Dominant clonotypes in the repertoire of peripheral CD4+ T cells in rheumatoid arthritis

Clonal expansion of T cell specificities in the synovial fluid of patients has been taken as evidence for a local stimulation of T cells. By studying the T cell receptor (TCR) repertoire of CD4+ T cells in the synovial and peripheral blood compartments of patients with early rheumatoid arthritis (RA), we have identified clonally expanded CD4+ populations. Expanded clonotypes were present in the peripheral blood and the synovial fluid but were not preferentially accumulated in the joint. Dominant single clonotypes could not be isolated from CD4+ cells of HLA-DRB1*04+ normal individuals. Clonal expansion involved several distinct clonotypes with a preference for V beta 3+, V beta 14+, and V beta 17+CD4+ T cells. A fraction of clonally related T cells expressed IL-2 receptors, indicating recent activation. The frequencies of clonally expanded V beta 17+CD4+ T cells fluctuated widely over a period of one year. Independent variations in the frequencies of two distinct clonotypes in the same patient indicated that different mechanisms, and not stimulation by a single arthritogenic antigen, were involved in clonal proliferation. These data support the concept that RA patients have a grossly imbalanced TCR repertoire. Clonal expansion may result from intrinsic defects in T cell generation and regulation. The dominance of expanded clonotypes in the periphery emphasizes the systemic nature of RA and suggests that T cell proliferation occurs outside of the joint.     

Selective accumulation of related CD4+ T cell clones in the synovial fluid of patients with rheumatoid arthritis

The role of T cells in the pathogenesis of rheumatoid arthritis (RA), especially in the perpetuation of advanced disease, remains unclear. Previous studies have focused on the TCR repertoire of synovial T cells in an attempt to determine whether the pattern of expression is characteristic of Ag-stimulated populations. However, the results of past studies have been conflicting. In the present work, we have undertaken an extensive analysis of the TCRs expressed by CD4+ T cells freshly isolated from synovial fluid of different joints and blood in three patients with established RA. Despite marked heterogeneity of synovial TCR expression, the results showed that 20 to 30% of the TCR beta-chain gene (TCRB) sequences found in one joint were also expressed in a second joint, but not in peripheral blood T cells of the same individual. Analysis of expressed TCRB complementarity-determining region 3 sequences showed the presence of multiple expanded clonal populations that were not predicted by quantitation of beta-chain variable region (Vbeta) expression by immunofluorescence staining. These studies also demonstrated sets of related, but different, complementarity-determining region 3 nucleotide sequences that encoded identical or highly homologous beta-chain amino acid sequences. Analysis of matching T cell clones derived from the joint by limiting dilution culture confirmed coexpression of highly homologous TCR alpha-chain gene (TCRA) and TCRB sequences. Together, these studies suggest that a significant proportion of synovial CD4+ T cells has been selected and expanded by conventional Ag(s) in this disease.     

Comparative sequence analysis of the human T cell receptor beta chain in juvenile rheumatoid arthritis and juvenile spondylarthropathies: evidence for antigenic selection of T cells in the synovium

OBJECTIVE: To identify features of the T cell receptors (TCRs) present on clonally expanded T cells in the joints of patients with similar types of childhood rheumatic disease. Vbeta8 and Vbeta20 TCRs were selected as prototypic for polyarticular juvenile rheumatoid arthritis (JRA) and pauciarticular/juvenile spondylarthropathy (SpA), respectively. METHODS: The portion of the TCR beta chain involved in antigen recognition in the synovial tissue, synovial fluid, and peripheral blood from patients with JRA and juvenile SpA was cloned and sequenced. The frequency of expanded clonotypes, size of expansions, the Jbeta region, and sequence motifs were determined for >2,000 sequences. RESULTS: The majority of Vbeta20 and Vbeta8 clonal expansions were found in the joint rather than the peripheral blood. While instances of both Vbeta8 and Vbeta20 clonal expansion were detected in all disease types, the features of these expanded clonotypes were specific for disease type and Vbeta family. For example, Vbeta20 clonal expansion was characterized by many small expanded clonotypes in samples from patients with pauciarticular JRA and juvenile SpA while single large Vbeta8-specific expansions were found only in patients with polyarticular disease. Motifs specific to individual patients were identified, and for Vbeta20 clonotypes, a motif was found in synovial tissue samples. CONCLUSION: Identification of common TCR features in oligoclonal expansions within individual patients and between patients with the same type of JRA suggests the recognition of a common or limited group of antigens in these diseases.     

