Characterization of human αβTCR repertoire and discovery of D-D fusion in TCRβ chains

The characterization of the human T-cell receptor (TCR) repertoire has made remarkable progress, with most of the work focusing on the TCRβ chains. Here, we analyzed the diversity and complexity of both the TCRα and TCRβ repertoires of three healthy donors. We found that the diversity of the TCRα repertoire is higher than that of the TCRβ repertoire, whereas the usages of the V and J genes tended to be preferential with similar TRAV and TRAJ patterns in all three donors. The V-J pairings, like the V and J gene usages, were slightly preferential. We also found that the TRDV1 gene rearranges with the majority of TRAJ genes, suggesting that TRDV1 is a shared TRAV/DV gene (TRAV42/DV1). Moreover, we uncovered the presence of tandem TRBD (TRB D gene) usage in ~2% of the productive human TCRβ CDR3 sequences.

Imunorregulação central e periférica em pacientes com Síndrome de Down e autoimunidade / Central and peripheral immunoregulation in patients with Down syndrome and autoimmunity

Introdução: A Síndrome de Down (SD) é uma doença genética de alta prevalência, com várias alterações imunológicas decorrentes da disfunção tímica associada à doença. Neste estudo, avaliou-se a associação entre presença de autoimunidade e disfunção do timo em pacientes com SD. Métodos: Foram avaliados 22 pacientes com SD (11 com autoimunidade e 11 sem), que preenchiam os critérios de inclusão: diagnóstico clinico e genético, idade > a 10 anos e sem uso de drogas imunossupressoras. Estes pacientes foram comparados a um grupo controle formado por adolescentes saudáveis (n=11) e outro de pacientes com doenças autoimunes, caracterizados por manifestações clínicas e presença de autoanticorpos (n=11). Todos os grupos foram pareados por idade e sexo. Os parâmetros laboratoriais avaliados foram: número de leucócitos, linfócitos CD3+, CD4+, CD8+, CD19+, CD21+, CD4+CD28null , células T reguladoras (CD4+CD25+Foxp3+), linfócitos T naive (CD4+CD45RA+CD62L+) e linfócitos T de memória (CD4+CD45RO+CD62L- ) e célula NK (CD3-CD16+, CD56+) por citometria de fluxo, Foi também avaliada a concentração de sjTREC (T receptor excision circles) em sangue total por qRT-PCR .Resultados: Nos pacientes com SD, observou-se redução das concentrações séricas de sjTREC, do número de linfócitos B e aumento do número de células CD4+CD28null. Na análise concomitante entre os grupos formados (SD com e sem autoimunidade, controle e autoimunidade sem SD), após correção de Bonferroni, observou-se que o grupo SD com autoimunidade apresentou redução de linfócitos T CD4, linfócitos naive e linfócitos B. Quando comparados os grupos SD com e sem autoimunidade observou-se redução significativa das concentrações de TREC no primeiro grupo. Não houve alterações das Células NK. Em valores percentuais, os pacientes com SD e autoimunidade apresentaram elevação da subpopulação de células T reguladoras. Conclusões: Este estudo mostra que pacientes com SD apresentam disfunção tímica quando avaliados pela...

