T-cell Receptor Beta-chain Gene Research Articles

Evaluation of T Cell Receptor Testing in Lymphoid Neoplasms: Results of a Multicenter Study of 29 Extracted DNA and Paraffin-Embedded Samples

To evaluate current diagnostic methods used for the evaluation of T cell receptor (TCR) gene rearrangements, 24 different laboratories analyzed 29 lymphoid neoplasm samples of extracted DNA and paraffin-embedded tissue and were asked to complete a technical questionnaire related to the testing. Participating laboratories performed Southern blot and polymerase chain reaction (PCR) testing for rearrangements of the TCRbeta chain gene and PCR for the TCRgamma chain gene rearrangements. Of 14 laboratories performing TCRbeta Southern blot analysis, there was complete agreement in 10 of 14 cases, with some false negative results obtained in 4 cases. No false positive results were obtained by Southern blot analysis. TCRbeta PCR analysis was only performed by two laboratories, and only 47.1% of positive samples were detected. Twenty-one laboratory results were obtained for TCRgamma PCR. This method showed an overall detection rate of 77.9% for T cell gene rearrangements with a 4.1% false positive rate, as compared to both TCRgamma Southern blot analysis results and immunophenotyping. The detection rate for TCRgamma PCR, however, significantly differed when extracted DNA samples from frozen tissue were compared to paraffin-embedded tissue (85.4% versus 65.9%; P = 0.0005). Significant differences in true positive results were obtained when laboratories using primers directed against multiple TCRgamma variable regions (V1-8 plus one to three other primer sets) were compared to laboratories that used only a single set of TCR primers directed against the V1-8 (P < 0.0001). Other technical factors significantly affecting results were also identified. These findings provide useful data on the current state of diagnostic TCR testing, highlight the risk of false negative results for TCR testing directed against only portions of the TCRgamma gene, and identify limitations of testing of paraffin-embedded tissues in some laboratories.

Molecular analysis of T-cell clonality with concomitant specific T-cell proliferation in vitro in nickel-allergic individuals.

The peripheral blood mononuclear cells (PBMC) of individuals with nickel contact allergy are reported to proliferate to a varying degree upon nickel stimulation in vitro. Different phenotypes of the T cells involved are described. With regard to preferential use of the T-cell receptor (TCR), analysis of the several families of the TCR-gamma gene allows rearrangement evaluation of all T cells regardless of predominant surface expression of TCR alpha/beta. The PBMC of 10 nickel-allergic and five nonallergic individuals were cultured for 4 days in the presence of either medium, PHA, NiSO4, or tetanus toxoid (TT). Proliferation was measured by radioactive thymidine uptake and expressed as stimulation index (SI). T-cell clonality was assessed by analysis of the TCR-gamma chain gene, including the use of PCR with a primer combination covering the four main groups (Vgamma1-8, Vgamma9, Vgamma10, and Vgamma11) of the variable region of the TCR-beta chain gene. In the allergic individuals, proliferation to NiSO4 was significantly (P<0.05) higher than in nonallergics (mean SI: 18.05/17.87 vs 0.67/2.27). In unstimulated and PHA-stimulated cultures, there was a random TCR spectrum in both groups. In contrast, in nickel-allergic individuals or individuals with recent TT-booster, oligoclonality could be observed in the correspondingly stimulated cultures. In addition to proliferation assay, analysis of T-cell clonality may be a further means to characterize clinical hypersensitivity reactions on the basis of antigen-dependent oligoclonal T-cell expansion, as in the case of tissue-infiltrating lymphocytes.

Peripheral T-cell lymphoma complicated by a proliferation of large B cells.

