunoglobulins and clonal effector/memory B cells that participate in antigen-specific decisions through their interactions with T cells. Here, we investigated the role of infiltrating B cells in bladder cancer via patient dataset analysis of intratumoral immunoglobulin repertoires. We showed that the IgG1/IgA ratio is a prognostic indicator for several subtypes of bladder cancer and for the whole IMVigor210 anti-PD-L1 immunotherapy study cohort. A high IgG1/IgA ratio associated with the prominence of a cytotoxic gene signature, T-cell receptor signaling, and IL21-mediated signaling. Immunoglobulin repertoire analysis indicated that effector B-cell function, rather than clonally produced antibodies, was involved in antitumor responses. From the T-cell side, we normalized a cytotoxic signature against the extent of immune cell infiltration to neutralize the artificial sampling-based variability in immune gene expression. Resulting metrics reflected proportion of cytotoxic cells among tumor-infiltrating immune cells and improved prediction of anti-PD-L1 responses. At the same time, the IgG1/IgA ratio remained an independent prognostic factor. Integration of the B-cell, natural killer cell, and T-cell signatures allowed for the most accurate prediction of anti-PD-L1 therapy responses. On the basis of these findings, we developed a predictor called PRedIctive MolecUlar Signature (PRIMUS), which outperformed PD-L1 expression scores and known gene signatures. Overall, PRIMUS allows for reliable identification of responders among patients with muscle-invasive urothelial carcinoma, including the subcohort with the low-infiltrated "desert" tumor phenotype.

Memory persistence and differentiation into antibody-secreting cells accompanied by positive selection in longitudinal BCR repertoires.

The stability and plasticity of B cell-mediated immune memory ensures the ability to respond to the repeated challenges. We have analyzed the longitudinal dynamics of immunoglobulin heavy chain repertoires from memory B cells, plasmablasts, and plasma cells from the peripheral blood of generally healthy volunteers. We reveal a high degree of clonal persistence in individual memory B cell subsets, with inter-individual convergence in memory and antibody-secreting cells (ASCs). ASC clonotypes demonstrate clonal relatedness to memory B cells, and are transient in peripheral blood. We identify two clusters of expanded clonal lineages with differing prevalence of memory B cells, isotypes, and persistence. Phylogenetic analysis revealed signs of reactivation of persisting memory B cell-enriched clonal lineages, accompanied by new rounds of affinity maturation during proliferation and differentiation into ASCs. Negative selection contributes to both persisting and reactivated lineages, preserving the functionality and specificity of B cell receptors (BCRs) to protect against current and future pathogens.

RNA-Seq-Based TCR Profiling Reveals Persistently Increased Intratumoral Clonality in Responders to Anti-PD-1 Therapy.

Substantial effort is being invested in the search for peripheral or intratumoral T cell receptor (TCR) repertoire features that could predict the response to immunotherapy. Here we demonstrate the utility of MiXCR software for TCR and immunoglobulin repertoire extraction from RNA-Seq data obtained from sorted tumor-infiltrating T and B cells. We use this approach to extract TCR repertoires from RNA-Seq data obtained from sorted tumor-infiltrating CD4+ and CD8+ T cells in an HKP1 (KrasG12Dp53-/-) syngeneic mouse model of lung cancer after anti-PD-1 treatment. For both subsets, we demonstrate decreased TCR diversity in response to therapy. At a later time point, repertoire diversity is restored in progressing disease but remains decreased in responders to therapy in both CD4+ and CD8+ subsets. These observations complement previous studies and suggest that stably increased intratumoral CD4+ and CD8+ T cell clonality after anti-PD-1/PD-L1 therapy could serve as a predictor of long-term response.

PiggyBac transposon tools for recessive screening identify B-cell lymphoma drivers in mice.

B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7, a transcription factor, and Phip, a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology.

Comparative Analysis of B-Cell Receptor Repertoires Induced by Live Yellow Fever Vaccine in Young and Middle-Age Donors.

Age-related changes can significantly alter the state of adaptive immune system and often lead to attenuated response to novel pathogens and vaccination. In present study we employed 5'RACE UMI-based full length and nearly error-free immunoglobulin profiling to compare plasma cell antibody repertoires in young (19-26 years) and middle-age (45-58 years) individuals vaccinated with a live yellow fever vaccine, modeling a newly encountered pathogen. Our analysis has revealed age-related differences in the responding antibody repertoire ranging from distinct IGH CDR3 repertoire properties to differences in somatic hypermutation intensity and efficiency and antibody lineage tree structure. Overall, our findings suggest that younger individuals respond with a more diverse antibody repertoire and employ a more efficient somatic hypermutation process than elder individuals in response to a newly encountered pathogen.

Preparing unbiased T-cell receptor and antibody cDNA libraries for the deep next generation sequencing profiling.

High-throughput sequencing has the power to reveal the nature of adaptive immunity as represented by the full complexity of T-cell receptor (TCR) and antibody (IG) repertoires, but is at present severely compromised by the quantitative bias, bottlenecks, and accumulated errors that inevitably occur in the course of library preparation and sequencing. Here we report an optimized protocol for the unbiased preparation of TCR and IG cDNA libraries for high-throughput sequencing, starting from thousands or millions of live cells in an investigated sample. Critical points to control are revealed, along with tips that allow researchers to minimize quantitative bias, accumulated errors, and cross-sample contamination at each stage, and to enhance the subsequent bioinformatic analysis. The protocol is simple, reliable, and can be performed in 1-2 days.

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.

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.

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.