Memory CD4+ T cells are generated in the human fetal intestine.

The fetus is thought to be protected from exposure to foreign antigens, yet CD45RO+ T cells reside in the fetal intestine. Here we combined functional assays with mass cytometry, single-cell RNA sequencing and high-throughput T cell antigen receptor (TCR) sequencing to characterize the CD4+ T cell compartment in the human fetal intestine. We identified 22 CD4+ T cell clusters, including naive-like, regulatory-like and memory-like subpopulations, which were confirmed and further characterized at the transcriptional level. Memory-like CD4+ T cells had high expression of Ki-67, indicative of cell division, and CD5, a surrogate marker of TCR avidity, and produced the cytokines IFN-γ and IL-2. Pathway analysis revealed a differentiation trajectory associated with cellular activation and proinflammatory effector functions, and TCR repertoire analysis indicated clonal expansions, distinct repertoire characteristics and interconnections between subpopulations of memory-like CD4+ T cells. Imaging mass cytometry indicated that memory-like CD4+ T cells colocalized with antigen-presenting cells. Collectively, these results provide evidence for the generation of memory-like CD4+ T cells in the human fetal intestine that is consistent with exposure to foreign antigens.

Ultrasensitive allele inference from immune repertoire sequencing data with MiXCR.

Allelic variability in the adaptive immune receptor loci, which harbor the gene segments that encode B cell and T cell receptors (BCR/TCR), is of critical importance for immune responses to pathogens and vaccines. Adaptive immune receptor repertoire sequencing (AIRR-seq) has become widespread in immunology research making it the most readily available source of information about allelic diversity in immunoglobulin (IG) and T cell receptor (TR) loci. Here we present a novel algorithm for extra-sensitive and specific variable (V) and joining (J) gene allele inference, allowing reconstruction of individual high-quality gene segment libraries. The approach can be applied for inferring allelic variants from peripheral blood lymphocyte BCR and TCR repertoire sequencing data, including hypermutated isotype-switched BCR sequences, thus allowing high-throughput novel allele discovery from a wide variety of existing datasets. The developed algorithm is a part of the MiXCR software. We demonstrate the accuracy of this approach using AIRR-seq paired with long-read genomic sequencing data, comparing it to a widely used algorithm, TIgGER. We applied the algorithm to a large set of IG heavy chain (IGH) AIRR-seq data from 450 donors of ancestrally diverse population groups, and to the largest reported full-length TCR alpha and beta chain (TRA; TRB) AIRR-seq dataset, representing 134 individuals. This allowed us to assess the genetic diversity within the IGH, TRA and TRB loci in different populations and to establish a database of alleles of V and J genes inferred from AIRR-seq data and their population frequencies with free public access through an online database.

Wnt/ß-Catenin Signaling Induces Integrin α4ß1 in T Cells and Promotes a Progressive Neuroinflammatory Disease in Mice.

The mechanisms leading to autoimmune and inflammatory diseases in the CNS have not been elucidated. The environmental triggers of the aberrant presence of CD4+ T cells in the CNS are not known. In this article, we report that abnormal ß-catenin expression in T cells drives a fatal neuroinflammatory disease in mice that is characterized by CNS infiltration of T cells, glial activation, and progressive loss of motor function. We show that enhanced ß-catenin expression in T cells leads to aberrant and Th1-biased T cell activation, enhanced expression of integrin α4ß1, and infiltration of activated T cells into the spinal cord, without affecting regulatory T cell function. Importantly, expression of ß-catenin in mature naive T cells was sufficient to drive integrin α4ß1 expression and CNS migration, whereas pharmacologic inhibition of integrin α4ß1 reduced the abnormal T cell presence in the CNS of ß-catenin-expressing mice. Together, these results implicate deregulation of the Wnt/ß-catenin pathway in CNS inflammation and suggest novel therapeutic strategies for neuroinflammatory disorders.

Comparative analysis of murine T-cell receptor repertoires.

