T-cell Antigen Receptor Repertoire Research Articles

Donor genetic determinant of thymopoiesis, rs2204985, and stem cell transplantation outcome in a multipopulation cohort

BackgroundA genetic polymorphism, rs2204985, has been reported to be associated with the diversity of T-cell antigen receptor repertoire and TREC levels, reflecting the function of the thymus. As the thymus function can be assumed to be an important factor regulating the outcome of stem cell transplantation (SCT), it was of great interest that rs2204985 showed a genetic association to disease-free and overall survival in a German SCT donor cohort. Tools to predict the outcome of SCT more accurately would help in risk assessment and patient safety. ObjectiveTo evaluate the general validity of the original genetic association found in the German cohort, we determined genetic associations between rs2204985 and the outcome of SCT in 1,473 SCT donors from four different populations. Study designGenetic associations between rs2204985 genotype AA versus AG/GG and overall survival (OS) and disease-free survival (DFS) in 1,473 adult, allogeneic SCT from Finland, the United Kingdom, Spain, and Poland were performed using the Kaplan-Meier analysis and log-rank tests. We adjusted the survival models with covariates using Cox regression. ResultsIn unrelated SCT donors (N = 425), the OS of genotype AA versus AG/GG had a trend for a similar association (p = 0.049, log-rank test) as previously reported in the German cohort. The trend did not remain significant in the Cox regression analysis with covariates. No other associations were found. ConclusionWeak support for the genetic association between rs2204985, previously also associated with thymus function, and the outcome of SCT could be found in a cohort from four populations.

T-cell receptor repertoire analysis of CD4-positive T cells from blood and an affected organ in an autoimmune mouse model.

One hallmark of some autoimmune diseases is the variability of symptoms among individuals. Organs affected by the disease differ between patients, posing a challenge in diagnosing the affected organs. Although numerous studies have investigated the correlation between T cell antigen receptor (TCR) repertoires and the development of infectious and immune diseases, the correlation between TCR repertoires and variations in disease symptoms among individuals remains unclear. This study aimed to investigate the correlation of TCRα and β repertoires in blood T cells with the extent of autoimmune signs that varies among individuals. We sequenced TCRα and β of CD4+ CD44high CD62Llow T cells in the blood and stomachs of mice deficient in autoimmune regulator (Aire) (AIRE KO), a mouse model of human autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. Data analysis revealed that the degree of similarity in TCR sequences between the blood and stomach varied among individual AIRE KO mice and reflected the extent of T cell infiltration in the stomach. We identified a set of TCR sequences whose frequencies in blood might correlate with extent of the stomach manifestations. Our results propose a potential of using TCR repertoires not only for diagnosing disease development but also for diagnosing affected organs in autoimmune diseases.

Maintenance of the human memory T cell repertoire by subset and tissue site

BackgroundImmune-mediated protection is mediated by T cells expressing pathogen-specific T cell antigen receptors (TCR) that are maintained at diverse sites of infection as tissue-resident memory T cells (TRM) or that disseminate as circulating effector-memory (TEM), central memory (TCM), or terminal effector (TEMRA) subsets in blood and tissues. The relationship between circulating and tissue resident T cell subsets in humans remains elusive, and is important for promoting site-specific protective immunity.MethodsWe analyzed the TCR repertoire of the major memory CD4+ and CD8+T cell subsets (TEM, TCM, TEMRA, and TRM) isolated from blood and/or lymphoid organs (spleen, lymph nodes, bone marrow) and lungs of nine organ donors, and blood of three living individuals spanning five decades of life. High-throughput sequencing of the variable (V) portion of individual TCR genes for each subset, tissue, and individual were analyzed for clonal diversity, expansion and overlap between lineage, T cell subsets, and anatomic sites. TCR repertoires were further analyzed for TRBV gene usage and CDR3 edit distance.ResultsAcross blood, lymphoid organs, and lungs, human memory, and effector CD8+T cells exhibit greater clonal expansion and distinct TRBV usage compared to CD4+T cell subsets. Extensive sharing of clones between tissues was observed for CD8+T cells; large clones specific to TEMRA cells were present in all sites, while TEM cells contained clones shared between sites and with TRM. For CD4+T cells, TEM clones exhibited the most sharing between sites, followed by TRM, while TCM clones were diverse with minimal sharing between sites and subsets. Within sites, TRM clones exhibited tissue-specific expansions, and maintained clonal diversity with age, compared to age-associated clonal expansions in circulating memory subsets. Edit distance analysis revealed tissue-specific biases in clonal similarity.ConclusionsOur results show that the human memory T cell repertoire comprises clones which persist across sites and subsets, along with clones that are more restricted to certain subsets and/or tissue sites. We also provide evidence that the tissue plays a key role in maintaining memory T cells over age, bolstering the rationale for site-specific targeting of memory reservoirs in vaccines and immunotherapies.

