BACKGROUND: The clonality of lymphoid infiltrates determined by polymerase chain reaction (PCR) for immunoglobulin heavy chain (IgH) or T cell receptor (TCR) genes is not only useful in confirming the diagnosis of malignant lymphoma but also in establishing the lineage of a clonal lymphoid proliferation. We analyzed the efficiency of PCR analyses for IgH and TCRgenes that have been routinely applied for the diagnosis of malignant lymphoma in our laboratory. METHODS: Paraffin sections of 200 cases were analyzed by seminested PCR. Primers were FRIIIA-LJH/VLJH consensus primer for IgH gene and V-J consensus primer for TCR gene. The cases showing negative results by PCR for TCR gene were further analyzed by multiplex V family primers with heteroduplex analysis. RESULTS: PCR approach for IgH gene allowed detection of clonality in 100% of cases with false positive rate of 0.3% and false negative rate of 0%. The combination of PCR for TCR consensus primers with multiplex V family primers allowed detection of clonality in 91% of cases with false positive rate of 0.6% and false negative rate of 10.3%. CONCLUSIONS:Combined analysis of IgH and TCR gene rearragnements by the PCR technique followed by heteroduplex analysis can be a useful diagnostic adjunct to determine the clonality of various lymphoproliferative diseases with high sensitivity. But clinical, morphological and immunophenotypical correlation should be considered to reach the final diagnosis due to a few false positive cases.
Consensus ; Diagnosis ; Gene Rearrangement* ; Genes, T-Cell Receptor ; Heteroduplex Analysis ; Humans ; Immunoglobulin Heavy Chains* ; Immunoglobulins* ; Lymphoma ; Paraffin ; Polymerase Chain Reaction ; Receptors, Antigen, T-Cell* ; T-Lymphocytes*

BACKGROUND: Recently, the molecular pathologic investigation for clonality in lymphomas has been introduced and has gained a role in the diagnosis of lymphomas. In fact, the clonality test using TCRGR phenomenon has been done by Southern blot analysis (SBA) and polymerase chain reaction (PCR) for molecular pathologic diagnosis of T cell lymphomas. However, it is difficult to perform SBA with paraffin embedded specimens or with samples of small skin biopsies. OBJECTIVE: We investigated the efficacy of PCR amplification of TCR gene in paraffin em-bedded cutaneous T cell lymphomas. METHODS: Iii this study, the clonality was assessed by polymerase chain reaction (PCR) analysis of T cell receptor gamma (TCR) gene from the DNA extracts obtained from paraffin em-bedded tissues (PET) of malignant T cells, B cell lymphomas, and benign cutaneous T cell proliferative disorders. Heteroduple-x-analyses were also performed to rule out the false positives. RESULTS: Among the total of 62 cases analyzed, monoclonality was observed in 4 out of 10 mycosis fungoides, 7 out of 9 cutaneous T cell lymphomas excluding mycosis fungoides, 1 out of 3 angiocentric lymphomas, 2 out of 2 lymphomatosis papulosis, 1 out of 7 large plaque parapsoriasis, and 1 out of 2 T cell lymphomas in other organs. No monoclonality was observed in 9 inflammatory cutaneous diseases, 5 small plaque parapsoriasis, 4 cutaneous B cell lymphomas, and 11 B cell lymphomas in lymph nodes. CONCLUSION: The results suggest that the PCR method and heteroduplex analysis used in this study were not only practical but also efficacious for the diagnosis of cutaneous T cell lymphomas using tissues embedded in paraffins.
Biopsy ; Blotting, Southern ; Diagnosis ; DNA ; Gene Rearrangement* ; Genes, T-Cell Receptor ; Heteroduplex Analysis* ; Lymph Nodes ; Lymphoma ; Lymphoma, B-Cell ; Lymphoma, T-Cell ; Lymphoma, T-Cell, Cutaneous ; Mycosis Fungoides ; Paraffin ; Parapsoriasis ; Polymerase Chain Reaction* ; Receptors, Antigen, T-Cell* ; Skin ; T-Lymphocytes

