Mikhail V. Pogorelyy

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.

tcR: an R package for T cell receptor repertoire advanced data analysis

BackgroundThe Immunoglobulins (IG) and the T cell receptors (TR) play the key role in antigen recognition during the adaptive immune response. Recent progress in next-generation sequencing technologies has provided an opportunity for the deep T cell receptor repertoire profiling. However, a specialised software is required for the rational analysis of massive data generated by next-generation sequencing.ResultsHere we introduce tcR, a new R package, representing a platform for the advanced analysis of T cell receptor repertoires, which includes diversity measures, shared T cell receptor sequences identification, gene usage statistics computation and other widely used methods. The tool has proven its utility in recent research studies.ConclusionstcR is an R package for the advanced analysis of T cell receptor repertoires after primary TR sequences extraction from raw sequencing reads. The stable version can be directly installed from The Comprehensive R Archive Network (http://cran.r-project.org/mirrors.html). The source code and development version are available at tcR GitHub (http://imminfo.github.io/tcr/) along with the full documentation and typical usage examples.

Distinctive properties of identical twins' TCR repertoires revealed by high-throughput sequencing

Significance The power of adaptive immunity in humans is realized through the hypervariable molecules: the T-cell receptors (TCRs). Each of those is built from genetically encoded parts with the addition of random nucleotides finally forming individual TCR repertoire. Despite that the individual TCR repertoire potentially can include 1011–1014 different variants, substantially less molecules are found in a single individual. The particular genetic impact on the final set of TCR molecules is still poorly understood. In this study, for the first time to the best of our knowledge, we compare deep TCR repertoires of genetically identical twins. We found that, although TCR repertoires of any pair of individuals have the same amount of identical receptors, twin repertoires share certain specific features. Adaptive immunity in humans is provided by hypervariable Ig-like molecules on the surface of B and T cells. The final set of these molecules in each organism is formed under the influence of two forces: individual genetic traits and the environment, which includes the diverse spectra of alien and self-antigens. Here we assess the impact of individual genetic factors on the formation of the adaptive immunity by analyzing the T-cell receptor (TCR) repertoires of three pairs of monozygous twins by next-generation sequencing. Surprisingly, we found that an overlap between the TCR repertoires of monozygous twins is similar to an overlap between the TCR repertoires of nonrelated individuals. However, the number of identical complementary determining region 3 sequences in two individuals is significantly increased for twin pairs in the fraction of highly abundant TCR molecules, which is enriched by the antigen-experienced T cells. We found that the initial recruitment of particular TCR V genes for recombination and subsequent selection in the thymus is strictly determined by individual genetic factors. J genes of TCRs are selected randomly for recombination; however, the subsequent selection in the thymus gives preference to some α but not β J segments. These findings provide a deeper insight into the mechanism of TCR repertoire generation.

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 TCRβ 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.

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 × 105 to 2 × 106 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.

Persisting fetal clonotypes influence the structure and overlap of adult human T cell receptor repertoires

The diversity of T-cell receptors recognizing foreign pathogens is generated through a highly stochastic recombination process, making the independent production of the same sequence rare. Yet unrelated individuals do share receptors, which together constitute a “public” repertoire of abundant clonotypes. The TCR repertoire is initially formed prenatally, when the enzyme inserting random nucleotides is downregulated, producing a limited diversity subset. By statistically analyzing deep sequencing T-cell repertoire data from twins, unrelated individuals of various ages, and cord blood, we show that T-cell clones generated before birth persist and maintain high abundances in adult organisms for decades, slowly decaying with age. Our results suggest that large, low-diversity public clones are created during pre-natal life, and survive over long periods, providing the basis of the public repertoire.

Huge Overlap of Individual TCR Beta Repertoires.

