Autoencoder based local T cell repertoire density can be used to classify samples and T cell receptors.

Recent advances in T cell repertoire (TCR) sequencing allow for the characterization of repertoire properties, as well as the frequency and sharing of specific TCR. However, there is no efficient measure for the local density of a given TCR. TCRs are often described either through their Complementary Determining region 3 (CDR3) sequences, or theirV/J usage, or their clone size. We here show that the local repertoire density can be estimated using a combined representation of these components through distance conserving autoencoders and Kernel Density Estimates (KDE). We present ELATE-an Encoder-based LocAl Tcr dEnsity and show that the resulting density of a sample can be used as a novel measure to study repertoire properties. The cross-density between two samples can be used as a similarity matrix to fully characterize samples from the same host. Finally, the same projection in combination with machine learning algorithms can be used to predict TCR-peptide binding through the local density of known TCRs binding a specific target.

Identifying the inheritable component of human thymic T cell repertoire generation in monozygous twins.

We have analyzed T cell receptor repertoires in a unique set of thymus samples from a pair of monozygotic twins. While genetics affect the V(D)J rearrangement and generation of junctional sequences, the thymic selections seem largely stochastic and import no detectable inheritable effect at clonal level.

Genomic and expression analyses of Tursiops truncatus T cell receptor gamma (TRG) and alpha/delta (TRA/TRD) loci reveal a similar basic public γδ repertoire in dolphin and human.

BACKGROUND: The bottlenose dolphin (Tursiops truncatus) is a mammal that belongs to the Cetartiodactyla and have lived in marine ecosystems for nearly 60 millions years. Despite its popularity, our knowledge about its adaptive immunity and evolution is very limited. Furthermore, nothing is known about the genomics and evolution of dolphin antigen receptor immunity. RESULTS: Here we report a evolutionary and expression study of Tursiops truncatus T cell receptor gamma (TRG) and alpha/delta (TRA/TRD) genes. We have identified in silico the TRG and TRA/TRD genes and analyzed the relevant mature transcripts in blood and in skin from four subjects. The dolphin TRG locus is the smallest and simplest of all mammalian loci as yet studied. It shows a genomic organization comprising two variable (V1 and V2), three joining (J1, J2 and J3) and a single constant (C), genes. Despite the fragmented nature of the genome assemblies, we deduced the TRA/TRD locus organization, with the recent TRDV1 subgroup genes duplications, as it is expected in artiodactyls. Expression analysis from blood of a subject allowed us to assign unambiguously eight TRAV genes to those annotated in the genomic sequence and to twelve new genes, belonging to five different subgroups. All transcripts were productive and no relevant biases towards TRAV-J rearrangements are observed. Blood and skin from four unrelated subjects expression data provide evidence for an unusual ratio of productive/unproductive transcripts which arise from the TRG V-J gene rearrangement and for a "public" gamma delta TR repertoire. The productive cDNA sequences, shared both in the same and in different individuals, include biases of the TRGV1 and TRGJ2 genes. The high frequency of TRGV1-J2/TRDV1- D1-J4 productive rearrangements in dolphins may represent an interesting oligo-clonal population comparable to that found in human with the TRGV9- JP/TRDV2-D-J T cells and in primates. CONCLUSIONS: Although the features of the TRG and TRA/TRD loci organization reflect those of the so far examined artiodactyls, genomic results highlight in dolphin an unusually simple TRG locus. The cDNA analysis reveal productive TRA/TRD transcripts and unusual ratios of productive/unproductive TRG transcripts. Comparing multiple different individuals, evidence is found for a "public" gamma delta TCR repertoire thus suggesting that in dolphins as in human the gamma delta TCR repertoire is accompanied by selection for public gamma chain.

Discovery of invariant T cells by next-generation sequencing of the human TCR α-chain repertoire.