Introduction: Down syndrome (DS) is a genetic disease of high prevalence, with many immunological alterations as a consequence of thymic disfunction associated to this disease. In this study, it was evaluated the association between the presence of thymic disfunction and autoimmunity in patients with DS. Methods: It was evaluated 22 patients with DS (11 with and 11 without autoimmunity) who fulfilled the inclusion criteria: clinical and genetic diagnosis, and age >10 years and no use of immunosuppressive drugs. These patients were compared to a control group composed by health adolescents (n=11) and patients with autoimmune diseases, characterized by clinical manifestations and autoantibodies (n=11). All groups were matched for age and sex. The laboratory parameters evaluated were: number of leukocytes, CD3+, CD4+, CD8+, CD19+, CD4+CD28null lymphocytes, regulatory T cells (CD4+CD25+Foxp3+), naive T lymphocytes (CD4+CD45RA+CD62L+), memory T lymphocytes (CD4+CD45RO+CD62L-) and NK cells (CD3-CD16+CD56+). The subpopulations of lymphocytes were determined by flow cytometry. It was also evaluated whole blood sjTREC (T receptor excision circles) concentrations by PCR. Results: In DS patients, there was reduction of sjTREC concentration, B lymphocytes number and increase of CD4+CD28null cells number. When compared all the groups formed (SD with and without autoimmunity, autoimmunity without SD and control group), after Bonferroni correction, the SD group with autoimmunity showed a reduction of T CD4+lymphocytes, naïve cells and B lymphocytes. When SD with and without autoimmunity groups were compared it was observed significant reduction in the TREC concentrations in the first group. There were no changes in NK cells. Patients with DS and autoimmune diseases had huge percentages of T reg cells comparing different groups. Conclusions: This study showed that DS patients presented thymic disfunction by reduced levels of whole blood sjTREC, and this condition is...

Diagnostic significance of TCR gene clonal rearrangement analysis in early mycosis fungoides / 癌症

Mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma, has various unspecific clinical and histological characteristics. Its early diagnosis is challenging. The application of T-cell receptor (TCR) gene clonal rearrangement to the diagnosis of MF has been widely studied. In this study, we used polymerase chain reaction (PCR) to investigate the diagnostic significance of detecting TCR-γ and -β gene clonal rearrangement in the early diagnosis of mycosis fungoides. PCR for TCR-γ and TCR-β gene rearrangement was performed on 19 patients with suspected early MF, 6 with typical MF, and 6 with chronic dermatitis. Of the 19 patients with suspected early MF, 13 had TCR-γ gene clonal rearrangement, whereas none had TCR-β gene clonal rearrangement. All patients with typical MF had TCR gene clonal rearrangement, in which 4 showed TCR-γ clonal rearrangement, 1 showed TCR-β gene clonal rearrangements, and 1 showed both. No patients with chronic dermatitis had TCR gene clonal rearrangement. These results indicate that TCR gene clonal rearrangement analysis is a useful tool in diagnosing early MF. TCR-γ gene is recommended to the routine analysis, whereas TCR-β gene has potential in combination toward intractable cases.

Significance of TCR gene clonal rearrangement analysis in diagnosis of mycosis fungoides / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the significance of detecting TCR gene clonal rearrangement in the diagnosis of mycosis fungoides (MF) and to optimize the primers used for detecting the TCR gene clonal rearrangement with PCR in paraffin embedded tissues of MF.</p><p><b>METHODS</b>Nineteen cases of MF were enrolled into the study. A panel of 10 antibodies were used for immunophenotypic analysis and polymerase chain reaction for TCR-γ and TCR-β gene rearrangement detection in this study.</p><p><b>RESULTS</b>TCR gene clonal rearrangements were detected in all 19 cases, in which 84.2% cases (16/19) had TCR-γ gene clonal rearrangements. The positive rates of the primers T(VG)/T(JX), V(2-5)/V(8-12)/JGT(1) and BIOMED-2-TCR-γ were 47.4%, 78.9% and 31.6%, respectively. The positive rate of V(2-5)/V(8-12)/JGT(1) was statistically significantly higher than that of T(VG)/T(JX) and BIOMED-2-TCR-γ (P < 0.05). No TCR gene clonal rearrangement was detected using the primers V(γ11)/V(γ101)/Jγ12 and V(γ11)/V(γ101)/J(p12). TCR-β gene clonal rearrangement was detected in 31.6% (6/19) cases.</p><p><b>CONCLUSIONS</b>TCR gene clonal rearrangement analysis is a useful tool in the diagnosis of MF and TCR-γ gene is a good target gene for the detection. The primers T(VG)/T(JX), V(2-5)/V(8-12)/JGT(1) and BIOMED-2-TCR-γ can be used in clinicopathologic detection for TCR gene clonal rearrangement and V(2-5)/V(8-12)/JGT(1) may be the first choice.</p>

Characteristics of T cell receptor beta gene rearrangements and its role in minimal residual disease detection in childhood T-cell acute lymphoblastic leukemia / 中华儿科杂志