We studied 14 cases that showed a morphologic appearance of peripheral T-cell lymphoma and contained substantial numbers of CD20+ large B cells. In all but 2 cases, the CD20+ large cells showed a mix of kappa and lambda light chain expression. Two cases showed a focal predominance of kappa expression. In situ hybridization using the EBER1 probe for detection of Epstein-Barr virus (EBV) RNA was performed on every case. EBV RNA was present in 10 cases. Of 8 cases with EBV RNA stained by immunohistochemistry for the latent membrane protein of EBV, 6 were positive. Double-labeling immunohistochemistry and in situ hybridization confirmed that EBV was present in the large B cells. Polymerase chain reaction (PCR) analysis showed a clonal rearrangement of the T-cell receptor (TCR)-gamma chain gene in 12 of 13 cases tested. One additional case showed a clonal rearrangement of the TCR-beta chain gene by Southern blot hybridization. PCR analysis showed a clonal immunoglobulin gene rearrangement in 5 cases, a suggestion of a clonal rearrangement in 1, an oligoclonal pattern in 4, and a polyclonal pattern in 4. The finding of large B and T cells may result in a misdiagnosis of a reactive process or of T-cell-rich B-cell lymphoma. The presence of EBV in some cases could cause further confusion with the reactive T- and B-immunoblastic proliferation of infectious mononucleosis.

Analysis of variable region of T-cell receptor beta chain usage in the human anti-porcine xenoresponse.

The human xenoreactive T-cell receptor (TCR) repertoire is not well documented. The aim of this study was to analyze the TCR repertoire in human anti-porcine xenoresponses. Peripheral blood lymphocytes (PBLs) from healthy donors were used to generate human T-cell lines against two different haplotypes of inbred Yucatan miniature swine (y/y and z/z). The variable region of TCR beta-chain (Vbeta) gene usage was determined by fluorescence CDR3 spectrotyping. TCR Vbeta usage of an established human antiporcine T-cell line analyzed at weeks 5, 7, and 9 showed a sequential increase in Vbeta 1, 2, 6.2, 11, and 19 as compared to unprimed peripheral blood lymphocytes, whereas the usage of other Vbetas decreased. The selection of limited Vbeta genes correlated with the sequential increase in the specific lysis of the T-cell line, suggesting a non-random clonal selection and expansion of T-cell clones that recognized porcine targets. Different Vbeta restriction was found using the same peripheral blood lymphocytes against a different haplotype of swine, indicating this selection of Vbeta gene was swine leukocyte antigen-dependent. There is restricted TCR Vbeta usage in the human anti-porcine response, suggesting that a limited number of xenogeneic epitopes are recognized by human T cells. The selection of particular TCR Vbeta clonotypes depends on the swine leukocyte antigen background.

Sequence and diversity of T-cell receptor beta-chain V and J genes of the owl monkey Aotus nancymaae.

The New World primate Aotus nancymaae is susceptible to infection with the human malaria parasite Plasmodium falciparum and has therefore been recommended by the World Health Organization as a model for the evaluation of malaria vaccine candidates. Recently, we have shown that Aotus TCRVA genes and TCRJA segments exhibit a high degree of similarity to human counterparts. In the present report we used reverse transcription polymerase chain reaction to analyze the sequences of A. nancymaae TCR beta-chain gene rearrangements. Alignment with human sequences and phylogenetic comparison identified 18 distinct Aotus TCRBV genes homologous to the human TCRBV gene families 2, 4, 5, 6, 7, 9, 12, 15, 24, and 28. Multiple Aotus genes were found in the TCRBV4, 5, 6, and 7 families. Some of these TCRBV genes aligned best to the same human gene and thus do not seem to have separate human homologues. Amino acid sequences of the Aotus TCRBV genes were 77 to 90% identical to their closest human counterparts. Ten distinct Aotus TCRBJ segments homologous to the human segments J1-1, J1-2, J1-4, J1-5, J1-6, J2-1, J2-2, J2-3, J2-4, J2-5 were found. In some cases the amino acid sequences of Aotus and human TCRBJ segments were completely identical. A comparison of the proportion of synonymous and non-synonymous substitutions in Aotus vs human beta-chain-encoding genes revealed a dominance of synonymous substitutions in TCRBJ segments and of nonsynonymous substitutions in TCRBV segments. Dominance of nonsynonymous substitutions was more pronounced in TCRBV CDR1 and CDR2 regions than in the framework regions. No evidence for the emergence of new TCRBJ segments or TCRBV families was found. These results confirm that the TCR repertoire in primates is remarkably stable and support the concept of using Aotus monkeys as an infection model for the evaluation of future subunit vaccine candidates.

