OBJECTIVETo 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.

METHODSNineteen 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.

RESULTSTCR gene clonal rearrangements were detected in all 19 cases, in which 84.2% cases (16/19) had TCR-&gamma; 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-&gamma; 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-&gamma; (P < 0.05). No TCR gene clonal rearrangement was detected using the primers V(&gamma;11)/V(&gamma;101)/J&gamma;12 and V(&gamma;11)/V(&gamma;101)/J(p12). TCR-&beta; gene clonal rearrangement was detected in 31.6% (6/19) cases.

CONCLUSIONSTCR gene clonal rearrangement analysis is a useful tool in the diagnosis of MF and TCR-&gamma; 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-&gamma; 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.

Adolescent ; Adult ; Aged ; Antigens, CD7 ; metabolism ; Base Sequence ; CD2 Antigens ; metabolism ; CD3 Complex ; metabolism ; CD4 Antigens ; metabolism ; Child ; Child, Preschool ; Female ; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor ; Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor ; Humans ; Leukocyte Common Antigens ; metabolism ; Male ; Middle Aged ; Molecular Sequence Data ; Mycosis Fungoides ; diagnosis ; genetics ; metabolism ; pathology ; Paraffin Embedding ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; Receptors, Antigen, T-Cell, gamma-delta ; genetics ; Skin Neoplasms ; diagnosis ; genetics ; metabolism ; pathology ; Young Adult

BACKGROUNDIn general, it is very important to understand the state of T cell immune response against tumor cells in leukemia patients and it is especially critical to assess the T cell repertoire of untreated patients. As we know, few studies have dealt with the distribution of oligoclonal T cells in leukemia, so we investigated the distribution and clonality of TCR Vbeta repertoire of T cells in patients with chronic myelogenous leukemia (CML) in chronic phase.

METHODSThe complementarity determining region 3 (CDR3) of TCR Vbeta24 subfamily genes were amplified in peripheral blood mononuclear cells from 27 cases with CML using reverse transcription-polymerase chain reaction (RT-PCR). In order to observe the distribution of TCR Vbeta repertoire, the PCR products were further analyzed by genescan technique to evaluate clonality of the detectable TCR Vbeta T cells. The PCR products of the oligoclonal T cells from three cases were analyzed by direct sequencing to define the sequence of CDR3.

RESULTSThe expression pattern of TCR Vbeta repertoire in different individuals are different. Vbeta2-21 subfamilies could be detected in CML cases. The frequent usage Vbeta repertoire in CML was Vbeta1, Vbeta2 or Vbeta13. Most of the PCR products from 27 patients displayed polyclonality, while a part of the PCR products from 21 out of 27 samples displayed clonal expansion pattern. The clonal expanded T cells in CML could be found in Vbeta16 subfamilies. The frequent usage of Vbeta genes in clonal expansion was Vbeta3, Vbeta13 or Vbeta21. Multiple Vbeta clonal expansion was a general phenomenon in the same patient. The CDR3 sequence of Vbeta21 oligoclonal T cells from 3 cases showed some difference in splice regions and in the usage of J segments.

CONCLUSIONSThese results indicated that clonal expanded T cells could be found in patients with CML and were tendentious in Vbeta3, Vbeta13 and Vbeta21 subfamilies that may be related to the specific immune response for leukemia cell associated antigen.

Clone Cells ; Complementarity Determining Regions ; analysis ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; immunology ; Receptors, Antigen, T-Cell, alpha-beta ; analysis ; T-Lymphocytes ; immunology ; pathology

OBJECTIVETo analyze the drift of the complementarity-determining region 3 (CDR3) of T cell receptor beta (TCRbeta) chain variable region in T cells of healthy volunteers cultured with interleukin-2 (IL-2).

METHODST cells were isolated from the peripheral blood and cultured in vitro in the presence of IL-2. The non-specific killing effect of the cells was analyzed by LDH releasing assay, and the distribution of TCRbeta chain CDR3 in healthy volunteers by immunoscope spectratyping method to evaluate the clonality of the T cells.

RESULTSThe results showed Gaussian distribution of TCR Vbeta gene CDR3 in healthy volunteers. The T cell cultured with IL-2, however, displayed some anomalous and oligoclonal expansion in different TCR Vbeta families without killing effect against nasophargngal carcinoma cell line CNE2.

CONCLUSIONIL-2 may affect TCRbeta chain CDR3 distribution in T cells cultured in vitro.

