Dominant T-cell receptor beta-chain gene rearrangements indicate clonal expansion in the rheumatoid joint

T-cell receptor (TcR) beta and delta gene rearrangements were studied in anti-CD3 expanded T-cell populations cultured from the synovial membrane (SM) (n = 5) or synovial fluid (SF) (n = 2) of rheumatoid arthritis (RA) patients. Dominant TcR beta-chain gene rearrangements to C beta 1 were demonstrated in all the patients tested and 1-3 expanded clones per patient were found. Clonal rearrangements to C beta 2 were detected in one SM sample (two clones) and one SF sample (one clone). The TcR delta gene was deleted in all the samples tested. We conclude that clonal dominance may be found in expanded T-cell populations from SM and SF of RA patients. Multiple clones may be present, either using the C beta 1 or C beta 2 gene segment.     

Role of CD4 molecule in intrathymic T-cell development.

We have investigated the role of CD4 molecules in intrathymic T-cell repertoire selection. The administration of monoclonal antibody (mAb) to CD4 in organ culture of murine foetal thymus (FTOC) completely inhibited the development of CD4+8- cells, and additional treatment with anti-class II MHC (Ia) mAb caused no further effects on this inhibition. On the other hand, when the potentially autoreactive cells in Mls-1a mice were monitored by expression of the Mls-1a-reactive V beta 6 gene product of T-cell receptor alpha beta (TcR alpha beta), the treatment with anti-CD4 resulted in the appearance of V beta 6-bearing cells to some extent, but this effect was considerably reinforced by the combinatory use of anti-Ia mAb with anti-CD4. In a model system where the bacterial superantigen staphylococcal enterotoxin B serves as self-antigen to deplete V beta 8-bearing cells in FTOC, the depletion of V beta 8+ cells was restored partially by anti-CD4 alone but completely by the combination with anti-Ia. These results suggest that CD4 is indispensable for positive selection of all CD4+8- thymocytes, whereas participation of CD4 in negative selection is only partial. It was also observed that the development of TcR alpha beta-bearing cells in the CD4-8- population was inhibited by the treatment with anti-CD4 mAb. In Mls-1a mice, V beta 6-bearing cells were developed in CD4-8+, CD4+8+, and also in CD4-8- populations after anti-CD4 mAb treatment. It is suggested that TcR alpha beta-bearing CD4-8- cells are possibly originated from CD4+ cells and undergo CD4-mediated thymic selection.     

Analysis of T-cell receptor delta chain gene in hematological malignancies

We analyzed the rearrangement of TcR delta chain gene in 179 cases of hematological malignancies. In 17 T-cell lines, RPMI 8402, DND41, Peer, and Molt 13 had delta rearranged band (s). Except for RPMI 8402, these cell lines expressed functional delta gene. All of those gamma delta-T-cell lines had short message (1 kb) of TcR beta gene. These findings suggest differences between alpha beta-T-cells and gamma delta-T-cells. All 9 cases of T-ALL/LBL, of which 4 had neither gamma nor beta gene rearrangement, had a new rearranged band of TcR delta locus. This rearrangement was observed in 63% of B-lineage ALL/LBL. In the other T-lymphoproliferative disorders, only 2 cases of AILD and 1 of T-cell lymphoma had the rearranged band (s), showing derived T-cell neoplasm from gamma delta-T-cell as minority. In B-leukemia/lymphoma and myelocytic leukemia, 15% of the cases had the delta rearrangement. Heterogenous findings of TcR delta locus analysis were observed in ATLL without proviral HTLV-I DNA, T-cell lymphoma, AILD and HD. The J delta 1 region was frequently used and the J delta 2 region was rearranged in one AILD. It is suspected that J delta 3 was used in one T-ALL/LBL. There was no correlation between the phenotypic pattern of CD3, CD4, and CD8 in T cell disorders and the rearrangement of the TcR delta gene. These findings suggest that the newly identified TcR delta chain gene rearranges at a very early stage of T cell ontogeny; prior to the other TcR genes and perhaps at almost the same differentiation level as that of CD7 expression. The TcR delta gene is useful in evaluating clonality for the most immature T cell neoplasms not showing rearrangement of the other TcR genes. This gene is not lineage specific, however, when used in conjunction with IgHC gene, it may be a useful tool for the study of ALL/LBL.     