Enhanced sensitivity with a novel TCR γ PCR assay for clonality studies in 569 formalin-fixed, paraffin-embedded (FFPE) cases

Clonal rearrangement of genes encoding the immunoglobulins (Ig) and T-cell antigen receptors (TCR) are considered to be useful markers for the diagnosis of lymphoma and for determining the clonal origins of B- and T-cell populations in lymphoid neoplasms. Polymerase chain reaction-based clonality assays for TCRgamma, TCRbeta, and immunoglobulin (Ig) heavy chain (IgH) gene rearrangements were evaluated for diagnostic sensitivity and specificity in 569 formalin-fixed, paraffin-embedded (FFPE) tissues. Combined TCRbeta and TCRgamma assays enhanced the routine detection of TCR clonality to 90% of all peripheral T-cell lymphoma (PTCL) cases. IgH clonality was detected in 59% of 241 peripheral B-cell lymphoma (BCL) cases and 6% of 169 PTCL cases. Of 452 lymphomas, 5% could not be classified phenotypically as B or T lineage after immunohistochemical and clonality studies. Of all BCL cases analyzed, 24% had detectable TCRbeta and/or TCRgamma clonality. Of these BCL with biclonal results, 47% were extranodal lymphomas from skin and various tissues. Clonality assays were useful for distinguishing reactive or benign lymph nodes from neoplastic lymphoid infiltrates in most cases. The inclusion of TCRb and TCRg assays in the assessment of lymphomas results in a significant increase in the sensitivity of clonality detection, but is of limited utility in assessing the T- or B-cell phenotype of the tumor.

Hodgkin's disease expressing follicular dendritic cell marker CD21 without any other B-cell marker: a clinicopathologic study of nine cases.

Reed-Sternberg (RS) and Hodgkin's (H) cells are considered to be the neoplastic cells in Hodgkin's disease (HD). Although most data suggest their lymphoid origin, the nature of these cells still remains a subject of controversy. Recently, a number of RS cells have been found to express an antigen that is also present on follicular dendritic cells (FDCs), asserting FDCs as the possible progenitor cells of H-RS cells. This prompted us to investigate whether these CD21-positive cases had distinct clinicopathologic characteristics. In a series of 94 examined cases of HD, we identified 9 CD21-positive ones (4 of 37 cases of nodular sclerosis, 1 of 41 mixed cellularity, and 4 of 12 lymphocyte depletion HD) without any other B-cell marker on paraffin sections. The patients varied in age from 16 to 82 years (median, 50 years) and included six men and three women. They had superficial or mesenteric lymphadenopathy without hepatosplenomegaly. Peripheral blood leukocytosis was seen in three patients. The clinical course was indolent, and all patients but one achieved an initial complete response with HD-based treatment regimens, although three of them relapsed. Morphologically, two subgroups could be delineated. Six of the cases were characterized, besides by the classic RS cells, by a varying number of the cells with the distinctive walnutlike or cerebrumlike nuclei and cytologically with cytoplasmic processes. Their fine structural examination also revealed villous processes, but no desmosomes. The other three cases had multinucleated RS cells often with triangular nuclei, but not cytoplasmic processes. The percentage of CD21-positive tumor cells ranged from less than 10% to 60% among the H-RS cells. These RS cells were positive for CD30 (9 of 9), CD15 (7 of 9), CD68 (1 of 8), fascin (8 of 8), S-100 protein (1 of 7), and epithelial membrane antigen (2 of 8) on paraffin sections. Notably, of eight cases examined on frozen sections, two showed immunostaining for DRC1, CD35, R4/23, and Ki-M4p. Only CD35 was also detected in the other two cases. Genotypic investigation showed germline configuration of the T-cell receptor beta and gamma chain genes and the immunoglobulin heavy chain gene in all eight cases examined. In situ hybridization showed Epstein-Barr virus sequences in four cases, three of which were examined by the terminal region analysis and showed the Epstein-Barr virus to be monoclonal. We concluded that in a small proportion (9.6%) of HD, H-RS cells might be derived from FDCs and that they appear to represent a distinct pathologic variant based on morphologic and phenotypic traits within the framework of HD.

