Analysis of T cell receptor V(beta) gene expression and clonality in bronchoalveolar fluid lymphocytes from a patient with chronic eosinophilic pneumonitis

Chronic eosinophilic pneumonitis (CEP) is characterized by longstanding respiratory symptoms accompanied by a massive pulmonary eosinophil infiltration. T lymphocytes in bronchoalveolar lavage (BAL) from patients with chronic eosinophilic pneumonitis are considered to recognize unknown antigens. To analyze the pathogenesis of CEP, we examined the T cell receptor (TCR) repertoire and T cell clonotype of BAL lymphocytes and peripheral blood lymphocytes (PBLs) in a 66-year-old woman patient with CEP. The expression of TCR BV gene was analyzed by the family PCR method using specific primers for 20 TCR BV genes and BC gene. The clonotype of BAL and peripheral T cells was examined by the PCR-single-strand conformation polymorphism (SSCP) method. Functional sequences of some T cell clones were also carried out. A TCR repertoire of BAL T cells was heterogeneous as well as PBLs. However, SSCP analysis showed that distinct T cell clonotypes were detected in BAL T cells, TCR BV3, BV4, BV6, BV8, BV9, BV14, and BV18-positive T cell clones especially, expanded clonally in BAL from the patient. Sequencing analysis showed that GVD, LGG, RDXS, and SSG amino acid sequence motif were found in the CDR3 in lung-specific T cells. BAL-specific T cell clones accumulated in the patient with CEP. Thus, we can conclude that BAL T cells are induced by the antigen-driven stimulation and these cells might play a crucial role in the generation of CEP. Accepted for publication: 27 February 2001     

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

Naturally acquired immune responses against human cancers often include CD8⁺ T cells specific for the cancer testis antigen NY-ESO-1. Here, we studied T cell receptor (TCR) primary structure and function of 605 HLA-A*0201/NY-ESO-1_157–165-specific CD8 T cell clones derived from five melanoma patients. We show that an important proportion of tumor-reactive T cells preferentially use TCR AV3S1/BV8S2 chains, with remarkably conserved CDR3 amino acid motifs and lengths in both chains. All remaining T cell clones belong to two additional sets expressing BV1 or BV13 TCRs, associated with α-chains with highly diverse VJ usage, CDR3 amino acid sequence, and length. Yet, all T cell clonotypes recognize tumor antigen with similar functional avidity. Two residues, Met-160 and Trp-161, located in the middle region of the NY-ESO-1_157–165 peptide, are critical for recognition by most of the T cell clonotypes. Collectively, our data show that a large number of αβ TCRs, belonging to three distinct sets (AVx/BV1, AV3/BV8, AVx/BV13) bind pMHC with equal antigen sensitivity and recognize the same peptide motif. Finally, this in-depth study of recognition of a self-antigen suggests that in part similar biophysical mechanisms shape TCR repertoires toward foreign and self-antigens.     

Diminished levels of T cell receptor zeta chains in peripheral blood T lymphocytes from patients with systemic lupus erythematosus.

OBJECTIVE: To examine the expression of molecules known to participate in early T cell receptor (TCR)/CD3 signaling in peripheral blood (PB) T lymphocytes from patients with systemic lupus erythematosus (SLE). METHODS: Signaling molecules were analyzed by immunoprecipitation and Western blotting of unstimulated PB T lymphocyte cell lysates from SLE patients, non-SLE disease controls, and healthy controls. Flow cytometry was used for analysis of the expression of membrane markers in intact cells. RESULTS: PB T lymphocytes from SLE patients showed diminished levels of TCRzeta chains. This was not due to trapping of these molecules in the cytoskeleton, nor was it dependent on the presence of monocyte/macrophages. There was normal expression of CD3epsilon chains and normal assembly of TCR/CD3 complexes in membranes. We observed a lack of expression of TCRzeta chains in in vitro cultures of SLE T cells, and reversal of the defective expression in some patients by culturing T cells in the presence of NH4Cl. CONCLUSION: Blood lymphocytes from SLE patients have a diminished expression of TCRzeta chains that may be related to enhanced degradation in the lysosomal compartment. The defective expression of these molecules may alter signal transduction via the CD3 pathway and contribute to abnormal T cell responses in T lymphocytes from SLE patients.     

