Complementarity-determining Region 3 Spectratyping Research Articles

Monoclonal and oligoclonal TCR AV and BV gene usage in CD4+ T cells from pigs immunised with C-strain CSFV vaccine

The classical swine fever virus C-strain vaccine (C-strain vaccine) plays a vital role in preventing and controlling the spread of classical swine fever (CSF). However, the protective mechanisms of C-strain vaccine and cellular immunity conferred by T cell receptors (TCRs) are less well defined. We aimed to analyse the association between the complementarity determining region 3 (CDR3) spectratype of αβTCR in CD4+ T cells and C-strain vaccine; and to find conserved CDR3 amino acid motifs in specific TCR α- and β-chains. We found that the CDR3 spectratype showed dynamic changes correlating with C-strain vaccine immunisation and that TCR AV5S/8–3S/8–4S/14/38 and BV4S/6S/7S/15S/30 gene families showed clonal expansion in immunised pigs. The sequences of CDR3 from these clonally expanded T cells indicated a high frequency of the ‘KLX’ motif in the TCR α chain and the ‘GGX’ motif in β chain, and Jα39, Jα43, Jβ2.5 and Jβ2.3 genes were also found in high frequency. To the best of our knowledge, this is the first report describing the dynamic changes of αβTCRs and conserved CDR3 amino acid motifs in CD4+ T cells from C-strain vaccine-immunised pigs, which will provide a basis for the development of high-efficiency epitope vaccines.

T cell receptor β-chain CDR3 spectratyping and cytomegalovirus activation in allogeneic hematopoietic stem cell transplant recipients

Objective: To explore the association between T-cell receptor beta variable (TCR BV) complementarity determining region 3 (CDR3) spectratyping and CMV activation in the recipients of allogeneic hematopoietic stem cell transplantation (HSCT). Methods: Fluorescence quantitative PCR melting curve analysis was used to sequence 24 TCR BV families in 7 HSCT recipients and 3 healthy controls. CMV-pp65 antigenemia was measured by immunohistochemical staining. Plasma IgM specific for CMV was identified using ELISA. Relationship between TCR BV families and CMV activation was statistically analyzed.Results: Twenty-four TCR BV families were expressed in 3 healthy controls, while TCR BV CDR3 sequencing results in 7 recipients turned out to be BV9, BV11, BV17, BV20 and so on. Amino acid sequence features were as follows:TCR BV9 contained "QVRGGTDTQ", TCR BV11 contained "VATDEQ" and "LGDEQ", TCR BV17 contained "IGQGNTEA", and TCR BV20 contained "VGLAANEQ". Five recipients suffered from pp65 antigenemia in 3 month after transplantation, and pp65-positive cells ranged from 2 to 15 per 5×104 white blood cells. Three recipients were CMV-IgM positive. No significant differences were found in TCR BV families between pp65-positive recipients and pp65-negative recipients (all P>0.05). But there was statistically significant difference in frequency of TCR BV11 between CMV-IgM negative recipients and CMV-IgM positive recipients (P<0.05).Conclusion: T cell immune response was characterized by special TCR BV CDR3 spectratyping in HSCT recipients, and TCR BV11 expression may be associated with CMV activation.

Reduced T-cell repertoire restrictions in abatacept-treated rheumatoid arthritis patients.

BackgroundCD28neg T cells, which display functional characteristic of oligoclonally expanded cytotoxic memory T lymphocytes, are believed to be pathologically relevant in rheumatoid arthritis manifestation. The CD28 co-stimulation blockade by abatacept can prevent the generation of CD28neg T-cell populations in these patients.MethodsSamples were obtained before and after 12 months of abatacept therapy. T-cell phenotype and T-cell receptor diversity were evaluated by flow cytometry and complementarity-determining region-3 spectratyping, respectively, while telomerase reverse-transcriptase gene level was measured by real-time PCR.ResultsAbatacept induces a decrease of the percentage and number of CD4+CD28neg T cells and a reduction of T-cell repertoire restrictions; these features are directly correlated. Thymic output and telomerase activity are not modified by the therapy.ConclusionsAbatacept-induced decrease of peripheral T-cell repertoire restrictions can due to a reduced generation of senescent, chronically stimulated CD4+CD28neg T cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0363-2) contains supplementary material, which is available to authorized users.