Use of a murine T-cell hybridoma expressing human T-cell receptor alpha- and beta-gene products as a tool for the production of human T-cell receptor-specific monoclonal antibodies.

We describe the production of mouse monoclonal antibodies specific for the human TcR using as the immunogen transfected murine T-cell hybridoma cells coexpressing mouse CD3 with human Jurkat TcR alpha and beta chains. The shortage of monoclonal antibodies (mAbs) specific for the human TcR-V alpha and V beta families reflects the difficulties in their production by conventional methods using whole human T cells or purified soluble receptors as immunogens. As an alternative strategy to circumvent these difficulties, we have generated a transfected mouse T-cell line expressing a human (Jurkat) TcR alpha beta dimer in a complex with mouse CD3. The parental mouse T-cell line, TG40, is a cell surface TcR-negative, cytoplasmic CD3-positive variant of the mouse T-cell hybridoma 2B4. The human-TcR alpha beta expressing mouse transfectant was used to immunize mice with the same genetic background as the parent mouse T-cell line, and a human TcR-specific response was successfully achieved. MAb-producing hybridomas were generated by fusing spleen cells from the immunized mice with the mouse myeloma cell line NSO. Of 124 hybridoma supernatants screens, 72 showed reactivity to the human T-cell line Jurkat. Twenty-four of the hybridomas producing human (Jurkat) TcR-specific antibodies were cloned and screened for reactivity to Jurkat TcR. Several IgG2b and IgM mAbs specific for the Jurkat T cell line were selected on the basis of their ability to modulate surface CD3 expression on Jurkat cells. Most of the antibodies do not stain other TcR-expressing human T cell leukemia cell lines, implying specificity for the variable domains of the Jurkat TcR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Demonstration of clonality in T-cell lymphoma using an anti-T-cell receptor variable region antibody panel

Frozen sections from 35 T-cell lymphomas were stained with the Diversi-T alpha beta T-Cell Receptor panel which includes seven antibodies to T-cell receptor variable region gene products. In five cases a monoclonal population of T-cells could be demonstrated (one case V beta 5+, three cases V beta 8+ and one case V beta 6+) and in a further case a biclonal population (V beta 5+ and V beta 8+). We conclude that this antibody panel is of limited usefulness for the demonstration of clonality in T-cell lymphoma.     

T-cell receptor gene usage and expression in renal allograft-derived T-cell lines

Southern blot analyses indicate that the T-cell receptors of alloreactive T-cell lines derived from needle biopsies of human kidney allografts are selected based on beta-chain usage. In order to examine this selection at the level of T-cell-receptor expression, we have generated monoclonal antibodies directed toward the T-cell-receptors of three allograft-derived T-cell lines, MH3, WP3, and EH3. Monoclonal antibodies have been isolated which appear to react specifically with each of these three T-cell lines. One anti-MH3 antibody precipitates a molecule from the surface of MH3 cells that comigrates with the alpha/beta TcR on a polyacrylamide gel. Ten WP3-reactive monoclonal antibodies were identified which cause a modulation of CD3 from the surface of WP3 T cells, although none as yet precipitates a molecule from lysates of surface 125I-labeled WP3 cells. Since Northern blot analysis of EH3 RNA has revealed that a member of the V beta 6 gene family is expressed by this T-cell line, we are attempting to identify a monoclonal antibody reactive with this V beta 6 gene product.     

Cutaneous T-cell lymphoma cells employ a restricted range of T-cell antigen receptor variable region genes.

Monoclonal antibodies that recognize the proteins specified by two families of T-cell antigen receptor variable region genes were assessed as clonal markers in cutaneous T-cell lymphoma (mycosis fungoides and Sezary syndrome). About 3% to 5% of tumors were expected to react with each antibody. However, 10 of 16 cases studied reacted with MAbs specific for the V beta 8 gene family. All the positive cases were examples of the plaque or tumor stages of CTCL. None of the cases of eczematoid or premycotic CTCL showed uniform reactivity with these antibodies. Unexpected use of one particular V gene family raises the possibility that CTCL derives from a distinct subpopulation of antigen- or virus-selected T cells.     