Evidence for selective in vivo expansion of synovial tissue-infiltrating CD4+ CD45RO+ T lymphocytes on the basis of CDR3 diversity

In this study we have analyzed the TCR V alpha and V beta regions at the DNA level in the CD4+CD45RO+ memory T cell population of synovial tissue infiltrating T lymphocytes of three rheumatoid arthritis (RA) patients and one patient with chronic arthritis. Cell lines of CD4+CD45RO+, CD4+CD45RO-, CD8+CD45RO+ and CD8+CD45RO- T lymphocyte populations were generated following FACS cell sorting of freshly isolated synovial tissue mononuclear cell infiltrates (STMC) and of freshly isolated peripheral blood mononuclear cells (PBMC) of these patients. The phenotypic and molecular analyses have revealed the following. (i) The TCR repertoires of tissue infiltrating T lymphocytes in the various subsets were extensive on the basis of TCR V gene family usage. (ii) Furthermore, each patient displayed individual specific TCR V gene expression patterns in the various STMC and PBMC derived T cell subsets. However, the majority of these arthritis patients manifested increased expression of multiple TCR V gene families in the synovial tissue derived CD4+CD45RO+ T cell population when compared with the peripheral blood derived CD4+CD45RO+ subset. Of these gene families, we found enhanced expression of the TCR V alpha 7 and V beta 11 gene segments in synovial tissue to be shared by all four patients analyzed. (iii) Nucleotide sequence analysis of the CDR3 regions of a number of TCR V regions in the CD4+CD45RO+ T cell subsets has revealed that the CDR3 regions comprised within synovial tissue derived TCR V regions differed from those found in peripheral blood derived TCR V regions. These differences in CDR3 diversity might be the consequence of a specific interaction with particular MHC-peptide complexes expressed at the site of inflammation. (iv) The CDR3 region analysis also showed individual specific amino acid motifs within the N-D-N regions of all analyzed TCR V beta genes derived from PBMC as well as STMC.     

Gender differences in use of stress testing and coronary heart disease mortality: a population-based study in Olmsted County, Minnesota

The distribution of CD57+ T and CD56+ T cells in patients with RA was examined. In control osteoarthritis patients, these cells exist as a minor population in the peripheral blood. Our data show that in patients with RA, CD57+ T cell levels are elevated in peripheral blood, knee joint fluid, knee synovial membrane and bone marrow (BM), compared with peripheral blood of controls. CD57+ T cells are especially high in knee joint fluid and joint-adjacent BM, while CD56+ T cells show no such increase. CD57+ T cells contain a major population of CD8+ cells and higher proportions of CD4-8- cells and gammadelta T cells than do CD57- T cells. CD57+ T cells in peripheral blood and joint fluid increase with the duration of disease. Erythrocyte sedimentation rate (ESR) is inversely correlated with the proportion of CD57+ T cells in the joint fluid. Although RA frequently occurs in patients with CD3+57+ cell leukaemia, and some CD57+ T cells are likely to be involved in the onset of RA, we suggest that CD57+ T cells may rather suppress inflammation of RA, and other cellular components (e.g. granulocytes) may govern the severity of the inflammation of RA. These CD57+ T cells are probably generated extrathymically in the adjacent BM or joint space.     

Analysis of type 1 and type 2 T cells in synovial fluid and peripheral blood of patients with rheumatoid arthritis