Cells, Cultured ; Complementarity Determining Regions ; genetics ; Genetic Drift ; Humans ; Interleukin-2 ; metabolism ; Leukocytes, Mononuclear ; metabolism ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; T-Lymphocytes ; immunology ; metabolism

OBJECTIVES: To investigate the diversity of peripheral blood T cell receptor (TCR) β chain complementarity-determining region 3 (CDR3) based on immune repertoire sequencing in neonates with sepsis and the possible pathogenesis of neonatal sepsis. METHODS: A total of 12 neonates with sepsis were enrolled as the case group, and 9 healthy full-term infants, matched for gestational age, birth weight, and age, were enrolled as the control group. Omega nucleic acid purification kit (SQ blood DNA Kit II) was used to extract DNA from peripheral blood samples, TCR β chain CDR3 was amplified by multiplex PCR, and then high-throughput sequencing was performed for the products to analyze the diversity of TCR β chain CDR3 and the difference in expression. RESULTS: The length and type of TCR β chain CDR3 were similar between the case and control groups, and Gaussian distribution was observed in both groups. With D50 and Shannon-Wiener index as the evaluation indices for diversity, the case group had a significantly lower diversity of TCR β chain CDR3 than the control group ( CONCLUSIONS There is a significant change in the diversity of TCR β chain CDR3 in the peripheral blood of neonates with sepsis, suggesting that it might be associated with the immune pathogenesis of neonatal sepsis.
Complementarity Determining Regions/genetics* ; High-Throughput Nucleotide Sequencing ; Humans ; Multiplex Polymerase Chain Reaction ; Neonatal Sepsis ; Receptors, Antigen, T-Cell, alpha-beta/genetics*

OBJECTIVETo observe the TCR V gamma gene repertoire diversity in the peripheral blood mononuclear cells of the patients with the chronic benzene poisoning.

METHODSComplementarity determining region 3 (CDR3) of TCR V gamma subfamily genes were amplified in 10 patients with the chronic benzene poisoning using RT-PCR. The PCR products were further analyzed by genescan to evaluate clonality of T cells. 8 healthy persons served as control.

RESULTSAll V gamma subfamilies were detected in the 8 healthy donors. (1.30 +/- 0.48) V gamma subfamilies were detected in 10 patients. The number of detectable V gamma subfamilies present in the patients with the chronic benzene poisoning was significantly lower than in the control group (P < 0.01). The most frequently used V gamma genes in clonally expanded T-cells were V gamma II.

CONCLUSIONSkewed distribution and clonal expansion of TCR V gamma subfamily T cells could be found in the patients with the chronic benzene poisoning. This is the first report of clonal expansion TCR V gamma T cells in patients with chronic benzene poisoning. The bias pattern of TCR V alpha T cells may be due to the immune cytotoxicity from benzene.

Adult ; Benzene ; poisoning ; Chronic Disease ; Complementarity Determining Regions ; genetics ; Female ; Humans ; Leukocytes, Mononuclear ; immunology ; Male ; Middle Aged ; Occupational Diseases ; genetics ; immunology ; Receptors, Antigen, T-Cell, gamma-delta ; genetics ; immunology ; Young Adult

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.
Complementarity Determining Regions ; genetics ; DNA Primers ; chemistry ; genetics ; Female ; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor ; genetics ; Gene Rearrangement, delta-Chain T-Cell Antigen Receptor ; genetics ; Genes, T-Cell Receptor beta ; genetics ; Genetic Variation ; High-Throughput Nucleotide Sequencing ; Humans ; Immunoglobulin Joining Region ; genetics ; Immunoglobulin Variable Region ; genetics ; Male ; Receptors, Antigen, T-Cell, alpha-beta ; genetics

OBJECTIVETo analyze the drift of T cell receptor (TCR) V&alpha; and V&beta; gene family CDR3 spectratype in response to ovarian carcinoma cells mediated by an anti-human ovarian carcinoma/CD3 bispecific single-chain antibody (BHL-1), and explore the mechanism of the bispecific single-chain antibody-mediated T cell immune response.

METHODSImmunoscopic spectratyping technique was used to analyze the TCR repertoire diversity (CDR3 spectratype distribution) of the T cells from 6 healthy donors before and after stimulation of the cells with human ovarian carcinoma in the presence of BHL-1. The predominant usage of TCR &alpha; and V&beta; chain CDR3 was analyzed after the stimulation, and sequence analysis was performed for the CDR3 region of the monoclonal T cells.

RESULTSThe spectratypes of V&alpha; and V&beta; gene family TCR CDR3 region showed a Gaussian distribution before stimulation of the T cells from the 6 donors. After stimulation of the T cells, CDR3 spectratype drift occurred in the T cells, and some TCR V&alpha; and V&beta; families showed an anomalous and oligoclonal expansion. Different CDR3 sequences of the V&alpha; and V&beta; gene family TCR were found in the monoclonal T cells stimulated with BHL-1.

CONCLUSIONCDR3 spectratype drift occurs in TCR &alpha; and V&beta; chains of T cells after stimulation with human ovarian carcinoma cells and BHL-1, indicating that the predominant usage of TCR V&alpha; and V&beta; families is associated with the specific T cell immune response mediated by BHL-1.

