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.     

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.     

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.     

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.     

Polymerase chain reaction amplification analyses of clonality in T-cell malignancy including peripheral T-cell lymphoma

Clonality in T-cell malignancy was investigated using T-cell receptor (TcR) V beta 1-20 family primers and polymerase chain reaction amplification (PCR) of cDNA prepared from tissue biopsies and blood involved with tumour. Secondary PCR amplification of the VDJ joints of primary PCR products was performed to distinguish clonal from polyclonal products, and clonal V beta gene products were confirmed by direct PCR sequencing in the majority of cases. In eight T-cell malignancies including T-cell acute lymphoblastic leukaemia (T-ALL) and T-cell chronic lymphocytic leukaemia (T-CLL) shown to be clonal by Southern blot analysis, one or two primary PCR products were identified and shown to be clonal. In five cases of peripheral T-cell lymphoma (PTCL) all V beta 1-20 families were identified after primary PCR amplification, and clonal products were identified in two cases after secondary amplification; TcR V beta clonal families could not be demonstrated in the remaining three cases. These data were in agreement with previous Southern blot analysis of these cases, and confirmed the presence of reactive T cells in PTCL as well as providing further evidence for the genotypic heterogeneity of this entity. In the remaining case, a blood lymphocytosis, primary PCR amplification produced predominant TcR V beta 6 and V beta 12 family products, of which the V beta 6 family proved clonal after secondary PCR amplification. There was no evidence for overrepresentation of TCR V beta families by the tumour populations in this study, furthermore the data confirm the involvement of reactive cells in T-cell malignancy and the genetic heterogeneity of PTCL.     

The clonal composition of a peptide-specific oligoclonal CTL repertoire selected in response to persistent EBV infection is stable over time

The TCR repertoire of a peptide-specific HLA A11-restricted CTL response to persistent infection with EBV was followed for a period of 57 mo. Sequencing of TCR V alpha and V beta chains and alanine scanning mutagenesis analysis of 83 CTL clones isolated in five reactivation experiments demonstrated that this repertoire is composed of at least four distinct CTL clonotypes that are constantly reactivated from donor's blood and express structurally heterogeneous TCRs. Target cell recognition and CD8 blocking experiments indicate that the four clonotypes possess different avidity and TCR affinity for the specific Ag. This demonstrates that at least in some individuals a heterogeneous peptide-specific memory CTL repertoire selected by a persistent Ag can be remarkably stable in time and accommodate a range of TCR affinities and T cell avidities. Our results suggest that competition for the specific Ag may be not the major force driving the maintenance of memory CTLs and that the nature of the first antigenic challenge may largely determine the clonal composition of memory.     

T-cell receptor repertoire in matched MART-1 peptide-stimulated peripheral blood lymphocytes and tumor-infiltrating lymphocytes

Characterization of tumor-associated antigens (TAAs) recognized by CTLs makes the consideration of therapeutic strategies based on peptide stimulation of peripheral blood lymphocytes (PBLs) feasible. Several such approaches are adoptive transfer of peptide-stimulated PBLs, ex vivo peptide stimulation of dendritic cells, and direct vaccination with TAA-derived peptides. A critical component of any of these peptide-based strategies is the requirement that the patient's PBLs are able to react productively against the presented TAA. The purpose of this study, through the study of T-cell receptor (TCR) usage, was to evaluate the T-cell response in matched MART-1(27-35) peptide-stimulated PBLs and tumor-infiltrating lymphocytes (TILs). MART-1(27-35)-reactive PBL and TIL cultures were generated from three patients by in vitro stimulation with an immunodominant peptide of MART-1 (MART-1(27-35)). All cultures had a human leukocyte antigen A2-restricted, MART-1(27-35)-specific CTL response. The TCR usage of each was assessed by the DNA sequence analysis of 50 TCR beta clones obtained by rapid amplification of cDNA ends per culture. TCR analysis suggests a TCR repertoire that differed from patient to patient (8-16 subfamilies were used) and a predominant usage of a different variable beta chain (BV) by each of these MART-reactive T cells. These predominant BV rearrangements were derived from multiple clonotypes because different variable, diversity, and junctional regions were observed. However, a similar pattern of expansion was present for both PBLs and TILs; the relative usage of each prevailing BV was more marked in TILs (36, 50, and 78% of TILs versus 26, 20, and 24% of PBLs, respectively), a broader TCR repertoire was used by PBLs (P > 0.05), and similar TCR subfamily usage was noted when TIL and PBL cultures from the same patient were compared (8 of 11, 7 of 9, and 7 of 8 for patients 1, 2, and 3, respectively). Furthermore, the exact same clonotypes derived from predominant TCR subfamilies in the PBLs and TILs were present in each patient, suggesting peptide-stimulated expansion in both biological compartments. These studies suggest that there will not be a limited and predictable TCR subfamily response to a specific TAA, although reproducible patterns of PBL and TIL expansion are present from patient to patient. Additionally, identical T-cell clonotypes having the same potential for antigen-driven expansion were present in a patient's PBLs and TILs. As such, our data support the conceptualization of approaches using adoptive transfer or vaccination based on TAA-derived peptide stimulation of PBLs.     