<p><b>OBJECTIVE</b>To explore the characteristics of T cell receptor beta (TCRbeta) gene rearrangements in children with T-cell acute lymphoblastic leukemia (T-ALL) and establish a system of quantitative detection of MRD with real-time quantitative (RQ-PCR) targeted at TCRbeta gene rearrangement.</p><p><b>METHODS</b>Multiplex polymerase chain reaction (PCR) designed by BIOMED-2 was used to detect TCRbeta gene rearrangements in the bone marrow samples of 26 children with T-ALL. Sequence of junction region were then compared and analyzed in IMGT database. Allele specific oligonucleotide (ASO) upstream primers were designed complementary to the V-D-J or D-J junctional region of TCRbeta gene rearrangements. Samples at diagnosis were serially diluted in DNA obtained from mononuclear cells (MNC) from a pool of 20 healthy donors to generate the patient specific standard curves. Subsequently, a TCRbeta RQ-PCR assay to quantify MRD with germline Jbeta primer/probe combinations was applied in six patients. To check the quantity and quality of DNA, the investigators used RQ-PCR analysis for the N-ras gene.</p><p><b>RESULTS</b>Clonal rearrangements were identified in 92.3% childhood T-ALL (Vbeta-Dbeta-Jbeta rearrangements in 84.6%, Dbeta-Jbeta rearrangements in 50%). Comparative sequence analysis of 42 TCRbeta recombinations revealed two downstream Vbeta families (BV5, BV6) were preferentially used. The segment Jbeta2.7 in childhood T-ALL was preferentially used. Jbeta1.3, Jbeta2.4, and Jbeta2.6 were not found to be used. The slope of the standard curves was from -3.54 to -3.37 and the intercepts were from 19.35 to 20.51. The correlation coefficients of all 6 standard curves were excellent (> or = 0.98). All the RQ-PCR quantitative range reached 10(-4). MRD analysis of follow up samples showed that MRD increased before relapse.</p><p><b>CONCLUSION</b>RQ-PCR analysis of TCRbeta gene rearrangements was highly sensitive and specific, it will be of high value for future T-ALL MRD studies. And quantitative and serial study of MRD may be of prognostic importance.</p>

Effects of T cell receptor gene rearrangement on BV CDR3 in Jurkat cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of T cell receptor (TCR) BD2-BJ2 gene rearrangement on the complementary-determining region (CDR) 3 of TCR beta chain (TCR BV CDR3) in Jurkat cells.</p><p><b>METHODS</b>TCR BV gene subfamilies were detected by RT-PCR in Jurkat cells during proliferation and after induction with non-specific T cell activators and SEA, respectively. To determine the clonality of TCR BV subfamilies and the lengths of CDR3, the PCR products were analyzed by TCR GeneScan technique, and the sequences of CDR3 were further analyzed by DNA sequencer.</p><p><b>RESULTS</b>No new TCR BV subfamilies were found in Jurkat cells, a monoclonal BV8(+)cell line, either during cell proliferation or after stimulation with different treatments, nor were any differences found in CDR3 size or sequences.</p><p><b>CONCLUSION</b>TCR BD2-BJ2 rearrangement in Jurkat cells may not play a role in modification of TCR BV CDR3 domains or the consequent antigen immunorecognition of BV CDR3, but the possibility of TCR modification can not be excluded.</p>

