Skewed T-cell receptor BV14 and BV16 expression and shared CDR3 sequence and common sequence motifs in synovial T cells of rheumatoid arthritis

T-lymphocytes play an important role in rheumatoid arthritis (RA). In this study, we evaluated the hypothesis that common T-cell receptor (TCR) structural features may exist among infiltrating T cells of different RA patients, if the TCR repertoire is shaped by interaction with common self or microbial antigens in the context of susceptible HLA genes in RA. Synovial lesion tissue (ST), synovial fluid (SF) and blood specimens from RA patients and controls were analyzed for TCR V gene repertoire by real-time PCR. There was highly skewed BV14 and BV16 usage in synovial T cells of RA as opposed to those of controls, which was accompanied with a trend for correlation between skewed BV16 and DRB1(*)0405. Immunoscope analysis of the V-D-J region of ST-derived T cells demonstrated oligoclonal and polyclonal expansion of BV14(+) and BV16(+) T cells. Detailed characterization using specific BV and BJ primers further revealed common clonotypes combining the same BV14/BV16, BJ and CDR3 length. DNA cloning and sequence analysis of the clonotypes confirmed identical CDR3 sequences and common CDR3 sequence motifs among different RA patients. The findings are important in the understanding of BV gene skewing and CDR3 structural characteristics among synovial infiltrating T cells of RA.     

Influence of antigenic experience on BJ gene segment usage in human CD4+ T cells

HLA molecules influence the selection of naive CD4+ T cells as demonstrated by HLA-DR-dependent differences in BV8-BJ frequencies. The repertoire of mature peripheral T cells utilized in antigen responses is shaped by additional factors such as antigens encountered in the environment. To identify mechanisms underlying the formation of the memory repertoire, differences in the BV8-BJ repertoire of CD45RO- and CD45RO+ CD4+ T cells were examined in 21 normal donors. The naive and memory CD4+ compartments displayed unique BV8-BJ repertoires in all individuals, demonstrating that the recruitment of CD4+ T cells into the memory population is a non-random process. The frequencies of selected BV8-BJ combinations were increased among CD45RO+ T cells. Size fractionation of such expanded BV8-BJ populations demonstrated that most of them were polyclonal in nature. Twenty-five percent of the expanded BV-BJ combinations included a dominant TCR sequence, indicating monoclonal proliferation. Selection of BV8-BJ combinations for preferential use among memory T cells was HLA dependent. HLA-DR1/4+ individuals were characterized by an increased usage of BV8-BJ2S7+ TCR, and decreased usage of BV8-BJ2S1 + and BV8-BJ2S2+ TCR, whereas HLA- DR3/7+ individuals preferentially recruited BV8-BJ2S5+ T cells, and disfavored BV8-BJ2S3+ and BV8-BJ2S7+ T cells. HLA-imposed effects on the naive and memory repertoire were distinct. The BV-BJ frequencies of CD45RO+ T cells could not be predicted from the pattern of TCR found in naive CD4+ T cells, suggesting that the HLA-DR polymorphisms influence thymic selection processes differently than peripheral selection forces.      

Clonal analysis of T cell infiltrates in synovial tissue of patients with rheumatoid arthritis

To investigate the possibility of the presence of disease-relevant, antigen-specific immune reactions in rheumatoid arthritis (RA), clonal diversity of the T cells in the synovial tissue was examined. T cells were directly cloned by in vitro stimulation with phytohemagglutin and interleukin 2 from both the peripheral blood and inflammatory synovial tissue. Their clonotypes were defined by analyzing rearrangement patterns of T cell receptor (TCR) beta and gamma chain genes using Southern blotting. In total, 111 clones from the synovial tissue (four patients) and 45 from the peripheral blood (one patient) were studied. Although most of the clones were unique in their TCR gene rearrangement patterns, 2 clones from the synovial tissue of one patient had identical patterns. These 2 clones were CD3+, 4-, 8+. Since phenotypic analysis of 82 clones from the synovial tissues revealed that CD8+ T cell clones were less frequent (24%) than CD4+ clones, the clonal identity observed here in 2 clones may not be negligible. Furthermore, 1 CD8+ clone from the peripheral blood of the same patient also had the same clonotype. These results may suggest selective trafficking or proliferation of CD4-, CD8+ T cells in RA synovial tissue.     