Antibodies, Bispecific ; immunology ; Cell Line ; Cell Line, Tumor ; Complementarity Determining Regions ; immunology ; Female ; Humans ; Monocytes ; immunology ; Ovarian Neoplasms ; immunology ; Receptors, Antigen, T-Cell, alpha-beta ; immunology ; Single-Chain Antibodies ; immunology

OBJECTIVETo study the CDR3 spectratyping and clonal expansion of T cells in the peripheral blood mononuclear cells (PBMCs) of patients with beta-mediterranean anemia patient undergoing allogeneic peripheral blood stem cell (PBSC) transplantation.

METHODSThe total RNA was isolated from the PBMCs of a nine-year-old boy with beta-mediterranean anemia before and after PBSC transplantation, and at the time of occurrence of graft-versus-host disease (GVHD). The CDR3 length was analyzed using immunoscope technique, and the characteristics of the T cell receptor (TCR) on the T cells with clonal expansion were examined by gene sequencing.

RESULTSThe 24 TCR BV CDR3 repertoire showed Gaussian distribution in the PBMCs isolated before the transplantation, and some of the TCR BV family CDR3 showed skewing in the PBMCs isolated 23 days after transplantation and at the onset of GVHD (28 days after transplantation), suggesting the clonal expansion of the donor PBSCs.

CONCLUSIONThe host PBMCs show muti-clonal expansion 23 days after PBSC transplantation, and in the stage of GVHD, some of the TCR BV family T cells show significant monoclonal expansion. Analysis of TCR CDR3 spectratyping and the molecular characteristics of specific TCR may help evaluate the immune reconstitution following the transplantation and indicate specific treatment for potential GVHD.

Child ; Complementarity Determining Regions ; genetics ; immunology ; Graft vs Host Disease ; etiology ; immunology ; Humans ; Male ; Peripheral Blood Stem Cell Transplantation ; adverse effects ; methods ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; immunology ; beta-Thalassemia ; immunology ; surgery

Humans ; Liver Neoplasms ; genetics ; immunology ; pathology ; Lymphoma, T-Cell ; genetics ; immunology ; pathology ; Receptors, Antigen, T-Cell, alpha-beta ; metabolism ; Receptors, Antigen, T-Cell, gamma-delta ; metabolism ; Splenic Neoplasms ; genetics ; immunology ; pathology

OBJECTIVEThe complementarity determining region 3 (CDR3) of T cell receptor (TCR) is the place through which T cells connect to the antigen. The lengths and DNA sequences of CDR3s are different according to different T cell clones. This leads to a diverse TCR CDR3 repertoires which can reflects the functional status of T cells precisely. This study aimed at elucidating the abnormality of TCR beta chain variable region (BV) CDR3 repertoires of children with acute B lymphoblastic leukemia, the pathogenesis of leukemia associated with T cell dysfunction and the immuno-reconstruction of T cells after the chemotherapy.

METHODSTwelve children aged from 3 to 13 years (average 4.50 +/- 3.78 years) with acute B lymphoblastic leukemia before chemotherapy and 8 healthy control donors aged from 6 to 16 years (average 10.30 +/- 3.00 years) were enrolled. Four of 12 patients were studied for the second time 3 months after complete remission (CR). Reverse transcription-polymerase chain reaction (RT-PCR) and polyacrylamide sequencing gel electrophoresis were used to detect the diversity of TCR BV CDR3 repertoires of these children.

RESULTS(1) The expression of BV2 and BV3 in 12 children increased and BV17 and BV18 decreased before the chemotherapy as compared with controls (P < 0.05). There were 4 children with a lower level expression of BV21 before the chemotherapy, and much lower level expression was found after the remission. (2) Normally each lane contained eight to ten bands, which represented unique CDR3 sizes for a given TCR BV family. Bands differed in size by 3 nucleotides and generally form a Gaussian distribution. There were 14% TCR BV families with abnormal CDR3 length distribution in 12 patients before therapy, which was significantly higher than that of controls (5.5%, P < 0.05). Restricted CDR3 length distribution was observed in BV14, BV1, BV16, BV20, BV13.1, BV13.2 and BV6. Every abnormal BV family recovered to normal Gaussian distribution 3 months after CR.

CONCLUSIONT lymphocytes in children with acute B lymphoblastic leukemia revealed markedly skewed repertoires, which suggests the abnormality of T cell functions. Most abnormal T cell repertoires recovered to normal Gaussian distribution 3 months after the first CR, which suggests the immunological reconstitution of T cell repertoires.

Adolescent ; Burkitt Lymphoma ; genetics ; immunology ; Child ; Child, Preschool ; Complementarity Determining Regions ; genetics ; DNA, Complementary ; chemistry ; genetics ; Electrophoresis, Polyacrylamide Gel ; Female ; Humans ; Male ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA