T-cell Receptor Variable Region Gene Research Articles

Identification and characterization of the T cell receptor (TCR) repertoire of the cynomolgus macaque (Macaca Fascicularis)

BackgroundCynomolgus macaque (Macaca fascicularis) is an attractive animal model for the study of human disease and is extensively used in biomedical research. Cynomolgus macaques share behavioral, physiological, and genomic traits with humans and recapitulate human disease manifestations not observed in other animal species. To improve the use of the cynomolgus macaque model to investigate immune responses, we defined and characterized the T cell receptor (TCR) repertoire.ResultWe identified and analyzed the alpha (TRA), beta (TRB), gamma (TRG), and delta (TRD) TCR loci of the cynomolgus macaque. The expressed repertoire was determined using 22 unique lung samples from Mycobacterium tuberculosis infected cynomolgus macaques by single cell RNA sequencing. Expressed TCR alpha (TRAV) and beta (TRBV) variable region genes were enriched and identified using gene specific primers, which allowed their functional status to be determined. Analysis of the primers used for cynomolgus macaque TCR variable region gene enrichment showed they could also be used to amplify rhesus macaque (M. mulatta) variable region genes.ConclusionThe genomic organization of the cynomolgus macaque has great similarity with the rhesus macaque and they shared > 90% sequence similarity with the human TCR repertoire. The identification of the TCR repertoire facilitates analysis of T cell immunity in cynomolgus macaques.

Clonal Expansion of ∝β-T Lymphocytes With Inverted Jβ1 Bias in Familial Hemophagocytic Lymphohistiocytosis

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare but fatal disease in infancy. There are no previous reports on the clonality of T cells in FHL patients. We analyzed here the clonality of alphabeta-T cells in 5 FHL patients using an inverse reverse transcriptase-polymerase chain reaction (RT-PCR) of the T-cell receptor variable region gene (TCR V), a joining region gene of the beta chain (Jbeta)-PCR, a single-strand conformation polymorphism (SSCP), and sequence analysis. A high frequency (15%) of Vbeta and Valpha families was observed in 3 of 5 and 4 of 4 patients examined, respectively. In 19 Vbeta repertoires, including all highly frequent Vbeta, the Jbeta-PCR analysis showed restricted usage of the Jbeta family, indicating a marked bias to Jbeta1 subsets (the mean rate of Jbeta1:Jbeta2 was 87:13 in 65% of the alphabeta-T cells) in widespread alphabeta-T cells (in all patients but 1). In all patients, the clonality of specific Vbeta-Jbeta fragment expanded was confirmed by SSCP and sequence analysis. These results suggest that the existence of clonal expansion and restricted Jbeta1 usage of T cells in FHL is genetically associated with the pathogenesis and the immunodysfunction of the disease. These results help to explain some of the abnormal functional behaviors of T cells in FHL and raise new questions regarding the mechanisms responsible for the restricted clonal diversity.

Epigenetic regulation of antigen receptor gene rearrangement.

V(D)J recombination assembles antigen-specific immunoglobulin and T-cell receptor variable region genes from germline V, D, and J segments during lymphocyte development. Regulation of this site-specific DNA rearrangement process occurs with respect to the cell type and stage of differentiation, order of locus recombination, and allele usage. Many of these controls are mediated via the modulation of gene accessibility to the V(D)J recombinase. Here, we summarise recent advances regarding the impact of nuclear organisation and epigenetic-based mechanisms on the regulation of V(D)J recombination.

