BV Gene Usage Research Articles

Monoclonal and oligoclonal TCR AV and BV gene usage in CD4+ T cells from pigs immunised with C-strain CSFV vaccine

The classical swine fever virus C-strain vaccine (C-strain vaccine) plays a vital role in preventing and controlling the spread of classical swine fever (CSF). However, the protective mechanisms of C-strain vaccine and cellular immunity conferred by T cell receptors (TCRs) are less well defined. We aimed to analyse the association between the complementarity determining region 3 (CDR3) spectratype of αβTCR in CD4+ T cells and C-strain vaccine; and to find conserved CDR3 amino acid motifs in specific TCR α- and β-chains. We found that the CDR3 spectratype showed dynamic changes correlating with C-strain vaccine immunisation and that TCR AV5S/8–3S/8–4S/14/38 and BV4S/6S/7S/15S/30 gene families showed clonal expansion in immunised pigs. The sequences of CDR3 from these clonally expanded T cells indicated a high frequency of the ‘KLX’ motif in the TCR α chain and the ‘GGX’ motif in β chain, and Jα39, Jα43, Jβ2.5 and Jβ2.3 genes were also found in high frequency. To the best of our knowledge, this is the first report describing the dynamic changes of αβTCRs and conserved CDR3 amino acid motifs in CD4+ T cells from C-strain vaccine-immunised pigs, which will provide a basis for the development of high-efficiency epitope vaccines.

Quantification of total T-cell receptor diversity by flow cytometry and spectratyping

BackgroundT-cell receptor diversity correlates with immune competency and is of particular interest in patients undergoing immune reconstitution. Spectratyping generates data about T-cell receptor CDR3 length distribution for each BV gene but is technically complex. Flow cytometry can also be used to generate data about T-cell receptor BV gene usage, but its utility has not been compared to or tested in combination with spectratyping.ResultsUsing flow cytometry and spectratype data, we have defined a divergence metric that quantifies the deviation from normal of T-cell receptor repertoire. We have shown that the sample size is a sensitive parameter in the predicted flow divergence values, but not in the spectratype divergence values. We have derived two ways to correct for the measurement bias using mathematical and statistical approaches and have predicted a lower bound in the number of lymphocytes needed when using the divergence as a substitute for diversity.ConclusionsUsing both flow cytometry and spectratyping of T-cells, we have defined the divergence measure as an indirect measure of T-cell receptor diversity. We have shown the dependence of the divergence measure on the sample size before it can be used to make predictions regarding the diversity of the T-cell receptor repertoire.

Restricted T cell receptor BV gene usage in the lungs and muscles of patients with idiopathic inflammatory myopathies

To investigate T cell receptor (TCR) expression in 3 different compartments that could be involved in patients with myositis: muscle, lung, and peripheral blood. Nine patients with polymyositis (PM), dermatomyositis, or inclusion body myositis underwent bronchoscopy and bronchoalveolar lavage (BAL) as well as muscle biopsy and blood sampling. A panel of 19 monoclonal antibodies specific for TCR V(beta) (BV) and V(alpha) (AV) were used to characterize the TCR profile in CD4(+) and CD8(+) T cell populations in BAL fluid and peripheral blood by flow cytometry. Muscle biopsy tissues were analyzed by immunohistochemistry. Patients were also typed for HLA-DRB1 and DRB3 alleles. A total of 17 T cell expansions were detected in BAL fluid, 6 in the CD4(+) T cell population and 11 in the CD8(+) T cell population. Four T cell expansions were detected in peripheral blood. A selective TCR V usage was found in muscle. Two PM patients, both of whom had BAL fluid BV3(+) T cell expansions in the CD4 population and in whom BV3 was also a prominent TCR V segment in muscle tissue, shared the HLA-DRB1*03 allele. These 2 patients were the only ones who were positive for anti-Jo-1 antibody. We found a restricted accumulation of T lymphocytes expressing selected TCR V-gene segments in the target organ compartments (i.e., lung and muscle). The occurrence of shared TCR gene segment usage in muscle and lungs could suggest common target antigens in these organs.