Potential of the immune complex transfer enzyme immunoassay for antigens and antibodies to improve the sensitivity and its limitations

To better characterize the cellular immune response taking place in the MS central nervous system, we investigated the blood and CSF T cell receptor (TCR) V beta 5 and V beta 17 repertoire in HLA-typed patients with recently diagnosed MS or other neurological diseases (OND). Using a RT-PCR based technique, we analysed directly ex vivo the CDR3 size of TCR beta chains utilizing V beta 5 (eight patients with MS and one with OND) or V beta 17 (eight patients with MS and six with OND) gene segments on paired blood-CSF samples. Globally, the analysis of V beta 5-J beta and V beta 17-J beta repertoire showed a less diverse pattern in the CSF samples than in the corresponding peripheral blood lymphocytes both in MS and in OND patients. However, we did not detect any recurrent clonal expansion within the V beta 5+ T cells in MS patients, underlining the potential limits of V beta 5-based immunotherapy in MS. We found an expanded T cell population using the same V beta 17-J beta 1.6 combination with identical CDR3 length in the CSF of three MS patients and none of the control patients. These results suggest selective expansion of T cells expressing this segment gene in the MS central nervous system.     

Molecular analysis of T-cell receptor repertoire in bone marrow transplant recipients: evidence for oligoclonal T-cell expansion in graft-versus-host disease lesions

We have analyzed the T-cell receptor (TCR) V beta repertoire using polymerase chain reaction (PCR) in a cohort of eight patients receiving allogeneic bone marrow transplantation (BMT) from related and unrelated donors at the City of Hope. Results of PCR studies from graft-versus-host disease (GVHD) skin lesions show a bias in the usage of TCR V beta families, whereas examination of peripheral blood (PB) withdrawn at the same time did not reveal a similar phenomenon. In one such family, TCR V beta 2 is predominantly expressed in 7 of 7 biopsy specimens examined. V beta 2 TCR expression from these patients was analyzed more extensively using a combination of individual TCR gene cloning, followed by sequence analysis. We found evidence of oligoclonal expansion of single V beta 2-bearing TCRs in GVHD lesions, and in the PB of some patients after diagnosis of GVHD. In contrast, GVHD-negative biopsy samples showed no evidence for clonotypic TCR amplification. Sequence-specific TCR CDR3 region probes were derived from analysis of the predominant expressed TCR in GVHD lesions, and used to probe Southern blots of amplified V beta 2 TCR mRNA from PB and tissue from BMT recipients and their respective donors. In most cases the probes are highly specific in detecting TCR expression from GVHD lesions alone, although in several instances expression could be detected in PB after GVHD diagnosis. These data provide supporting evidence for the hypothesis that acute GVHD is associated with expansion of T-cell clones expressing antigen-specific TCRs that may contribute to the disease pathology.     