During infection and autoimmune disease, activation and expansion of T cells take place. Consequently, the TCR repertoire contains information about ongoing and past diseases. Analysis and interpretation of the human TCR repertoire are hampered by its size and stochastic variation and by the diversity of Ags and Ag-presenting molecules encoded by the MHC, but are highly desirable and would greatly impact fundamental and clinical immunology. A subset of the TCR repertoire is formed by invariant T cells. Invariant T cells express interdonor-conserved TCRs and recognize a limited set of Ags, presented by nonpolymorphic Ag-presenting molecules. Discovery of the three known invariant T cell populations has been a tedious and slow process, identifying them one by one. Because conservation of the TCR α-chain of invariant T cells is much higher than the ß-chain, and because the TCR α-chain V gene segment TRAV1-2 is used by two of the three known invariant TCRs, we employed next-generation sequencing of TCR α-chains that contain the TRAV1-2 gene segment to identify 16 invariant TCRs shared among many blood donors. Frequency analysis of individual clones indicates these T cells are expanded in many donors, implying an important role in human immunity. This approach extends the number of known interdonor-conserved TCRs and suggests that many more exist and that these TCR patterns can be used to systematically evaluate human Ag exposure.

The feature of distribution and clonality of TCR γ/δ subfamilies T cells in patients with B-cell non-Hodgkin lymphoma.

Restricted T-cell receptor (TCR) Vα/Vß repertoire expression and clonal expansion of αß T cells especially for putative tumor-associated antigens were observed in patients with hematological malignancies. To further characterize the γδ T-cell immune status in B-cell non-Hodgkin lymphoma (B-NHL), we investigated the distribution and clonality of TCR Vγ/Vδ repertoire in peripheral blood (PB), bone marrow (BM), and lymph node (LN) from patients with B-NHL. Four newly diagnosed B-NHL cases, including three with diffuse large B-cell lymphoma (DLBCL) and one with small lymphocytic lymphoma (SLL), were enrolled. The restrictive expression of TCR Vγ/Vδ subfamilies with different distribution patterns could be detected in PB, BM, or LN from all of four patients, and partial subfamily T cells showed clonal proliferation. At least one clonally expanded Vδ subfamily member was found in PB from each patient. However, the expression pattern and clonality of TCR Vγ/Vδ changed in different immune organs and showed individual feature in different patients. The clonally expanded Vδ5, Vδ6, and Vδ8 were detected only in PB but neither in BM nor LN while clonally expanded Vδ2 and Vδ3 could be detected in both PB and BM/LN. In conclusion, the results provide a preliminary profile of distribution and clonality of TCR γ/δ subfamilies T cells in PB, BM, and LN from B-NHL; similar clonally expanded Vδ subfamily T cells in PB and BM may be related to the same B-cell lymphoma-associated antigens, while the different reactive clonally expanded Vγ/Vδ T cells may be due to local immune response.

Revealing individual signatures of human T cell CDR3 sequence repertoires with Kidera Factors.

The recent development of High Throughput Sequencing technologies has enabled an individual's TCR repertoire to be efficiently analysed at the nucleotide level. However, with unique clonotypes ranging in the tens of millions per individual, this approach gives a surfeit of information that is difficult to analyse and interpret in a biological context and gives little information about TCR structural diversity. Using publicly available TCR CDR3 sequence data, we analysed TCR repertoires by converting the encoded CDR3 amino acid sequences into Kidera Factors, a set of orthogonal physico-chemical properties that reflect protein structure. This approach enabled the TCR repertoire from different individuals to be distinguished and demonstrated the close similarity of the repertoire in different samples from the same individual.

T cell receptor αß diversity inversely correlates with pathogen-specific antibody levels in human cytomegalovirus infection.

A diverse T cell receptor (TCR) repertoire capable of recognizing a broad range of antigenic peptides is thought to be central to effective pathogen-specific immunity by counteracting escape mutations, selecting high-avidity T cells, and providing T cell specificities with comprehensive functional characteristics. However, evidence that TCR diversity is important for the successful control of human infections is limited. A single-cell strategy for the clonotypic analysis of human CD8⁺ TCRαß repertoires was used to probe the diversity and magnitude of individual human cytomegalovirus (CMV)-specific CD8⁺ T cells recovered directly ex vivo. We found that CD8⁺ TCRαß repertoire diversity, but not the size of the CD8⁺ T cell response, was inversely related to circulating CMV-specific antibody levels, a measure that has been correlated epidemiologically with differential mortality risks and found here to be higher in persons with detectable (versus undetectable) CMV viral loads. Overall, our findings indicate that CD8⁺ T cell diversity may be more important than T cell abundance in limiting the negative consequences of CMV persistence, demonstrate high prevalence of both TCRα and TCRß public motif usage, and suggest that a highly diverse TCRαß repertoire may be an important benchmark and target in the success of immunotherapeutic strategies.