OBJECTIVE: It has been reported that CD4+ helper T cells play an important role in the pathogenesis of rheumatoid arthritis (RA). We evaluated the presence of intracellular cytokines interleukin 4 (IL-4) and interferon-gamma (IFN-gamma) produced by CD4+ and CD8+ T cells in the synovial fluid and peripheral blood of patients with RA at the single cell level. METHODS: We used 3 color flow cytometric analysis. Synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) were stimulated with phorbol myristate acetate (PMA) and calcium ionophore. The stimulated SFMC and PBMC were triple stained with conjugated mononuclear antibodies (Mab) against cytokines and surface antigens after fixation and permeabilization with a saponine buffer solution. The cells were analyzed for intracellular cytokines (IFN-gamma, IL-4) and surface antigens (CD3, CD4, CD8) using a flow cytometer. RESULTS: The CD4/CD8 ratio was significantly lower in SFMC than in PBMC. The positive rates of IFN-gamma producing cells among CD4+ T cells were significantly higher than those of IL-4 producing cells in both the SFMC and the PBMC of patients with active RA. In the SF of these patients, we also found CD8+ T cells that produce IL-4 alone, or both IL-4 and IFN-gamma. CONCLUSION: In the SF of patients with RA, CD4+ type 1 T cells, which may infiltrate into the synovium and cause pathogenic immune responses in the tissue, are predominant. We believe this cell type also induces migration and activation of CD8+ type 2 T cells into the active site of inflammation, which appears to downregulate the activity of CD4+ type 1 T cells, modulating the excess immune response.     

Inflammatory infiltrate and interleukin-8 expression in the synovium of psoriatic arthritis--an immunohistochemical and mRNA analysis

Psoriatic arthritis is an inflammatory arthropathy that ultimately can lead to joint destruction. In this study, we investigated the immunophenotypes of the inflammatory cells and the expression of interleukin-8 (IL-8), which is the hallmark chemoattractant cytokine of psoriasis in synovial membranes from patients exhibiting active psoriatic synovitis (n = 9). The tissue samples were examined by immunohistochemistry, Western blot analysis and in situ hybridisation. The inflammatory infiltrate consisted predominantly of CD3+ T lymphocytes, with a higher proportion of CD4+ than CD8+ T lymphocytes in six cases. CD3+ T lymphocytes were focally distributed near small blood vessels and the enlarged synovial intima. CD1+ interdigitating reticulum cells were not detected. CD22+ B lymphocytes and plasma cells were found in small aggregates without KiM4+ follicular dendritic cells. KiM8+ macrophages were located in the synovial intima and were distributed in a diffuse pattern near the synovial lining cells. CD15+ neutrophil granulocytes were detected in four cases. They were preferentially located in the vicinity of blood vessels and the synovial intima. IL-8 was found at a high level in the synovial lining cells and to a lesser extent in cells located in the perivascular areas. Immunofluorescence double staining showed IL-8 to be expressed in KiM8+ multinucleated giant cells, KiM8+ macrophages and CD3+ T lymphocytes. IL-8 receptor A was demonstrated in the synovial lining and in macrophages and lymphocytes. IL-8 was detected by immunoblot analysis of the synovial tissue at 8.4 kD. Employing in situ hybridisation, IL-8 mRNA was strongly and preferentially expressed in the synovial intima, as well as in macrophages and lymphocytes. The immunophenotype of the psoriatic arthritis inflammatory cells shows great similarity to the inflammatory infiltrate found in the synovial tissue of patients with rheumatoid arthritis. The preferential expression of IL-8 and IL-8 mRNA in the enlarged synovial intima and in lymphocytes and macrophages suggests that IL-8 exerts its action through activated mononuclear cells and T lymphocytes. It seems to play a role in regulating leucocyte traffic into the enlarged synovial intima and may contribute to the aggressive synovitis of patients with psoriatic arthritis.     

Identification of autoimmune T cells among in vivo expanded CD25+ T cells in multiple sclerosis

Although clonal expansion of autoimmune T cells has been reported in multiple sclerosis (MS), very limited information is available on specificities, clonal size, or activation state of the expanded clones. Here we address the issue of clonal expansion by using a novel technique demonstrating clonotypes defined by single-strand conformation polymorphism of TCR beta-chain cDNAs. Examination of activated T cells (CD3+CD25+) isolated from the peripheral blood of MS revealed limited numbers (20 approximately 82) of expanded clones defined by single-strand conformation polymorphism clonotype. To estimate the Ag specificities of dominant clonotypes in the activated T cells, these samples were examined in parallel with Th1 T cell clones specific for myelin basic protein or proteolipid protein (PLP) derived from the same patients. Analysis of two patients demonstrated that the dominant clonotypes would contain those specific for myelin basic protein or PLP. Although the majority of the clonotypes could be detected only transiently, a PLP95-116-specific clonotype was found to persist for over 1 yr. Thus, single-strand conformation polymorphism clonotype analysis allows us to monitor the kinetics of given T cell clones in vivo and could provide useful information for designing clonotype (Id)-specific manipulation of human diseases such as MS.     