Dynamic T cell receptor clonotype changes in synovial tissue of patients with early rheumatoid arthritis: effects of treatment with cyclosporin A (Neoral)

OBJECTIVE: To study T cell receptor (TCR) repertoire changes in synovial membrane over a 16 week period in patients with early rheumatoid arthritis (RA); and to study the influence of cyclosporin A (CSA) on TCR repertoire in a subgroup of these patients. METHODS: Synovial tissue biopsies and paired blood samples were obtained from 12 patients with early RA at 2 time points. Seven patients were treated with CSA (Neoral-Sandimmun, 3 mg/kg/day) and 5 patients with placebo for 16 weeks. TCR V gene repertoires were analyzed by semiquantitative PCR-ELISA. CDR3 spectratyping and sequence analysis was used to compare TCR clonotype distributions. RESULTS: TCR-specific mRNA was detected in all synovial tissue biopsies at the first sampling, but in only 8/12 biopsies 16 weeks later (4/7 CSA group, 4/5 placebo group). Overrepresented TCR BV genes were found in biopsies of 10/12 patients at the first time point, and in 7/12 patients after 16 weeks (3/7 CSA, 4/5 placebo). CDR3 sequence analysis revealed dynamic repertoire changes with only a few persisting clonotypes in the synovial tissue of placebo controls. Persisting T cell clonotypes were more frequently found in the synovial tissue of CSA treated patients compared to the placebo group. CONCLUSION: These data suggest a dynamic process of T cell recruitment in the joints of RA patients. This process, possibly due to activation and subsequent infiltration of new T cell clones, apparently is influenced by CSA treatment. Synovial tissue T cells were no longer detected after 16 weeks' CSA treatment in 3 patients. In the other CSA treated patients, new T cell clones infiltrated, while other clones were persistently represented in the joints. These data may have important consequences for the design of T cell targeted therapies for RA.     

Clonal expansion of mycobacterial heat-shock protein–reactive T lymphocytes in the synovial fluid and blood of rheumatoid arthritis patients

Objective. To examine the reactivity pattern and T cell receptor (TCR) characteristics of mycobacterial heat-shock protein 65 (hsp65)-reactive T cells generated from paired synovial fluid (SF) and peripheral blood (PB) samples obtained from rheumatoid arthritis (RA) patients and from healthy subjects. Methods. The reactivity pattern of hsp65-reactive T cell clones generated under limiting-dilution conditions was analyzed in ³H-thymidine incorporation assays. The TCR variable regions of these hsp65-reactive T cells were characterized by polymerase chain reaction with TCR AV- and BV-specific primers and by DNA sequence analysis of the third complementarity-determining region (CDR3). Results. The hsp65-reactive T cells derived both from RA patients and controls preferentially recognized the 1–170 and 303–540 regions of hsp65 and did not cross-react with human hsp60. The hsp65-reactive T cell clones derived from RA patients displayed a restricted TCR AV and BV gene usage, which can be attributed to the limited clonal origin(s) of the independent T cell clones, as evidenced by CDR3 sequence analysis. These clonally expanded T cells were found in both PB and SF and in different inflamed joints of RA patients. Conclusion. Our study suggests that there is in vivo clonal activation and expansion of mycobacterial hsp65-reactive T cells in patients with RA.     

Molecular analysis of T cell clonotypes in muscle-infiltrating lymphocytes from patients with human T lymphotropic virus type 1 polymyositis

Epidemiological studies have shown that a correlation may exist between human T cell lymphotropic virus type 1 (HTLV-1) infection and a form of polymyositis (PM). To characterize muscle-infiltrating lymphocytes (MILs) from patients with HTLV-1 PM, we examined the T cell receptor (TCR) beta-chain variable region repertoire and clonotype of MILs and peripheral blood mononuclear cells (PBMC) from 3 patients, using TCR complementarity-determining region 3 (CDR3) length spectratyping and DNA sequencing. Immunohistochemical studies showed that MILs from patients with HTLV-1 PM contain both CD4(+) and CD8(+) T cells. Although some clonotypes observed in PBMC were also found in MILs in all patients examined, MILs consisted predominantly of locally expanded clones. One clonotype in MILs was derived from human leukocyte antigen (HLA)-A*02/Tax11-19 tetramer-positive cells, the CDR3 motif of which contains amino acid residues for HLA-A*02/Tax peptide-TCR interaction. We conclude that certain T cell clones proliferate in the muscle lesions of HTLV-1 PM and may contribute to the pathogenesis of the disease.     