Assignment1 of TCRB encoding the T-cell receptor beta chain gene to cat chromosome A2q25–q26 by fluorescence in situ hybridization

Assignment<footref rid="foot01">1</footref> of TCRB encoding the T-cell receptor beta chain gene to cat chromosome A2q25–q26 by fluorescence in situ hybridization

Improved polymerase chain reaction detection of clonally rearranged T-cell receptor beta chain genes.

A new method for the detection of all known possible rearrangements at the variable (V), diversity (D), and joining (J) segments of the T-cell receptor beta chain (TcR beta) gene in tissue DNA extracts is described that involves two polymerase chain reactions (PCRs). The first PCR round (screening PCR) allowed the identification of the J beta segment involved in a clonal rearrangement. A J beta-primer was used for the second PCR (J beta-specific PCR), recognizing the J beta segment identified in the screening PCR in combination with a consensus V beta primer. This PCR generated prominent and short amplificates suitable for direct sequence analysis because of their low background. Using this approach, clonal TcR beta gene rearrangements were able to be demonstrated in all T-cell lines (n = 7) and in all peripheral T-cell lymphomas (n = 33) analyzed. No clonal TcR beta gene rearrangements were found in any of the normal tissues studied nor in any B-cell non-Hodgkin lymphomas. This method is applicable to DNA from fresh frozen tissues, and, after the TcR beta rearrangement of a patient's malignant T-cell clone has been identified by the screening PCR, DNA can also be detected in follow-up formalin-fixed paraffin-embedded samples by the J beta-specific PCR with high sensitivity and specificity.

Regulation of Vbeta germline transcription in RAG-deficient mice by the CD3epsilon-mediated signals: implication of Vbeta transcriptional regulation in TCR beta allelic exclusion.

During the thymic development of alphabeta lineage T cells, maturation of the CD4- CD8- double-negative (DN) cells into the CD4+ CD8+ double-positive cells is accompanied by the induction of TCR beta allelic exclusion. Recent studies have shown that these events are regulated by the signals through the pre-TCR complex which consists of the TCR beta, pre-TCR alpha and CD3 components. The Vbeta germline transcripts are detected prior to the TCR beta chain gene rearrangements in the DN thymocytes. To examine the effects of the pre-TCR-mediated signals on Vbeta germline transcription, we analyzed thymocytes from RAG-2-deficient mice treated with anti-CD3epsilon antibody. The germline transcripts of all Vbeta we examined, except for Vbeta14, were down-regulated by the anti-CD3epsilon antibody treatment. These data indicate that the regulation of Vbeta germline transcription by the signals through the pre-TCR complex may reflect the modulation of Vbeta accessibility to the VDJ recombinase, which contributes to TCR beta allelic exclusion.

Analysis of T-cell receptor gene rearrangement in lymph nodes of patients with mycosis fungoides. Prognostic implications.

To determine the prognostic value of analyzing lymph node (LN) DNA from patients with mycosis fungoides for the presence of a monoclonal T-cell population. Inception cohort study. A tertiary care referral center in Seattle, Wash. Fifty-five uniformly staged patients with the diagnosis of mycosis fungoides and who had a lymph node biopsy, 21 with clinically abnormal nodes and 34 with normal nodes. Lymph nodes were evaluated by Southern blot analysis for T-cell receptor beta-chain (TCRB) gene rearrangement and by histopathologic examination for the LN classification using the National Cancer Institute system. Patients were observed clinically for a mean (+/- SD) of 4.7 +/- 3.4 years. Patients with detectable TCRB gene rearrangement in lymph node DNA had an increased likelihood of a poor clinical outcome and a decreased probability of survival (P < .001 for both) compared with patients with the TCRB germline. Although patients with clinically enlarged nodes were more likely to have the TCRB gene rearranged, those with normal nodes and the TCRB gene rearranged also had a poor clinical outcome and a decreased probability of survival. Similar to those with the TCRB gene rearranged, most patients with advanced histopathologic changes (LN3 and LN4) had a poor prognosis. The presence of a rearranged TCRB gene, however, correctly predicted some patients with intermediate LN scores (LN2) who had a poor clinical outcome. Detection of a monoclonal T-cell population, as demonstrated by a rearranged TCRB gene on Southern blot analysis, in LNs of patients with mycosis fungoides is predictive of a poor clinical outcome and a reduced probability of survival. Lymph node TCRB gene analysis provides additional prognostic information for patients with mycosis fungoides with intermediate LN histopathology.