The Complementarity Determining Region 2 of BV8S2 (Vβ8.2) Contributes to Antigen Recognition by Rat Invariant NKT Cell TCR

Invariant NKT cells (iNKT cells) are characterized by a semi-invariant TCR comprising an invariant alpha-chain paired with beta-chains with limited BV gene usage which are specific for complexes of CD1d and glycolipid Ags like alpha-galactosylceramide (alpha-GalCer). iNKT cells can be visualized with alpha-GalCer-loaded CD1d tetramers, and the binding of mouse CD1d tetramers to mouse as well as to human iNKT cells suggests a high degree of conservation in recognition of glycolipid Ags between species. Surprisingly, mouse CD1d tetramers failed to stain a discrete cell population among F344/Crl rat liver lymphocytes, although comprised iNKT cells are indicated by IL-4 and IFN-gamma secretion after alpha-GalCer stimulation. The arising hypothesis that rat iNKT TCR recognizes alpha-GalCer only if presented by syngeneic CD1d was then tested with the help of newly generated rat and mouse iNKT TCR-transduced cell lines. Cells expressing mouse iNKT TCR reacted to alpha-GalCer presented by rat or mouse CD1d and efficiently bound alpha-GalCer-loaded mouse CD1d tetramers. In contrast, cells expressing rat iNKT TCR responded only to alpha-GalCer presented by syngeneic CD1d and bound mouse CD1d tetramers only poorly or not at all. Finally, CD1d-dependent alpha-GalCer reactivity and binding of mouse CD1d tetramers was tested for cells expressing iNKT TCR comprising either rat or mouse AV14 (Valpha14) alpha-chains and wild-type or mutated BV8S2 (Vbeta8.2) beta-chains. The results confirmed the need of syngeneic CD1d as restriction element for rat iNKT TCR and identified the CDR2 of BV8S2 as an essential site for ligand recognition by iNKT TCR.

T-cell receptor BV gene usage in colorectal carcinoma patients immunised with recombinant Ep-CAM protein or anti-idiotypic antibody.

The tumour-associated antigen, Ep-CAM, is over-expressed in colorectal carcinoma (CRC). In the present study, a recombinant Ep-CAM protein or a human anti-idiotypic antibody (anti-Id) mimicking Ep-CAM, either alone or in combination, was used for vaccination of CRC patients (n=9). GM-CSF was given as an adjuvant cytokine. A cellular immune response was assessed by measuring anti-Ep-CAM lymphoproliferation, IFN-gamma production (ELISPOT) and by analysing the TCR BV gene usage within the CD4+ and CD8+ T-cell subsets followed by CDR3 fragment analysis. A proliferative and/or IFN-gamma T-cell response was induced against the Ep-CAM protein in eight out of nine patients, and against Ep-CAM-derived peptides in nine out of nine patients. Analysis of the TCR BV gene usage showed a significantly higher usage of BV12 family in CD4+ T cells of patients both before and after immunisation than in those of healthy control donors (p<0.05). In the CD8+ T-cell subset, a significant (p<0.05) increase in the BV19 usage was noted in patients after immunisation. In individual patients, a number of TCR BV gene families in both CD4+ and CD8+ T cells were over-expressed mainly in post-immunisation samples. Analysis of the CDR3 length polymorphism revealed a higher degree of clonality in post-immunisation samples than in pre-immunisation samples. In vitro stimulation with Ep-CAM protein confirmed the expansion of anti-Ep-CAM T-cell clones. The results indicate that immunisation with the Ep-CAM protein and/or anti-Id entails the induction of an anti-Ep-CAM T-cell response in CRC patients, and suggest that BV19+ CD8+ T cells might be involved in a vaccine-induced immune response.

Restricted BV gene usage by factor VIII-reactive CD4+ T cells in inhibitor-positive patients with severe hemophilia A.

In the present study, we have analyzed the T cell receptor (TCR) repertoires of CD4+ T cells isolated from peripheral blood of 10 inhibitor-positive patients with severe hemophilia A. The distribution of complementarity determining region (CDR3) lengths of the beta chain of the TCRs was analyzed by spectratyping prior to and following in vitro stimulation of the cells with human factor VIII (FVIII). The repertoires of CD4+ T cells of patients were perturbed when compared to those of healthy blood donors. The perturbations of T cell repertoires were heterogeneous among patients with respect to the number and the nature of V-beta (BV) families that exhibited expansion following incubation with FVIII. Some patients showed alterations in one or two BV families, others exhibited more perturbed repertoires affecting 5 to 8 of the 14 BV families tested. Alterations of BV2, BV5 and/or BV9 were consistently found after incubation of CD4+ T cells in the presence of FVIII in 80% of the patients. These findings indicate that the presence of FVIII inhibitors in patients with severe hemophilia A is associated with measurable perturbations of the CD4+ T cell repertoire that results from oligoclonal expansion of FVIII-specific cells and may be relevant for the design of strategies aimed at modulating the anti-FVIII immune responses by T cell-targeted therapy

T cell receptor BV gene usage in interstitial cellular infiltrates in active Heymann nephritis.

