Psoriatic arthritis joint fluids are characterized by CD8 and CD4 T cell clonal expansions appear antigen driven

The CD8 alphabetaT cell receptor repertoire in joint fluid of individuals with active psoriatic arthritis contained an average of 32 major oligoclonal expansions in many variable genes of the TCR beta chain (BV) families, as shown by beta-chain CDR3 length analysis. Interestingly, a small number of oligoclonal expansions were shared between simultaneous samples of joint fluid and blood; however, most expansions found in joint fluid were not identifiable in blood emphasizing the immunologic specificity of the clonal events for the inflamed joint at a given point of time. The CD4 T cell joint fluid repertoire contained fewer and smaller oligoclonal expansions also largely restricted to the joint, suggesting that CD4 T cells participate perhaps by interacting cognitively to generate the CD8 clones. The inferred amino acid sequence of a single CD8 oligoclonal expansion revealed that they usually are composed of one or a few structurally related clones at the amino acid sequence level with beta-chains that encode identical or highly homologous CDR3 motifs. These were not shared among patients. Moreover, several clones that encoded the same amino acid sequence were found to be structurally distinct at the nucleotide level, strongly implying clonal selection and expansion is operating at the level of specific TCR-peptide interactions. The findings support a model of psoriatic arthritis inflammation involving extensive and selective Ag, likely autoantigen, driven intra-articular CD4, and CD8 T cell clonal expansions.     

Identification of pathogenic T cells in patients with beryllium-induced lung disease.

Chronic beryllium disease (CBD) is caused by beryllium exposure and is characterized by granulomatous inflammation with accumulation of CD4+ T cells in the lung. We analyzed TCR beta-chain and alpha-chain genes expressed by these CD4+ T cells. In the lungs of individual patients, as well as among four of five CBD patients studied, different oligoclonal expansions within the Vbeta3 subset were found to express homologous or even identical CDR3 amino acid sequences. These related expansions were specific for CBD patients, were compartmentalized to lung, and persisted at high frequency in patients with active disease. Limiting dilution cloning and analysis of coexpressed TCR alpha-chain genes confirmed that these TCRs were selectively expanded by a common Ag involving beryllium. Overall, homologous TCR beta- and alpha-chains showed identical V regions and invariant charged residues within the CDR3 but considerable variability in TCRJ usage. Remarkably, CBD patients expressing nearly identical TCRs did not share common HLA-DRB1 or DQ alleles. These results implicate particular CD4+ cells in the pathogenesis of CBD and provide insight into how beryllium is recognized in human disease.     

TCR bias of in vivo expanded T cells in pancreatic islets and spleen at the onset in human type 1 diabetes

Autoreactive T cells, responsible for the destruction of pancreatic β cells in type 1 diabetes, are known to have a skewed TCR repertoire in the NOD mouse. To define the autoreactive T cell repertoire in human diabetes, we searched for intraislet monoclonal expansions from a recent onset in human pancreas to then trace them down to the patient's peripheral blood and spleen. Islet infiltration was diverse, but five monoclonal TCR β-chain variable expansions were detected for Vβ1, Vβ7, Vβ11, Vβ17, and Vβ22 families. To identify any sequence bias in the TCRs from intrapancreatic T cells, we analyzed 139 different CDR3 sequences. We observed amino acid preferences in the NDN region that suggested a skewed TCR repertoire within infiltrating T cells. The monoclonal expanded TCR sequences contained amino acid combinations that fit the observed bias. Using these CDR3 sequences as a marker, we traced some of these expansions in the spleen. There, we identified a Vβ22 monoclonal expansion with identical CDR3 sequence to that found in the islets within a polyclonal TCR β-chain variable repertoire. The same Vβ22 TCR was detected in the patient's PBMCs, making a cross talk between the pancreas and spleen that was reflected in peripheral blood evident. No other pancreatic monoclonal expansions were found in peripheral blood or the spleen, suggesting that the Vβ22 clone may have expanded or accumulated in situ by an autoantigen present in both the spleen and pancreas. Thus, the patient's spleen might be contributing to disease perpetuation by expanding or retaining some autoreactive T cells.     