Profiling the T-cell receptor repertoire of patient with pleural tuberculosis by high-throughput sequencing

Pleural tuberculosis (PLTB), a major cause of morbidity and mortality, is the most common extrapulmonary manifestation of active Mycobacterium tuberculosis (Mtb) in developing countries. Gamma delta T-cell receptor (TCR) repertoire of peripheral blood mononuclear cells (PBMCs) and pleural effusion mononuclear cells (PEMCs) and beta TCR repertoire from peripheral blood mononuclear cells (PBMCs) have been reported. However, a detailed different characteristic of beta TCR repertoire of mononuclear cells isolated from peripheral blood and pleural fluid in the immune response to Mtb infection should be further revealed. The TCR β-chain (TRB) from PBMCs and PEMCs from an untreated pleural tuberculosis patient was sequenced by the Illumina sequencing platform. A total of 96,758 and 124,130 unique complementarity-determining region 3 (CDR3) sequences were identified at the nucleotide level, encoding 69,488 and 99,095 peptide sequences, respectively. TCR profiling showed that TRBV20-1 family and TRBV20-1/TRBJ1-5 gene combination had a dominant expression in PEMCs, but not in PBMCs. Expansive expression of common CDR3 clonotypes was observed in PEMCs. CDR3 spectratyping analysis showed that few TRBV families had a significantly skewed pattern, with one peak or a few prominent peaks in the PBMCs. By contrast, some TRBV families showed oligoclonal or clonal expansion in the PEMCs. Here, we firstly profiled the TRB repertoire differences of PBMCs and PEMCs from one PLTB patient using high-throughput sequencing. And this study may provide new insight for the detailed and efficient study of TCR repertoire of PEMCs in the future.

Analysis of T Cell Receptor Vβ Diversity in Peripheral CD4+ and CD8+ T Lymphocytes Obtained From Patients With Chronic Severe Hepatitis B

Background:The hepatitis B virus (HBV) antigen-induced cellular immune response plays an important role in HBV clearance. Changes in the diversity of complementarity determining region 3 (CDR3) and T-cell receptor (TCR) sequences are used to monitor the response of T cells to antigens.Objectives:The aim of the present study was to determine whether the TCR Vβ repertoire of patients with chronic severe hepatitis B (CSHB) undergoes increased stimulation, and to identify conserved motifs in specific TCR Vβ families.Patients and Methods:Peripheral blood mononuclear cells (PBMCs) from 18 patients with CSHB were sorted into CD4+ and CD8+ T subsets, using monoclonal antibody-coated magnetic beads. The TCR Vβ CDR3 was subsequently characterized using immune spectratyping. The TCR Vβ families exhibiting a CDR3 spectratype that underwent monoclonal expansion were sequenced.Results:The number of oligoclonal or monoclonal expansion TCR Vβ families detected in the analyzed CD8+ T cells was significantly higher than the number detected in CD4+ T cells. The CDR3 spectratype analysis showed predominant usage of TCR Vβ5, Vβ7, Vβ9, Vβ12, and Vβ18 families in CD8+ T cell subsets of CSHB patients. Furthermore, conserved amino acid motifs were found to be associated with the monoclonal expansion of CD8+ TCR Vβ families. In addition, JB1S1 and JB2S7 region genes were present at a high frequency.Conclusions:The CD4+ and CD8+ TCR Vβ gene families undergo clonal expansion in CSHB patients, and CD8+ T cells play a major role in the pathogenesis of CSHB. Moreover, the conserved motifs and limited use of joining region genes observed in the CSHB patients of this cohort indicated that similar antigenic epitopes are recognized.