The T-cell receptor repertoires expressed by CD4+ and CD4- large granular lymphocytes derived from the same patients suggest the persistent action of an immune-mediated selection process

The lymphoproliferative syndrome with large granular lymphocytes (LGL) is an heterogeneous disorder of unknown etiology. The analysis of T-cell receptor (TCR) genes rearrangements has shown that, in most cases, the disease is associated with clonal proliferation of CD8+CD57+ LGL. However, the putative neoplastic nature of these expansions remains questionable because clonal proliferations of CD8+ cells have recently been found also in physiologic conditions. To obtain more precise information on the mechanisms responsible for LGL expansions, we decided to compare the molecular characteristics of TCRBV chains expressed by LGL with different phenotype and function, but derived from the same patients. To this end, we characterized, at the molecular level, the TCR repertoires of fractionated T-cell populations of two unusual patients with concurrent expansions of CD4+CD57+ and CD4-CD57+ LGL. Our results show that the dominant TCRBV chains expressed by the different CD4+ and CD4- LGL populations were strictly oligoclonal. However, the molecular characteristics of the dominant V-D-J rearrangements also imply that the selection of these clones was not due to a neoplastic event. Rather, our data suggest that these particular LGL proliferations can be ascribed to a chronic T-cell-mediated immune response that involves recognition by the engaged TCR of antigens that are not necessarily presented to immune system in the classical major histocompatibility complex-restricted pathway.     

Hazards of decontextualised accounts of public perceptions of radio frequency radiation (RFR) risk

We report a case of large granular lymphocytic leukaemia (LGLL) with mixed T-cell/B-cell phenotypes. The LGLL cells expressed T-cell markers such as CD1, CD2, CD3, CD5, CD7, CD8 and CD57. The CD8+ LGLL cells coexpressed B-cell markers including CD20 and PCA-1, and a fraction of purified CD8+ LGLL cells secreted double isotypes of immunoglobulins (IgG-kappa and IgA-kappa). Both TCRB and IGH genes were clonally rearranged. The LGLL cells could be divided into at least three subpopulations that were cytogenetically distinct, and all subpopulations involved the 11q23. The expression of both T- and B-cell markers on the LGLL cells suggests the involvement of a putative common lymphoid progenitor in leukaemic transformation.     

Matrix protein of rabies virus is responsible for the assembly and budding of bullet-shaped particles and interacts with the transmembrane spike glycoprotein G

Patients with Omenn's syndrome have a form of severe immune deficiency that is associated with pathological features of graft-versus-host disease, except for the lack of foreign engraftment. It has been hypothesized that the disease's unique clinical features are mediated by an expanded population of autologous self-reactive T cells of limited clonality. In the current study, an investigation of the T-cell receptor (TCR) repertoire was undertaken to identify defects in T-cell rearrangement and development. The TCR repertoire in this group of patients was exquisitely restricted in the number of different TCR clonotypes, and some of these clonotypes seemed to have similar recognition motifs in the antigen-binding region, indicating antigen-driven proliferation of T lymphocytes. The TCRs from some patients lacked N- or P-nucleotide insertions and used proximal variable and joining gene segments, suggesting abnormal intrathymic T-cell development. Finally, abnormal assembly of gene segments and truncated rearrangements within nonproductive alleles suggested abnormalities in TCR rearrangement mechanisms. Overall, the findings suggest that inefficient and/or abnormal generation of TCRs may be a consistent feature of this disease.     

Evidence for superantigen involvement in insulin-dependent diabetes mellitus aetiology

Insulin-dependent diabetes mellitus (IDDM) is a T-cell-mediated autoimmune disease whose onset is believed to be triggered by unknown environmental factors acting on a predisposing genetic background. Islet-infiltrating T (IIT) cells from two IDDM patients, who had died at the onset of the disease from brain swelling as a complication of ketoacidosis, were analysed. The results provided evidence for the involvement of a pancreatic islet cell membrane-bound superantigen as a diabetes aetiopathogenetic factor. There was a selective expansion of a T-cell receptor (TCR) variable segment of the beta-chain (V beta 7) in these IIT cells in association with unselected V alpha-chain segments; extensive junctional diversity of the TCR V beta 7 chains; and evidence of positive selection, after exposure to diabetic islet cell membrane preparations, of V beta 7+ T-cell clones among peripheral blood lymphocytes from non-diabetic individuals.