Dominant and shared T cell receptor beta chain variable regions of T cells inducing synovial hyperplasia in rheumatoid arthritis.

Previously, we demonstrated the presence of at least two distinct subpopulations of patients with rheumatoid arthritis (RA) employing a cell-transfer experiment using severe combined immunodeficient (SCID) mice. One group of patients, whose T cells derived from the rheumatoid joints, induced synovial hyperplasia (SH) in the SCID mice (the positive group). The other group did not display the induction of SH (the negative group). TCR/Vbeta gene usage analysis indicated that some dominant T cell subpopulations were oligoclonally expanding only in the rheumatoid joints, and not in the periphery of the patients of the positive group. Moreover, these T cell subpopulations were not seen in the joints of patients in the negative group or in non-RA patients. In addition, the preferential uses of certain TCR/Vbetas (Vbeta8, Vbeta12, Vbeta13, and Vbeta14) genes were demonstrated in these T cells. In this study, to investigate whether these T cells are driven by a certain antigen(s), the third complementarity determining regions (CDR3s) of TCR/Vbeta, especially Vbeta8 and Vbeta14 PCR products, were cloned and sequenced. As a result, a dominant CDR3 sequence, CASS-PRERAT-YEQ, was found in Vbeta14+ T cells from the rheumatoid joint of a patient (Patient 1) of the positive group with a Vbeta14 skew. The identical CDR3 sequence also predominated in Vbeta14+ T cells from the rheumatoid joint of another patient (Patient 7) of the positive group with a Vbeta14 skew. In addition, in the patients (Patients 4, 7, 8) of the positive group with a Vbeta8 skew, other dominant CDR3 sequences, CASS-ENS-YEQ and CASS-LTEP-DTQ, were found as in the case of Vbeta14. However, no identical CDR3 sequences were detected dominantly in the joints of the patients in the negative group or in non-RA patients. A Vbeta14+ T cell clone (TCL), named G3, with the identical CDR3 sequence, CASS-PRERAT-YEQ, was isolated successfully from Patient 1, and cell transfer of G3 with autologous irradiated peripheral mononuclear cells induced SH in the SCID mice. Taken together, these results suggest that T cells inducing SH, thought to be pathogenic for RA, might be driven by a certain shared antigen(s).     

T cell receptor gene segment utilization by HLA-DR1-alloreactive T cell clones.

Transplantation of histoincompatible tissues leads to allograft rejection, which involves recognition of allogeneic MHC molecules by Ag-specific receptors expressed on T cells. The interaction of these molecules is highly specific yet poorly understood. We have investigated the relationship between TCR gene utilization and allo-MHC restriction patterns by using a one-way polymerase chain reaction to amplify the alpha- and beta-chain mRNA from a panel of 10 HLA-DR1-alloreactive T lymphocyte clones. Two previously unreported V alpha and five J alpha gene sequences were obtained. Although a few V alpha, V beta, and J alpha genes were utilized more than once, no correlation between TCR gene usage and DR1 alloreactivity was identified. At the sequence level, the presumed TCR alpha- and beta-chain CDR1 and CDR2 regions displayed limited diversity, whereas the CDR3 or junctional sequences were highly variable. Although most TCR probably interact with subtly different surface features of the DR1 alloantigen, we predict that TCR with similar CDR1 and CDR2 sequences would contact essentially identical regions of the DR1 molecule. The lack of sequence conservation in the junctional regions suggests that different endogenous peptides also may be recognized. Thus, alloreactive T cells may recognize not only allogeneic MHC molecules but perhaps also bound endogenous peptides.     