A disease-associated mutation that weakens ZAP70 autoinhibition enhances responses to weak and self-ligands.

The cytoplasmic kinase ZAP70 is critical for T cell antigen receptor (TCR) signaling. The R360P mutation in ZAP70 is responsible for an early-onset familial autoimmune syndrome. The structural location and biochemical signaling effects of the R360P mutation are consistent with weakening of the autoinhibitory conformation of ZAP70. Mice with a ZAP70 R360P mutation and polyclonal TCR repertoires exhibited relatively normal T cell development but showed evidence of increased signaling. In addition, the R360P mutation resulted in enhanced follicular helper T cell expansion after LCMV infection. To eliminate the possibility of a TCR repertoire shift, the OTI transgenic TCR was introduced into R360P mice, which resulted in enhanced T cell responses to weaker stimuli, including weak agonists and a self-peptide. These observations suggest that disruption of ZAP70 autoinhibition by the R360P mutation enables increased mature T cell sensitivity to self-antigens that would normally be ignored by wild-type T cells, a mechanism that may contribute to the break of tolerance in human patients with R360P mutation.

Reverse TCR repertoire evolution toward dominant low-affinity clones during chronic CMV infection.

Adaptive evolution is a key feature of T cell immunity. During acute immune responses, T cells harboring high-affinity T cell antigen receptors (TCRs) are preferentially expanded, but whether affinity maturation by clonal selection continues through the course of chronic infections remains unresolved. Here we investigated the evolution of the TCR repertoire and its affinity during the course of infection with cytomegalovirus, which elicits large T cell populations in humans and mice. Using single-cell and bulk TCR sequencing and structural affinity analyses of cytomegalovirus-specific T cells, and through the generation and in vivo monitoring of defined TCR repertoires, we found that the immunodominance of high-affinity T cell clones declined during the chronic infection phase, likely due to cellular senescence. These data showed that under conditions of chronic antigen exposure, low-affinity TCRs preferentially expanded within the TCR repertoire, with implications for immunotherapeutic strategies.

Skin Associated Staphylococcus Aureus Contributes to Disease Progression in CTCL

It has been proposed that bacteria play a direct role in progression of cutaneous T cell lymphoma (CTCL), although definitive evidence is missing, and the underlying mechanism of how microbes contribute to disease progression remains unknown. The skin of CTCL patients is frequently colonized with Staphylococcus aureus (S. aureus) strains and infections with hospital and community associated strains of S. aureus are a frequent cause of morbidity and mortality among patients with advanced CTCL. Here we provide a comprehensive analysis of the association between CTCL and S. aureus colonization, and use our unique pre-clinical animal model of CTCL to determine the cause-effect relationship between skin-associated S. aureus and CTCL progression. To understand the relationship between bacterial colonization and CTCL we collected skin swabs from active lesions, unaffected skin and nares of CTCL patients to perform S. aureus cultures and 16S rRNA gene sequencing. Skin swabs of psoriasis patients and healthy donors served as controls. The frequency of S. aureus colonization determined by culture based techniques revealed that >65% of advanced stage patients had S. aureus present at lesional/tumor sites, while corresponding sites in patients with psoriasis and in healthy controls rarely had detectable S. aureus. Colonization rates correlated positively with the disease stage. Unbiased, 16s sequencing based analysis of the skin microbiome from advanced CTCL patients revealed that the overall skin microbiome of these patients is distinct from that of healthy individuals and patients with psoriasis. A lower phylogenetic diversity and significantly higher relative abundance of Staphylococcus species was found in CTCL patients. To determine the causal relationship between skin flora and progression of CTCL we used our mouse model of CTCL and assessed disease progression in both conventionally housed specific-pathogen-free (SPF) conditions and in germ free (GF) isolators using a standardized clinical score. The CD4CreSTAT3stopfl/+ mice express a hyper-active STAT3C mutant protein selectively in T lymphocytes and virtually all mutant mice develop T cell infiltration in the epidermis causing skin lesions resembling CTCL, by eight months of age. In contrast to the SPF housed animals, GF mice remained disease free or developed only a mild phenotype (clinical score 1 out of 5) after 11 months of follow-up. Notably, when GF CD4CreSTAT3stopfl/+ mice were transitioned to SPF conditions they all developed advanced disease. Finally, we examined the role of T cell antigen receptor (TCR) signaling in mediating the transformation of T lymphocytes. R26STAT3Cstopfl/+CD4Cre Rag2KO OTII mice express only OVA-specific TCRs. T cells from R26STAT3Cstopfl/+ CD4Cre Stim1fl/fl mice express a normal TCR repertoire, but exhibit defective T cell receptor signaling due to compromised calcium influx. Both strains failed to develop typical skin lesions, suggesting an essential role for TCR interaction with tumor microenvironment and microbial antigens in the pathogenesis of CTCL. In conclusion, we demonstrate a strong correlation between CTCL staging and rates of S. aureus colonization. Our study supports a cause-effect relationship between skin flora and CTCL oncogenesis. We propose that CTCL represents an antigen driven malignancy. Further studies using mono-colonization with single bacterial strains are needed to further interrogate the role of specific bacteria. Disclosures Hymes: Celgene: Consultancy. Odum:Micreos human Health B.V: Consultancy. Geskin:Merck: Other: Supported/Contracted Research; UpToDate: Patents & Royalties: Royalty, Receipt of Intellectual Property Rights / Patent Holder; Mallinckrodt: Consultancy, Honoraria, Other: Supported/Contracted Research; Helsinn: Consultancy, Honoraria, Other: Supported/Contracted Research; Stratpharma: Other: Supported/Contracted Research; Medivir: Consultancy, Honoraria; Medscape: Speakers Bureau; Actelion: Other: Supported/Contracted Research.