For understanding the rules and laws of adaptive immunity, high-throughput profiling of T-cell receptor (TCR) repertoires becomes a powerful tool. The structure of TCR repertoires is instructive even before the antigen specificity of each particular receptor becomes available. It embodies information about the thymic and peripheral selection of T cells; the readiness of an adaptive immunity to withstand new challenges; the character, magnitude and memory of immune responses; and the aetiological and functional proximity of T-cell subsets. Here, we describe our current analytical approaches for the comparative analysis of murine TCR repertoires, and show several examples of how these approaches can be applied for particular experimental settings. We analyse the efficiency of different metrics used for estimation of repertoire diversity, repertoire overlap, V-gene and J-gene segments usage similarity, and amino acid composition of CDR3. We discuss basic differences of these metrics and their advantages and limitations in different experimental models, and we provide guidelines for choosing an efficient way to lead a comparative analysis of TCR repertoires. Applied to the various known and newly developed mouse models, such analysis should allow us to disentangle multiple sophisticated puzzles in adaptive immunity.

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.

VDJviz: a versatile browser for immunogenomics data.

BACKGROUND: The repertoire of T- and B-cell receptor sequences encodes the antigen specificity of adaptive immunity system, determines its present state and guides its ability to mount effective response against encountered antigens in future. High throughput sequencing of immune repertoires (Rep-Seq) is a promising technique that allows to profile millions of antigen receptors of an individual in a single experiment. While a substantial number of tools for mapping and assembling Rep-Seq data were published recently, the field still lacks an intuitive and flexible tool that can be used by researchers with little or no computational background for in-depth analysis of immune repertoire profiles. RESULTS: Here we report VDJviz, a web tool that can be used to browse, analyze and perform quality control of Rep-Seq results generated by various pre-processing software. On a set of real data examples we show that VDJviz can be used to explore key repertoire characteristics such as spectratype, repertoire clonality, V-(D)-J recombination patterns and to identify shared clonotypes. We also demonstrate the utility of VDJviz in detection of critical Rep-Seq biases such as artificial repertoire diversity and cross-sample contamination. CONCLUSIONS: VDJviz is a versatile and lightweight tool that can be easily employed by biologists, immunologists and immunogeneticists for routine analysis and quality control of Rep-Seq data. The software is freely available for non-commercial purposes, and can be downloaded from: https://github.com/antigenomics/vdjviz .

DNA repair in Mycoplasma gallisepticum.

BACKGROUND: DNA repair is essential for the maintenance of genome stability in all living beings. Genome size as well as the repertoire and abundance of DNA repair components may vary among prokaryotic species. The bacteria of the Mollicutes class feature a small genome size, absence of a cell wall, and a parasitic lifestyle. A small number of genes make Mollicutes a good model for a "minimal cell" concept. RESULTS: In this work we studied the DNA repair system of Mycoplasma gallisepticum on genomic, transcriptional, and proteomic levels. We detected 18 out of 22 members of the DNA repair system on a protein level. We found that abundance of the respective mRNAs is less than one per cell. We studied transcriptional response of DNA repair genes of M. gallisepticum at stress conditions including heat, osmotic, peroxide stresses, tetracycline and ciprofloxacin treatment, stationary phase and heat stress in stationary phase. CONCLUSIONS: Based on comparative genomic study, we determined that the DNA repair system M. gallisepticum includes a sufficient set of proteins to provide a cell with functional nucleotide and base excision repair and mismatch repair. We identified SOS-response in M. gallisepticum on ciprofloxacin, which is a known SOS-inducer, tetracycline and heat stress in the absence of established regulators. Heat stress was found to be the strongest SOS-inducer. We found that upon transition to stationary phase of culture growth transcription of DNA repair genes decreases dramatically. Heat stress does not induce SOS-response in a stationary phase.

A Rapid Method for Detection of Antigen-Specific B Cells.

The global SARS-CoV-2 pandemic has united the efforts of many scientists all over the world to develop wet-lab techniques and computational approaches aimed at the identification of antigen-specific T and B cells. The latter provide specific humoral immunity that is essential for the survival of COVID-19 patients, and vaccine development has essentially been based on these cells. Here, we implemented an approach that integrates the sorting of antigen-specific B cells and B-cell receptor mRNA sequencing (BCR-seq), followed by computational analysis. This rapid and cost-efficient method allowed us to identify antigen-specific B cells in the peripheral blood of patients with severe COVID-19 disease. Subsequently, specific BCRs were extracted, cloned, and produced as full antibodies. We confirmed their reactivity toward the spike RBD domain. Such an approach can be effective for the monitoring and identification of B cells participating in an individual immune response.