Oligoclonal expansion of αβ T lymphocytes in Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis with abnormal karyotypes

We observed a fatal case of Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) with an abnormal karyotype. Increased levels of αβ T cells of the patient were investigated using an inverse polymerase chain reaction (PCR) of the T-cell receptor variable region gene, followed by Jβ-PCR and single-strand conformation polymorphism (SSCP) to confirm the clonality of specific αβ-T cell subsets. A high frequency (>15%) was recognized in Vβ9 at onset, but not in any Vβ and Vα families 2 weeks after chemotherapy. High levels (>20%) of some Jβ genes were detected in all Vβ families investigated, and the predominant bias of the Jβ2 gene relative to the Jβ1 gene (86.1% versus 13.9% at onset, and 77.4% versus 23.5% after chemotherapy) was recognized in pan-αβ T cells. When each Vβ–Jβ fragment was compared among the samples at onset and after chemotherapy by SSCP analysis, several distinct bands were observed that indicate a clonal evolution. Thus, the findings suggest that some of the αβ T cell clones could be associated with abnormal karyotypes in EBV-HLH. The present findings provide molecular evidence of the presence of oligoclonal T cells in pan-αβ-T cells and clonal evolution during a short clinical course in EBV-HLH with abnormal karyotypes.

Diversity and evolution of T-cell receptor variable region genes in mammals and birds.

The receptor of a T lymphocyte (TCR) recognizes nonself antigens in the company of major histocompatibility complex (MHC) molecules presented to it by the antigen-presenting cell. The variable region of TCR is encoded by either a concatenation of variable region (TCR-V), diversity region (TCR-D), and joining region (TCR-J) genes, or a concatenation of TCR-V and TCR-J genes. The TCR-V genes exist as a multigene family in vertebrate species. Here we study the evolutionary relationships of TCR-V genes from humans, sheep, cattle, rabbits, mice, and chicken. These six species can be classified into two groups according to the frequency of gamma(delta) T-cells in their peripheral T-cell populations. The "gamma(delta) low" group of species includes humans and mice, in which gamma(delta)T-cells constitute very limited portion of the T-cell population. The "gamma(delta) high" group includes sheep, cattle, rabbits, and chicken, in which gamma(delta) T-cells comprise up to 60% of the T-cell population. Here, we compiled TCR-V sequences from the six species and conducted a phylogenetic analysis. We identified various TCR-V gene subgroups based on the analysis. We found that humans and mice have representatives from nearly all of the subgroups identified, while other species have lost subgroups to different extent. Therefore, the gamma(delta) low species have a high degree of diversity of TCR-V genes, while gamma(delta) high species all have limited diversity of TCR-V genes. This pattern is similar to that found for immunoglobulin variable region (IGV) genes.

T-cell receptor variable region genes in cutaneous T-cell lymphomas.

The genes encoding the T-cell receptor (TCR) variable beta (TCRBV) regions were studied in skin biopsy samples from 24 patients with cutaneous T-cell lymphomas (CTCL), i.e. mycosis fungoides (n = 7), Sézary syndrome (n = 4), lymphomatoid papulosis (n = 3) and large cell CTCL with (n = 3) or without (n = 7) CD30 expression. A panel of 24 primers specific for TCRBV families 1-24 was designed and applied to cDNA using a semiquantitative reverse transcriptase coupled polymerase chain reaction (PCR) method. Three patients showed restricted expression of a limited number (1-3) of TCRBV families. In the remaining patients, an average of 17 (range: 13-22) different families was expressed. All patients showed elevated (> 10%) expression of individual families significantly higher than that seen in normal blood lymphocytes, but no preferential usage of particular gene families was observed. Direct sequence analysis of more than 60 PCR products revealed clonal TCR transcripts in 18 patients. A single T-cell clone, constituting 9-100% (mean: 26%) of the TCRBV mRNA, was present in 12 patients; two T-cell clones, constituting 13-72% (mean: 21.5%) of the TCRBV mRNA, were present in five patients; and three T-cell clones, accounting for < 0.5-13% of the TCRBV mRNA, were present in one patient. In the remaining six patients, clonal TCR transcripts could not be identified. It is concluded that most CTCL contain both clonal and non-clonal (reactive) T cells, and that the latter cells are more numerous than anticipated. These cells may be engaged in tumour immune reactions, although prospective studies and/or serial investigations are needed to elucidate this issue fully.