Restoration of peripheral blood T cell repertoire complexity during remission in advanced cutaneous T cell lymphoma

In advanced stages, cutaneous T cell lymphomas (CTCL) are associated with increased mortality from infections and also increased susceptibility to skin malignancies. In this study, we analyzed the complexity of the peripheral blood T cell repertoire with a sensitive b-variable (BV) complementarity-determining region 3 (CDR3) spectratyping analysis and flow cytometry in three-stage IV CTCL/Sezary syndrome patients who achieved complete clinical remission after therapy. The T cell repertoire of peripheral blood T cells before treatment was profoundly abnormal across multiple BV subfamilies. Following treatment, CDR3 spectratype patterns showed dramatic restoration of normal diversity and complexity. However, absolute CD4 counts across multiple BV families remained low for many months, even after identifiable circulating malignant T cell populations were eliminated. These data suggest that the diversity of the T cell repertoire can be recovered after successful treatment of even advanced CTCL.

Dominance of Nonmalignant T-Cell Clones and Distortion of the TCR Repertoire in the Peripheral Blood of Patients with Cutaneous CD30+ Lymphoproliferative Disorders

The CD30-positive cutaneous lymphoproliferative disorders (CLPD) include lymphomatoid papulosis (LyP) and primary cutaneous anaplastic large T-cell lymphoma (cALCL). Despite the malign-appearing histology, an excellent prognosis and spontaneous regression of single lesions characterize LyP. Even after years of clinical remission newly erupting lesions often harbor a T-cell clone identical to the initial one. This fact raises the question whether the clonal T-cell population persists in the peripheral blood. Therefore we investigated genomic DNA of 126 samples of lesional skin and peripheral blood from 31 patients with CLPD, obtained during both active disease and clinical remission. We performed molecular genetic analysis by combining T-cell receptor (TCR)-gamma PCR with the GeneScan technique and assessed the TCR repertoire in selected blood samples by beta-variable complementarity-determining region 3 (CDR3) spectratyping qualitatively and quantitatively. We were able to detect a clonal T-cell population in 36/43 (84%) skin samples and in 35/83 (42%) blood samples. Comparison of the compartments in each patient demonstrated different T-cell clones in skin and blood, suggesting a reactive nature of the clonal T cells in the blood. Moreover, CDR3 spectratyping revealed a restricted T-cell repertoire in the blood, suggesting T-cell stimulation by an unknown antigen.

Analysis of T cell receptor Vβ diversity in peripheral CD4+ and CD8+ T lymphocytes in patients with autoimmune thyroid diseases

Autoimmune thyroid diseases are characterized by intrathyroidal infiltration of CD4(+) and CD8(+) T lymphocytes reactive to self-thyroid antigens. Early studies analysing T cell receptor (TCR) Valpha gene usage have shown oligoclonal expansion of intrathyroidal T lymphocytes but not peripheral blood T cells. However, TCR Vbeta diversity of the isolated CD4(+) and CD8(+) T cell compartments in the peripheral blood has not been characterized fully in these patients. We performed complementarity-determining region 3 (CDR3) spectratyping as well as flow cytometric analysis for the TCR Vbeta repertoire in peripheral CD4(+) and CD8(+) T cells from 13 patients with Graves' disease and 17 patients with Hashimoto's thyroiditis. Polyclonal TCR Vbeta repertoire was demonstrated by flow cytometry in both diseases. In contrast, CDR3 spectratyping showed significantly higher skewing of TCR Vbeta in peripheral CD8(+) T cells but not CD4(+) T cells among patients with Hashimoto's thyroiditis compared with healthy adults. We found trends towards a more skewed CDR3 size distribution in those patients having disease longer than 5 years and requiring thyroid hormone replacement. Patients with Graves' disease exhibited no skewing both in CD4(+) and CD8(+) T cells. These findings indicate that clonal expansion of CD8(+) T cells in Hashimoto's thyroiditis can be detected in peripheral blood and may support the role of CD8(+) T cells in cell-mediated autoimmune attacks on the thyroid gland in Hashimoto's thyroiditis.