Abnormalities of the alphabeta T-cell receptor repertoire in advanced myelodysplastic syndrome.

OBJECTIVE: Analysis of the alphabeta T-cell receptor (TCR) repertoire in patients with myelodysplastic syndrome (MDS) using the technique of TCR beta-chain spectratyping has provided valuable insight into the pathophysiology of cytopenias in a subset of patients with this heterogeneous disorder. TCR beta-chain spectratypes are complex data sets, however, and statistical tools for their comprehensive analysis are limited. The objective of the present work was to develop a method to enable quantitative evaluation and global comparison of spectratype data from different individuals and to study the prevalence of TCR beta repertoire abnormalities in MDS patients. MATERIALS AND METHODS: We developed a robust statistical method based on k-means clustering analysis, and applied this method to analysis of the alphabeta TCR repertoires in 50 MDS patients and 23 age-matched healthy controls. RESULTS: Cluster analysis identified a subset of 11 MDS patients with profoundly abnormal alphabeta TCR repertoires. This group of patients was characterized by advanced disease by International Prognostic Scoring System and World Health Organization criteria, increased expression of the Wilms' tumor-1 oncogene, increased bone marrow myeloblast count, and older age. CONCLUSIONS: We have developed a robust analytic algorithm that enables the comparison of alphabeta TCR repertoires between individuals and have shown that abnormal alphabeta TCR repertoire is a feature of a subset of patients with advanced MDS.

Genomic structure of the whole D-J-C clusters and the upstream region coding V segments of the TRB locus in pig.

In the vertebrate immune system, T cells play a central role in host defense against microbial or viral infection. Previous studies suggested that at least two sets of TRBD-J-C clusters are harbored in the porcine genome. In this study, we determined 212,193 bp of a continuous porcine genomic sequence covering the entire TRBC region. EPHB6, TRPV6, TRY, and ten TRBV genes were conserved in the vicinity of the TRBD-J-C clusters. Interestingly, three TRBD-J-C clusters were identified in this sequence; each TRBD-J-C cluster consisted of one TRBD and seven TRBJ segments, with one TRBC region composed of four exons. The distribution of repetitive sequences and phylogenetic analysis indicated that the TRBD-J-C cluster, located at the center of the three clusters identified, had a structure combined with the others. Most of the TRBJ segments were available in public databases, suggesting that all three TRBD-J-C clusters are functional in pigs.

Distinct sets of alphabeta TCRs confer similar recognition of tumor antigen NY-ESO-1157-165 by interacting with its central Met/Trp residues.

Naturally acquired immune responses against human cancers often include CD8(+) T cells specific for the cancer testis antigen NY-ESO-1. Here, we studied T cell receptor (TCR) primary structure and function of 605 HLA-A*0201/NY-ESO-1(157-165)-specific CD8 T cell clones derived from five melanoma patients. We show that an important proportion of tumor-reactive T cells preferentially use TCR AV3S1/BV8S2 chains, with remarkably conserved CDR3 amino acid motifs and lengths in both chains. All remaining T cell clones belong to two additional sets expressing BV1 or BV13 TCRs, associated with alpha-chains with highly diverse VJ usage, CDR3 amino acid sequence, and length. Yet, all T cell clonotypes recognize tumor antigen with similar functional avidity. Two residues, Met-160 and Trp-161, located in the middle region of the NY-ESO-1(157-165) peptide, are critical for recognition by most of the T cell clonotypes. Collectively, our data show that a large number of alphabeta TCRs, belonging to three distinct sets (AVx/BV1, AV3/BV8, AVx/BV13) bind pMHC with equal antigen sensitivity and recognize the same peptide motif. Finally, this in-depth study of recognition of a self-antigen suggests that in part similar biophysical mechanisms shape TCR repertoires toward foreign and self-antigens.

