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 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

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

OBJECTIVETo investigate the distribution and clonality of T cell receptor (TCR) Vγ and Vδ subfamily in peripheral blood of patients with allergic rhinitis before and after 1 year treatment with immunotherapy.

METHODSThe specific IgE and the complementary determinant region 3 (CDR3) of TCR V γ (I-III) and Vδ(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 Vγ and Vδ 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 Vγ and Vδ T cells. Peripheral blood of 10 healthy adults served as controls.

RESULTSAll 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 Vγ 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 VγI-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 Vδ (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 Vδ 1, 2, 3 and 6. The TCR Vδ 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 Vδ 6 oligoclonal was identified in T cells in patients with allergic rhinitis after 1 year treatment.

CONCLUSIONSThere was difference in the expression levels of the TCR Vγ I-III subfamily genes and distribution and clonality of TCR Vγ and Vδ 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 γδ T cells, especially the TCR Vδ6 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

BACKGROUNDWe distinguished graft-versus-host disease (GVHD) from graft-versus-leukemia (GVL) effects and to investigate the distribution of T-cell receptor (TCR) V beta gene repertoire in individuals with leukemia before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT).

METHODSPeripheral blood mononuclear cells (PBMC) were obtained from 10 normal individuals, 8 donors and 11 patients with leukemia before and after transplantation. Polymerase chain reaction (PCR) amplification of complementarity-determining region 3 (CDR3) of 24 TCR V beta genes was used to examine serial samples of PBMC. The PCR products were further analyzed by genescan to evaluate clonality of T cells.

RESULTSThe 24 TCR V beta gene repertoire displayed highly diverse and polyclonal spectratypes in all normal individuals and 4 of 8 donors. Another 4 donors expressed part of the 24 TCR V beta subfamily and 1 donor had oligoclonality. The expressions of the 24 TCR V beta subfamilies were skewed and restricted in 11 leukemia patients before and after transplantation. Some absences of 24 TCR V beta subfamily expression were quite similar between the recipients pro-transplantation and related donors. The number of subfamilies expressed increased over time post-transplantation, but the restricted expressions of the subfamily could last 6 - 30 months after transplantation. All patients with GVHD and some without GVHD exhibited T cell clonal expansion. The expansive T cell clone was distributed in V beta 2-3, 16-17, 18-19, 21 and V beta 23 in patients with GVHD and in V beta 7, 9, 16 and 19 in patients without GVHD. One patient with syngeneic-HSCT (syn-HSCT) had V beta 15 and 16 T cell expansion after transplantation. One patient displayed V beta 18 T cell expansion after donor lymphocyte infusion (DLI).

CONCLUSIONSNormal individuals express the entire 24 TCR V beta gene repertoire and have polyclonal distribution. However, the TCR V beta gene repertoire is only partially expressed in some donors. The TCR V beta gene repertoire is restrictedly expressed in a skew fashion in patients with leukemia before and after transplantation. The number of TCR V beta gene subfamilies increases over time post-transplantation. GVHD and GVL effects may induce the proliferation of T cell clones. Clinical GVL response may be distinguished from GVHD alloreactivity through the host MHC antigen.

Graft vs Host Disease ; genetics ; Graft vs Leukemia Effect ; genetics ; Hematopoietic Stem Cell Transplantation ; Humans ; Leukemia ; genetics ; therapy ; Polymerase Chain Reaction ; Receptors, Antigen, T-Cell, alpha-beta ; genetics

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 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

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;, 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 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

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-&alpha;/&beta; 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