Aetiology and pathogenesis of reactive arthritis: role of non-antigen-presenting effects of HLA-B27

Spondyloarthropathies are inflammatory diseases closely associated with human leukocyte antigen (HLA)-B27 by unknown mechanisms. One of these diseases is reactive arthritis (ReA), which is typically triggered by Gram-negative bacteria, which have lipopolysaccharide as an integral component of their outer membrane. Several findings in vivo and in vitro obtained from patients with ReA and from different model systems suggest that HLA-B27 modulates the interaction between ReA-triggering bacteria and immune cells by a mechanism unrelated to the antigen presentation function of HLA-B27. In this review we piece together a jigsaw puzzle from the new information obtained from the non-antigen-presenting effects of HLA-B27.     

The evolutionarily conserved ribosomal protein L23 and the cationic urease beta-subunit of Yersinia enterocolitica O:3 belong to the immunodominant antigens in Yersinia-triggered reactive arthritis: implications for autoimmunity.

BACKGROUND: Reactive arthritis (ReA) is a T cell mediated inflammatory process. The immune response is primarily directed against a triggering organism, although autoimmunity has been invoked in long-lasting, antibiotic-resistant disease. Although a variety of different species are known to trigger Reactive arthritis, the clinical manifestations are strikingly similar as well as closely associated to the HLA-B27 (70%). MATERIALS AND METHODS: Various antigenic fractions and single antigens of Yersinia enterocolitica were prepared, and their immunological activity was assessed by proliferation of synovial fluid mononuclear cells from 10 Reactive arthritis patients. The gene encoding one hitherto unknown antigen has been sequenced. Nonapeptides deduced from sequences of the target antigens were tested in an assembly assay. RESULTS: Two immunodominant proteins of Yersinia enterocolitica were found, one being the urease beta-subunit and the other the 50 S ribosomal protein L23. The latter has been sequenced and belongs to the evolutionarily conserved ribosomal proteins with homology to procaryotes and eucaryotes. One nonapeptide derived from the urease beta-subunit was identified as a possible epitope for HLA-B27-restricted cytotoxic T cells by its high affinity. This epitope is also highly conserved. CONCLUSION: Sharing of conserved immunodominant proteins between different disease triggering microorganisms could provide an explanation of the shared clinical picture in Reactive arthritis. Moreover, autoimmunity in Reactive arthritis might be mediated by antigen mimicry between evolutionarily conserved epitopes of ribosomal proteins and their host analogs.     

Novel HLA-B27-restricted Epitopes from Chlamydia trachomatis Generated upon Endogenous Processing of Bacterial Proteins Suggest a Role of Molecular Mimicry in Reactive Arthritis

Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na⁺-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27⁺ cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330–338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211–223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211–223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA.     

Molecular analysis of TCR clonotypes in LGL: a clonal model for polyclonal responses

Large granular lymphocytic (LGL) leukemia is a clonal lymphoproliferative disorder of CTL associated with cytopenias resulting from an immune and cytokine attack on hemopoietic progenitor cells. Extreme clonality of CTL expansions seen in LGL leukemia makes it an ideal model to study the role of the T cell repertoire in other less-polarized immune-mediated disorders. Complementarity-determining region 3 (CDR3) of the TCR is a unique Ag-specific region that can serve as a molecular marker, or clonotype, of the disease-specific T cells. We studied the variable portion of the beta-chain spectrum in a cohort of LGL leukemia patients. The CDR3 sequences were determined for the immunodominant clones and used to design clonotype-specific primers. By direct and semi-nested amplification, clonotype amplicons were found to be shared by multiple patients and controls. Analysis of the generated sequences demonstrated that the original clonotypes are rarely encountered in normal control samples; however, high levels of homology were found in both controls and patients. Clonotypes derived from individual LGL patients can be used as tumor markers for the malignant clone. More generally, clonotypic analysis and comparison of the variable portion of the beta-chain CDR3-specific sequences from a large number of patients may lead to better subclassification of not only LGL but also other immune-mediated disorders.     

Antigen-dependent B cell differentiation in the synovial tissue of a patient with reactive arthritis.