Decreased number of T cells bearing TCR rearrangement excision circles (TREC) in active recent onset systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is characterized by several T lymphocyte abnormalities. An indirect assessment of recent thymus emigrants (RTE) has been recently been made available by measuring the number of TCR recombination excision circles (TREC) in peripheral T cells. We studied TREC levels in peripheral blood mononuclear cells (PBMC) of 32 SLE patients with active disease and 32 normal age- and sex-matched controls. Signal-joint TREC concentration was determined by real-time quantitative-PCR as the number of TREC copies/microg PBMC DNA. SLE patients had lower TREC levels (4.1+/-3.9 x 10(4) TREC/microg DNA) than controls (8.9+/-7.9 x 10(4)/microg DNA) (P = 0.004). There was an inverse correlation between age and TREC levels in controls (r = -0.41, P = 0.02) but not in SLE patients. No clinical association was observed between TREC levels and clinical and laboratory SLE manifestations. TREC levels tended to be lower in patients with SLEDAI above 20 than in the rest of the patients (P = 0.08). The decreased PBMC TREC levels is indicative of a low proportion of RTE in SLE and could be caused by decreased RTE output and/or by increased peripheral T cell proliferation in this disease. The under-representation of RTE in the peripheral T cell pool may play a role in the immune tolerance abnormalities observed in SLE.     

Analysis of the CDR3 region of alpha/beta T‐cell receptors (TCRs) and TCR BD gene double‐stranded recombination signal sequence breaks end in peripheral blood mononuclear cells of T‐lineage acute lymphoblastic leukemia

Recently, numerous reports have highlighted the restriction of the CDR3 length of T‐cell receptor (TCR) beta chain in T‐cells infiltrating solid tumors and hematological malignancies. However, these studies ignored the restriction of CDR3 length of TCR alpha chain and few of them attempted to reveal the mechanisms of the oligo‐clonal expansion of T cells in the tumors. The primary aims of this study were twofold to: (i) analyze the CDR3 length of TCR alpha and beta chain in peripheral blood mononuclear cells of T‐lineage acute lymphoblastic leukemia (T‐ALL); and (ii) discover the relationship between the clonality of T cells and the process of TCR rearrangement in peripheral T cells. To this end, we investigated the TCR BV and TCR AV family spectratypes of two T‐ALL patients and healthy controls using the immunoscope spectratyping technique. We found that the spectratypes exhibited a Gaussian distribution in healthy controls. However, the TCR repertoires of the two patients were highly restricted in the number of different TCR BV and TCR AV family members present. Furthermore, we found that the peripheral blood mononuclear cells (PBMC) of two T‐ALL patients had the recombination signal sequence (RSS) 5′‐ and 3′‐breaks end in the TCR BD2 gene using a specialized ligation‐mediated polymerase chain reaction, implying the ongoing recombination of the TCR beta gene. Analysis of the particular CDR3 length of TCR alpha/beta T cells might be helpful for further study of the individualized therapy of T‐ALL. This information will also be helpful in exploring new immunological pathogenesis and facilitating the design of a T‐ALL vaccine, as well as in improving our understanding of healthy human T‐cell development.     

Emergence of oligoclonal t cell populations following therapeutic t cell depletion in rheumatoid arthritis

