The rearrangement segments of TCR Valpha40 gene with Jdelta1, Ddelta3 or psi Jalpha were amplified in genomic DNA from peripheral blood mononuclear cells of 10 normal subjects, sorted CD3(+) cells from peripheral blood of 4 cases and thymocytes from 7 cases, by using nested PCR. Different amounts of DNA from all samples were amplified to estimate the frequency of Valpha40 gene rearrangements. The results indicated that the rearrangements of TCR Valpha40 gene with Jdelta1, Ddelta3 or psi Jalpha could be found respectively in the most samples of peripheral blood T cells or thymocytes. The frequencies of Valpha40 rearrangements were different in peripheral blood T cells and thymocytes by analysis of PCR with different amounts DNA. It is concluded that the TCR V alpha40-psi Jalpha was the most frequent rearrangement in mature and immature T cells, whereas TCR Valpha40-Ddelta3 was more frequently rearranged in immature T cells
Gene Rearrangement ; Humans ; Polymerase Chain Reaction ; Protein Subunits ; genetics ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; T-Lymphocytes ; physiology

In order to analyze the distribution and clonal expansion of TCR Vbeta subfamily T cells in patients with acute promyelocytic leukemia (APL) in vivo and in vitro after T cell culture, the peripheral blood mononuclear cells from 3 APL patients were expanded by rhIL-2 and anti-CD3 antibody using liquid T lymphocytes culture technique. The complementary determining region 3 (CDR3) of TCR beta with variable region genes was amplified in T cells from 3 APL cases before and after T cell culture by using RT-PCR. The positive products were further analyzed to identify the clonality of T cells by genescan. The results showed that only a part of 24 Vbeta subfamilies was detected in T cells from the patients, and some Vbeta subfamily T cells could be identified after T cells culture. The clonal expansion T cells in some TCR Vbeta subfamilies could be found in all patients. The similar oligoclonal expansion of Vbeta1, Vbeta3, Vbeta7, Vbeta16 and Vbeta20 T cells was detected in two cases at different time points after T cell culture. It is concluded that the restricted expression of TCR Vbeta subfamily in T cells from patients might be the common feature in leukemia. Some Vbeta subfamily T cells could be induced after T cells culture in vitro. The continual clonal expansion of TCR Vbeta subfamily T cells at different time points after T cells culture could be a specific immune response of patients T cells related to the specific APL cell associated antigen.
Humans ; Leukemia, Promyelocytic, Acute ; genetics ; immunology ; Lymphocyte Activation ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; T-Lymphocytes ; immunology

The T cell antigen receptor-CD3 (TCR/CD3) complex is assembled in the endoplasmic reticulum (ER) of T cells after synthesis of individual chains, and is transported to the cell surface for immune recognition and regulation. Partially assembled or unassembled TCR chains are retained and rapidly degraded in the ER. These processes are strictly regulated in the ER at post-translational level for the maintenance of cellular homeostasis. In order to identify the region responsible for the ER retention and rapid degradation of the TCR beta chain, number of mutants were engineered and their fates, after synthesis in the ER of the HeLa cells, were investigated. Extensive mutagenic analysis of TCR beta chain, including changing the charged amino acid residues and two tyrosine residues of the transmembrane region into hydrophobic amino acid residues, did not alter the ER retention and rapid degradation. Soluble TCR beta chain and cytoplasmic tail truncation mutant were also rapidly degraded in the ER. However, N-glycosylation rate of soluble TCR beta chain in the ER was significantly increased possibly due to the increased exposure of the N-glycosylation site. These results suggest that the ER retention of TCR beta chain is mediated through its extracellular and transmembrane-cytoplasmic regions and that the rapid ER degradation can be caused by an exposure of unassembled subregion of TCR beta chain, either extracellular domain or hydrophobic transmembrane region to the hydrophilic environment (lumen of the ER) rather than by presence of a specific degradation signal.
Cytoplasm/metabolism ; Endoplasmic Reticulum/metabolism* ; Hela Cells/metabolism ; Human ; Receptors, Antigen, T-Cell, alpha-beta/metabolism* ; Receptors, Antigen, T-Cell, alpha-beta/genetics* ; Recombinant Proteins/metabolism ; Recombinant Proteins/genetics

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

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

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

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

To understand the characteristics of T cell receptors recognizing antiphospholipid syndrome associated antigen, the characteristics of T cells were analyzed using T cell receptor beta variable region (TCRbetaV) gene spectrotyping in a case of antiphospholipid syndrome (APS). The results indicated that in the case of APS there were 2 dominant T cell clones. The TCRbetaVs sequences of the 2 T cell clones showed the TCRbetaVs belonged to 8 and 23 gene families respectively. The peptides of third complementarity-determining regions (CDR3) in the TCRbetaVs were CASSLLVAGGPRAYNEQFFGPG and CASSLAGFGQPQHFGDG. Comparing the motifs in CDR3 with another autoimmune disease, the motif YNEQFFGPG in TCRbetaV8 and motif QHFGDG in TCRbetaV23 were identical with that of idiopathic thrombocytopenic purpura and systemic lupus erythematosus reported before. In conclusion, some T cell clones proliferating in these autoimmune diseases may recognize the same antigens.
Adult ; Antiphospholipid Syndrome ; immunology ; Autoantigens ; immunology ; Female ; Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor ; immunology ; Genes, T-Cell Receptor beta ; genetics ; immunology ; Humans ; Immunoglobulin Variable Region ; immunology ; Receptors, Antigen, T-Cell ; immunology

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 β-chain (TCR BV), CD3, CD4 and CD8, as well as TCRαβ, TCRγδ 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 TCRα/β(+). 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

OBJECTIVETo investigate the cytotoxicity of normal CD8(+) T lymphocytes retrovirally transduced with WT1 peptide-specific T-cell receptor (TCR) genes against human lung cancer cells.

METHODSHLA-A*2402-restricted and WT1 peptide-specific TCR-α/β genes were cloned from a cytotoxic T lymphocyte clone and inserted into a retroviral TCR expression vector. The cytotoxicity of normal peripheral CD8⁺ T cells transduced with the WT1-TCR genes against human lung cancer cells was evaluated using a standard ⁵¹Cr release assay.

RESULTSThe WT1-TCR gene-modified T cells recognized the peptide-pulsed target cells but not the non-pulsed cells. TCR-redirected CD8⁺ T cells lysed WT1-overexpressing human lung cancer cells in an HLA-A*2402-restricted manner, but did not kill normal cells positively expressing HLA-A*2402.

CONCLUSIONThese data demonstrate the feasibility of adoptive immunotherapy with TCR-redirected T cell for the treatment of lung cancer.

CD8-Positive T-Lymphocytes ; cytology ; Cell Line, Tumor ; Genes, T-Cell Receptor ; Humans ; Immunotherapy, Adoptive ; Lung Neoplasms ; pathology ; Peptides ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; Retroviridae ; T-Lymphocytes, Cytotoxic ; cytology ; Transduction, Genetic ; WT1 Proteins ; genetics