BACKGROUND: Reactive arthritis (ReA) can develop as a consequence of a bacterial infection with organisms such as Chlamydia trachomata, Shigella flexneri, or Yersinia enterocolitica. Although the mechanism underlying the induction of a chronic synovitis is unknown, the expression of HLA-B27 seems to play a crucial role in the etiology of the disease. Bacterial antigens induce a humoral immune response, but little is know about the impact of B cells on the inflammatory processes developing in the synovial membrane. MATERIALS AND METHODS: Cryostat sections were prepared from the synovial tissue (ST) of patients with ReA and stained with antibodies specific for T, B, and follicular dendritic cells. Lymphoid infiltrates were directly isolated by microdisection and DNA was prepared from them. The rearranged V genes were amplified by polymerase chain reaction (PCR), cloned, and sequenced. RESULTS: Histological staining showed that germinal, center-like structures develop in the ST of patients with ReA. B cells with a heterogenous repertoire were isolated from these lymphoid infiltrates. The majority of V regions carried somatic mutations indicating that sequences are derived from memory B cells. Genealogical trees demonstrate clonal expansion and diversification of the B cell repertoire in the ST. CONCLUSIONS: The finding of local V-region diversification suggests that in the ST of patients with ReA, an antigen-driven, T cell-dependent differentiation of B cells occurs. This local B cell response may contribute to the progress of the disease. Whether B cells are specific for the bacteria inducing the synovitis or for self-determinants present in the ST remains to be determined.     

Public T cell receptor beta-chains are not advantaged during positive selection

Studies of human and murine T cells have shown that public TCR beta-chain rearrangements can dominate the Ag-specific and naive repertoires of distinct individuals. We show that mouse T cells responding to the minor histocompatibility Ag HYDbSmcy share an invariant Vbeta8.2-Jbeta2.3 TCR gene rearrangement. The dominance of this rearrangement shows that it successfully negotiated thymic selection and was highly favored during clonal expansion in all animals examined. We hypothesized that such beta-chains are advantaged during thymic and/or peripheral selection and, as a result, may be over-represented in the naive repertoire. A sequencing study was undertaken to examine the diversity of Vbeta8.2-Jbeta2.3 CDR3 loops from naive T cell repertoires of multiple mice. Public TCR beta-chain sequences were identified across different repertoires and MHC haplotypes. To determine whether such public beta-chains are advantaged during thymic selection, individual chains were followed through T cell development in a series of novel bone marrow competition chimeras. We demonstrate that beta-chains were positively selected with similar efficiency regardless of CDR3 loop sequence. Therefore, the establishment and maintenance of public beta-chains in the periphery is predominantly controlled by post-thymic events through modification of the primary, thymus-derived TCR repertoire.     

A novel HLA-B27 allele maps B27 allospecificity to the region around position 70 in the alpha 1 domain.

There are six known HLA-B alleles that share the HLA-B27 allospecificity, yet differ by one to six amino acid substitutions. Each of these B27 alleles can be readily assigned by one of the six representative IEF patterns. Two unrelated individuals, LH and HS, express B27 Ag that appear to be identical by IEF, but an HLA-B27 alloreactive CTL clone I-73 was found to react differently with these cells, suggesting these B27 molecules are not identical. We sequenced polymerase chain reaction-amplified B27 cDNA clones obtained from HS and compared its deduced amino acid sequence (B27-HS) with the B27 sequence of LH (B27-LH) which was previously designated the B*2701 allele. B27-HS and B27-LH differ by eight amino acids; three in alpha 1 domain and five in alpha 2 domain. These amino acid substitutions of B27-HS altered T cell recognition but not the B27 serologic epitope or IEF pattern. B27-HS differs from the six known B27 alleles by five to eight amino acid substitutions, and thus it represents the seventh allele of the HLA-B27 Ag family. This novel B27 allele might have been derived from a gene conversion event. Previously, two amino acid residues at positions 70 and 97 were suggested to be specific for B27 Ag family. B27-HS now reveals that Lys at position 70 is specific for B27 but Asn at position 97 is not. We propose that the region around position 70 might be crucial in determining the B27 serologic epitope and possibly in peptide Ag binding. This study also demonstrates that class I molecules of the same Ag specificity sharing an indistinguishable IEF pattern are not necessarily identical, and indicates that only the definitive determination of primary structure would identify all the class I alleles that are functionally relevant in regard to alloreactivity, T cell restriction, and disease association.     