4 Pathology and ‘molecular histology’ of Hodgkin's disease and the border to non-Hodgkin's lymphomas

HD is characterized by a small amount of tumour cells (the H- and RS-cells) in a background of many, probably reactive, lymphocytes and histiocytes. Lymphocyte predominant HD seems to be a distinct clinicopathological entity of germinal centre cell origin and has a relatively favourable prognosis. This type has to be differentiated from mixed cellularity and nodular sclerosis HD, which show a different marker profile of the H- and RS-cells and their variants. Differentiation of progressively transformed germinal centres from lymphocyte predominant HD may be problematical and is based on the detection of L&H-cells in the latter entity. Diffuse paragranuloma may be difficult to differentiate from classic HD as well as non-Hodgkin's lymphomas, especially T-cell-rich B-cell lymphomas and peripheral T-cell lymphomas. In rare cases a distinction between HD and non-Hodgkin's lymphomas may be impossible. Similar to lymphocyte predominant HD, lymphocyte depletion HD may provide difficulties in its differential diagnosis from non-Hodgkin's lymphomas. The border with anaplastic large cell lymphoma cannot be clearly drawn and may be blurred. The border between HD and non-Hodgkin lymphomas may be especially blurred in composite lymphomas, if single cell studies can demonstrate a common clonal origin of both parts of the lymphoma. Analysis of immunoglobulin or T-cell receptor variable region genes amplified by PCR from single cells picked from histological sections ("molecular histology') combines morphological characterization and identification of H- and RS-cells with a molecular analysis of those cells. This enables one not only to determine the nature of H- and RS-cells and their clonality but also to answer questions about the relationship between Hodgkin and non-Hodgkin's lymphomas.

Limited heterogeneity in the T-cell receptor V-gene usage in lymphocytes infiltrating human colorectal tumours

The presence of T lymphocytes in solid tumours may reflect an ongoing immune response against the transformed cells. We have used polymerase chain reaction (PCR) technology to investigate the T-cell receptor variable-region gene (V-gene) usage in freshly isolated tumour-infiltrating lymphocytes (TILs) to look for a possible oligoclonality of T cells in the tumour area. We used 19 different V beta-family-specific primers. Peripheral blood lymphocytes and lamina propria lymphocytes from the same patients were also tested by PCR. Our results demonstrate a limited heterogeneity in the V-gene usage of TILs from seven patients with colorectal cancers, suggesting a local antigen-driven immune response at the tumour site.

T-cell receptor variable region gene usage by CD4+ and CD8+ T cells in bronchoalveolar lavage fluid and peripheral blood of sarcoidosis patients.

Sarcoidosis is a chronic noncaseating granulomatous disease of unknown etiology. An accumulation of CD4+ T cells in the alveolar space of the lungs is a characteristic feature of the disease. We have in this study analyzed T-cell receptor (TCR) variable region (V) gene usage by CD4+ and CD8+ lung and peripheral blood T cells of 29 sarcoidosis patients and 15 control subjects. In the patient group, we found a 100% positive correlation between TCR V alpha 2.3+ CD4+ lung T-cell expansions and the expression of the HLA-DR3(17),DQ2 haplotype. The remaining TCR V alpha/V beta gene products analyzed in this study--V alpha 12, V beta 2, V beta 3, V beta 5.1, V beta 5.2/5.3, V beta 5.3, V beta 6.7, V beta 8.1, and V beta 12--were in general normally expressed by CD4+ T cells, although some of them were used to a significantly higher or lower degree by lung T cells compared to peripheral blood T cells. We also performed repeated TCR V gene analyses on some HLA-DR3+ patients and found an association between the ratio bronchoalveolar lavage fluid/peripheral blood V alpha 2.3+ CD4+ T cells and clinical signs of disease activity. Finally, when analyzing TCR V gene usage by CD8+ bronchoalveolar lavage fluid and peripheral blood T cells, a normal V alpha 2.3 usage was found in all cases, but lung-restricted T-cell expansions using other TCR V gene segment products were identified.

Preferential T-cell receptor beta-chain variable gene use in myelin basic protein-reactive T-cell clones from patients with multiple sclerosis.