It has been reported that human TCR repertoires commonly carry so-called public clonotypes – CDR3 variants that are often shared between individuals. Cross-comparison of individual immune repertoires has previously revealed the existence of a population of TCR beta CDR3 variants that are identical at the amino acid level for any two donors (1–3). The lower bound for the total overlap between any two given donors’ TCR beta repertoires within their CD8+ naive T cell subset has been estimated as ~14,000 identical amino acid CDR3 variants based on comparison of 200,000–600,000 individual TCR beta clonotypes (1). Here, we have used deep profiling data consisting of 1–2 × 106 individual TCR beta clonotypes that we obtained from healthy donors (4) to better estimate the total overlap between TCR beta repertoires for any two individuals. The apparent paradox is, that the deeper we sequence, the larger is the percentage of observed overlapping clonotypes between the two repertoires, since the number of possible element pairs between the two sets grows geometrically. To demonstrate this, we analyzed TCR beta repertoires for 12 unrelated pairs assembled from a total of nine human donors [adults and children, see Ref. (4) for details]. We plotted the number of identical variants found in samples of increasing size, with up to 106 unique CDR3 sequences randomly drawn from the repertoires of each individual in a given pair (Figure ​(Figure1).1). For every pair, the number of shared clonotypes grew geometrically with the arithmetic growth of the sample size (Figures ​(Figures1A–C,1A–C, colored lines); at maximum sequencing depth (~1 × 106 unique sequences/donor), we observed an average of ~72,000, 68,000, and 6,000 CDR3 variants that were respectively identical at the amino acid, amino acid only/non-nucleotide and nucleotide level. This exceeds previous estimates (1) by several-fold. The greatest overlap was between two donors from whom we obtained ~1 × 106 and 1.7 × 106 CDR3 variants, where we observed 113,000, 108,000, and 11,000 identical clonotypes at the amino acid, amino acid only/non-nucleotide and nucleotide level, respectively. Figure 1 Overlap of individual TCR beta CDR3 repertoires grows geometrically with the number of sequence pairs sampled. Plots indicate the number of shared sequences for 12 unrelated donor pairs in relation to sample size at the level of (A) all amino acid sequences, ... The lower bound on total individual TCR beta repertoire diversity has previously been estimated to be 5 × 106 unique clonotypes [Ref. (5) and our unpublished data]. With that in mind, we extrapolated our intersection curves by fitting them to a power law model [Y = aXb, as in Ref. (1)], which yielded coefficient “b” close to 2.0 and R2 > 0.999 for all cases (Figures ​(Figures1A–C,1A–C, dashed lines). We estimated that the total overlap of the TCR beta CDR3 repertoires for two individuals constitutes ~2,200,000, 2,060,000, and 180,000 variants, i.e. 44.1, 41.3, and 3.6% of a given individual’s sequence diversity at the amino acid, amino acid only/non-nucleotide, and nucleotide level, respectively. Thus, the real paradox is that nearly half of the TCR beta CDR3 repertoire is functionally identical between any two individuals, in spite of the fact that the theoretical diversity that can be achieved by TCR beta variants has been estimated to be ~5 × 1011 sequences (1, 6). The results from our extrapolation are direct and evident. We took numerous precautions to exclude contamination in our work, including sequencing of pair-analyzed donor repertoires in separate Illumina lanes (4). Even if contaminations were present, these would not affect overlap at the amino acid only/non-nucleotide level (Figure ​(Figure1B).1B). Furthermore, we performed CDR3 extraction and error correction with MiTCR (http://mitcr.milaboratory.com/) using the stringent ETE algorithm, which eliminates 98% of PCR and sequencing errors with minimal loss of natural TCR beta diversity (7). Such large overlap between individuals suggests the existence of a rather limited pool of frequently used functional CDR3 sequences. To further investigate this, we calculated the lower and upper bounds of the Chao richness estimate as described in Ref. (8) based on the numbers of singletons and doubletons (sequences observed in one and two individuals, respectively) in 12 paired donors’ samples. From this model, we obtained a confidence interval of 1.2 × 107 to 5.4 × 107 unique amino acid CDR3 sequences, at a significance level of α = 0.001. These findings represent a shift in our understanding of human adaptive immunity. It now appears likely that recombinatorial biases (3, 9) and thymic selection (4, 10, 11) shape our repertoires so tightly that the majority of TCR beta CDR3 variants expressed by naive T cells leaving the thymus are chosen from a “short-list” of just under 108 amino acid variants – even shorter than the 2 × 109 “effective sequence space” estimated by Robins and colleagues (1). Nevertheless, the repertoire has a complex structure and those clonotypes that are characterized as low-complexity [see figure 7 in Ref. (4)] predominantly form the backbone of the shared clonotype pool. Interestingly, when we examined the intersection of all nine donor samples, we found that the number of donors in which a given clonotype can be detected is distributed according to a power law, with a degree of −2.95 and R2 = 0.99 (Figure ​(Figure1D).1D). These findings confirm the fractal structure of the human TCR beta repertoire that determines the landscape of shared clonotypes (1–3, 12), and may reveal a more complex picture with the deeper profiling experiments.

The Evidence for Increased L1 Activity in the Site of Human Adult Brain Neurogenesis

Retroelement activity is a common source of polymorphisms in human genome. The mechanism whereby retroelements contribute to the intraindividual genetic heterogeneity by inserting into the DNA of somatic cells is gaining increasing attention. Brain tissues are suspected to accumulate genetic heterogeneity as a result of the retroelements somatic activity. This study aims to expand our understanding of the role retroelements play in generating somatic mosaicism of neural tissues. Whole-genome Alu and L1 profiling of genomic DNA extracted from the cerebellum, frontal cortex, subventricular zone, dentate gyrus, and the myocardium revealed hundreds of somatic insertions in each of the analyzed tissues. Interestingly, the highest concentration of such insertions was detected in the dentate gyrus—the hotspot of adult neurogenesis. Insertions of retroelements and their activity could produce genetically diverse neuronal subsets, which can be involved in hippocampal-dependent learning and memory.

Method for identification of condition-associated public antigen receptor sequences

Diverse repertoires of hypervariable immunoglobulin receptors (TCR and BCR) recognize antigens in the adaptive immune system. The development of immunoglobulin receptor repertoire sequencing methods makes it possible to perform repertoire-wide disease association studies of antigen receptor sequences. We developed a statistical framework for associating receptors to disease from only a small cohort of patients, with no need for a control cohort. Our method successfully identifies previously validated Cytomegalovirus and type one diabetes responsive TCRβ sequences .

Precise tracking of vaccine-responding T-cell clones reveals convergent and personalized response in identical twins

T-cell receptor (TCR) repertoire data contain information about infections that could be used in disease diagnostics and vaccine development, but extracting that information remains a major challenge. Here we developed a statistical framework to detect TCR clone proliferation and contraction from longitudinal repertoire data. We applied this framework to data from three pairs of identical twins immunized with the yellow fever vaccine. We identified 500-1500 responding TCRs in each donor and validated them using three independent assays. While the responding TCRs were mostly private, albeit with higher overlap between twins, they could be well predicted using a classifier based on sequence similarity. Our method can also be applied to samples obtained post-infection, making it suitable for systematic discovery of new infection-specific TCRs in the clinic.