Changes in T-cell receptor repertoire in aplastic anemia and effects of Shengxue Mixture / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the immune pathogenesis of aplastic anemia (AA) and the therapeutic effects of Shengxue Mixture (SM) through the gene expressions of subfamilies of T-cell receptor variable region beta (TCR Vbeta) using immunologic and molecular biologic technology.</p><p><b>METHODS</b>Gene expressions of TCR Vbeta sub-families in peripheral blood mononuclear cells from 20 AA patients were detected before and after treatment with SM using RT-PCR and gene scanning method.</p><p><b>RESULTS</b>TCR Vbeta gene repertoire of the 24 subfamily genes deviated in AA patients, and the oligoclonal gene expressions increased obviously compared with those in healthy people (P < 0.01), including Vbeta2, 5, 6, 15, 16, 22, and 23 were found in 30%-50% AA patients, and Vbeta8, 21 were in more than 50% patients. These oligoclonal genes reduced significantly after treatment with SM compared with those before treatment (P < 0.05).</p><p><b>CONCLUSION</b>Multiple TCR Vbeta subfamilies of clonal proliferation participate in the pathogenesis of AA. SM can rectify the deviation of TCR Vbeta gene repertoire, reduce the abnormal clonal proliferation of T cells, thus to alleviate the immune injury to hematopoietic tissue, and thus to benefit the recovery of hematopoiesis of bone marrow.</p>

Analysis of T cell receptor BV dominant usage and CDR3 sequences during acute exacerbation in patients with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To understand the role cellular immunology plays in the pathogenesis of chronic hepatitis B (CHB) through analysis of T cell receptor (TCR) beta chain variable region gene (BV) family dominant usage and beta chain complementarity determining region3 (CDR3) sequences of peripheral blood mononuclear cells of the patients.</p><p><b>METHODS</b>TCR BV families were amplified by inverse polymerase chain reaction (RT-PCR), and the dominant usage of BV families and CDR3 repertoire were analyzed by immunoscope technology for 8 CHB patients during their acute exacerbations and for 4 healthy blood donors who served as controls. The clonality of the T cells suspected by immunoscope was further confirmed by CDR3 sequencing.</p><p><b>RESULTS</b>The TCR BV CDR3 repertoire of the 4 healthy blood donors showed a Gaussian distribution. In the 8 CHB patients, however, the clonal expansion of T cells showed different TCR BV families with each patient. The T cells of the clonal expansion shared different CDR3 sequences.</p><p><b>CONCLUSION</b>The peripheral blood T cells of CHB patients during their acute exacerbation showed significantly a clonal expansion and their T cell clonal expansion may be stimulated by several HBV epitopes. These results indicate that cellular immunology is involved in the pathogenesis of the liver inflammation process of CHB.</p>

Identification of clonally rearranged T-cell receptor beta chain genes in HTLV-I carriers as a potential instrument for early detection of neoplasia

We analyzed the genetic recombination pattern of the T-cell receptor beta-chain gene (TCR-beta) in order to identify clonal expansion of T-lymphocytes in 17 human T-lymphotropic virus type I (HTLV-I)-positive healthy carriers, 7 of them with abnormal features in the peripheral blood lymphocytes. Monoclonal or oligoclonal expansion of T-cells was detected in 5 of 7 HTLV-I-positive patients with abnormal lymphocytes and unconfirmed diagnosis by using PCR amplification of segments of TCR-beta gene, in a set of reactions that target 102 different variable (V) segments, covering all members of the 24 V families available in the gene bank, including the more recently identified segments of the Vbeta-5 and Vbeta-8 family and the two diversity beta segments. Southern blots, the gold standard method to detect T-lymphocyte clonality, were negative for all of these 7 patients, what highlights the low sensitivity of this method that requires a large amount of very high quality DNA. To evaluate the performance of PCR in the detection of clonality we also analyzed 18 leukemia patients, all of whom tested positive. Clonal expansion was not detected in any of the negative controls or healthy carriers without abnormal lymphocytes. In conclusion, PCR amplification of segments of rearranged TCR-beta is reliable and highly suitable for the detection of small populations of clonal T-cells in asymptomatic HTLV-I carriers who present abnormal peripheral blood lymphocytes providing an additional instrument for following up these patients with potentially higher risk of leukemia.