A common TCR V-D-J sequence in V beta 13.1 T cells recognizing an immunodominant peptide of myelin basic protein in multiple sclerosis.

T cell responses to the immunodominant peptide (residues 83-99) of myelin basic protein are potentially associated with multiple sclerosis (MS). This study was undertaken to examine whether a common sequence motif(s) exists within the TCR complementarity-determining region (CDR)-3 of T cells recognizing the MBP83-99 peptide. Twenty MBP83-99-reactive T cell clones derived from patients with MS were analyzed for CDR3 sequences, which revealed several shared motifs. Some V beta 13.1 T cell clones derived from different patients with MS were found to contain an identical CDR3 motif, V beta 13.1-LGRAGLTY. Oligonucleotides complementary to the shared CDR3 motifs were used as specific probes to detect identical target CDR3 sequences in a large panel of T cell lines reactive to MBP83-99 and unprimed PBMC. The results revealed that, in contrast to other CDR3 motifs examined, the LGRAGLTY motif was common to T cells recognizing the MBP83-99 peptide, as evident by its expression in the majority of MBP83-99-reactive T cell lines (36/44) and PBMC specimens (15/48) obtained from randomly selected MS patients. The motif was also detected in lower expression in some PBMC specimens from healthy individuals, suggesting the presence of low precursor frequency of T cells expressing this motif in healthy individuals. This study provides new evidence indicating that the identified LGRAGLTY motif is preferentially expressed in MBP83-99-reactive T cells. The findings have important implications in monitoring and targeting MBP83-99-reactive T cells in MS.     

肿瘤浸润性γδT细胞（γδTIL）CDR3δ1区的序列分析

目的对肿瘤浸润性γδT细胞（γδTIL）CDR3δ1区进行序列分析。方法用固相包被抗体,体外扩增14例肿瘤组织（胃癌、肾癌、卵巢癌、膀胱癌、食管癌、肺癌、嗜铬细胞瘤）和2例肿瘤腹水（浆乳癌）的γδ肿瘤浸润淋巴细胞（tumor infiltration lymphocytes,γδTIL）,RT-PCR法扩增CDR3δ1基因片段,测序分析其序列特征。结果 16例γδTIL的CDR3δ1确实存在优势序列,不完全相同。其中4例胃癌来源的γδTIL中3例的CDR3δ1优势序列相同,3例食管癌来源的γδTIL中2例的CDR3δ1优势序列相同。结论体外扩增培养的γδTIL的TCR CDR3δ1序列具有相对优势特征。不同个体的同种组织来源的γδTIL的CDR3δ1优势序列趋于相同。     

HLA-Bw22: a family of molecules with identity to HLA-B7 in the alpha 1-helix.

Various HLA-B molecules exhibit serologic cross-reactions with HLA-B7, including HLA-B27, B40, Bw42, and Bw22. Of this group, primary structures for the three serologic subdivisions of HLA-Bw22, HLA-Bw54, Bw55, and Bw56, have yet to be determined. Here, we describe the nucleotide sequences of five distinctive HLA-Bw22 alleles isolated from cells of different ethnic origins typed either for Bw54, Bw55, or Bw56. Heterogeneity in molecules typed as Bw55 and Bw56 was defined. The five HLA-Bw22 alleles form a closely related family that appears to have evolved by a series of simple gene conversion events, all of which alter the antigen recognition site of the encoded proteins. Of note, HLA-Bw54 is the product of a gene conversion between HLA-B and C alleles. All the Bw22 alleles encode an alpha 1-helix identical in amino acid sequence to that of HLA-B7 and Bw42, a feature almost certainly responsible for the serologic cross-reactivity of these molecules. Shared substitutions in the alpha 1-helix can also explain the cross-reactivity of Bw22 and B7 with B27. Patterns of amino acid substitution in the alpha 2-domain of Bw22 heavy chains correlate with certain antibody and T cell cross-reactivities, thereby implicating particular amino acids in their target epitopes.     

