T Cell Receptor Repertoire in Rheumatoid Arthritis

CD4⁺ T cells are a major component of the inflammatory infiltrate in rheumatoid synovitis. Within synovial lesions, clonal CD4⁺ T cell populations are detectable, supporting the notion of an antigen specific recognition event in the joint. In general, the clonal size of individual T cell clones is small and does not lead to a marked distortion of the synovial T cell receptor (TCR) repertoire. Comparison of TCR sequences derived from different patients has not provided evidence for common sequences. Either multiple antigens are recognized or the TCR repertoire is sufficiently plastic with a multitude of different TCR structures responding to the same antigen(s). However, within one individual, the repertoire of clonal T cell populations is restricted. Identical T cell clones can be identified in different joints and at different timepoints of the disease, emphasizing that the spectrum of antigens recognized is conserved over time and that the T cell response pattern is not subject to evolution. Characterization of antigens involved in the latter stages of the disease may thus provide critical information on disease-initiating events.

Recent data have led to the new concept that the role of T cells in rheumatoid arthritis (RA) is not limited to synovial inflammation. Evidence has been provided that the premorbid TCR repertoires of RA patients and normal controls can be distinguished. The T cell repertoire in RA patients is prone to recognize certain microbial products and autoantigens. The selection of this response pattern can only partially be attributed to the disease associated HLA-DRB1 alleles. Additional factors common in RA patients but not in HLA-DR matched control individuals seem to be important in shaping the TCR repertoire. Furthermore, the repertoire of mature T cells in RA patients is characterized by oligoclonality which involves T cells in the peripheral blood compartment. Possibly, these clonal T cell populations react to widespread autoantigens, raising the possibility that RA patients have a defect in controlling peripheral tolerance and an anomaly of lymphoproliferation. In contrast to joint residing CD4⁺T cells, expanded clonotypes isolated from the blood of different patients have been described to share TCRβ chain structures. How these characteristic features of the global TCR repertoire in RA patients translate into mechanisms of disease remains to be elucidated.     

Characterization of peripheral blood TCR repertoire in patients with ankylosing spondylitis by high-throughput sequencing

Ankylosing spondylitis (AS) is a chronic and progressive autoimmune disease affecting the invasion of the spine, sacroiliac joints and peripheral joints. T cells play a vital role in the underlying pathogenesis of AS, which mediated autoimmune and inflammatory responses via specific recognition of autoantigen peptides presented by susceptibility HLA. Antigen-specific T cells triggered by HLA/antigen complexes will undergo a massive expansion that forming an uneven T cell repertoire. To enhance our understanding of T-cell-mediated autoimmune in AS, we applied TCR β chains high-throughput sequencing to AS patients for in-depth TCR repertoire analysis. A significantly lower TCR repertoire diversity was observed in peripheral blood of AS patients relative to controls. And severe patients in our AS cohort have a more restricted TCR repertoire than mild patients, suggesting that the TCR repertoire diversity might be associated with the clinical severity of disease. No V, J and VJ pairs with significant biased usage were identified, which indicated that the usage frequency deviation of certain V/J/V-J genes in AS patients is little. This is a pilot study with potentially interesting observation on reduced diversity of T cells repertoire in peripheral blood of AS patients and further studies are needed.     

乙肝疫苗无应答者CD4~+T细胞TCR Vβ基因克隆化特征的研究

目的:研究乙肝疫苗接种者CD4+T细胞TCR Vβ基因克隆化特征,分析乙肝疫苗有应答者和无应答者TCR Vβ基因单克隆改变的差异性。方法:采用多引物PCR技术扩增80例乙肝疫苗接种者CD4+T细胞TCR Vβ基因22个家族的CDR3区基因片段,对PCR产物分别应用琼脂糖凝胶电泳和基因扫描两种方法检测。结果:80例乙肝疫苗接种者中有58例产生抗体,有22例未产生抗体。TCR Vβ基因单克隆改变主要集中在Vβ2、Vβ8、Vβ9、Vβ11和Vβ17五个家族上,乙肝疫苗无应答组这五个Vβ基因单克隆改变频率明显低于乙肝疫苗有应答组(P<0.01或P<0.05)。结论:CD4+T细胞TCR Vβ基因克隆化改变是影响机体对乙肝疫苗免疫应答效果的重要因素之一。     

Altered naive CD4 and CD8 T cell homeostasis in patients with relapsing-remitting multiple sclerosis: thymic versus peripheral (non-thymic) mechanisms