Multiple sclerosis is an autoimmune disease in which T lymphocytes reactive to myelin basic protein (BP) could play a central role. T cells specific for BP were cloned from the blood of multiple sclerosis patients and normal individuals, and expression of T-cell receptor variable region genes was analyzed. A remarkable bias for use of beta-chain variable region (V beta) 5.2 and, to a lesser extent, V beta 6.1 was seen among BP-specific clones from patients but not from controls. The preferential use of V beta 5.2 for BP recognition did not reflect altered expression of this V beta in the peripheral repertoire. Interestingly, shared V beta 5.2 usage was apparent for clones specific for different BP determinants, even when derived from the same individual. The concurrent demonstration by others (J. R. Oksenberg, M. A. Panzara, A. B. Begovich, H. Erlich, R. Murray, M. Sherritt, S. Stuart, C. C. Bernard, and L. Steinman, personal communication) that T cells within demyelinating areas of multiple sclerosis brains preferentially express V beta 5.2 and V beta 6.1 suggests that the BP-specific clones derived from blood may be relevant to disease pathogenesis. These findings may have important implications for the treatment of multiple sclerosis.

Further localization of a multiple sclerosis susceptibility gene on chromosome 7q using a new T cell receptor beta-chain DNA polymorphism

Multiple sclerosis (MS) has been associated with particular HLA haplotypes and has recently been reported to also be associated with the T cell receptor (TCR) β-chain complex. We have tried to determine the source of the TCR-β/MS association by exploiting the pattern of linkage disequilibrium within the TCR-β complex. We describe a new DNA polymorphism with the TCR variable region gene segment V β15 which appears to localize between the constant region and V β11. When the distribution of V β11–V β15 haplotypes in MS patients was compared to healthy controls, the strength of the V β11–V β15 MS association ( p = 0.107) was much less than the MS association with the adjacent V β8–V β11 haplotype ( p = 0.0010). On this basis we exclude an MS susceptibility gene telomeric to V β11. The reported MS association with the TCR-β gene complex therefore does not appear to be due to genes within the diversity, joining, or constant region but more likely involves a specific gene(s) within the variable region.

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.

An analysis of T-cell receptor variable region gene expression in major histocompatibility complex disparate mice.

To define the impact of major histocompatibility complex (MHC)-encoded glycoproteins on the selection of the T-cell receptor repertoire, we have determined the frequency with which T-cell receptor variable region (V alpha and V beta) genes are expressed in T cells from MHC disparate mice. Approximately 500 T-cell hybridomas were generated from each of three strains of MHC congenic mice [B10 (H-2b), B10.BR (H-2k), and B10.Q (H-2q)] by fusing mitogen-stimulated lymph node T cells with the thymoma BW5147. RNA was prepared from 1629 individual hybridomas and analyzed for the expression of 10 V alpha and 16 V beta gene families. These experiments reveal significant differences in the relative contributions of 1 V alpha gene family (V alpha 3) and several V beta gene segments (V beta 5.1, -5.2, -11, and -12) to the T-cell receptor repertoire of MHC disparate mice.

T-cell receptor variable region gene usage in T-cell populations

T-cell receptor variable region gene usage in T-cell populations

T-cell receptor variable region gene usage in T-cell populations.

We have examined T-cell receptor alpha- and beta-chain variable (V) region gene usage in T-cell populations predicted to have different major histocompatibility complex-restriction specificities. Using a sensitive ribonuclease protection assay to measure T-cell receptor mRNA levels, we found no striking differences in the usage of three V alpha genes and three V beta genes in T-cell populations from three congeneic H-2-disparate strains of mice and between the mutually exclusive Ly2+ L3T4- and Ly2- L3T4+ T-cell subpopulations. These results suggest that major histocompatibility complex restriction cannot be explained by the differential usage of nonoverlapping V alpha or V beta gene pools. In contrast, striking but unpredictable differences were seen in V gene usage in populations of T cells selected by activation with particular alloantigens.