Ultrasensitive allele inference from immune repertoire sequencing data with MiXCR.

Allelic variability in the adaptive immune receptor loci, which harbor the gene segments that encode B cell and T cell receptors (BCR/TCR), has been shown to be of critical importance for immune responses to pathogens and vaccines. In recent years, B cell and T cell receptor repertoire sequencing (Rep-Seq) has become widespread in immunology research making it the most readily available source of information about allelic diversity in immunoglobulin (IG) and T cell receptor (TR) loci in different populations. Here we present a novel algorithm for extra-sensitive and specific variable (V) and joining (J) gene allele inference and genotyping allowing reconstruction of individual high-quality gene segment libraries. The approach can be applied for inferring allelic variants from peripheral blood lymphocyte BCR and TCR repertoire sequencing data, including hypermutated isotype-switched BCR sequences, thus allowing high-throughput genotyping and novel allele discovery from a wide variety of existing datasets. The developed algorithm is a part of the MiXCR software ( https://mixcr.com ) and can be incorporated into any pipeline utilizing upstream processing with MiXCR. We demonstrate the accuracy of this approach using Rep-Seq paired with long-read genomic sequencing data, comparing it to a widely used algorithm, TIgGER. We applied the algorithm to a large set of IG heavy chain (IGH) Rep-Seq data from 450 donors of ancestrally diverse population groups, and to the largest reported full-length TCR alpha and beta chain (TRA; TRB) Rep-Seq dataset, representing 134 individuals. This allowed us to assess the genetic diversity of genes within the IGH, TRA and TRB loci in different populations and demonstrate the connection between antibody repertoire gene usage and the number of allelic variants present in the population. Finally we established a database of allelic variants of V and J genes inferred from Rep-Seq data and their population frequencies with free public access at https://vdj.online .

Targeted depletion of TRBV9+ T cells as immunotherapy in a patient with ankylosing spondylitis.

Autoimmunity is intrinsically driven by memory T and B cell clones inappropriately targeted at self-antigens. Selective depletion or suppression of self-reactive T cells remains a holy grail of autoimmune therapy, but disease-associated T cell receptors (TCRs) and cognate antigenic epitopes remained elusive. A TRBV9-containing CD8+ TCR motif was recently associated with the pathogenesis of ankylosing spondylitis, psoriatic arthritis and acute anterior uveitis, and cognate HLA-B*27-presented epitopes were identified. Following successful testing in nonhuman primate models, here we report human TRBV9+ T cell elimination in ankylosing spondylitis. The patient achieved remission within 3 months and ceased anti-TNF therapy after 5 years of continuous use. Complete remission has now persisted for 4 years, with three doses of anti-TRBV9 administered per year. We also observed a profound improvement in spinal mobility metrics and the Bath Ankylosing Spondylitis Metrology Index (BASMI). This represents a possibly curative therapy of an autoimmune disease via selective depletion of a TRBV-defined group of T cells. The anti-TRBV9 therapy could potentially be applicable to other HLA-B*27-associated spondyloarthropathies. Such targeted elimination of the underlying cause of the disease without systemic immunosuppression could offer a new generation of safe and efficient therapies for autoimmunity.

Adoptive Immunotherapy Based on Chain-Centric TCRs in Treatment of Infectious Diseases.

Complications after vaccination, lack of vaccines against certain infections, and the emergence of antibiotic-resistant microorganisms point to the need for alternative ways of protection and treatment of infectious diseases. Here, we proposed a therapeutic approach to control salmonellosis based on adoptive cell therapy. We showed that the T cell receptor (TCR) repertoire of salmonella-specific memory cells contains 20% of TCR variants with the dominant-active α-chain. Transduction of intact T lymphocytes with the dominant salmonella-specific TCRα led to their enhanced in vitro proliferation in response to salmonella. Adoptive transfer of transduced T cells resulted in a significant decrease in bacterial loads in mice infected with salmonella before or after the adoptive transfer. We demonstrated that adoptive immunotherapy based on T cells, transduced with dominant-specific TCRα could be successfully applied for treatment and prevention of infectious diseases and represent a useful addition to vaccination and existing therapeutic strategies.