Frequent contribution of T cell clonotypes with public TCR features to the chronic response against a dominant EBV-derived epitope: application to direct detection of their molecular imprint on the human peripheral T cell repertoire

In an attempt to provide a global picture of the TCR repertoire diversity of a chronic T cell response against a common Ag, we performed an extensive TCR analysis of cells reactive against a dominant HLA-A2-restricted EBV epitope (hereafter referred to as GLC/A2), obtained after sorting PBL or synovial fluid lymphocytes from EBV-seropositive individuals using MHC/peptide multimers. Although TCR beta-chain diversity of GLC/A2+ T cells was extensive and varied greatly from one donor to another, we identified in most cell lines several recurrent Vbeta subsets (Vbeta2, Vbeta4, and Vbeta16 positive) with highly conserved TCRbeta complementarity-determining region 3 (CDR3) length and junctional motifs, which represented from 11 to 98% (mean, 50%) of GLC/A2-reactive cells. While TCR beta-chains expressed by these subsets showed limited CDR1, CDR2, and CDR3 homology among themselves, their TCR alpha-chains comprised the same TCRAV region, thus suggesting hierarchical contribution of TCR alpha-chain vs TCR beta-chain CDR to recognition of this particular MHC/peptide complex. The common occurrence of T cell clonotypes with public TCR features within GLC/A2-specific T cells allowed their direct detection within unsorted PBL using ad hoc clonotypic primers. These results, which suggest an unexpectedly high contribution of public clonotypes to the TCR repertoire against a dominant epitope, have several implications for the follow-up and modulation of T cell-mediated immunity.     

Use of HLA-B27 tetramers to identify low-frequency antigen-specific T cells in <Emphasis Type="Italic">Chlamydia</Emphasis>-triggered reactive arthritis

Reports of the use of HLA-B27/peptide tetrameric complexes to study peptide-specific CD8⁺ T cells in HLA-B27⁺-related diseases are rare. To establish HLA-B27 tetramers we first compared the function of HLA-B27 tetramers with HLA-A2 tetramers by using viral epitopes. HLA-B27 and HLA-A2 tetramers loaded with immunodominant peptides from Epstein–Barr virus were generated with comparable yields and both molecules detected antigen-specific CD8⁺ T cells. The application of HLA-B27 tetramers in HLA-B27-related diseases was performed with nine recently described Chlamydia-derived peptides in synovial fluid and peripheral blood, to examine the CD8⁺ T cell response against Chlamydia trachomatis antigens in nine patients with Chlamydia-triggered reactive arthritis (Ct-ReA). Four of six HLA-B27⁺ Ct-ReA patients had specific synovial T cell binding to at least one HLA-B27/Chlamydia peptide tetramer. The HLA-B27/Chlamydia peptide 195 tetramer bound to synovial T cells from three of six patients and HLA-B27/Chlamydia peptide 133 tetramer to synovial T cells from two patients. However, the frequency of these cells was low (0.02–0.09%). Moreover, we demonstrate two methods to generate HLA-B27-restricted T cell lines. First, HLA-B27 tetramers and magnetic beads were used to sort antigen-specific CD8⁺ T cells. Second, Chlamydia-infected dendritic cells were used to stimulate CD8⁺ T cells ex vivo. Highly pure CD8 T cell lines could be generated ex vivo by magnetic sorting by using HLA-B27 tetramers loaded with an EBV peptide. The frequency of Chlamydia-specific, HLA-B27 tetramer-binding CD8⁺ T cells could be increased by stimulating CD8⁺ T cells ex vivo with Chlamydia-infected dendritic cells. We conclude that HLA-B27 tetramers are a useful tool for the detection and expansion of HLA-B27-restricted CD8⁺ T cells. T cells specific for one or more of three Chlamydia-derived peptides were found at low frequency in synovial fluid from HLA-B27⁺ patients with Ct-ReA. These cells can be expanded ex vivo, suggesting that they are immunologically functional.