Objective. To examine the compartment of CD4+ T cells in patients with rheumatoid arthritis (RA) who have developed persistent lymphopenia following antibody-mediated T cell depletion and to investigate why T cell depletion is of limited therapeutic efficacy. Methods. Circulating T lymphocytes from 10 patients with seropositive RA treated with the monoclonal antibody (MAb) CAMPATH-1H were longitudinally monitored by fluorescence-activated cell sorter analysis with MAb. To assess the molecular diversity of repopulating T cells, random samples of T cell clones from the peripheral blood of 3 patients were analyzed by sequencing the T cell receptor (TCR) β chains. At the time of recurring disease, the synovial tissue was examined by immunohistochemistry, and the repertoires of peripheral and synovial tissue T cells were compared by TCR β-chain sequencing and by semiquantitative hybridization with oligonucleotides specific for the V–D–Jβ junctional region of selected clones. Results. The reconstitution of the peripheral T cell compartment was very slow. A mean CD4+ T cell count of 105/μl was reached 34 weeks following MAb treatment. After treatment, the percentage of CD4+ T cells with the CD45RO+ phenotype was significantly increased (P = 0.001), indicating the expansion of antigen-primed memory T cells. TCR β-chain sequences revealed a marked restriction in the diversity of repopulating T cells with the emergence of dominant clonotypes. Despite the low counts of peripheral CD4+ T cells, the synovial tissue was infiltrated by CD4+ T cells to a similar extent as that in RA patients not treated with MAb. Selected clonotypes that had emerged in the peripheral blood compartment dominated the repertoire of tissue-infiltrating T cells in the synovium. Conclusion. In patients with RA, T cell depletion induces a long-term imbalance in T cell homeostasis. Clonal proliferation of CD4+ T cells severely restricts the diversity of available T cell specificities and results in the emergence of dominant clonotypes, which accumulate in the synovial tissue despite peripheral lymphopenia.     

Prevalence of T cell receptor zeta chain deficiency in systemic lupus erythematosus

T cells from patients with systemic lupus erythematosus (SLE) display antigen receptor-mediated signaling aberrations associated with defective T cell receptor (TCR) zeta chain expression. We determined the prevalence of TCR zeta chain deficiency in SLE from a large cohort of unselected racially diverse patients with different levels of clinical disease activity as determined by SLE Disease Activity Index (SLEDAI). Our data show that the occurrence of TCR zeta chain deficiency is 78% in SLE patients. There was no relationship between the deficiency of TCR zeta chain and the SLEDAI scores or theapy. TCR zeta chain deficiency was also not associated with age, race or gender and persisted over a 3 year follow-up period. Thus, there is a high prevalence of TCR zeta chain deficiency in SLE patients that is independent of disease activity, and persists over time indicating an important role for TCR zeta chain deficiency in SLE pathogenesis.     

Hypoxanthine-guanine phosphoribosyltransferase reporter gene mutation for analysis of in vivo clonal amplification in patients with HTLV type 1-associated Myelopathy/Tropical spastic paraparesis

We tested a surrogate selection approach utilizing mutation at a reporter gene [hypoxanthine-guanine phosphoribosyltransferase (hprt)] as a probe for in vivo cell division, for detection of clonal T cell expansion in human T lymphotropic (HTLV-1) carriers. Peripheral blood samples from HTLV-1-infected individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) were tested to determine the hprt mutant frequency (Mf). Wild-type and hprt mutant T cell clones were isolated, and clonal identity determined by multiplex PCR and DNA sequencing of T cell receptor (TCR) variable region beta-chain (TCR BV) and third complementarity determining regions (CDR3). Seven samples from HAM/TSP patients were tested, and Mfs were within the normal range for adults (mean 11.3 x 10(-6), max 22.4 x 10(-6), min 5.6 x 10(-6)). The frequency of HTLV-1 infection in wild-type and hprt mutant T cells from HAM/TSP patients was determined to identify enrichment in the mutant fraction of cells. This analysis was performed on 196 isolates from 6 individuals with HAM/TSP. In each case, there is enrichment for virally infected cells in the hprt mutant fraction of isolates. Ten mutant and eight wild-type isolates from sample LS42A (Mf 8.4 x 10(-6)) were tested for clonality by TCR BV PCR and sequencing. Of the 10 hprt mutants, there were two in vivo-expanded clones (four isolates with two identical TCRs, or 80% unique TCR sequences). These studies may provide new insights into the precise mechanism of HTLV-1 leukemogenesis, and aid in the study of mutator phenotypes generated by a combination of Tax-mediated in vivo expansion and mutagenesis.     

T cell signaling abnormalities in systemic lupus erythematosus are associated with increased mutations/polymorphisms and splice variants of T cell receptor zeta chain messenger RNA.