Functionally specialized human CD4+ T-cell subsets express physicochemically distinct TCRs.

The organizational integrity of the adaptive immune system is determined by functionally discrete subsets of CD4+ T cells, but it has remained unclear to what extent lineage choice is influenced by clonotypically expressed T-cell receptors (TCRs). To address this issue, we used a high-throughput approach to profile the αß TCR repertoires of human naive and effector/memory CD4+ T-cell subsets, irrespective of antigen specificity. Highly conserved physicochemical and recombinatorial features were encoded on a subset-specific basis in the effector/memory compartment. Clonal tracking further identified forbidden and permitted transition pathways, mapping effector/memory subsets related by interconversion or ontogeny. Public sequences were largely confined to particular effector/memory subsets, including regulatory T cells (Tregs), which also displayed hardwired repertoire features in the naive compartment. Accordingly, these cumulative repertoire portraits establish a link between clonotype fate decisions in the complex world of CD4+ T cells and the intrinsic properties of somatically rearranged TCRs.

Measuring Intratumoral Heterogeneity of Immune Repertoires.

There is considerable clinical and fundamental value in measuring the clonal heterogeneity of T and B cell expansions in tumors and tumor-associated lymphoid structures-along with the associated heterogeneity of the tumor neoantigen landscape-but such analyses remain challenging to perform. Here, we propose a straightforward approach to analyze the heterogeneity of immune repertoires between different tissue sections in a quantitative and controlled way, based on a beta-binomial noise model trained on control replicates obtained at the level of single-cell suspensions. This approach allows to identify local clonal expansions with high accuracy. We reveal in situ proliferation of clonal T cells in a mouse model of melanoma, and analyze heterogeneity of immunoglobulin repertoires between sections of a metastatically-infiltrated lymph node in human melanoma and primary human colon tumor. On the latter example, we demonstrate the importance of training the noise model on datasets with depth and content that is comparable to the samples being studied. Altogether, we describe here the crucial basic instrumentarium needed to facilitate proper experimental setup planning in the rapidly evolving field of intratumoral immune repertoires, from the wet lab to bioinformatics analysis.

The Changing Landscape of Naive T Cell Receptor Repertoire With Human Aging.

Human aging is associated with a profound loss of thymus productivity, yet naïve T lymphocytes still maintain their numbers by division in the periphery for many years. The extent of such proliferation may depend on the cytokine environment, including IL-7 and T-cell receptor (TCR) "tonic" signaling mediated by self pMHCs recognition. Additionally, intrinsic properties of distinct subpopulations of naïve T cells could influence the overall dynamics of aging-related changes within the naïve T cell compartment. Here, we investigated the differences in the architecture of TCR beta repertoires for naïve CD4, naïve CD8, naïve CD4+CD25-CD31+ (enriched with recent thymic emigrants, RTE), and mature naïve CD4+CD25-CD31- peripheral blood subsets between young and middle-age/old healthy individuals. In addition to observing the accumulation of clonal expansions (as was shown previously), we reveal several notable changes in the characteristics of T cell repertoire. We observed significant decrease of CDR3 length, NDN insert, and number of non-template added N nucleotides within TCR beta CDR3 with aging, together with a prominent change of physicochemical properties of the central part of CDR3 loop. These changes were similar across CD4, CD8, RTE-enriched, and mature CD4 subsets of naïve T cells, with minimal or no difference observed between the latter two subsets for individuals of the same age group. We also observed an increase in "publicity" (fraction of shared clonotypes) of CD4, but not CD8 naïve T cell repertoires. We propose several explanations for these phenomena built upon previous studies of naïve T-cell homeostasis, and call for further studies of the mechanisms causing the observed changes and of consequences of these changes in respect of the possible holes formed in the landscape of naïve T cell TCR repertoire.