It is often problematic to diagnose T-cell lymphoproliferative disorders of the skin because of the difficulty in establishing clonality in paraffin-embedded tissue. We used polymerase chain reaction single strand conformational polymorphism (PCR-SSCP) and heteroduplex analysis in paraffin embedded tissue to detect clonal rearrangement of T-cell receptor gamma (TCRgamma) gene in 17 T-cell lymphoproliferative disorders and 6 atypical lymphoproliferative diseases. We used polymerase chain reaction to detect TCR beta gene rearrangement in 8 of 17 cases which did not show TCRgamma gene rearrangement. Jurkat cell lines were used as monoclonal controls. DNA was extracted from 5 biopsies of T-cell lymphomas, 10 biopsies of mycosis fungoides, 2 biopsies of lymphomatoid papulosis, and 6 biopsies of atypical lymphoproliferative lesions. We detected monoclonality in 5 of 5 T-cell lymphoma cases, 2 of 2 lymphomatoid papulosis cases, 6 of 10 mycosis fungoides cases, and 2 of 6 atypical lymphoproliferative disease cases. We conclude that nonradioactive PCR-SSCP for TCR gene rearrangement analysis is a useful adjunct to routine histological and immunophenotypic methods in the diagnosis of cutaneous T cell lymphoproliferative disorders in paraffin embedded tissue.
Biopsy ; Diagnosis* ; DNA ; Gene Rearrangement ; Genes, T-Cell Receptor ; Genes, T-Cell Receptor beta ; Heteroduplex Analysis ; Humans ; Jurkat Cells ; Lymphoma, T-Cell ; Lymphomatoid Papulosis ; Lymphoproliferative Disorders ; Mycosis Fungoides ; Paraffin ; Polymerase Chain Reaction ; Receptors, Antigen, T-Cell ; Skin ; T-Lymphocytes

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

The purpose of this study was to set up an approach for expansion of the peripheral blood gammadeltaT cells from normal subjects in order to explore the characteristics of gammadeltaT cells. Peripheral blood mononuclear cells (PBMNC) were separated from 5 - 10 ml peripheral blood and stimulated by the low molecular peptide derived from Mycobacterium tuberculosis (MTb-Ag), and expanded in rIL-2-containing medium. The relative amount of gammadeltaT cells were measured by anti TCR gammadelta-PE staining and flow cytometry. The Cytotoxicity were detected by gammadeltaT assay. The results showed that after stimulation and expansion for 10 days, gammadeltaT cells increased to 69.2% of the total PBMNC and demonstrated significant cytotoxicity against K562 cells. In conclusion, this is a simple, rapid and specific method for expansion of peripheral blood gammadeltaT cells in vitro.
Antigens, Bacterial ; immunology ; Cell Separation ; methods ; Flow Cytometry ; Humans ; Mycobacterium tuberculosis ; immunology ; Receptors, Antigen, T-Cell, gamma-delta ; analysis ; T-Lymphocyte Subsets ; cytology

Recent studies in man and animal models have demonstrated that TCR-gamma delta-bearing T cells (gamma delta T cells) are activated by mycobacteria and accumulate in the sites of mycobacterial infection. Although the function of gamma delta T cells remains unclear, some data suggest a potential role for these cells in the granulomatous immune response. To address the presence of gamma delta T cells within the BCG granulomas, we have characterized the TCR phenotype of T-lymphocytes present in the BCG granulomatous lesion immunohistochemically using a monoclonal antibody to TCR delta 1 and others. Fairly large numbers of gamma delta T cells were located at the periphery of the BCG granulomas without necrosis and most of them also expressed CD8. However, gamma delta T cells were rarely present in the granulomas with central caseous necrosis, calcification and fibrotic changes. With these results, it might be speculated that the CD8+ gamma delta T lymphocytes participate in the BCG granuloma formation mainly in the early stage.
Female ; Granuloma/immunology/*pathology ; Human ; Infant ; Lymph Nodes/pathology ; Male ; *Mycobacterium bovis ; Receptors, Antigen, T-Cell, gamma-delta/*analysis ; T-Lymphocytes/*immunology ; Tuberculosis/immunology/*pathology

OBJECTIVETo investigate the percentages of peripheral blood γδ T cells and regulatory T cells (Treg) and the expression of associated cytokines, interleukin 17 (IL-17) and transforming growth factor-β1 (TGF-β1), in infants with human cytomegalovirus (HCMV) infection.

METHODSTwenty-two infants with HCMV infection (HCMV group) and 22 healthy infants who underwent physical examination (control group) were enrolled in this study. The percentages of peripheral blood γδ T cells and Treg cells were determined by flow cytometry. The levels of IL-17 and TGF-β1 in plasma were measured using ELISA.

RESULTSCompared with the control group, the HCMV group had significantly higher percentage of γδ T cells and IL-17 level (P<0.01) and significantly lower percentage of Treg cells and TGF-β1 level (P<0.01). In the HCMV group, the percentage of γδ T cells was negatively correlated with the percentage of Treg cells and TGF-β1 level (P<0.05), but positively correlated with IL-17 level (P<0.05); the percentage of Treg cells was positively correlated with TGF-β1 level (P<0.05), but negatively correlated with IL-17 level (P<0.05); there was no correlation between IL-17 level and TGF-β1 level (P>0.05).