Large granular lymphocyte leukemia with pure red cell aplasia in a renal transplant recipient

Neoplastic disorders sometimes accompany a renal transplant. Herein, we report a large granular lymphocyte (LGL) leukemia patient with pure red cell aplasia (PRCA) after renal transplantation. A 36-year-old female was presented to our department with anemia in February 1996. She had undergone hemodialysis because of pregnancy in December 1981. She received a renal transplantation from her mother in April 1986. After the transplantation, she received cyclosporin A (CyA) at 2 mg/kg/day, mizoribine at 1 mg/kg/day, and methylprednisolone at 0.1 mg/kg/day for 8 years. In July 1995, her hemoglobin level dropped to 9.3 g/dl, and anemia developed gradually. In January 1996, her hemoglobin was 5.8 g/dl, and she was given a red blood cell transfusion. Laboratory findings were as follows: RBC 1.46 x 10(12)/L; hemoglobin 5.8 g/dl; hematocrit 17.8%; leucocytes 5.2 x 10(9)/L with 62.4% neutrophils, 34.1% lymphocytes, 2.6% monocytes; platelets 50.8 x 10(10)/L; reticulocytes 0.4%. Bone marrow aspirate smears and biopsy sections revealed normal myeloid and megakaryocyte differentiation with few erythroid precursors. The lymphocytes were of medium size with granules in the cytoplasm. More than 90% of lymphocytes were of the LGL type. Surface markers of peripheral blood mononuclear cells demonstrated increases in the CD2+, CD3+, CD4-, and CD8+ populations. A monoclonal rearrangement of T-cell receptor (TCR)-beta chain gene was found by Southern blot analysis of the mononuclear cells in peripheral blood. A diagnosis of LGL leukemia with PRCA was made. During the next 4 months, she received six red blood cell transfusions, a total of 12 U. In March 1996, the patient was treated with cyclophosphamide (1 mg/kg/day). After 1 month of treatment, serum GPT levels increased to 60 IU/l. The dose of cyclophosphamide was reduced to 0.5 mg/kg/day. Two months after initiation of the therapy, the patient developed reticulocytosis and blood transfusion was not needed thereafter. During remission, the number of CD2+, CD3+, CD4-, and CD8+ lymphocytes decreased. Large granular lymphocytes decreased to less than 10% of peripheral blood. The monoclonal rearrangement of the TCR-beta chain gene in peripheral blood disappeared.

Molecular analysis of skewed Tcra-V gene use in T-cell receptor β-chain transgenic mice

The influence of beta-chain diversity on the expressed T-cell receptor (TCR) alpha-chain repertoire was investigated using transgenic mice which exclusively express a single rearranged TCR beta-chain gene. Analysis of these mice using alpha-chain-specific recombinant cDNA libraries showed that expression of the transgene-encoded beta chain results in significant skewing in Tcra-V gene segment usage vs nontransgenic mice. Skewing was most pronounced towards alpha chains using TCRA-V segments. Sequence analysis of Tcra-V8-containing genes from transgenic T cells revealed predominant use of a single Tcra-J segment (Tcra-J24), which was not detected in Tcra-V8 containing genes isolated from nontransgenic T cells. Further analysis revealed that co-expression of Tcra-V8 with Tcra-J24 in beta-transgenic mice is exhibited almost exclusively by CD4+ T cells, and is associated with a limited number of closely related N-regions. Analysis of transgenic CD8+ T cells demonstrated predominant co-expression of Tcra-V8 with another Tcra-J (Tcra-J30), together with a different, limited N-region sequence. We conclude that the composition of expressed beta chains can profoundly influence the selection of companion alpha chains expressed in the periphery, and that alpha-chain N and J regions play a crucial role in discriminating between class I vs class II major histocompatibility complex (MHC)-restricted recognition. Further, these results are in agreement with recent data concerning the crystal structure of the TCR, and most consistent with a model for TCR structure in which the complementarity determining region (CDR)3alpha domain participates in direct contact with the MHC.