Analysis of T lymphocytic clones in diseases by spectrotyping of T cell beta variable region - review / 中国实验血液学杂志

T cells recognize antigens through TCRs (T cell receptor). T cell clones can be sorted into 24 gene subfamilies based on the usage of the segments of TCR BV in gene rearrangement. Application of the various segments of TCR BV may establish TCR BV spectrotyping that can be used to analyze and recognize the different functional T cell clones, and understand the function and proliferation of various T cell clones in malignancy and autoimmune disease. In vitro expansion of a great deal of the specific antitumor T cells and transfusing them to patients will be able to develop a new method for tumor immunotherapy. Through analyze the character of the TCR BV gene, McAb against TCR or DNA vaccine to inhibit the growth of T cell clones associated-autoimmune disease and tumors might be developed. The McAb and vaccine may be used to cure these diseases. The commo T cells can also be modifed to specific antitumor T cells by method of TCR gene transfer. In this review, the characteristics of TCR, analysis method for gene spectrotyping of TCR BV, segments of TCR BV and autoimmue distase, T cell clones in hematologic maligrancies, recognition of T cell oligoclone expansion of T cells, and application of TCR BV gene spectrotyping in bone marrow transplantation were discussed and summarised.

Pathologic diagnoses of core needle biopsies of the mediastinum / 中华病理学杂志

<p><b>OBJECTIVES</b>To assess the diagnostic accuracy and to study the histologic typing of mediastinal lesions using core needle biopsies.</p><p><b>METHODS</b>The histopathology and immunophenotype of 65 mediastinal core needle biopsy specimens were studied retrospectively by light microscopy and immunohistochemical staining (ABC method). Gene rearrangement studies were performed in some of the non-Hodgkin's lymphomas cases using PCR. Follow-up records were also analyzed.</p><p><b>RESULTS</b>Morphologically, all specimens showed a combination of epithelioid cells, lymphoid cells and fibrous tissue in different proportions. The pathologic diagnoses included lymphoma (21 cases), pulmonary carcinoma (20 cases), thymoma (14 cases), thymic carcinoma (4 cases), seminoma (3 cases) and chronic inflammation (1 case). Definitive diagnosis was not possible in 2 cases due to insufficient material. The tumor cells in lymphoma (21 cases) expressed CD20, CD3, TDT, CD30, CD15 or EMA, depending on their histologic subtypes. Tumor cells in the 17 pulmonary carcinoma cases expressed cytokeratin (CK), except 3 cases of small cell carcinoma of lung. Synaptophysin, chromogranin A and neuron-specific enolase were all positive in the 10 cases of small cell carcinoma of lung and 1 case of thymic small cell carcinoma (which was also CD5 negative). The 3 cases of adenocarcinoma of lung showed positivity for thyroid transcription factor-1 (TTF-1) and they were negative for CD5. The 14 thymoma cases expressed CK, CD3 or CD20. The 3 thymic carcinoma cases expressed CK and CD5. Placental-like alkaline phosphatase (PLAP) was positive in 3 seminoma cases which were CK-negative. Immunoglobulin heavy chain gene was rearranged in the 3 cases of diffuse large B-cell lymphoma and 1 B-cell anaplastic large cell lymphoma case. T-cell receptor beta gene was rearranged in 5 T-cell lymphoblastic lymphoma cases.</p><p><b>CONCLUSIONS</b>Microscopic assessment of tissue samples from mediastinal core needle biopsies should be made in combination with clinical and radiologic information. Ancillary investigations, including immunohistochemical staining and/or gene rearrangement studie, are needed in both non-lymphoma and lymphoma cases of mediastinum.</p>

High frequency of specific T cell receptor rearrangements in rheumatoid arthritis: analysis of synovial fluid T lymphocytes

1. We have searched for rearrangements in the beta chain T cell receptor genes to identify clonal T lymphocyte populations in the synovial fluid of 10 patients with well established rheumatoid arthritis, using a T cell population unbiased by preselection. 2. Analysis of the restriction fragments with the beta chain constant region probe C beta 2 disclosed a rearranged band in 50 of cases (5/10). No significant differences in age, duration of the disease, treatment employed and presence of articular deformities or erosion upon X-ray examination were observed when patients with or without rearrangements were compared. 3. The rearranged band observed after BamH I digestion was of the same size in the 5 patients (14 kb). In addition, two patients presented a 10-kb rearranged band upon restriction with Hind III. 3. These data indicate that a significant number of rheumatoid arthritis patients probably present oligoclonal T cell proliferation of their synovial fluid lymphocytes.