Cysteine and hydrophobic residues in CDR3 serve as distinct T-cell self-reactivity indices

Cysteine and hydrophobic residues in CDR3 serve as distinct T-cell self-reactivity indices

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.

Increased cathepsin S in Prdm1-/- dendritic cells alters the TFH cell repertoire and contributes to lupus.

Aberrant expansion of follicular helper T (TFH) cells occurs in lupus patients. An unanswered question is whether an altered T cell receptor (TCR) repertoire is associated with this expansion. Here, we demonstrate that Blimp-1 repressed expression of the cathepsin S gene (Ctss) which encodes a cysteine protease that cleaves invariant chain and produces antigenic peptides for MHC class II loading. The increased CTSS in dendritic cells (DCs) of female Prdm1 conditional knockout (CKO) mice altered antigen presentation to CD4+ T cells. Analysis of Vβ CDR3s demonstrated a more diverse repertoire of TFH from female CKO mice. In vivo treatment of CKO mice with a CTSS inhibitor abrogated lupus-related phenotypes and reduced the diversity of the TFH TCR repertoire. Thus, Blimp-1 deficiency in DCs leads to a loss of appropriate regulation of Ctss expression in female mice thereby modulating antigen presentation and TFH repertoire to contribute to autoimmunity.

Class II major histocompatibility complex mutant mice to study the germ-line bias of T-cell antigen receptors

The interaction of αβ T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class II MHC amino acids that often are involved in interactions with the germ-line-encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC.

Mutation of the Traj18 gene segment using TALENs to generate Natural Killer T cell deficient mice.

Invariant Natural Killer T (iNKT) cells are a unique subset of T lymphocytes that have been implicated in both promoting and suppressing a multitude of immune responses. In mice, iNKT cells express T cell antigen receptors (TCRs) comprising a unique TCRα rearrangement between the Trav11 and Traj18 gene segments. When paired with certain Trbv TCRβ chains, these TCRs recognize lipid antigens presented by the major histocompatibility complex (MHC) class I-like molecule, CD1d. Until recently, the sole model of iNKT deficiency targeted the Jα18, which is absolutely required to form the TCR with the appropriate antigenic specificity. However, these mice were demonstrated to have a large reduction in TCR repertoire diversity, which could confound results arising from studies using these mice. Here, we have created a new NKT-deficient mouse strain using transcription activator-like effector nuclease (TALEN) technology to only disrupt the expression of Jα18, leaving the remaining Jα repertoire unperturbed. We confirm that these mice lack iNKT cells and do not respond to lipid antigen stimulation while the development of conventional T cells, regulatory T cells, and type Ib NKT cells is normal. This new mouse strain will serve as a new model of iNKT cell deficiency to facilitate our understanding of iNKT biology.