Cloning of a T-cell receptor beta-chain enhancer binding protein.

Several eukaryotic DNA binding proteins have been isolated by screening lambda expression libraries with DNA probes containing their binding site. This strategy has been employed to isolate clones of the factor that interacts with the T-cell receptor beta-chain enhancer motif. A cDNA clone encoding a protein similar to YB-1 has been isolated with this. It seems probable that this protein YB, might interact with other proteins and regulate the transcription of the T-cell receptor beta-chain gene.

CD56-positive cutaneous lymphoma: a poorly recognized entity in the spectrum of primary cutaneous disease

CD56-positive (CD56+) lymphomas, characterized by the expression of the neural cell adhesion molecule on pathological lymphocytes, share a frequent extranodal involvement and a generally aggressive course. Five CD3- CD56+ lymphoma patients presenting with nodular lesions were identified among 180 immunophenotyped cutaneous lymphomas. All the patients were men, with ages ranging from 55 to 78 years. After staging, two patients were diagnosed as having primary cutaneous lymphomas; the remaining three had the secondary cutaneous type. The clinical course was aggressive and four patients died within 8 months from diagnosis. The remaining patient is still alive after a 17-month follow-up. The histological diagnosis was immunoblastic lymphoma in two patients, and medium and large cell pleomorphic lymphoma in three. The angiocentric infiltrate was located mainly in the dermis; azurophilic granules were present in three of the five patients. Immunogenotypic analyses suggested the natural killer cell origin of these neoplasias: all cases exhibited a CD56+ CD3- CD5- T-cell receptor (TCR) silent phenotype, and Southern blot analysis showed a germline configuration of the TCR beta-chain gene.

Analysis of the T-cell receptor repertoire in human atherosclerosis.

Analysis of T-cell receptor (TCR) beta-chain gene expression in atherosclerotic lesions of human aorta. TCR diversity was studied using non-radioactive polymerase chain reaction for quantitative assessment of TCRBV gene transcripts, together with size and sequence analysis of the beta-chain third complementarity-determining region (CDR3). Samples represent a wide range of atheromatous histology, allowing evaluation of the T-cell repertoire at different stages of disease. Diverse TCRBV family usage was observed in the majority of the samples, as the 25 different TCRBV products were detected at levels exceeding background. The data also showed that TCRBV transcripts expressed in the diseased aorta tissue displayed considerable size heterogeneity and no repetition of CDR3 nucleotide motifs. The early presence of T-lymphocytes in the atheromatous blood vessel has been interpreted as an indication of specific immunological reactions operating during the course of the atherosclerotic process. Although a T-cell infiltrate characterized by limited usage of TCRAV genes cannot be excluded the unrestricted usage of TCRBV genes argues against a local T-cell clonal expansion in atherogenesis.

The alpha beta T cell receptor clustering upon superantigen/MHC recognition.

Antigen recognition by T cells is the key event for the antigen specific immune responses to be triggered. This recognition is initiated by the binding of the T cell receptor (TCR) to antigen peptide/major histocompatibility complex (MHC) on the surface of the antigen presenting cells. TCR on most of the T cells is a heterodimer composed of alpha and beta chains which are associated with CD3 gamma delta epsilon as well as zeta chains, the signal transmission molecules. The dynamics of this TCR complex upon antigen/MHC recognition, however, has not been well understood. In this paper the authors analyse the configuration of TCR complex on T cells from a TCR beta chain gene transgenic mouse (TGM) strain. Unlike many other TGM strains reported, a considerable proportion of T cells from this TGM expresses both transgene-encoded (V beta 3) and endogenous TCR beta chains on their surface. By immunoprecipitation and immunoblotting analysis of T cells stimulated with a superantigen, staphylococcal enterotoxin B (SEB), the authors found that V beta 3 was coprecipitated with V beta 8, demonstrating the clustering of TCR alpha beta upon superantigen/MHC recognition.