T-Cell Receptor Complementarity Determining Region Analysis of Peripheral Blood and Bone Marrow T-Lymphocyte Subsets and Quantitative Evaluation of T-Regulatory Cells in Patients with Chronic Idiopathic Neutropenia

Clonal/oligoclonal expansions of cytotoxic T-lymphocytes (CTLs) and quantitative/qualitative abnormalities of T-regulatory cells (Tregs) have been implicated in the pathophysiology of acquired bone marrow (BM) failure syndromes such as aplastic anemia, myelodysplastic syndromes and large granular lymphocyte proliferative disease. Chronic Idiopathic Neutropenia (CIN) is an acquired disorder of granulopoiesis characterised by increased apoptosis of BM CD34+/CD33+ granulocytic progenitor cells. We have previously provided evidence for oligoclonal T-cell expansions with possible pathogenetic significance in CIN. The aim of the current study is (a) to define and compare the magnitude of CD4+ and CD8+ T-cell responses and (b) to evaluate the number of Tregs, in the peripheral blood (PB) and BM of CIN patients. Eighty five CIN patients and 85 healthy controls, age- and sex-matched to the patients, were studied after informed consent. All patients had PB neutrophil counts below 1800/μL (mean 1410 ± 330 neutrophils/μL, range 100–1799 neutrophils/μL), displayed negative anti-neutrophil antibody activity and were satisfying the previously reported diagnostic criteria for the disease.The T-cell receptor (TCR)-Vβ repertoire was analysed by flow cytometry (Vβ spectratyping) in the PB and BM CD3+, CD4+ and CD8+ cells. The size distribution of the TCR-Vβ complementarity determining region 3 (CDR3) was analysed in immunomagnetically sorted PB and BM CD4+ and CD8+ cells by PCR according to the -2 protocol using a fluorescence-based DNA sequencer (CDR3 spectratyping). Vβ family expansions were defined as above of 2 standard deviations from the mean values of the 85 healthy controls. CDR3 oligoclonal/monoclonal patterns were defined upon comparison with the normal Gaussian-type size distribution. The Treg cell frequency was defined as the proportion of FOXP3+ cells within the CD4+/CD25high PB and BM cell fraction by flow cytometry. We found that 69.41% and 82.61% of CIN patients displayed one or more predominant TCR-Vβ family expansions within the CD3+ cell fraction of PB and BM, respectively. None of the controls displayed clonal/oligoclonal expansions in either PB or BM (P<0.001 and P<0.001, respectively). Further analysis showed that 58.62% and 74.19% of CIN patients displayed a skewed TCR-Vβ repertoire in PB CD4+ and CD8+ cells, respectively. A higher proportion of patients i.e. 93.75% and 75.0% displayed a skewed TCR-Vβ repertoire in BM CD4+ and CD8+ cells, respectively. The total sum of predominant TCR-Vβ clones was statistically significant increased in patients' BM compared to PB in all subpopulations studied, namely the CD3+, CD4+ and CD8+ cells (P<0.05, P<0.05, P<0.05, respectively). Interestingly, CDR3 spectratyping identified a skewed (oligoclonal) pattern in PB and/or BM CD8+ cells in 100% of patients. PCR analysis of PB and BM CD4+ cells revealed a normal Gaussian-like CDR3 size distribution in all but three patients. None of the controls displayed a skewed CDR3 profile in either CD4+ or CD8+ cells. The percentage of FOXP3+ cells within the PB CD4+CD25high cell fraction was significantly decreased in CIN patients (57.77%±15.77%) compared to controls (72.95±12.23%; P=0.0004). A parallel analysis of Treg cell proportion in patients' PB and BM showed statistically significant increased percentage of FOX3+ cells within the CD4+/CD25high T-cells of BM (68.51%±11.88%) compared to PB (52.20%±12.77%) (P=0.0005). No statistically significant difference was found in the percentage of FOX3+ cells within the CD4+/CD25high T-cells of BM compared to PB in healthy controls. These data suggest that oligoclonal CTL expansions are uniformly identified in CIN patients. The polarized cell subsets are more frequent in BM than the PB suggesting the possible existence of “target” within the BM. Whether, however, the CD34+/CD33+ BM cells represent the target or the innocent bystanders of this immune process, remains elusive. The low frequency of Tregs in the PB and the accumulation of these cells in patients' BM represents probably a compensatory control mechanism aiming to suppress the local immune reactions. All these data substantiate further the concept that CIN belongs to the spectrum of T-cell mediated BM failure syndromes.