We have reported previously that naive T cells from relapsing-remitting multiple sclerosis (RRMS) patients have T cell receptor (TCR) repertoire shifts, but the basis of these TCR repertoire shifts was uncertain. Here, we questioned whether RRMS patients have altered naive CD4 and CD8 T cell homeostasis by studying homeostatic proliferation and thymic production in RRMS patients and healthy controls. We measured thymic production by quantifying signal joint T cell receptor excision circles (sjTRECs). Both naive T subsets from controls showed an age-associated decrease in sjTRECs, i.e. evidence of progressive thymic involution, but we detected no age-associated decrease in sjTRECs in RRMS patients. Instead, naive CD8 T cells from patients had lower sjTRECs (P = 0.012) and higher Ki-67 proliferation levels (P = 0.04) than controls. Naive CD4 T cell sjTRECs did not differ between patients and controls. However, in RRMS these sjTRECs correlated strongly with CD31, a marker expressed by newly generated CD4 T cells but not by naive CD4 T cells that have undergone homeostatic proliferation. HLA-DR2 positivity correlated negatively with naive CD4 T cell CD31 expression in RRMS (P = 0.002). We conclude in RRMS that naive T subsets have homeostatic abnormalities due probably to peripheral (non-thymic) mechanisms. These abnormalities could have relevance for MS pathogenesis, as naive T cell changes may precede MS onset.     

αβ T cell receptors as predictors of health and disease

The diversity of antigen receptors and the specificity it underlies are the hallmarks of the cellular arm of the adaptive immune system. T and B lymphocytes are indeed truly unique in their ability to generate receptors capable of recognizing virtually any pathogen. It has been known for several decades that T lymphocytes recognize short peptides derived from degraded proteins presented by major histocompatibility complex (MHC) molecules at the cell surface. Interaction between peptide-MHC (pMHC) and the T cell receptor (TCR) is central to both thymic selection and peripheral antigen recognition. It is widely assumed that TCR diversity is required, or at least highly desirable, to provide sufficient immune coverage. However, a number of immune responses are associated with the selection of predictable, narrow, or skewed repertoires and public TCR chains. Here, we summarize the current knowledge on the formation of the TCR repertoire and its maintenance in health and disease. We also outline the various molecular mechanisms that govern the composition of the pre-selection, naive and antigen-specific TCR repertoires. Finally, we suggest that with the development of high-throughput sequencing, common TCR ‘signatures'' raised against specific antigens could provide important diagnostic biomarkers and surrogate predictors of disease onset, progression and outcome.     

Flexibility of the Thyroiditogenic T Cell Repertoire for Murine Autoimmune Thyroiditis in CD8-Deficient (B2m -/-) and T Cell Receptor VBC Congenic Mice

In murine experimental autoimmune thyroiditis (EAT), previous studies have revealed a highly adaptable thyroiditogenic T cell repertoire which involves both CD4⁺ and CD8⁺ T cells in the susceptible H2k strain. To further test this flexibility, congenic B10.K mice lacking CD8⁺ T cells (B2m -/-) or harboring 70% T cell receptor (TCR) V6 gene deletions (V6^C) were immunized with mouse thyroglobulin (MTg) and evaluated for EAT 28 days later. All 62m -/- mice developed moderate antibodies to MTg, and thyroidal inflammation was comparable to B10.K mice, averaging 35-40%. Spleen cells (SC) from MTg-immunized mice were then injected into syngeneic recipients after stimulation in vitro with MTg or with conserved, thyroxine (T4)- or thyronine (TO)- containing 12mer peptides, hT4(5), hT0(2553), or hT4(2553), derived from the primary hormonogenic sites at position 5 or 2553 of human Tg. As previously shown in another H2^k strain (CBA/J), all three peptides activated MTg-primed SC to transfer EAT in B10.K mice. hT4(5) and hT4(2553) were further tested in B10.K-V6^C and ß2m^- B10.K mice. Both peptides expanded thyroiditogenic T cells in either strain, resulting in severe thyroiditis in syngeneic recipients. That EAT can develop in the absence of CD8⁺ T cells or in the presence of a severely restricted TCR repertoire underscores the remarkable flexibility of the thyroiditogenic T cell profile in the susceptible k haplotype.     

