<b>OBJECTIVEb>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.

<b>METHODSb>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-&gamma; and TCR-&beta; gene rearrangement detection in this study.

<b>RESULTSb>TCR 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.

<b>CONCLUSIONSb>TCR 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

<b>OBJECTIVEb>To 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).

<b>METHODSb>T 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.

<b>RESULTSb>The 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.

<b>CONCLUSIONb>IL-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

<b>BACKGROUNDb>In 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.

<b>METHODSb>The 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.

<b>RESULTSb>The 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.

<b>CONCLUSIONSb>These 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

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*

<b>OBJECTIVEb>To observe the TCR V gamma gene repertoire diversity in the peripheral blood mononuclear cells of the patients with the chronic benzene poisoning.

<b>METHODSb>Complementarity 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.

<b>RESULTSb>All 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.

<b>CONCLUSIONb>Skewed 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

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

<b>OBJECTIVEb>To investigate the distribution and clonality of T cell receptor (TCR) V&gamma; and V&delta; subfamily in peripheral blood of patients with allergic rhinitis before and after 1 year treatment with immunotherapy.

<b>METHODSb>The specific IgE and the complementary determinant region 3 (CDR3) of TCR V &gamma; (I-III) and V&delta;(1-8) subfamily genes in mononuclear cells were amplified from 10 effective cases of allergic rhinitis before and after 1 year treatment with immunotherapy, to observe the distribution and utilization of TCR V&gamma; and V&delta; repertoire. The positive PCR products were further labeled with RT-PCR and analyzed by gene scan technique to determine the CDR3 size and evaluate the clonality of the detectable TCR V&gamma; and V&delta; T cells. Peripheral blood of 10 healthy adults served as controls.

<b>RESULTSb>All symptoms were significantly improved after 1 year specific immunotherapy, but no changes were seen in specific IgE [(22.89 ± 9.60) kU/L before treatment, (19.62 ± 7.63) kU/L after treatment, Z = 1.051, P > 0.05]. No statistically significant differences of expression levels of the TCR V&gamma; I-III subfamily genes were found between patients with allergic rhinitis normal control group (t value were -0.679, -0.516, -0.808, all P > 0.05), but significantly decreased after 1 year treatment. There were statistically significant differences of expression levels of the TCR V&gamma;I-II subfamily genes before and after treatment (t value were -2.904, -2.217, all P < 0.05). 5.30 ± 0.82, 4.90 ± 0.57 and 5.20 ± 1.40 out of TCR V&delta; (1-8) subfamilies were selectively expressed in T cells in patients with allergic rhinitis before and after 1 year treatment and normal control group, predominantly for TCR V&delta; 1, 2, 3 and 6. The TCR V&delta; 6 subfamily was found to have statistically significant differences in these groups (Fisher's Exact Test, P < 0.05). Compared with the normal control group and the allergic rhinitis group before treatment, a significant higher frequency of V&delta; 6 oligoclonal was identified in T cells in patients with allergic rhinitis after 1 year treatment.

<b>CONCLUSIONSb>There was difference in the expression levels of the TCR V&gamma; I-III subfamily genes and distribution and clonality of TCR V&gamma; and V&delta; subfamily T cells in peripheral blood of patients with allergic rhinitis before and after 1 year treatment. Specific immunotherapy can be effective in alleviation of the symptom in patients with allergic rhinitis during the early stage, possibly by inducing TCR &gamma;&delta; T cells, especially the TCR V&delta;6 subfamily, and possibly no significant relativity between symptom and specific IgE.

Adolescent ; Adult ; Case-Control Studies ; Female ; Genes, T-Cell Receptor ; Humans ; Immunoglobulin E ; blood ; Immunotherapy ; Male ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; immunology ; Receptors, Antigen, T-Cell, gamma-delta ; genetics ; immunology ; Rhinitis ; genetics ; immunology ; therapy ; Young Adult