Analysis of the interindividual conservation of T cell receptor α- and β-chain variable regions gene in the peripheral blood of patients with systemic lupus erythematosus

The aim of this study was to find conserved motifs in specific T cell receptor (TCR) alpha- and beta-chains, and to analyse the association between complementarity determining region 3 (CDR3) spectratype and systemic lupus erythematosus (SLE) activity. TCR alpha-and beta-chain CDR3 spectratypes were analysed in 20 SLE patients. The CDR3 spectratypes of three patients were monitored over time, and the CDR3 regions of clonally expanded T cells were sequenced. CDR3 spectratype analysis showed prominent usage of TCR AV8, AV14, AV23, AV30, AV31, BV2, BV8, BV11, BV14, BV16, BV19 and BV24 families in SLE patients. The CDR3 spectratype showed dynamic change correlating with SLE activity. The sequence of the CDR3 region in clonally expanded T cells suggested a conserved GGX amino acid motif in both alpha- and beta-chains. The Ja34 and Jb2s1 region genes were found in high frequency. Both TCR Valpha and Vbeta gene usage is highly restricted in SLE, suggesting that the TCRs recognize a limited number of antigenic epitopes. The conserved motifs and limited use of joining region genes may indicate the recognition of similar antigenic epitopes in multiple individuals.

A novel approach for the analysis of T-cell reconstitution by using a T-cell receptor β-based oligonucleotide microarray in hematopoietic stem cell transplantation

Analysis of T-cell population diversity is important to hematopoietic stem cell transplantation (HSCT). The millions of specificities in T-cell receptor (TCR) hypervariable complementarity- determining region 3 (CDR3) precludes detection of all T-cell populations by antibody-based flow cytometry. An alternative method, the TCR CDR3 spectratyping assay, involves multiple polymerase chain reaction (PCR) analyses and is interpreted only qualitatively. In this study, we designed the first TCRbeeta-based oligonucleotide microarray and investigated its specificity, clonality discrimination, sensitivity of detection, and feasibility for monitoring T-cell population diversity in HSCT. The array contains 27 TCR Vbeta probes and 13 Jbeta probes. TCRbeta repertoire diversity was detected with single PCR, microarray hybridization system, and Spotfire analysis software. TCRO-based microarray provides specific sequence-based information and can distinguish T-cell monoclonal expansion within a polyclonal population. We successfully used this microarray to quantitatively and qualitatively analyze T-cell population diversity in recipients of hematopoietic stem cell transplants. This success suggests broad potential applications of the microarray for use in many other areas, including anti-tumor immunity, vaccination, autoimmunity, infectious diseases, and leukemia. By providing a single PCR-based assay to quantify multiple T-cell populations in parallel, this device will allow clinicians and researchers to rapidly perform high-throughput surveys.

High Frequencies of Increased Interferon-γ-Producing and/or Skewed CD8+ T Lymphocyte Subfamilies in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH).