Ex vivo monitoring of antigen-specific CD4+ T cells after recall immunization with tetanus toxoid

To monitor antigen-specific CD4+ T cells during a recall immune response to tetanus toxoid (TT), a sequential analysis including ex vivo phenotyping and cytokine flow cytometry, followed by cloning and T-cell-receptor (TCR) spectratyping of cytokine-positive CD4+ T cells, was performed. Grossly, twice as many TT-specific CD4+ T-cell clones, ex vivo derived from the CCR7+/- CD69+ interleukin-2-positive (IL-2+) CD4+ subsets, belonged to the central memory (T(CM); CD62L+ CD27+ CCR7+) compared to the effector memory population (T(EM); CD62L- CD27- CCR7-). After the boost, a predominant expansion of the T(CM) population was observed with more limited variations of the T(EM) population. TCR beta-chain-variable region (BV) spectratyping and sequencing confirmed a large concordance between most frequently expressed BV TCR-CDR3 from ex vivo-sorted CCR7+/- CD69+ IL-2+ CD4+ subsets and BV usage of in vitro-derived TT-specific CD4+ T-cell clones, further demonstrating the highly polyclonal but stable character of the specific recall response to TT. Taken together, ex vivo flow cytometry analysis focused on the CCR7+/- CD69+ IL-2+ CD4+ subsets appears to target the bulk of antigen-specific T cells and to reach an analytical power sufficient to adequately delineate in field trials the profile of the antigen-specific response to vaccine.     

The role of T cells in rheumatoid arthritis

In rheumatoid arthritis (RA), T cells infiltrate into the synovial membrane where they initiate and maintain activation of macrophages and synovial fibroblasts, transforming them into tissue-destructive effector cells. The diversity of the disease process and the formation of complex lymphoid microstructures indicate that multiple T cell activation pathways are involved. This model is supported by the association of distinct disease patterns with different variants and combinations of HLA class II molecules. T cell pathology in RA, however, is not limited to the joint. Affected patients have major abnormalities in the T cell pool, with a marked contraction in T cell receptor diversity and an outgrowth of large clonal populations. Clonally expanded CD4+ T cells lose expression of the CD28 molecule and gain expression of perforin and granzyme. Consequently, the functional profile of expanded CD4(+)CD28null T cells is fundamentally changed and is shifted towards tissue-injurious capabilities. CD4(+)CD28null T cells are particularly important in patients with extra-articular manifestations of RA, where they may have a direct role in vascular injury. Understanding the mechanisms underlying the loss of T cell diversity and the emergence of pro-inflammatory CD4(+)CD28null T cell clonotypes may have implications for other autoimmune syndromes.     

胶原诱导的关节炎大鼠关节中浸润的T细胞TCR Vβ克隆型分析

目的：研究胶原诱导的关节炎（CIA）大鼠关节中局部浸润的T细胞TCR Vβ克隆型。方法：用Ⅱ型胶原（CⅡ）诱导Lewis大鼠发生关节炎后，经流式细胞术分析大鼠外周血CD4^＋和CD8^＋细胞的变化。提取CIA大鼠关节组织的总RNA，通过RT-PCR／SSCP方法分析大鼠两后肢足关节中TCR Vβ克隆型的一致率。将CIA大鼠脾淋巴细胞转移至同系正常大鼠，观察发病情况。结果：从注射CⅡ的第30天起，大鼠后爪出现红、肿等症状。流式细胞术分析显示，与正常大鼠相比较，CIA大鼠外周血CD8^＋T细胞的降低有显著性意义（P〈0．01），CD4^＋T／CD8^＋T的比值大于2（P〈0．01），提示CIA大鼠体内存在T淋巴细胞亚群的比例失调。CIA大鼠两后肢足关节中聚集的TCR Vβ克隆型的一致率，以TCR Vβ5．2和8．2最高，分别为78．89％和72．23％。脾淋巴细胞转移试验显示，受者发病的大鼠关节中，出现与供者CIA大鼠一致的TCR Vβ克隆型条带，表明关节局部聚集的TCR Vβ克隆型与关节炎（RA）的发病有关。结论：CIA大鼠关节局部存在以TCR Vβ5．2和8．2为主的致病性的T细胞TCR Vβ克隆型，可将其作为治疗CIA的靶物质，进行实验动物RA治疗的研究。     

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 even 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 TCR beta chain structures. How these characteristic features of the global TCR repertoire in RA patients translate into mechanisms of disease remains to be elucidated.     