OBJECTIVE: T cells from patients with systemic lupus erythematosus (SLE) display antigen receptor-mediated signaling aberrations associated with defective T cell receptor (TCR) zeta chain protein and messenger RNA (mRNA) expression. This study was undertaken to explore the possibility that coding-region mutations/polymorphisms of the TCR zeta chain could account for its decreased expression and altered signaling in SLE T cells. METHODS: TCR zeta chain mRNA from 48 SLE patients, 18 disease controls, and 21 healthy volunteers was reverse transcribed, amplified by polymerase chain reaction, and cloned, and complementary DNA (cDNA) was sequenced. DNA sequences from multiple clones were analyzed for silent single-nucleotide polymorphisms, mutations, and splice variations, to promote the identification of heterozygosity. RESULTS: DNA sequence analysis revealed several widely distributed missense mutations and silent polymorphisms in the coding region of the TCR zeta chain, which were more frequent in SLE patients than in patients with other rheumatic diseases or healthy controls (P < 0.0001). Several of the missense mutations were located in the 3 immunoreceptor tyrosine activation motifs or the GTP binding domain, and this could lead to functional alterations in the TCR zeta chain. A splice variant of the TCR zeta chain with a codon CAG (glutamine) insertion between exons IV and V was found in half of the SLE and control samples. Two larger spliced isoforms of the TCR zeta chain, with an insertion of 145 bases and 93 bases between exons I and II, were found only in SLE T cells. We also identified various alternatively spliced forms of the TCR zeta chain resulting from the deletion of individual exons II, VI, or VII, or a combined deletion of exons V and VI; VI and VII; II, III, and IV; or V, VI, and VII in SLE T cells. The frequency of the deletion splice variants was significantly higher in SLE than in control samples (P = 0.004). These variations were observed in cDNA and may not reflect the status of the genomic DNA. CONCLUSION: These findings demonstrate that heterogeneous mutations/polymorphisms and alternative splicing of TCR zeta chain cDNA are more frequent in SLE T cells than in T cells from non-SLE subjects and may underlie the molecular basis of known T cell signaling abnormalities in this disease.     

Dysfunction of T cell receptor AV24AJ18+, BV11+ double-negative regulatory natural killer T cells in autoimmune diseases

OBJECTIVE: We examined the reduction of T cell receptor (TCR) AV24+,BV11+ CD4-,CD8- (double-negative [DN]) natural killer T (NKT) cells in peripheral blood lymphocytes (PBLs) from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and Sjogren's syndrome (SS) to analyze why NKT cells are selectively reduced in autoimmune diseases, and to examine whether nonresponse to alpha-galactosylceramide (alpha-GalCer) is due to an abnormality in the antigen-presenting cells (APCs) or NKT cells. METHODS: Peripheral blood from patients with RA (n = 20), SLE (n = 18), SSc (n = 13), and SS (n = 17), as well as from healthy donors (n = 13) and patients with Beh?et's disease (BD; n = 20), was examined by flow cytometry to determine the number of TCR AV24+,BV11+ DN T cells. PBLs from 10 RA, 10 SLE, 8 SSc, and 9 SS patients, as well as from 7 healthy subjects, were cultured in vitro with alpha-GalCer, and the number of TCR AV24+,BV11+ DN NKT cells was estimated. APCs from responder and nonresponder patients were cocultured with NKT cells from responder and nonresponder patients. RESULTS: The mean +/- SEM number of TCR AV24+,BV11+ DN NKT cells per ml of whole blood was found to be 48.8+/-10.0 in RA patients, 50.6+/-12.9 in SLE patients, 80.8+/-30.6 in SSc patients, and 40.0+/-11.7 in SS patients, while 290.0+/-69.6 and 321.2+/-103.4 NKT cells were present in healthy subjects and BD patients, respectively (P < 0.01). Three of 10 RA patients, 5 of 10 SLE patients, 4 of 8 SSc patients, and 6 of 9 SS patients (a total of 18 of 37 patients, or 48.6%) responded to alpha-GalCer, indicating that patients could be divided into two groups: alpha-GalCer responders and nonresponders. In contrast, NKT cells from all healthy subjects proliferated against alpha-GalCer. APCs from all nonresponder patients were found to function as alpha-GalCer-presenting cells, while NKT cells from nonresponders did not expand even in the presence of APCs from normal responders. CONCLUSION: These findings strongly suggest that patients with autoimmune diseases can be divided into two groups (alpha-GalCer responders and nonresponders). They also suggest that the reduced numbers of NKT cells in patients with autoimmune diseases may be due to an inadequate amount of alpha-GalCer-like natural ligands (i.e., adequate in only 48.6% of patients) for the induction of NKT cells in vivo, or to a dysfunction in the NKT cells themselves (in 51.4% of patients).