Effects of cyclosporin A on T-cell development in organ-cultured foetal thymus.

The effects of cyclosporin A (CsA) on T-cell development were assessed in an organ culture of murine foetal thymus. Applying three-colour flow cytometric analysis, we showed that the agent inhibits the development of mature CD3/T-cell receptor alpha beta (TcR alpha beta)+ cells both in CD4+8- and CD4-8+ populations. CD4-8- cells appeared to be accumulated by CsA. We examined the heterogeneity of CD4-8- cells generated in the organ culture, and defined five subpopulations by the expression of the cell-surface molecules CD3/TcR, J11d and CD25. It has been demonstrated that only the CD3/TcR alpha beta+ J11d- CD25- subpopulation is susceptible to the suppressive effects of CsA among CD4-8- cells, whereas all the other four subpopulations, including CD3/TcR gamma delta+ cells, are resistant. Thus, all of the TcR alpha beta-bearing cells, including CD4-8- cells but none of the TcR alpha beta- cells, are CsA sensitive. Because it is known that CsA inhibits the TcR-mediated signalling events in mature T cells and that signallings mediated via the interaction of TcR with major histocompatibility complex (MHC) molecules on thymic stroma cells are crucial for thymic selection of T cells, these results indicate that TcR alpha beta-bearing CD4-8- cells but not TcR gamma delta-bearing CD4-8- cells undergo thymic positive selection.     

Simultaneous occurrence of peripheral T-cell lymphoma unspecified and B-cell small lymphocytic lymphoma. Report of 2 cases

We report on 2 composite lymphomas occurring in elderly patients, morphologically characterized by the combination of peripheral T-cell lymphoma (PTCL) unspecified and B-cell small lymphocytic lymphoma. Immunohistochemistry provided objective confirmation of the coexistence of the 2 malignancies, as did molecular biology by revealing clonal T-cell receptor gamma and immunoglobulin heavy chain gene rearrangements. One of the patients had no history of indolent lymphoma either at the personal and family level, whereas the other showed a strong familial predisposition, his mother and sister having suffered from B-cell chronic lymphocytic leukemia. Epstein-Barr virus was detected in the PTCL component of 1 case, but was negative in the other. To the best of our knowledge, the simultaneous occurrence of PTCL unspecified and B-cell small lymphocytic lymphoma is an exceptional event; the possible pathogenetic correlations between the 2 neoplasms are discussed.     

Immunophenotyping and gene rearrangement analysis provide additional criteria to differentiate between cutaneous T-cell lymphomas and pseudo-T-cell lymphomas.

Differentiation between cutaneous pseudo-T-cell lymphomas and cutaneous T-cell lymphomas (CTCLs) may be extremely difficult. In this study, it was investigated whether demonstration of an aberrant phenotype and detection of clonal T-cell receptor gamma (TCR gamma) gene rearrangements can be used as additional differential diagnostic criteria. Immunohistochemical studies and TCR gamma gene rearrangement analysis using a polymerase chain reaction with primers specific for V gamma 1-8 and V gamma 9 gene segments in combination with denaturing gradient gel electrophoresis (PCR/ DGGE) were performed on frozen material of 11 pseudo-T-cell lymphomas and 17 CTCLs, including 9 cases of mycosis fungoides (MF) and 8 pleomorphic CTCLs. Clonal TCR gamma gene rearrangements were found in 66% of patch/plaque-stage MF and 100% of tumor-stage MF and pleomorphic CTCL, but not in any of 10 pseudo-T-cell lymphomas studied. Aberrant expression of CD2, CD3, and/or CD5 antigens was noted in 3 of 6 (50%) cases of patch/plaque-stage MF, all three cases of tumor-stage MF, and 5 of 8 (62%) pleomorphic CTCLs, but not in any of the 11 pseudo-T-cell lymphomas. Moreover, in pseudo-T-cell lymphomas exhibiting a nodular or diffuse growth pattern, a considerable admixture with reactive CD8+ T cells (15 to 60%), B cells (up to 20%), and macrophages was a characteristic finding. In conclusion, the results of this study suggest that demonstration of clonal TCR gene rearrangements and an aberrant phenotype, as well as demonstration of many admixed CD8+ T cells and B cells can be considered as useful additional criteria in the differentiation between pseudo-T-cell lymphomas and CTCLs.     