The frequency of double-positive thymocytes expressing an alphabeta TCR clonotype regulates peripheral CD4 T cell compartment homeostasis

The present study aimed to determine whether the frequency of double positive (DP) thymocytes expressing alphabeta T-cell receptor (TCR) clonotypes at the time of selection regulates peripheral CD4 T-cell compartment size. Scid recipients were inoculated with various ratios of TCR Calpha(0/0) and wild-type bone marrow (BM) stem cells. Increasing the frequency of TCR Calpha(0/0) thymocytes at steady-state introduced a graded decrease in the maturation probability of the total DP thymocyte pool. At 12-14 weeks following BM inoculation, the frequency of TCR Calpha(0/0) DP thymocytes was inversely correlated with that of CD4 single positive (SP) thymocytes. Notwithstanding, a decreased frequency of wild-type DP thymocytes led to a marked increase in their transit efficiency from the DP to SP compartments. The frequency-dependent increase in thymocyte transit efficiency was associated with a CD4 SP cell surface phenotype indicative of increased antigenic experience. Importantly, the frequency of DP thymocytes capable of expressing TCR clonotypes dictated the steady-state size of the peripheral CD4 T cell compartment and its potential for homeostatic proliferation. Collectively, these results indicate that the efficiency of DP to CD4 SP transit is a frequency dependent process, which determines (1) the steady-state size of the peripheral T cell compartment and (2) the threshold for homeostatic expansion of peripheral CD4 T cells.     

Immunization of volunteers with Salmonella enterica serovar Typhi strain Ty21a elicits the oligoclonal expansion of CD8+ T cells with predominant Vbeta repertoires

CD8(+) T cells are likely to play an important role in host defense against Salmonella enterica serovar Typhi by several effector mechanisms, including lysis of infected cells (cytotoxicity) and gamma interferon (IFN-gamma) secretion. In an effort to better understand these responses, we studied the T-cell receptor (TCR) repertoire of serovar Typhi-specific CD8(+) T cells in humans. To this end, we determined the TCR beta chain (Vbeta) usage of CD8(+) T cells from three volunteers orally immunized with Ty21a typhoid vaccine by flow cytometry using a panel of monoclonal antibodies. Although TCR Vbeta usage varied among volunteers, we identified oligoclonal Vbeta subset expansions in individual volunteers (Vbeta 2, 5.1, 8, 17, and 22 in volunteer 1; Vbeta 1, 2, 5.1, 14, 17, and 22 in volunteer 2; and Vbeta 3, 8, 14, and 16 in volunteer 3). These subsets were antigen specific, as shown by cytotoxicity and IFN-gamma secretion assays on Vbeta sorted cells and on T-cell clones derived from these volunteers. Moreover, eight-color flow cytometric analysis showed that these clones exhibited a T effector memory phenotype (i.e., CCR7(-) CD27(-) CD45RO(+) CD62L(-)) and coexpressed gut homing molecules (e.g., high levels of integrin alpha4beta7, intermediate levels of CCR9, and low levels of CD103). In conclusion, our results show that long-term T-cell responses to serovar Typhi in Ty21a vaccinees are oligoclonal, involving multiple TCR Vbeta families. Moreover, these serovar Typhi-specific CD8(+) T cells bearing defined Vbeta specificities are phenotypically and functionally consistent with T effector memory cells with preferential gut homing potential.     

The impact of Metastasis Suppressor-1, MTSS1, on oesophageal squamous cell carcinoma and its clinical significance

Background

Non Obese Diabetic mice lacking B cells (NOD.Igμ^null mice) do not develop diabetes despite their susceptible background. Upon reconstitution of B cells using a chimera approach, animals start developing diabetes at 20 weeks of age.

Methods

We have used the spectratyping technique to follow the T cell receptor (TCR) V beta repertoire of NOD.Igμ^null mice following B cell reconstitution. This technique provides an unbiased approach to understand the kinetics of TCR expansion. We have also analyzed the TCR repertoire of reconstituted animals receiving cyclophosphamide treatment and following tissue transplants to identify common aggressive clonotypes.

Results

We found that B cell reconstitution of NOD.Igμ^null mice induces a polyclonal TCR repertoire in the pancreas 10 weeks later, gradually diversifying to encompass most BV families. Interestingly, these clonotypic BV expansions are mainly confined to the pancreas and are absent from pancreatic lymph nodes or spleens. Cyclophosphamide-induced diabetes at 10 weeks post-B cell reconstitution reorganized the predominant TCR repertoires by removing potential regulatory clonotypes (BV1, BV8 and BV11) and increasing the frequency of others (BV4, BV5S2, BV9, BV16-20). These same clonotypes are more frequently present in neonatal pancreatic transplants under the kidney capsule of B-cell reconstituted diabetic NOD.Igμ^null mice, suggesting their higher invasiveness. Phenotypic analysis of the pancreas-infiltrating lymphocytes during diabetes onset in B cell reconstituted animals show a predominance of CD19⁺ B cells with a B:T lymphocyte ratio of 4:1. In contrast, in other lymphoid organs (pancreatic lymph nodes and spleens) analyzed by FACS, the B:T ratio was 1:1. Lymphocytes infiltrating the pancreas secrete large amounts of IL-6 and are of Th1 phenotype after CD3-CD28 stimulation in vitro.