Distinct features of human CD8 T cell TCR repertoire specific to influenza A virus matrix protein M1

Abstract CD8 T cells play a vital role in the immune response to viral infection. Diversity of the T cell antigen receptor (TCR) is believed to be essential for the host to survive in this microorganism-rich world. However, there is limited information regarding the characteristics of the TCR repertoire to defined viral antigens in humans. Here, we report a comprehensive analysis of CD8 TCR repertoire diversity (size, V and J gene usage), the consensus binding motifs in CDR3, affinity, and structure of selected a/b paired TCRs to influenza A virus matrix protein M1 (GILGFVFTL, GIL) on HLA-A2+ healthy individuals. Using high-throughput sequencing coupled with the unique molecular identifier (UMI) method, we identified several hundred GIL-binding TCRa and TCRb. TRAV13-1 (63.2%) and TRAJ42 (72.5%) were most frequently found in alpha chain whereas TRBV19 (94.9%) and TRBJ2-5 (54.8%) were frequently found in beta chain. Furthermore, the length of the CDR3 amino acid region was highly restricted with over 70% of alpha (10) and of beta (11). CDR3 sequence analysis revealed shared motifs among different TCRs (a and b chains). We isolated paired expressed TCRa/b from single cells for further structural analysis. The affinity between TCRs and GIL-HLA-A2 complexes showed a wide range (1.8–191 mM). Finally, we resolved two crystal structures of TCRs-GIL-HLA-A2 complexes and found that the TCRs exhibited similar patterns of interactions with the antigen, even though the V, J, and CDR3 lengths were different. Together, our findings revealed the size of the antigen-specific CD8 TCR repertoire, the consensus binding motifs in CDR3, binding affinity of TCR and pMHC, and shared patterns of TCR-pMHC interactions.

Narrowing the Gap: Preserving Repertoire Diversity Despite Clonal Selection during the CD4 T Cell Response.

T cell immunity relies on the generation and maintenance of a diverse repertoire of T cell antigen receptors (TCRs). The strength of signaling emanating from the TCR dictates the fate of T cells during development, as well as during the immune response. Whereas development of new T cells in the thymus increases the available TCR repertoire, clonal selection during the immune response narrows TCR diversity through the outgrowth of clonotypes with the fittest TCR. To ensure maintenance of TCR diversity in the antigen-selected repertoire, specific mechanisms can be envisaged that facilitate the participation of T cell clonotypes with less than best fit TCRs. Here, we summarize the evidence for the existence of such mechanisms that can prevent the loss of diversity. A number of T cell-autonomous or extrinsic factors can reverse clonotypic hierarchies set by TCR affinity for given antigen. Although not yet complete, understanding of these factors and their mechanism of action will be critical in interventional attempts to mold the antigen-selected TCR repertoire.

TCR ITAM multiplicity is required for the generation of follicular helper T-cells.

The T-cell antigen receptor (TCR) complex contains 10 copies of a di-tyrosine Immunoreceptor-Tyrosine-based-Activation-Motif (ITAM) that initiates TCR signalling by recruiting protein tyrosine kinases. ITAM multiplicity amplifies TCR signals, but the importance of this capability for T-cell responses remains undefined. Most TCR ITAMs (6 of 10) are contributed by the CD3ζ subunits. We generated ‘knock-in' mice that express non-signalling CD3ζ chains in lieu of wild-type CD3ζ. Here we demonstrate that ITAM multiplicity is important for the development of innate-like T-cells and follicular helper T-cells, events that are known to require strong/sustained TCR–ligand interactions, but is not essential for ‘general' T-cell responses including proliferation and cytokine production or for the generation of a diverse antigen-reactive TCR repertoire.

The human T-cell repertoire grows up.

The human T-cell repertoire grows up.

Transcriptional control of the development and function of Vα14i NKT cells.

The majority of T lymphocytes, sometimes referred to as as mainstream or conventional T cells, are characterized by a diverse T cell antigen receptor (TCR) repertoire. They require antigen priming in order to become memory cells capable of mounting a rapid effector response. It has become established, however, that there are several distinct T cell lineages that exhibit a memory phenotype in the absence of antigen priming, even as they differentiate in the thymus. These lymphocytes typically express a markedly restricted TCR repertoire and their rapid response kinetics has led to their being described as innate-like T cells. In addition, several of these subsets typically express surface markers commonly found on natural killer cells, which has led to the moniker natural killer T cells (NKT cells). This review will describe our current understanding of the unique ways whereby transcription factors control the development and function of an abundant and widely studied lineage of NKT cells that recognizes glycolipid antigens.