Molecular detection of a B cell clone in a case of PTCL in the absence of T cell clonality

In this paper we document a case of peripheral T cell lymphoma (PTCL) that exhibited variable T cell histology at presentation and follow-up. Southern blot analysis for T cell receptor (TCR) and immunoglobulin (Ig) receptor gene rearrangements failed to reveal clonal T or B cell populations. TCR gamma (TCRG) and beta (TCRB) chain gene polymerase chain reaction (PCR) amplification of DNA isolated from biopsies was also consistent with polyclonal T cell populations, however Ig PCR revealed clonal Ig rearrangements in follow-up biopsies but not in the presentation biopsy. There was no histological evidence for a neoplastic B cell population in these biopsies although occasional EB virus positive blasts were present. The significance of a cryptic B cell clone is unknown but suggests a relationship with the proliferating polyclonal T cells in this case of PTCL. These data reflect the complexity of PTCL with implications for treatment and patient management.

Age-associated thymic atrophy in the mouse is due to a deficiency affecting rearrangement of the TCR during intrathymic T cell development.

Involution of the thymus is a feature of age and precedes inefficient functioning of the immune system. C57BL/10 mice show an 83% reduction in number of thymocytes between 3 and 20 mo of age, with a significant decline in each of the thymic subsets defined by their expression of CD4 and CD8. The similar percentage contribution of each subset to the whole at 3, 12, and 20 mo suggests a lesion in the T cell developmental pathway within an early subset. The CD3- CD4- CD8- subset showed a significant decline in number by 20 mo of age, but despite this reduction, no significant difference was noted in the number of CD44+ CD25- cells, the earliest stage of this subset between 3 and 20 mo of age. A significant decline in the number of their progeny, the CD44+ CD25+, and the progeny of these cells, the CD44- CD25+ cells, was noted by 12 mo of age. Expression of CD25 within this subset is associated with rearrangement of TCR beta-chain genes. F5 transgenic mice, carrying a complete TCR-alphabeta transgene under the control of a CD2 minigene cassette on a C57BL/10 background, showed no age-associated thymic atrophy in any of the defined thymic subsets over the same period as the normal C57BL/10 mice. Similar results were noted with mice carrying the same transgene but which in addition were also RAG-1-. The results indicate that age-associated thymic involution was associated with problems with rearrangement of the TCR beta-chain genes affecting the production of thymocytes.

Highly restricted TCR-alpha beta usage by autoreactive human T cell clones specific for DNA topoisomerase I: recognition of an immunodominant epitope.

Autoantibody responses to DNA topoisomerase I (Topo I) are highly specific to patients with systemic sclerosis (SSc). We recently demonstrated that Topo I-specific T cells are components of the T cell repertoire of patients with SSc and healthy individuals. These autoreactive T cells were essential for the Ag-specific activation of B cells resulting in anti-Topo I Ab production in vitro and therefore are believed to play a central role in autoantibody production. To characterize the Topo I-specific T cell, 15 T cell clones reactive with Topo I were generated from two patients with SSc and three healthy donors, all of whom shared the MHC class II allele DR11. All clones expressed a CD3+CD4+CD8- phenotype and were restricted by HLA-DR. When eight rTopo I fragments were tested individually as Ags, all clones responded to F5, which encodes amino acids 209 through 386 of Topo I, but not to F10, which encodes amino acids 209 through 276, indicating that one or more immunodominant epitopes on Topo I is located between amino acids 276 and 386. Analysis of TCR gene usage showed that the predominant V(alpha) segment of the functionally rearranged TCR-alpha gene was Vdelta5, which was used by seven clones. Most strikingly, all except one T cell clone had functional rearrangements of TCR beta-chain genes using the Vbeta120.la and Jbeta1.1 gene segments. Comparison of the CDR3 sequences of the TCRs revealed limited diversity, and, of note, all clones contained the amino acid motif PGGN (or minor variations) in the CDR3 of their TCR beta-chains. Furthermore, identical beta-chain CDR3 amino acid sequences were encoded by cDNAs generated from T cell clones derived from multiple individuals, including patients with SSc and healthy donors.