Age-related modifications of the human alphabeta T cell repertoire due to different clonal expansions in the CD4+ and CD8+ subsets

We have studied the effects of a life-long antigen stimulation on the clonal heterogeneity of human peripheral T cell subsets, as defined by their CD45 isoform expression. CD4+ or CD8+ T cells were obtained from healthy donors ranging in age from 20 to 100 years, and sorted into CD45RA+ and CD45RO+ populations. A modified PCR-heteroduplex analysis was then used to directly compare the TCR Vbeta clonal make up of either compartment pair. We find that the CD4+ T cell repertoire remains largely polyclonal throughout life, since CD4+ expanded clones are rare and accumulate predominantly in the CD45RO+ compartment of exceptionally old donors (100 years old). In contrast, the CD8+ T cell subset contains expanded clones which are already detectable in young adults and become very frequent in 70- to 75-year-old donors in both CD45RA+ and CD45RO+ compartments analyzed. Interestingly, some expanded clones are detectable in the CD45RA+ or in both CD45RA+ and CD45RO+ compartments of either CD4+ or CD8+ T cells. These results indicate that the age-dependent accumulation of expanded clones starts earlier and is more pronounced in the CD8+ than in the CD4+ T cell subset, reinforcing the concept that clonal expansion in the two subsets is controlled by substantially different mechanisms. Furthermore, whereas the finding of expanded CD45RO+ T cell clones is explained by antigen-driven proliferation, the detection of expanded clones in the CD45RA+ or in both CD45RA+ and CD45RO+ compartments would support the hypothesis of reversion from the CD45RO+ to the CD45RA+ phenotype after antigen encounter.     

Oligoclonal accumulation of T cells in peripheral blood from patients with idiopathic thrombocytopenic purpura

To determine whether clonal T cells accumulate in idiopathic thrombocytopenic purpura (ITP), we performed single-strand conformation polymorphism (SSCP) analysis to detect T-cell receptor (TCR) beta-chain usage of peripheral T cells. We detected significantly more oligoclonal T cells (15.5 +/- 8.9 bands representative for clonal T-cell expansions) in peripheral blood from ITP patients than from healthy donors (2.8 +/- 2.6 bands). Frequently used V beta genes in these accumulated T cells in ITP were V beta 3, 6, 10, 13.1 and 14. To determine whether these bands were derived from clonal T cells, presumably in a preactivated state, we established some T-cell clones (expressing CD4 and TCR V beta 6. 13.1. or 14) by nonspecific stimulation from patients peripheral mononuclear cells, and examined their clonotypes. Clonal identities for three out of seven clones tested were confirmed using SSCP analyses to compare the migration of their beta-chain complementarity determining region 3 (CDR3) cDNAs, expanded by polymerase chain reaction (PCR) with those from peripheral blood. Therefore, distinctive T-cell clones accumulated in the periphery in ITP and they may be related to the autoimmune-mediated destruction of platelets.     

How tumor stage affects surgeons'' surveillance strategies after surgery for carcinoma of the upper aerodigestive tract

OBJECTIVE: To compare the expression of adhesion molecules on synovial T cells from patients with early spondyloarthropathy (SpA) and rheumatoid arthritis (RA), with special reference to the beta7 integrins alpha4beta7 and alphaEbeta7 in view of their intimate association with intestinal tissue. METHODS: Twenty-five synovial cell lines were generated by interleukin 2 (IL-2) expansion from synovial biopsies of patients with early SpA and RA, obtained from macroscopically inflamed synovial tissue by needle arthroscopy, and subsequently characterized by flow cytometry for CD3, CD4, CD8, L-selectin, CD11a, CD31, CD44, and alpha4beta7 and alphaEbeta7 integrin. RESULTS: In SpA, the beta7 integrin expression was increased, compared to RA. Furthermore, an inverse relation between alpha4beta7 and alphaEbeta7 was present in SpA (r = -0.75, p < 0.02), as on many mucosal T cells. In contrast, an opposite correlation was noted in RA (r = +0.84, p < 0.01), as similarly described on a subset of circulating T cells. CONCLUSION: Increased expression of beta7 integrins was noted on synovial T cell lines from SpA compared to RA, with discriminative correlations between alpha4beta7 and alphaEbeta7. This suggests a different origin of the synovial T cells in these diseases.     