Further characterization of T cell receptor chains of marsupials.

cDNA clones encoding T cell receptor alpha (TCRalpha) and beta (TCRbeta) from the South American opossum, Monodelphis domestica were isolated and characterized. A single clone isolated encoding a TCRalpha chain was full length, containing the complete V (variable), J (joining) and C (constant) regions. Three partial cDNA clones were isolated for TCRbeta which contained complete C sequences. Phylogenetic analysis of the TCR Valpha revealed that the M. domestica sequence and a sequence from the Australian brushtail possum, Trichosurus vulpecula, belong to separate Valpha families and intersperse with sequences from eutherian mammals. Similar to results described for marsupial and eutherian light chains, diversity at the V region of the TCR is ancient and maintained. In contrast phylogenetic analysis of the TCR Calpha and Cbeta sequences from M. domestica, T. vulpecula, and other vertebrates revealed that the marsupial TCR C grouped together forming a sister group to eutherian mammals.

Evolutionary dynamics of the T-cell receptor VB gene family as inferred from the human and mouse genomic sequences.

The diversity of T-cell receptors is generated primarily by the variable-region gene families, each of which is composed of a large number of member genes. The entire genomic sequence of the variable region (VB) of the T- cell receptor beta chain from humans and mice has become available. To understand the evolutionary dynamics of the VB gene family, we conducted a phylogenetic analysis of all VB genes from humans and mice, as well as a detailed analysis of internal DNA duplications in the human genomic VB region. The phylogenetic tree obtained shows that human and mouse VB genes intermingle extensively rather than forming two separate clusters and that many gene duplications occurred both before and after the divergence between primates and rodents. Analyzing the genomic maps of transposable elements (e.g., LINEs and SINEs) and relic VB genes in the VB gene region, we present evidence that a 20-kb VB region duplicated tandemly four times in the human lineage during the last 32 Myr, and 6 out of the 15 VB genes in this region have become nonfunctional during this period. Our results show that the VB gene family is subject to evolution by a birth-and-death process rather than to concerted evolution.

T-cell receptor Vbeta repertoire in mite-allergic subjects after sublingual immunotherapy.

Sublingual immunotherapy has been recognized as an alternative to injected immunotherapy for the treatment of allergic diseases. Even if compelling clinical evidence supports such a view, few studies are available on its mechanisms of action. This study was carried out to investigate the peripheral lymphocyte Vbeta repertoire of subjects with mite-allergic respiratory allergy who were either not treated or treated for 2 years with mite-specific sublingual immunotherapy. The T-cell receptor Vbeta distribution was studied by flow-cytometric techniques in three subject groups. Group A (untreated) included 19 subjects with symptomatic, mite-allergic, low to moderate asthma and/or rhinitis. Group B (treated) was made up of 10 asymptomatic subjects treated for 2 years with mite-specific sublingual-swallow immunotherapy for low to moderate asthma and/or rhinitis. Group C (controls) included 10 healthy subjects. The Vbeta usage was investigated with monoclonal antibodies specific to the diverse beta segments V3, V5a, V5b, V5c, V6a, V8a, V8b and V12a. The comparison between the group A and group C repertoires showed a lower expression (p < 0.05) of the beta V8b+ T-cell subset. The group B repertoire, when compared with group A, showed a significantly greater usage of the beta V5a (p <0.05), 8a (p <0.05) and 12a (p <0.01) segments. The significantly lower expression of beta V8b observed in the symptomatic untreated group was not present in the group that was asymptomatic after treatment. The oligoclonal expansion observed in the treated group was consistent with the development of suppressor T-cell and/or of Th1 clones but not with deletion mechanisms of induced tolerance.

Analysis of T cell repertoire in the liver of patients with chronic hepatitis C.

Many T cells infiltrate into the liver of patients with chronic hepatitis C (CH-C). They are believed to play a crucial role in the immunopathogenesis of hepatic inflammation, but their clonality and specificity are unknown. The aim of this study was to clarify the characteristics of these T cells. We analysed the complementarity-determining region (CDR)3 size lengths of T cell receptor (TCR) beta-chains by size spectratyping, and determined the sequences of Vbeta CDR3 after subcloning Vbeta-specific polymerase chain reaction products. Spectratyping showed clonal expansions in all liver specimens, most of which showed more than two T cell clones. Moreover, many non-clonal T cells also accumulated in the liver. Clonality of the T cells suspected by spectratyping was confirmed by CDR3 sequencing. Although the sequences revealed no whole CDR3-shared clones among different patients, some common motif sequences were observed. Our data suggest that T cells are stimulated by several hepatitis C virus (HCV) epitopes, then accumulate in the liver of CH-C patients. Shared motifs of expanded T cell clones suggest that they might recognize the same regions of HCV peptides, but have differences due to HCV peptide mutational changes. These clones might also interact with non-clonal T cells and play a crucial role in the immunopathogenesis of CH-C.