Phenotypic heterogeneity of intraepithelial T lymphocytes from mouse small intestine.

We have used two-colour flow cytometry to examine the heterogeneity of intraepithelial lymphocytes (IEL) from mouse small intestine. We have confirmed the predominance of CD3+ Thy 1- CD8+ IEL and show that a substantial but variable proportion of CD8+ IEL does not express the alpha beta T-cell receptor (TcR) for antigen. Simultaneous analysis of the co-expression of the alpha and beta chains of the CD8 heterodimer and of the alpha beta TcR revealed three populations of CD8+IEL. The first of these expressed both CD8 alpha and beta chains and had normal expression of V beta families and so represented conventional CD8+ alpha beta TcR+ T cells. The second population comprised alpha beta TcR- T cells (presumed gamma delta TcR+) which expressed only the alpha chain of the CD8 molecule. Finally, we identified a second, unique population of alpha beta TcR+ CD8+ IEL which were also CD8 beta-. Gamma delta + IEL predominated in mice aged less than 8 weeks, but there was a rapid increase in both populations of alpha beta TcR+ CD8+ IEL in older mice. CD8+ IEL were similar to peripheral CD8+ T cells in having high expression of the CD45RB molecule, but CD4+ IEL had generally lower expression of CD45RB than their peripheral counterparts, despite having normal expression of TcR. These findings emphasize the heterogeneity of IEL and underline the need to study phenotypically defined populations.     

Peripheral T-cell lymphoma with a nodular growth pattern

Peripheral T-cell lymphomas (PTCL) with nodular growth patterns are very rare, with only 17 cases reported previously. Here, we report a case of PTCL with a nodular growth pattern. The patient was an 81-year-old Japanese woman who complained of malaise, fever and generalized lymph node swelling. Cervical lymph node biopsy was performed, and histological examination revealed proliferation of medium- to large-sized atypical lymphoid cells with indented to irregular nuclei, distinct nucleoli and clear cytoplasm. The nodular growth pattern of the lymphoma cells was obvious. On immunohistochemistry, the atypical lymphoid cells proved to be of T-helper cell origin (CD2+CD3CD4+CD5+CD7+ CD8-CD10-CD25-CD30-CD57-). Polymerase chain reaction analysis of the T-cell receptor gamma-chain revealed a monoclonal rearrangement band. This unusual growth pattern should be distinguished among PTCL, as such cases could be confused with reactive nodular hyperplasia, nodular lymphoma, mantle cell lymphoma and marginal zone lymphoma with nodular colonization.     

Differential expression and regulation of the human CD8α and CD8β chains

The CD8 glycoprotein is expressed by thymocytes, mature T cells and natural killer (NK) cells and has been implicated in the recognition of monomorphic determinants on major histocompatibility complex (MHC) Class I antigens, and in signal transduction during the course of T-cell activation. Both human and rodent CD8 antigens are comprised of two distinct polypeptide chains, alpha and beta. The majority of monoclonal antibodies (mAb) reactive with the human CD8 antigen bind the CD8 alpha chain, while a single mAb, T8/2T8-5H7, has been identified which binds to the CD8 alpha/beta heterodimer. While the two chains of CD8 have been presumed to be coordinately expressed in normal T cells, this is not always the case. Northern blot analysis of a panel of T-cell leukemias and normal cells demonstrate that CD8 alpha and CD8 beta are not invariably co-transcribed and phenotypic analysis of fresh and interleukin 2 (IL-2) expanded peripheral blood mononuclear cells (PBMC) confirm that the CD8 alpha and CD8 beta chains are differentially expressed at the cell surface. Four distinct subpopulations of CD8+ cells have been identified based on the expression of CD8 alpha/alpha or CD8 alpha/beta complexes: (1) T-cell receptor (TcR) alpha beta+ T cells which are CD8 alpha+/beta+; (2) TcR alpha beta+ T cells which are CD8 alpha+/beta-; (3) TcR gamma delta+ T cells which are CD8 alpha+/beta- and (4) natural killer (NK) cells which are CD8 alpha+/beta-. We also demonstrate the down-regulation of the CD8 alpha/beta heterodimers from the surface of a CD8+ T-cell clone following treatment with phorbol myristate acetate (PMA) while CD8 alpha/alpha homodimers remain on the cell surface. This observation demonstrates that a) a CD8+ T-cell clone can express both CD8 alpha/alpha homodimers and CD8 alpha/beta heterodimers and b) these two complexes do not have identical biological properties. Together, these data suggest that CD8 alpha/alpha and CD8 alpha/beta dimers may not subserve identical functions. The differential contribution of these two CD8 complexes should be considered in models of T-cell-mediated cytotoxicity and T-cell activation.     