TCR function analysis using a novel system reveals the multiple unconventional tumor-reactive T cells in human breast cancer-infiltrating lymphocytes

Tumor-infiltrating lymphocytes (TILs) are a potent source for obtaining tumor-reactive T cell receptors (TCRs). Although comprehensive methods to analyze the TCR repertoire in TILs have been reported, the evaluation system for TCR-reactivity to endogenously expressed antigen in tumor cells remains laborious and time consuming. Consequently, very limited numbers of TCRs in TILs have been analyzed for their reactivity to tumor cells. In this study, we developed an efficient evaluation system for TCR function designated c-FIT (c omprehensive f unctional i nvestigation of T CRs) to analyze TCR reactivity. The c-FIT system enabled us to analyze up to 90 TCRs for their reactivity to tumor cells by a single assay within a month. Using c-FIT, we analyzed 70 TCRs of CD8⁺ TILs derived from two breast cancer patients and obtained 23 TCRs that reacted to tumor cells. Surprisingly, although two TCRs were HLA class I-restricted, the remaining 21 TCRs were non-HLA-restricted. Thus, c-FIT can be applied for monitoring multiple conventional and unconventional antigen-specific killer T cells in TILs, leading to the development of new designs for more effective T-cell-based immunotherapies.     

Post-thymic in vivo proliferation of naive CD4+ T cells constrains the TCR repertoire in healthy human adults

In spite of thymic involution early in life, the numbers of naive CD4(+) T cells only slowly decline in ageing humans implying peripheral post-thymic naive CD4(+) T cell expansion. This proliferation may compensate for continuous activation and death of naive CD4(+) T cells but may also have negative consequences for protective immunity. Here we show that naive CD4(+) T cells that have proliferated in the periphery are characterized by a highly restricted oligoclonal TCR repertoire. Additionally these cells, which constitute the majority of naive CD4(+) T cells in the elderly, display signatures of recent TCR engagement. Our results demonstrate for the first time that peripheral post-thymic proliferation of naive CD4(+) T cells in healthy human individuals causes a significant contraction of the peripheral TCR repertoire. This age-dependent deterioration of CD4(+) T cell immunity could entail ageing-associated autoimmunity, increased susceptibility to infection or cancer and decreased efficiency of vaccination.     

TCR tuning of T cell subsets

After selection in the thymus, the post-thymic T cell compartments comprise heterogenous subsets of naive and memory T cells that make continuous T cell receptor (TCR) contact with self-ligands bound to major histocompatibility complex (MHC) molecules. T cell recognition of self-MHC ligands elicits covert TCR signaling and is particularly important for controlling survival of naive T cells. Such tonic TCR signaling is tightly controlled and maintains the cells in a quiescent state to avoid autoimmunity. Here, we review how naive and memory T cells are differentially tuned and wired for TCR sensitivity to self and foreign ligands.     

CD28协同刺激分子对T细胞受体介导的胸腺细胞亚群凋亡的影响

目的：分析anti-TCRαβmAb anti-CD28mAb诱导小鼠胸腺淋巴细胞不同亚群的凋亡及凋亡程度，分析CD28协同刺激分子对TCR受体介导的胸腺细胞亚群凋亡的影响。方法：新鲜分离胸腺细胞，加入anti-TCRαβmAb-anti-TCRαβmAb anti-CD28mAb等培养20h，进行多重染色，流式细胞仪分析。结果：与胸腺细胞自发凋亡的结果相比较；（1）双信号刺激可明显增加胸腺细胞凋亡的数目，尤其是CD4^ CD8^ 胸腺细胞的凋亡数目。（2）凋亡的CD4^ CD8^ 亚群，CD4^ CD8^-亚群细胞表面CD28的表达均增多。结论：CD28共刺激分子对TCR受体介导的胸腺细胞亚群凋亡的影响与细胞的成熟程度有关，CD28共刺激分子能明显增强不成熟皮质胸腺细胞的凋亡。     

T Cell Receptor Gene in Synovial Tissues of Rheumatoid Arthritis

Rheumatoid arthritis is a chronic and destructive autoimmune joint disease characterized by inflammation of synovial tissue of unknown aetiology. Studies on TCR genes expressed by infiltrating T cells in synovial tissues have attempted to identify mechanism and specificity of the recruitment. T cell infiltrate in rheumatoid arthritis appears to be an association of a polyclonal non specific infiltrate with dominant clones or clonotypes. T cell repertoire in synovial tissue is biased compared to peripheral blood but no TCR V gene can be identified as commonly over-used. Comparison of motifs found in the CDR3 region of dominant clones from different studies has currently failed to identified a commonly motif. The fact that a number of dominant clones or clonotypes is present in different joints and at different times of the disease suggests a selective expansion of T lymphocytes in rheumatoid arthritis synovial membrane. Further investigations are needed to characterize the specificity of these dominant clonotypes.