NKT cells in the rat: organ-specific distribution of NK T cells expressing distinct V alpha 14 chains.

Rat invariant TCR alpha-chains and NKT cells were investigated to clarify whether CD1d-mediated recognition by NKT cells is conserved further in evolution. Rats had multiple-copies of TRAV14 genes, which can be categorized into two types according to the diversity accumulated in the CDR2 region. Rats retained invariant TCR alpha forms with the homogeneous junctional region similar to mouse invariant TRAV14-J281. The proportion of invariant TCR among V alpha 14+ clones was 12.9% in the thymus and increased in the periphery, 31% in the spleen and 95% in hepatic sinusoidal cells. The invariant TRAV14-J281 was expressed by liver sinusoidal and splenic NKT cells with CD8, CD44high, and TCR V beta 8. Type 1 invariant TCR alpha was expressed more frequently in hepatic lymphocytes, while type 2 invariant TCR alpha was expressed predominantly in the spleen. Both types of cells cytolyzed to and were stimulated to proliferate by CD1d-expressing cells in a CD1d-restricted manner. These results suggested that rat NKT cells bearing distinct V alpha 14 chains are distributed in a tissue-specific pattern. NKT cell populations in rats were more variable than those in mice, indicating that they play novel roles in nature. The implication of the molecular interaction between the structurally diverse invariant TCR alpha and CD1d/ligand complex in different organs is discussed.

Transient T cell receptor beta-chain variable region-specific expansions of CD4+ and CD8+ T cells during the early phase of pediatric human immunodeficiency virus infection: characterization of expanded cell populations by T cell receptor phenotyping.

T cell receptor (TCR) repertoire perturbations are commonly detected in CD8+ T cells during adult primary human immunodeficiency virus (HIV) infection and have been associated with HIV-specific cytotoxic T cell responses. By use of flow cytometry, transient high-level TCR beta-chain variable region-specific expansions of CD4+ and CD8+ T cells were observed more frequently in HIV-infected children than in children exposed to HIV who remained uninfected. TCR beta-chain diversity analysis and diversity-specific polymerase chain reaction were used to study the clonality of expanded CD4+ and CD8+ subsets. In CD8+ T cells, structural features of the complement-determining regions 3 were altered during the course of the expansion, and persistent TCR clonotypes were observed, consistent with antigen-driven selection. In contrast, TCR beta-chain variable region-specific expansions without clonotypic overrepresentation or persistence were observed in CD4+ T cells, possibly related to HIV-specific helper T cell responses or to the progressive destruction of the CD4+ cell compartment.

Predominance of one T-cell antigen receptor BV haplotype in African populations.

The human T-cell antigen receptor (TCR) is the counter-receptor for the HLA/peptide complex displayed on the surface of antigen-presenting cells. It confers antigen specificity on T lymphocytes and therefore plays a central role in pathogen recognition and host response. The most frequently used form of the TCR is a heterodimer composed of variable alpha and beta chains. We investigated allele frequencies for four variable-region gene segments of the beta chain (2S1, 3S1, 8S3, and 15S1) in 146 Caucasians and 165 Africans. The results reveal significant unexpected differences between the two populations for allele frequencies, phenotypes, genotypes, and haplotypes. Among Caucasians, there are 43 phenotypes, whereas there are 31 among the Africans studied. There are 17 haplotypes in the Caucasian sample but only 10 in Africans. This loss of diversity is largely due to the high frequency of one haplotype in the African sample which represents 65% of the informative chromosomes. At least one copy of this haplotype is present in 90% of informative individuals. As a result, 29% of Africans are homozygous for the common haplotype. Less genetic diversity at TCRBV is unexpected, since Africans usually show greater genetic diversity than other ethnic groups. For example, there are approximately twice as many HLA haplotypes in Africans compared to Caucasians. Homozygosity is also unexpected because it reduces the number of TCR variants available to recognize HLA pathogen-derived peptide complexes.