Conclusions

Diabetes in NOD.Igμ^null mice appears to be caused by a polyclonal repertoire of T cell accumulation in pancreas without much lymphoid organ involvement and is dependent on the help by B cells.     

Cytokine profile,HLA restriction and TCR sequence analysis of human CD4+ T clones specific for an immunodominant epitope of Mycobacterium tuberculosis 16-kDa protein

The identification of immunodominant and universal mycobacterial peptides could be applied to vaccine design and have an employment as diagnostic reagents. In this paper we have investigated the fine specificity, clonal composition and HLA class II restriction of CD4+ T cell clones specific for an immunodominant epitope spanning amino acids 91-110 of the 16-kDa protein of Mycobacterium tuberculosis. Twenty-one of the tested 28 clones had a Th1 profile, while seven clones had a Th0 profile. None of the clones had a Th2 profile. While the TCR AV gene usage of the clones was heterogeneous, a dominant TCR BV2 gene family was used by 18 of the 28 clones. The CDR3 regions of BV2+ T cell clones showed variation in lengths, but a putative common motif R-L/V-G/S-Y/W-E/D was detected in 13 of the 18 clones. Moreover, the last two to three residues of the putative CDR3 loops, encoded by conserved BJ sequences, could also play a role in peptide recognition. Antibody blockade and fine restriction analysis using HLA-DR homozygous antigen-presenting cells established that 16 of 18 BV2+ peptide-specific clones were DR restricted and two clones were DR-DQ and DR-DP restricted. Additionally, five of the 18 TCRBV2+ clones recognized peptide 91-110 in association with both parental and diverse HLA-DR molecules, indicating their promiscuous recognition pattern. The ability of peptide 91-110 to bind a wide range of HLA-DR molecules, and to stimulate a Th1-type interferon (IFN)-gamma response more readily, encourage the use of this peptide as a subunit vaccine component.     

Immune reconstitution following autologous transfers of CD3/CD28 stimulated CD4(+) T cells to HIV-infected persons

We have previously shown that adoptive transfer of in vitro CD3/CD28 activated autologous CD4(+) T cells results in increased CD4 counts and CD4/CD8 ratios in HIV+ subjects. In this report, analysis of variable beta (Vbeta) chain T cell receptor (TCR) repertoire showed that CD3/CD28 stimulation was able to increase polyclonality within skewed spectra types in vitro. In vivo, two of eight subjects showed increase in TCR diversity and importantly, in no subject did a highly skewed in vivo repertoire emerge. Measurement of proliferative response to alloantigen showed increases following infusions. Response to pharmacological stimulus and lectin via Interferon-gamma ELISpot assay showed increases in a subset of subjects following infusions. However, interferon-gamma response to HIV antigens and peptides declined concurrent with stable or diminishing latent infectious viral load in CD4(+) T cells. These data provide further evidence that adoptive transfer of activated autologous CD4(+) T cells can augment the immune system.     

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.     

Single cell analysis of T cells infiltrating labial salivary glands from patients with Sj?gren's syndrome.

Sj?gren's syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration into the lacrimal and salivary glands leading to symptomatic dry eyes and mouth. To analyze the function of T cells infiltrating the labial salivary glands, we analyzed T cell receptor (TCR) beta and alpha chains, the expression of various cytokine mRNAs, and apoptosis associated genes in predominant TCR BV2+ T cells in the labial salivary glands of patients with SS at the single cell level. TCR BV2+ T cells in the labial salivary glands were sorted as single cells by flow-cytometry, and then examined by a single cell polymerase chain reaction (PCR). We isolated 18 TCR BV2+ T cell clones from three patients with SS. In six clones, there were highly conserved amino acid motifs (RDxG, GNT, QGxxQETQ) in the CDR3 region of the TCR beta chain. Three of the six clones showed conserved amino acids (EDxTG, or ExxTG) in the CDR3 region of the TCR alpha chain, suggesting restricted T cell epitopes. All TCR BV2+ clones expressed IL-2 mRNA, and six clones were able to produce IL-4, indicating that the cells were Th0 type T cells. All TCR BV2+ clones in the labial salivary glands were CD4+ T cells, and ten clones overexpressed Fas antigen at the mRNA level. In contrast, only one clone expressed Fas-ligand (Fas-L) mRNA, and neither perforin nor granzyme A/B was expressed. In conclusion, these findings support the notion that TCR BV2+ T cells that infiltrate labial salivary glands recognize restricted epitopes and function as CD4+ Th0 type T cells in the induction phase of autoimmunity.     

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.     

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.