CONCLUSIONSThere is an imbalance between γδ T cells and Treg cells in the peripheral blood of infants with HCMV infection, and γδ T cells may be involved in the secretion of IL-17.

Cytokines ; blood ; Cytomegalovirus Infections ; immunology ; Female ; Humans ; Infant ; Interleukin-17 ; blood ; Male ; Receptors, Antigen, T-Cell, gamma-delta ; analysis ; T-Lymphocytes, Regulatory ; immunology ; Transforming Growth Factor beta1 ; blood

Hepatosplenic gammadelta T cell lymphoma represents rare, often aggressive type of malignant peripheral T-cell lymphoma, which is characterized by expressing T-cell-associated markers CD2, CD3 and gammadelta T-cell receptor, and nonactivated cytotoxic cell phenotype (TIA-1+, granzyme B-). The pathological findings of a liver biopsy specimen revealed the diffuse infiltration of lymphocytes in the sinusoids and the aspiration biopsy from spleen revealed the diffuse infiltration of lymphocytes in the red pulp, not shaped to the nodes, often resulted in the misdiagnosis. Recently, by analyzing the immunophenotype and TCR rearrangement from liver, spleen and bone marrow, a case of adult hepatosplenic gammadelta T cell lymphoma was diagnosed. In combination with references, It is belived that immunophenotype and TCR rearrangement are necessary means to diagnosis hepatosplenic gammadelta T cell lymphoma.
Adult ; Antigens, CD20 ; metabolism ; CD2 Antigens ; analysis ; CD3 Complex ; metabolism ; Humans ; Immunohistochemistry ; Ki-1 Antigen ; metabolism ; Liver Neoplasms ; metabolism ; pathology ; Lymphoma, T-Cell ; metabolism ; pathology ; Male ; Receptors, Antigen, T-Cell, gamma-delta ; metabolism ; Splenic Neoplasms ; metabolism ; pathology

Aneuploidy ; DNA, Neoplasm ; analysis ; genetics ; Flow Cytometry ; methods ; Humans ; Lymphoma ; diagnosis ; genetics ; immunology ; Lymphoma, B-Cell ; diagnosis ; genetics ; immunology ; Lymphoma, T-Cell ; diagnosis ; genetics ; immunology ; Receptors, Antigen, T-Cell ; analysis ; genetics

BACKGROUND: Current management strategies attempt to diagnose rheumatoid arthritis (RA) at an early stage. Transcription profiling is applied in the search for biomarkers for detecting early-stage disease. Even though gene profiling has been reported using several animal models of RA, most studies were performed after the development of active arthritis, and conducted only on the peripheral blood and joint. Therefore, we investigated gene expression during the initial phase of collagen-induced arthritis (CIA) before the arthritic features developed in the thymus in addition to the peripheral blood and synovium. METHODS: For gene expression analysis using cDNA microarray technology, samples of thymus, blood, and synovium were collected from CIA, rats immunized only with type II collagen (Cll), rats immunized only with adjuvant, and unimmunized rats on days 4 and 9 after the first immunization. Arrays were scanned with an Illumina bead array. RESULTS: Of the 21,910 genes in the array, 1,243 genes were differentially expressed at least 2-fold change in various organs of CIA compared to controls. Among the 1,243 genes, 8 encode T-cell receptors (TCRs), including CD3zeta, CD3delta, CD3epsilon, CD8alpha, and CD8beta genes, which were down-regulated in CIA. The synovium was the organ in which the genes were differentially expressed between CIA and control group, and no difference were found in the thymus and blood. Further, we determined that the differential expression was affected by adjuvant more than Cll. The differential expression of genes as revealed by real-time RT-PCR, was in agreement with the microarray data. CONCLUSION: This study provides evidence that the genes encoding TCRs including CD3zeta, CD3delta, CD3epsilon, CD8alpha, and CD8beta genes were down-regulated during the initial phase of CIA in the synovium of CIA. In addition, adjuvant played a greater role in the down-regulation of the CD3 complex compared to CII. Therefore, the down-regulation of TCR gene expression occurred dominantly by adjuvant could be involved in the pathogenesis of the early stage at CIA.
Animals ; Antigens, CD3 ; Arthritis ; Arthritis, Experimental ; Arthritis, Rheumatoid ; Biomarkers ; Collagen Type II ; Down-Regulation ; Gene Expression ; Genes, T-Cell Receptor ; Immunization ; Joints ; Models, Animal ; Oligonucleotide Array Sequence Analysis ; Rats ; Receptors, Antigen, T-Cell ; Synovial Membrane ; T-Lymphocytes ; Thymus Gland ; Transcriptome