A new isolation method for rat intraepithelial lymphocytes.

Intraepithelial lymphocytes (IELs) play critical roles in gut immunity. In mice, gammadelta T cells are a large component of the IEL population. In the rat, gammadelta IELs are reportedly much less common, but technical issues suggest that previous analyses should be interpreted cautiously. The study of IELs in rats has been impeded by isolation procedures that are lengthy and complex, leading to small cell yields. For this reason, it is possible that rat IELs analyzed in previous studies have not been representative of the entire IEL compartment. We report a new method for the isolation of rat IELs that is based on the selective removal of intestinal epithelial cells under conditions that leave the basement membrane undisturbed. The method is rapid and requires neither enzymatic digestion, nor surgical removal of Peyer's patches, nor vigorous mechanical manipulation of the intestine. The yield of rat IELs using this method is 5- to 10-fold greater than that reported for other methods. Morphological and phenotypic analyses demonstrated that the purified cell population is comprised of IELs and is not contaminated with lamina propria or Peyer's patch lymphocytes. Phenotypic analysis revealed five major subsets of IELs based on differential cell surface expression of CD4, CD8, and alphabeta T cell receptor (TcR). Among the alphabetaTcR- cells was a population of gammadelta T cells present at levels not previously detected. The isolation of IEL sub-populations using this methodology should facilitate studies of the function of these cells in gut immunity.

The frequency of different T-cell receptor V-families in oral lichen planus and lichenoid contact lesions: an immunohistochemical study.

Oral lichen planus (OLP) and lichenoid contact lesions (CL) are recognized as different pathological conditions of the oral mucosa. Cutaneous delayed-type hypersensitivity to mercury displayed by patients with CL but not by OLP patients supports the concept of different etiological mechanisms behind the two lesions. It is not possible to reveal this difference by histopathological assessments, and differences in clinical appearances are at present the only way to discriminate between the two conditions. It has recently been observed that T cells in OLP lesions express T-cell receptors (TCR) belonging to the Vbeta3 family in a higher frequency than expected from a random distribution, suggesting an involvement of superantigens as an etiologic factor behind this condition. In an effort to discriminate more clearly between OLP and CL, and to provide clues to the etiological mechanisms behind the two lesions, the TCR V-family distributions in the inflammatory infiltrates of OLP and CL were compared. Biopsies were taken from 10 patients with manifest OLP and 10 patients with CL. Frozen sections were incubated with antibodies against TCR Vbeta3, Valpha2 and Vbeta5a utilizing a standard immunoperoxidase technique. The frequency of Vbeta3.1 (clone 8F10) was calculated as 7%, and for Valpha2 less than 3%, and the results did not reveal any differences between OLP and CL regarding the frequencies of T-cell V-families. Thus, it was not possible to discriminate between OLP and CL by immunohistochemistry staining for different V families.

Evidence for heterogeneous TCR V beta repertoire expression in mercury-induced immune disorders in rats.

Administration of subtoxic doses of HgCl2 affects differentially the immune system depending on the strain of rats tested. Susceptible Brown-Norway (BN) rats exhibit a CD4+ T cell-dependent polyclonal activation of B cells; in contrast, Lewis (LEW) rats are resistant and develop an immunosuppression mediated by CD8+ T cells recruited by CD4+ T cells. The mechanisms by which mercury induces immune disorders are poorly understood. We were interested in analyzing the diversity and mercury-mediated changes of the TCR Vbeta repertoire in the BN and LEW strains of rats at different times of HgCl2 exposure. Our results obtained after analysis of lymph node T cells by RNase protection assay, flow cytometry or immunoscope assay (i) were not consistent with a superantigen-like stimulus since we observed neither a V beta-selective expansion nor deletion that would have been expected and (ii) showed that in BN rats, as well as in LEW rats, an increase in the number of T cells was associated with the heterogeneous TCR V beta repertoire, thus supporting a polyclonal T cell activation. However, in BN rats the total number of T cells increased very rapidly, whereas in LEW rats only CD8+ T cells accumulated.