Functional analysis of rheumatoid factor-producing B cells from the synovial fluid of rheumatoid arthritis patients

OBJECTIVE: To understand the regulation of rheumatoid factor (RF) production in rheumatoid arthritis (RA), we studied IgM-RF production by B cells isolated from the synovial fluid (SF). METHODS: Highly purified SF and peripheral blood (PB) B cells were isolated by negative selection in a fluorescence-activated cell sorter (FACS) and then cultured with either L cells, CD40 ligand (CD40L)-transfected L cells, or type B synoviocytes in the presence or absence of interleukin-2 (IL-2), IL-4, or IL-10. Total IgM and IgM-RF were detected after 14 days by enzyme-linked immunosorbent assay. Enzyme-linked immunospot assays were performed to detect cells that spontaneously produced immunoglobulin. SF B cells were also phenotypically characterized by FACS analysis. RESULTS: Terminally differentiated CD20-,CD38+ synovial plasma cells (PC) present in the SF of RA patients secreted IgM-RF in the absence of a stimulus. IgM-RF production markedly increased when SF B cells were cultured in the presence of type B RA synoviocytes together with IL-10, but independently of CD40-CD40L interaction. Although CD20-,CD38+ PC could also be demonstrated in SF B cells from patients with other forms of arthritis, IgM-RF production was restricted to the SF B cell cultures of patients with seropositive RA. The frequency of IgM-RF-producing cells among IgM-producing PC in patients with seropositive RA was estimated to be as much as 50%. CONCLUSION: These data demonstrate that terminally differentiated CD20-,CD38+ IgM-RF-producing B cells are specifically present in the inflamed joints of patients with seropositive RA. There is evidence that the local environment in the rheumatoid joint favors RF production. The relatively high frequency of IgM-RF PC in the SF B cell population provides evidence of a dominant RA-specific antigen-driven response in the development of the synovial PC repertoire.     

Expression of the activation antigen CD27 in rheumatoid arthritis

Differentiation of CD4+ T-cells is reflected by the change from the CD45RA+CD27+ phenotype via CD45RO+CD27+ to the CD45RO+CD27- phenotype. To provide insight into the migration and activation of T-cells at the site of inflammation in rheumatoid arthritis (RA), CD27 expression by T-cells in peripheral blood (PB), synovial fluid (SF), and synovial tissue (ST) as well as the levels of the soluble form of CD27 (sCD27) in plasma and SF were studied in patients with RA. Since CD4+CD27+ T-cells are involved in providing helper activity for B-cells, we also investigated the levels of rheumatoid factors in serum and SF in relation to CD27 expression. The mean level of sCD27, which is produced by CD27+ cells, and the mean percentage of CD27 T-cells within the CD4+CD45RA- subset were higher in SF than in PB. SF sCD27 levels were higher in the patients with RA than in the patients with osteoarthritis, who served as controls. In ST infiltration by CD4+CD45RO+CD27+ T-cells, could be demonstrated in the rheumatoid perivascular lymphocytic aggregates with a relative increase in the percentage of CD27- T-cells in the diffuse lymphocytic infiltrate. The sCD27 levels and the percentages of CD4+CD27+ cells in SF correlated positively with the levels of rheumatoid factors in serum and SF. The findings presented in this study suggest a continuous influx of preactivated CD4+CD45RO+CD27+ cells from the PB into the rheumatoid ST and further activation and differentiation to CD4+CD45RO+CD27- cells in situ, followed by migration to the SF. These activated T-cells are likely to play a role in synovial inflammation.     