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematological disorder affecting all hematopoietic lineages, which lack glycosylphosphatidylinositol (GPI)-anchored membrane proteins due to somatic mutations in the phosphatidylinositol glycan-class A gene, and is one disorder of bone marrow failure (BMF) syndromes. Autoreactive T lymphocytes are implicated in some of the immune mechanisms involved in PNH. In fact, we reported recently that the HLA-DRB1*1501 allele and HLA-A*0206 allele is frequent and is related to grading of hemolysis, respectively, in PNH patients (Shichishima T et al, Blood, 2002 and Haematologica, 2006, respectively). However, some characteristics of CD4+ and CD8+ T lymphocytes, including GPI-negative CD4+ and CD8+ T lymphocytes, in PNH patients remain unknown. To know some characteristics of CD4+ and CD8+ T lymphocytes with and without expressions of GPI proteins in PNH, we examined preferential variable beta chain (Vβ) repertoires of the T-cell receptor (TCR) and expressions of interferon-γ (IFN-γ) by flow cytometry and the TCR Vβ complementarity-determining region 3 (CDR3) spectratypes by genetic methods at the same time in CD4+CD59+, CD4+CD59−, CD8+CD59+, and/or CD8+CD59− T lymphocytes from 10 Japanese patients, including 6 and 4 with the HLA-DRB1*1501 allele and HLA-A*0206 allele, respectively, and from 5 age-matched healthy individuals. In the analyses of TCR Vβ repertoires, over-expressed TCR Vβ subfamilies were found in any T lymphocytes subsets from all the patients. We found significantly higher numbers (mean ± standard deviation; 1.9 ± 1.2) of over-expressed TCR Vβ subfamilies in CD8+CD59+ T lymphocytes from PNH patients compared with those (0 ± 0, p <0.01) from healthy individuals. In the TCR Vβ CDR3 spectratyping, skewed TCR Vβ CDR3 spectatypes were found in more than one TCR Vβ subfamilies of CD3+CD4+CD59−, CD3+CD8+CD59+, and CD3+CD8+CD59− T lymphocytes from all the PNH patients. The numbers of skewed TCR Vβ CDR3 spectatypes in CD3+CD8+CD59+ (4.0 ± 3.3) and CD3+CD8+CD59− (7.5 ± 3.9) T lymphocytes from one PNH patient were significantly greater than those in CD3+CD4+CD59+ T lymphocytes (0.6 ± 1.0, p <0.005) and CD3+CD4+CD59− (2.5 ± 1.5, p <0.002), respectively. Skewed TCR Vβ CDR3 spectatypes were found commonly in Vβ 25 subfamily of CD3+CD8+CD59+ and CD3+CD8+CD59− T lymphocytes from all of 4 PNH patients with the HLA-A*0206 allele. In the analyses of IFN-γ expressions, more than one TCR Vβ subfamiliy with over-expression of IFN-γ was found in CD8+CD59+ and/or CD8+CD59− T lymphocytes from 9 patients and in CD4+CD59+ and/or CD4+CD59− T lymphocytes from 8 patients. The numbers of TCR Vβ subfamiliy with over-expression of IFN-γ in CD8+CD59+ T lymphocytes (5.2 ± 4.3), but not in the other T lymphocyte subsets, from one PNH patient were significantly greater than those from healthy individuals (0 ± 0, p <0.05). However, there were no specific Vβ subfamilies determined by any analyses, described above, in PNH. In conclusion, we found high frequencies of increased IFN-γ-producing and/or skewed CD8+ T lymphocyte subfamilies with and/or without CD59 expression in PNH patients, suggesting that these cells may contribute to the occurrence of BMF rather than negative selection of PNH clones through action of IFN-γ in PNH.

Tracking of V beta 8.2-positive encephalitogenic T cells by complementarity-determining region 3 spectratyping and subsequent Southern blot hybridization in Lewis rats after neuroantigen sensitization.