A conserved human T cell population targets mycobacterial antigens presented by CD1b

T cell receptors (TCRs) pair in millions of combinations to create complex and personally unique T cell repertoires. Using tetramers to analyze CD1b-reactive TCRs, we detected T cells with highly stereotyped TCR α chains present among genetically unrelated tuberculosis patients. These germline-encoded mycolyl-reactive (GEM) T cells were defined by CD4 expression and rearrangement of TRAV1-2 to TRAJ9 with few N-region additions. TCR analysis by high throughput sequencing, binding and crystallography showed linkage of TCR α sequence motifs to high affinity antigen recognition. Thus, the CD1-reactive TCR repertoire is composed of at least two compartments, high affinity GEM TCRs and more diverse TCRs with low affinity for CD1b-lipid complexes. These data demonstrate high inter-donor conservation of TCRs, which likely results from selection by a non-polymorphic antigen presenting molecule and an immunodominant antigen.

Sequence analysis of T-cell repertoires in health and disease.

T-cell antigen receptor (TCR) variability enables the cellular immune system to discriminate between self and non-self. High-throughput TCR sequencing (TCR-seq) involves the use of next generation sequencing platforms to generate large numbers of short DNA sequences covering key regions of the TCR coding sequence, which enables quantification of T-cell diversity at unprecedented resolution. TCR-seq studies have provided new insights into the healthy human T-cell repertoire, such as revised estimates of repertoire size and the understanding that TCR specificities are shared among individuals more frequently than previously anticipated. In the context of disease, TCR-seq has been instrumental in characterizing the recovery of the immune repertoire after hematopoietic stem cell transplantation, and the method has been used to develop biomarkers and diagnostics for various infectious and neoplastic diseases. However, T-cell repertoire sequencing is still in its infancy. It is expected that maturation of the field will involve the introduction of improved, standardized tools for data handling, deposition and statistical analysis, as well as the emergence of new and equivalently large-scale technologies for T-cell functional analysis and antigen discovery. In this review, we introduce this nascent field and TCR-seq methodology, we discuss recent insights into healthy and diseased TCR repertoires, and we examine the applications and challenges for TCR-seq in the clinic.

Specific T-cell activation in an unspecific T-cell repertoire.

T-cells are a vital type of white blood cell that circulate around our bodies, scanning for cellular abnormalities and infections. They recognise disease-associated antigens via a surface receptor called the T-cell antigen receptor (TCR). If there were a specific TCR for every single antigen, no mammal could possibly contain all the T-cells it needs. This is clearly absurd and suggests that T-cell recognition must, to the contrary, be highly degenerate. Yet highly promiscuous TCRs would appear to be equally impossible: they are bound to recognise self as well as non-self antigens. We review how contributions from mathematical analysis have helped to resolve the paradox of the promiscuous TCR. Combined experimental and theoretical work shows that TCR degeneracy is essentially dynamical in nature, and that the T-cell can differentially adjust its functional sensitivity to the salient epitope, "tuning up" sensitivity to the antigen associated with disease and "tuning down" sensitivity to antigens associated with healthy conditions. This paradigm of continual modulation affords the TCR repertoire, despite its limited numerical diversity, the flexibility to respond to almost any antigenic challenge while avoiding autoimmunity.

Regulatory T cells: roles of T cell receptor for their development and function

Naturally arising CD4(+)CD25(+) regulatory T cells (Treg cells), which specifically express the forkhead family transcription factor Foxp3, are essential for the maintenance of immunological self-tolerance and immune homeostasis. Stimulation of the T cell antigen receptor (TCR) via recognizing self-peptide/major histocompatibility complex (MHC) is required for their expression of Foxp3 in the course of their development in the thymus. The TCR repertoires displayed by Treg cells and naïve T cells are apparently distinct, suggesting that Treg cells with high reactivity to self-peptide/MHC ligands are somehow driven to Treg cell lineage in the thymus. Treg cells also require stimulation via TCR to exert suppression in the periphery. At the molecular level, assembly of Foxp3, Foxp3-interacting factors, and chromatin-remodeling factors is in part under the control of TCR signaling, and TCR stimulation alters Foxp3-dependent transcriptional regulation, protein-protein interaction, and Foxp3 recruitment to the specific genomic loci. These findings collectively indicate that the TCR signaling is essential for suppressive function of Treg cells and that TCR has a determinant role for driving developing T cells to the Foxp3(+)CD4(+)CD25(+) Treg cell lineage and differentiation.