Cadmium inhibits proteoglycan and procollagen production by cultured human lung fibroblasts

OBJECTIVE: Previously, we showed that 15-20% of patients with rheumatoid arthritis (RA) have oligoclonal expansions of peripheral blood CD8+ T cells expressing T cell receptors encoded by the V(alpha)12 (AV12S1) gene. To better understand the significance of these expansions, the present study was undertaken to determine their specificity. METHODS: We cloned and characterized V(alpha)12+,CD8+ T cells from the peripheral blood of 1 RA patient with a clonal expansion of these T cells. RESULTS: The T cell clones were autoreactive since they recognized autologous, but not allogeneic, antigen-presenting cells. Upon activation, these T cells secreted interleukin-4 and interleukin-10. The autoreactive T cell clones were class I major histocompatibility complex (MHC) restricted, by either HLA-B60 or HLA-Cw3. CONCLUSION: A large population of class I MHC-restricted CD8+ T cells in a patient with RA is clonally expanded and autoreactive. These cells define a novel immune aberration in RA and provide a tool for defining the autoantigens that activate expanded T cell populations in vivo.     

Detection of Borrelia burgdorferi-specific CD8+ cytotoxic T cells in patients with Lyme arthritis

T cells are believed to play an important role in the pathogenesis of Lyme arthritis (LA), an inflammatory joint disease caused by the spirochete Borrelia burgdorferi (Bb). The presence or absence of certain Bb-specific CD4+ T helper cells has been associated with prognosis. Since recent observations suggested the activation of CD8+ T cells during infection with Bb, we searched for CD8+ cytotoxic T lymphocytes in patients with LA. CD8+ T cell lines were generated from peripheral blood and synovial fluid of five patients with LA. In addition, CD8+ T cells were expanded by Ag-specific stimulation in bulk cultures. A cytotoxicity assay was established using target cells infected with recombinant vaccinia viruses expressing the borrelial proteins outer surface protein (Osp) A, OspB, or flagellin. We found Bb-specific CTL lines derived from the peripheral blood of three patients with LA with specificity for flagellin, OspA, and OspB. All Bb-specific CTL lines were CD3+, CD8+, and TCRalphabeta, and cytotoxic activity was HLA class I restricted. Moreover, CD8+ T cells expanded by Ag-specific stimulation in vitro demonstrated Bb-specific and HLA class I-restricted lysis toward individual borrelial proteins. Interestingly, Bb-specific lytic activity was only detected in patient samples obtained after the disappearance of arthritis. We report the detection of Bb-specific cytotoxic CD8+ T cells in patients with LA. The induction of specific CD8+ T cells may play an important role in disease control and may have important bearings for the development of effective vaccines against Lyme borreliosis.     

Redox regulation of ubiquitin-conjugating enzymes: mechanistic insights using the thiol-specific oxidant diamide

This study explores whether previous failures on antiretroviral drug regimens preclude the possibility of immune restoration. This was assessed by evaluating T cell subset changes in individuals who received a salvage regimen of highly active antiretroviral therapy (HAART) after initially failing protease inhibitor monotherapy. Ten HIV-1-infected asymptomatic patients received a regimen of indinavir, zidovudine, and 3TC after failing saquinavir monotherapy. Changes in absolute numbers of naive, memory, and activated CD4+ and CD8+ T cells expressing a selection of CD45RA, CD62L, CD45RO, HLA-DR, and CD38 markers were monitored prospectively over 6 months. These measurements were correlated with plasma viral load along with alterations in a selected CD8+ V alpha/Vbeta T cell receptor (TCR) repertoire. Over 6 months there was a progressive increase in numbers of CD4+ memory (CD45RA-CD62L+) and naive (CD45RA+CD62L+) T cells, which displayed a modest inverse correlation with viral load. Two phases of CD8+ memory cell changes were identified, consisting of a transient increase in CD45RA+CD62L- numbers after 2 months and thereafter a progressive rise in CD45RA-CD62L+ cells until 6 months. A strong correlation existed between reduced viral load and loss of activated CD8+CD38+HLA-DR+ cell numbers. There was also a temporary broadening of the CD8+ V alpha/Vbeta TCR repertoire at 8 weeks, which became skewed after 6 months in parallel with reduced viral suppression. Closer analysis of naive and memory cell subset proportions in individual patients revealed that enlarged pools of naive subsets were evident in those patients with rebounds in viral load. Overall, drug-experienced patients responding to HAART displayed increased numbers of naive and memory CD4+ subsets, and reduced CD8+ cell activation with a loss of TCR skewing.