BACKGROUND: T lymphocytes that bear CD3 but lack CD4 and CD8 (Double negative T cells, DN T cells) are normally present early in ontogeny in the fetal thymus, but constitute only a small proportion of adult thymocytes and peripheral blood. Since DN T TCR alpha beta+ cells have been found to accumulate in the lymphoid organs of lpr and gld mice and to be expanded in patients with autoimmune diseases, their functional properties are now of considerable interest. METHOD: Sixty four rheumatoid arthritis (RA) patients and 24 healthy volunteers were studied from Jan 1997 to Feb 1998. The whole blood from the patients and controls were analyzed by flow cytometry (Elite ESP, Coulter, USA) and XL-II software after the cells were stained with trifluorochrome monoclonal antibodies (anti-CD4-FITC/anti-CD8-PE/anti-CD3-PE-Cy5, anti-CD3-FITC/anti-CD19-PE/anti-CD45-PE-Cy5, anti-CD3-FITC/ CD16+56-PE) (Immunotech, Coulter, USA). We reviewed patient records to find out the inflammatory parameters and Ritchie index. RESULTS: We confirmed the presence of DN T cells in peripheral blood of healthy volunteers and RA patients. DN T cells were lower in RA patients (mean+/-SD; 6.44%+/-4.46), when compared to healthy volunteers (mean+/-SD; 9.97%+/-4.50) (p=0.001). There was no clinical correlations between DN T cells and inflammatory parameters. CONCLUSION: Our study showed that the DN T cells in normal control were about 10% of CD3 positive T cells and the cells were significantly lower in RA patients. Although we did not identify whether these cells have either TCR alpha beta or TCR gamma delta, we could conclude that these cells are not expanded in RA patients. We would like to continue this study further 1) to identify TCR the DN T cells have and 2) to monitor the changes after treatment.
Adult ; Animals ; Antibodies, Monoclonal ; Arthritis, Rheumatoid* ; Autoimmune Diseases ; Flow Cytometry ; Healthy Volunteers ; Humans ; Mice ; Receptors, Antigen, T-Cell, alpha-beta ; Receptors, Antigen, T-Cell, gamma-delta ; T-Lymphocytes* ; Thymocytes ; Thymus Gland

This study was aimed to investigate the immunogenetic diagnosis of large granular lymphocytic leukemia (LGLL) and therapeutic efficacy of sirolimus, and to analysis 256 cases of LGLL reported at home and abroad within 2000 - 2010. Besides the routine examination of peripheral blood and classification of bone marrow cell morphology, the expression of T cell receptor variable region of &beta;-chain (TCR BV), CD3, CD4 and CD8, as well as TCR&alpha;&beta;, TCR&gamma;&delta; were detected by flow cytometry; the RT-PCR was used to amplify and determine the TCR gene spectrotypes, and to analyze the clonality of abnormal cells. Sirolimus was first given to patients who did not gain efficacy from common agents. The results showed that lymphocytosis happened in all LGLL patients, but patients from West countries always displayed neutropenia while Chinese patients always displayed anemia. In 2 out of 4 patients from our hospital, the large granular lymphocytes (LGL) were difficult to be distinguished. In all 4 patients, almost all lymphocytes were CD3(+), CD8(+), and TCR&alpha;/&beta;(+). TCR BV 24 gene family clones showed monoclonal TRBV 23, TRBV 20, TRBV 13.6, and TRBV 13.6, respectively. FCM results were consistent with those of RT-PCR. When 4 patients had been given sirolimus (6 mg first dose, 2 mg once a day) for about 1 week, hemoglobin level and reticulocyte count increased significantly without any serious side effects. It is concluded that the detection of specific lymphocyte monoclonal TCR BV 24 gene family by FCM contributes to the diagnosis of LGLL. Sirolimus is an effective agent without serious side effect for LGLL patients, especially for patients who cannot tolerate common drugs.
Adult ; Aged ; Female ; Flow Cytometry ; Humans ; Immunogenetics ; Leukemia, Large Granular Lymphocytic ; diagnosis ; drug therapy ; Male ; Middle Aged ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; Receptors, Antigen, T-Cell, gamma-delta ; genetics ; Sirolimus ; therapeutic use ; Treatment Outcome

<b>OBJECTIVEb>To 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.

<b>METHODSb>Immunoscopic 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.

<b>RESULTSb>The 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.

<b>CONCLUSIONb>CDR3 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