Synovial T cell infiltrate in rheumatoid arthritis appears to be polyclonal and the majority of T cells are non-specifically recruited into sites of inflammation following tissue injury. But almost all the studies show the presence of a large number of dominant clones in the synovial tissue, using different TCR V genes, which certainly play an important role in the perpetuation of the disease. This, and the fact that a number of dominant clones or clonotypes is present in different joints and at different times of the disease, strongly suggest a selective expansion of T lymphocytes in synovial membrane. Comparison of motifs found in the CDR3 region of dominant clones from different studies is difficult as of the number of TCRs analysed is generally low and the HLA-DR differs from patient to other. To further investigate TCR in RA, identification of the pathogenic clonotypes would be the first step before characterization of CDR3 regions of dominant clonotypes. Although the target or the auto-antigens involved in the disease have not yet been identified, sequence analyses of the CDR3 regions of dominant clonotypes could, in the future, give important informations about peptides that are the targets of auto-reactive T cells.     

Epitope Context and Reshaping of Activated T Helper Cell Repertoire

In recent years, a growing interest in the study of peptide antigenicity in relation to the role of flanking sequences and protein topology in processing, presentation, and recognition has been observed. However, the information available on the antigenicity of recombinant fusion proteins and their effect on the selection of antigen receptor repertoires is limited. To analyze the role of molecular topology of T epitopes in a system relevant to human pathology, we have used the bacterially expressed Schistosoma japonicum glutathione S transferase (GST) to construct recombinant antigens containing HIV-1 derived T cell determinants, and human T cell clones specific for these determinants. We found that antigenicity of a given GST—peptide combination was not the same when T cells and antigen presenting cells from different individuals were tested. Our results show that differences in processing and presentation of chimeric proteins are not dictated by the use of diverse restriction elements. We also found that the context in which an antigenic peptide is delivered affects the recruited repertoire as defined according to T cell receptor Vβ usage and fine specificities of selected T cells.     

A T cell receptor (TCR) antagonist competitively inhibits serial TCR triggering by low-affinity ligands,but does not affect triggering by high-affinity anti-CD3 antibodies

It has been demonstrated that modified peptides which fail to induce detectable T cell responses can act as T cell receptor (TCR) antagonists when presented together with agonist by the same antigen-presenting cell (APC). We report that a TCR antagonist competitively inhibits TCR triggering induced by low-affinity ligands such as agonistic peptides or bacterial superantigens. However, the same antagonist cannot inhibit TCR triggering and T cell activation induced by high-affinity anti-CD3 antibodies that engage most TCR at once. These results indicate that TCR antagonists inhibit T cell responses by interfering with the ongoing process of serial triggering, rather than by delivering an inhibitory signal to T cells.     

Cross Reactivity of T Cell Receptor on Memory CD8+ Cells Isolated after Immunization with Allogeneic Tumor Cells

Experiments on mice deficient in expression of class I major histocompatibility complex molecules showed that memory CD8+ cells recognizing the alloantigen by the direct allogeneic recognition mechanism selectively proliferated in response to heated allogeneic cells. Adoptive transfer of memory cells from mice expressing green fluorescent protein transgene to wild-type animals showed for the first time that long-living memory cells suppress the response of naive T cells and abolish their involvement in the pool of memory cells. The pool of long-living memory T cells was obtained in vitro with heated allogeneic stimulators. Apart from immunizing alloantigen, this clone recognized foreign molecules of the major histocompatibility complex. Cloning and sequencing of rearranged regions in memory T cells showed that two alpha-chains and one functional beta-chain are rearranged in cells of this pool. Only one alpha-chain was capable of forming protein product, which determines expression of only one form of T cell receptor. Experimental data directly confirm the hypothesis about degeneracy of recognition of allelic products of major histocompatibility complex molecules by T cell receptors. Suppression of the response of naive cells by memory cells probably underlies a previously unknown type of polarization of the immune response and determines clonal dominance and peripheral selection of T lymphocytes.     

多发性硬化症脑脊液中活化T细胞抗原受体V_β基因的表达

应用定量逆转录聚合酶链反应（ＲＴ－ＰＣＲ）对４例多发性硬化症（ＭＳ）脑脊髓液（ＣＳＦ）细胞及末梢血单个核细胞（ＰＢＭＣ）中Ｔ细胞抗原受体（ＴＣＲ）Ｖ_β基因（Ｖ_β１～Ｖ_β２０）表达进行了探讨。结果表明：未经刺激的ＣＳＦ细胞和ＰＢＭＣ表达广谱Ｖ_β基因，经ＴＣＧＦ／ＩＬ－２／ＩＬ－４短期培养的ＣＳＦ细胞则表达限制性Ｖ_β基因，而经同样培养的ＰＢＭＣ各Ｖ_β基因表达的百分率同培养前ＰＢＭＣ相同。说明在ＭＳ疾病的中枢神经系统中存在着对某种抗原的特异性Ｔ细胞应答，为ＭＳ的免疫治疗提供了新的启示。     

Functional differences between influenza A-specific cytotoxic T lymphocyte clones expressing dominant and subdominant TCR.

We have shown that the dominance of CD8+ T cells expressing TCR Vbeta17 in the adult HLA-A*0201-restricted influenza A/M1(58-66)-specific response is acquired following first antigen exposure. Despite the acquired dominance of Vbeta17+ cells, subdominant M1(58-66)-specific clones expressing non-Vbeta17+ TCR persist in all individuals. To determine whether the affinity of the expressed TCR for the HLA-A*0201/M1(58-66) complex could influence functional properties, M1(58-66)-specific clones expressing subdominant (non-Vbeta17+) TCR were compared to cytotoxic T lymphocyte (CTL) clones expressing dominant (Vbeta17+) TCR. The Vbeta17+ CTL required up to 10,000-fold lower amounts of M1 peptide to mediate lysis compared to CTL clones expressing other Vbeta gene segments. All Vbeta17+ CTL clones tested bound HLA-A*0201/M1(58-66) tetramer, but two of three CTL clones expressing other TCR did not bind tetramer. The inability of non-Vbeta17+ CTL to bind tetramer did not correlate with phenotype, CD8 dependence or with cytokine production profiles. This suggests a limitation for the use of tetramers in examining subdominant T cell responses. Together these findings suggest that Vbeta17+ CTL which dominate the HLA-A*0201-restricted CTL response against influenza A are not functionally distinct from subdominant non-Vbeta17+ CTL. The dominance of Vbeta17+ CTL is likely to result from a competitive advantage due to superior CTL avidity for the HLA-A*0201/M1(58-66) complex.     

T Cell Receptor Excision Circles (TRECs) in Relation to Acute Cardiac Allograft Rejection

The purpose of this study was to quantify T cell receptor excision circles (TRECs) in blood mononuclear cells of cardiac transplant recipients and to investigate a possible relationship between TREC levels and rejection episodes. In addition, we investigated the correlation of TREC levels with age and also compared the levels between transplant recipients and healthy individuals. TREC levels were assessed by quantitative competitive PCR in 70 blood samples from 27 graft recipients and in 66 blood samples from 66 healthy individuals. The results showed: (1) higher TREC levels during rejection than during rejection-free periods (medians 9.0 vs. 0.3; p<0.001); (2) no suggestion of correlation with doses of prednisone or time after transplantation; (3) a negative correlation between TREC levels and age; and (4) lower TREC levels in cardiac recipients than in age-matched healthy controls. The value of blood TREC level measurements as an approach to rejection monitoring warrants future investigation.     

T Cell Repertoire Evolution after Allogeneic Bone Marrow Transplantation: An Organizational Perspective

High-throughput sequencing (HTS) of human T cell receptors has revealed a high level of complexity in the T cell repertoire, which makes it difficult to correlate T cell reconstitution with clinical outcomes. The associations identified thus far are of a broadly statistical nature, precluding precise modeling of outcomes based on T cell repertoire development following bone marrow transplantation (BMT). Previous work has demonstrated an inherent, mathematically definable order observed in the T cells from a diverse group of donors, which is perturbed in recipients following BMT. In this study, T cell receptor (TCR)-β sequences from HLA-matched related donor and recipient pairs are analyzed to further develop this methodology. TCR-β sequencing from unsorted and sorted T cell subsets isolated from the peripheral blood samples of BMT donors and recipients show conservation and symmetry of VJ segment usage in the clonal frequencies, linked to the organization of the gene segments along the TCR locus. This TCR-β VJ segment translational symmetry is preserved post-transplantation and even in cases of acute graft-versus-host disease (aGVHD), suggesting that GVHD occurrence represents a polyclonal donor T cell response to recipient antigens. The complexity of the repertoire is significantly diminished after BMT, and the T cell clonal hierarchy is altered post-transplantation. Low-frequency donor clones tended to take on a higher rank in the recipients following BMT, especially in patients with aGVHD. Over time, the repertoire evolves to a more donor-like state in the recipients who did not develop GVHD as opposed to those who did. The results presented here support new methods of quantifying and characterizing post-transplantation T cell repertoire reconstitution.