Clonal gene rearrangement patterns correlate with immunophenotype and clinical parameters in patients with angioimmunoblastic lymphadenopathy.

T cell receptor beta (TcR beta) chain gene rearrangements have been reported in cases of angioimmunoblastic lymphadenopathy (AILD) and provided evidence for the presence of clonal T cell proliferations in this disorder. Twenty-three cases of AILD and two cases of hyperimmune reaction (HR) were investigated. In the two HR cases, essentially the same histologic pattern was present as in AILD but lymph node follicles were hyperplastic. Both HR cases showed germline configuration for the TcR and immunoglobulin heavy chain (IgH) genes. All other patients diagnosed with AILD had clonal rearrangements for TcR gamma and beta chain genes. In addition, seven out of these cases had clonally rearranged their IgH genes. These two different rearrangement patterns (TcR with or without Ig gene rearrangement) correlated to immunohistochemical and clinical data. Cases with TcR but without Ig gene rearrangements (group I) exclusively showed CD4+ proliferating T cells, whereas those cases with TcR and Ig gene rearrangements had significantly elevated numbers of CD8+ proliferating cells (group II). Group II patients significantly more often presented with hemolytic anemia and went into transient remission spontaneously or under steroid treatment. Group I patients, however, had a higher response to chemotherapy and a longer survival time. These data show that, based on different rearrangement patterns, it is possible to divide AILD into two different groups with distinct immunophenotypic properties and differences in clinical parameters. Immunogenotyping in AILD thus will have prognostic and therapeutic implications.     

Phenotypical and Functional Characterization of Small Intestinal TcRγδ+ T Cells in Coeliac Disease

Increased numbers of TcR gamma delta + T cells are present in the small intestinal epithelium of patients with coeliac disease (CoD). Their function, however, is unknown. In order to facilitate detailed functional studies, intestinal gamma delta T cells have been isolated from small intestinal biopsies of patients with CoD (n = 18) and controls (n = 14). As expected, increased numbers of V delta 1+ TcR gamma delta + T cells were detected in freshly isolated intraepithelial cell suspensions (IEL) from CoD patients. Also, in the in vitro expanded IEL T-cell populations from CoD patients the numbers of V delta 1+ TcR gamma delta + T cells were increased compared with similar cell cultures from control patients. From IEL cultures derived from six CoD patients, 107 T-cell clones were generated by limiting dilution and analysed. Sixty of these clones were either CD4 or CD8 positive TcR alpha beta + clones. The remaining 47 clones expressed the TcR gamma delta. Further phenotypical analysis of the gamma delta T-cell clones indicated that the TcR gamma delta + T-cell population in the small intestinal epithelium of CoD patients is heterogeneous: four TcR gamma delta phenotypes could be detected and, although the majority of the TcR gamma delta + T cells were CD4 CD8, gamma delta T-cell clones expressing either a CD8 alpha alpha homodimer, a CD8 alpha beta heterodimer or CD4 were also identified. In contrast to the TCR alpha beta + IEL, most TcR gamma delta + IEL were CD5 negative. Furthermore, biochemical analysis indicated that the increase in V delta 1+ gamma delta T cells in the small intestinal epithelium of CoD patients was not the result of a monoclonal expansion. The small intestinal epithelium-derived gamma delta T-cell clones were functional in vitro since the majority of these clones were able to lyse target cell lines such as K562. Molt4 and Daudi. These novel findings therefore indicate that the gamma delta T cells in the small intestine of CoD patients represent a heterogeneous population and that such cells are functional in vitro. The isolation and the in vitro propagation and cloning of these cells may open new avenues for the study of the putative immune mechanisms leading to coeliac disease.     