Pathogenic T cells in organ-specific autoimmune diseases use a limited number of TCR alpha- and beta-chains. In experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats by immunization with myelin basic protein, encephalitogenic T cells mainly use Vbeta8.2 TCR and clonal expansion of the Vbeta8.2 spectratype containing the EAE-specific complementarity-determining region 3 (CDR3) sequence, DSSYEQYFGPG, is found in the spinal cord throughout the course of clinical EAE. In the present study we performed temporal and spatial analyses of Vbeta8.2 spectratype expansion by CDR3 spectratyping and subsequent DNA hybridization with a probe specific for the encephalitogenic CDR3 sequence to elucidate the kinetics of encephalitogenic T cells during the induction phase after neuroantigen sensitization. It was demonstrated that Vbeta8.2 spectratype expansion and/or the positive signal in Southern blot were first detected in the regional lymph nodes as early as day 3 postimmunization and was disseminated over the lymphoid organs by day 6. Because perfusion of immunized rats with PBS erased the positive signals on day 3 postimmunization, the majority of Vbeta8.2-positive encephalitogenic T cells at the very early stage would reside within the lymphatic or blood vessels. Furthermore, removal of the draining lymph node 1, 3, and 6 days after immunization in the foot pad did not ameliorate clinical EAE. These findings strongly suggest that encephalitogenic T cells disseminate throughout the whole body very rapidly after sensitization. Analysis of pathogenic T cells at the clonal level provides useful information for designing effective immunotherapy.

Identification of autoimmune encephalomyelitis-associated common CDR3 sequences by CDR3 spectratyping and subsequent DNA hybridization

In previous studies, we demonstrated that T cell receptor (TCR) Vβ8.2 and Vβ10, both of which are frequently used by encephalitogenic T cells, spectratypes expand oligoclonally in spinal cord lesions of Lewis rats with experimental autoimmune encephalomyelitis (EAE) and that the DSSYEQYF and WDGSGNVLYF sequences are predominantly found in the complementarity-determining region 3 (CDR3) of spectratype-derived TCR clones. However, it is unknown whether these CDR3 sequences are used only by Vβ8.2- and Vβ10-positive T cells or by encephalitogenic T cells bearing Vβs other than these Vβs. The present study was undertaken to address this issue using a new approach, i.e. CDR3 spectratyping and subsequent DNA hybridization with several probes corresponding to various parts of the CDR3 region. Consequently, we found that probes specific for the Vβ8.2 spectratype-derived Dβ and Jβ2.7 hybridized only with the Vβ8.2 spectratype in acute EAE and with the Vβ8.2 and Vβ12 spectratypes in chronic relapsing EAE. Similarly, a probe specific for the Vβ10 spectratype-derived Dβ hybridized only with the Vβ10 spectratype in both acute and chronic relapsing EAE. In contrast, a probe specific for Jβ1.3 hybridized with several Vβ spectratypes including Vβ8.2 and Vβ10 only during the early stage of the disease. These findings suggest that T cells bearing a few Vβs with a limited number of the CDR3 sequences are activated in acute and chronic relapsing EAE induced in Lewis rats. Characterization of the CDR3 region of pathogenic TCR by this approach may be of value for the screening of autoimmune disease-associated TCR and for the development of TCR-based specific immunotherapy.

Successful TCR-based immunotherapy for autoimmune myocarditis with DNA vaccines after rapid identification of pathogenic TCR.

The identification of TCRs of autoimmune disease-inducing T cells within a short period of time is a key factor for designing TCR-based immunotherapy during the course of the disease. In this study, we show that experimental autoimmune carditis-associated TCRs, Vbeta8.2 and Vbeta10, were determined by complementarity-determining region 3 (CDR3)-spectratyping analysis and subsequent sequencing of the CDR3 region of spectratype-derived TCR clones. Immunotherapy targeting both Vbeta8.2 and Vbeta10 TCRs using mAbs and DNA vaccines significantly reduced the histological severity of experimental autoimmune carditis and completely suppressed the inflammation in some animals. Since depletion or suppression of one of two types of effector cells does not improve the severity of the disease significantly, combined TCR-based immunotherapy should be considered as a primary therapy for T cell-mediated autoimmune diseases. TCR-based immunotherapy after rapid identification of autoimmune disease-associated TCRs by CDR3 spectratyping can be applicable, not only to animal, but also to human autoimmune diseases whose pathomechanism is poorly understood.

Analysis of T cell receptor beta chain CDR3 size using RNA extracted from formalin fixed paraffin wax embedded tissue.

AIMS: To isolate RNA and DNA simultaneously from formalin fixed paraffin wax embedded tissue to assess the clonality of enteropathy associated T cell lymphomas and to analyse it in detail...