Analysis of T-cell subpopulations in T-cell non-Hodgkin's lymphoma of angioimmunoblastic lymphadenopathy with dysproteinemia type by single target gene amplification of T cell receptor- beta gene rearrangements

Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is defined in the current lymphoma classifications as a T-cell non-Hodgkin's lymphoma. However, in approximately one third of the cases of this lymphoproliferative disease rearrangements of T-cell receptor (TCR) genes indicating clonal expansion of T cells are not detectable. It is currently believed that these cases may represent early stages of a lymphoma with a minor oligoclonal T-cell population. In the present study, 18 lymph nodes with the characteristic histology of AILD were investigated for clonal T-cell receptor gene rearrangements by analysis of DNA extracted from whole tissue sections. Dominant T-cell clones were detected in 12 of these cases. Single CD4(+) and CD8(+) T cells and proliferating Ki67(+) cells of seven cases were micromanipulated from frozen tissue sections. TCRbeta gene rearrangements were amplified from these cells by polymerase chain reaction and sequenced. In all informative cases, the clonal gene rearrangements were only detected among CD4(+), and not among CD8(+) T cells, indicating that the tumor clones in AILD usually derive from CD4(+) T cells. Minor clonal T-cell populations in those cases in which no clone was found by whole-tissue DNA analysis were not detectable even at single cell resolution. T-cell clones in 4 of 10 cases were found to express similar TCRbeta chains, indicating a potential role of (super) antigen triggering in at least some cases of AILD.     

T-cell antigen receptor beta-chain variable region families: a study of their distribution in normal and reactive tissues

The beta chain of the T-cell antigen receptor is constructed using a variable region gene from one of approximately 20 variable gene families. Antibodies that react with the products of these gene families have been used to demonstrate clonal proliferation of T cells. Here, we describe the expression of two beta-chain variable region families in normal and reactive lymphoid tissues. In all the tissues studied, expression was scattered throughout the T-cell areas, without any clustering of expression.     

Profiling the repertoire of T-cell receptor beta-chain variable genes in peripheral blood lymphocytes from subjects who have recovered from acute hepatitis B virus infection

The profile of T-cell receptor beta-chain variable （TRBV） genes usually skews in subjects with virus infection or cancer. The gene melting spectral pattern （GMSP） can be used to determine the profile of the TRBV gene family. To explore the portrait of the TRBV family in peripheral blood lymphocytes from subjects who have recovered from acute hepatitis B virus infection （AHI）, peripheral blood mononuclear cells （PBMCs） were separated and further sorted into CD4^＋ and CD8^＋ T-cell subsets. The molecular features of the TRBV complementary determining region 3 （CDR3） motifs were determined using GMSP analysis. When a GMSP profile showed a single peak, the monoclonally expanded TRBV gene was cloned and sequenced. Skewed expansions of multiple TRBV genes were observed among the CD4^＋ and CD8^＋ T-cell subsets and the PBMCs. The frequency of monoclonally expanded TRBV genes in the CD8^＋ T-cell subset was significantly higher than that of the CD4^＋ T-cell subset and the PBMCs. Compared to other members of the TRBV gene family, TRBV11, BV15 and BV20 were predominantly expressed in the repertoire of peripheral blood lymphocytes in recovered AHI subjects. The relatively conserved amino acid motifs of TRBV5.1 and BV20 CDR3 were also detected in the CD4^＋ and CD8^＋ T-cell subsets. These results demonstrate the presence of multiple biased TRBV families in recovered AHI subjects. TRBV11, BV15 and BV20, especially from the CD8＋ T-cell subset, may be relevant to the pathogenesis of subjects with AHh The preferentially selected TRBV5.1 and BV20 with the relatively conserved CDR3 motif may be potential targets for personalized treatments of chronic HBV infection.