Infiltration of the kidney by mononuclear cells is a prominent feature of active Heymann nephritis (HN). These cells could be present as a part of generalized inflammatory response, or could be proliferating in response to specific antigens. To examine these questions, we have analysed the T cell receptor (TCR) BV repertoire of T cells infiltrating the renal interstitium at regular time intervals throughout the course of the disease. HN was induced in Lewis rats by immunization with renal tubular antigen (Fx1A) in complete Freund's adjuvant (CFA). Kidneys were collected 8 and 12 weeks after immunization. Renal tissue was homogenized and RNA extracted. RT-PCR and sequencing were used to characterize expression of TCR BV genes. Preferential expression of TCR BV2 and BV16 gene families was seen at 8 weeks. By 12 weeks the diversity of the TCR BV gene repertoire had increased and was highly heterogeneous. Sequence analysis of BV2, and BV16 RT-PCR products from 8 week HN kidneys revealed conserved usage of CDR3 regions, and an over-representation of arginine residues in the CDR3 regions at a frequency of between 60 and 100% of clones sequenced in most of BV2 and BV16 subfamilies. The preferential usage of CDR3 region sequences in TCR BV2 and BV 16 families indicates clonal expansion of individual T cells in HN kidneys at 8 weeks. The conserved usage of arginine residues in the CDR3 regions may indicate recognition of select antigenic epitopes. By 12 weeks, the diverse TCR BV repertoire in the kidney may be due to epitope spreading or may represent a non-specific inflammatory response in the late phase of the disease.

Association of MHC and rheumatoid arthritis: Association of RA with HLA-DR4 - the role of repertoire selection

Most patients with rheumatoid arthritis (RA) express HLA-DR4, HLA-DR1 or HLA-DR10. These alleles share a common amino acid motif in their third hypervariable regions: the shared epitope. In normals and patients with RA, HLA-DR genes exert a major influence on the CD4 αβ T-cell repertoire, as shown by studies of AV and BV gene usage and by BV BJ gene usage by peripheral blood CD4 αβ T-cells. However, the rheumatoid T-cell repertoire is not entirely under HLA-DR influence, as demonstrated by discrepancies in VB JB gene usage between identical twins discordant for RA and by contraction of the CD4 αβ T-cell repertoire in RA patients. Shared epitope positive HLA-DR alleles may shape the T-cell repertoire by presenting self peptides to CD4 T cells in the thymus. Peptides processed from HLA-DR molecules and encompassing the shared epitope may also be presented by HLA-DQ and select CD4 αβ T cells in the thymus. Thus, shared epitope-positive alleles impose a frame on the T-cell repertoire. This predisposing frame is further modified (by unknown factors) to obtain the contracted rheumatoid repertoire.

Detection of clonotypic changes of T cells after stimulation with Porphyromonas gingivalis.

The aim of this study was to investigate whether Porphyromonas gingivalis stimulation would induce a selective activation and expansion of a limited T-cell receptor V beta repertoire or T-cell clonotype. Using samples from patients with chronic inflammatory periodontal diseases, we examined TCRBV gene usage and T-cell clonotypes of peripheral blood mononuclear cells incubated in the presence or absence of P. gingivalis outer membrane through a combination of reverse transcription polymerase chain reaction (RT-PCR) and subsequent single strand conformation polymorphism (SSCP) analysis. There was no difference in the mean expression for most BV families with or without P. gingivalis outer membrane in the culture. However, in individual cases, a few BV gene families did become overexpressed or underexpressed following stimulation, although a consistent pattern did not emerge. SSCP analysis showed that several new distinct bands appeared after stimulation, indicating distinct clonal accumulations, although the number of distinct bands decreased in most cases. These data suggest that clonotypic change occurred following stimulation with P. gingivalis outer membrane. Furthermore, the possibility of superantigen stimulation by P. gingivalis is unlikely to be due to the small change in BV gene usage and clonal T-cell accumulations with P. gingivalis outer membrane stimulation, as evidenced by SSCP. Thus, RT-PCR and SSCP analysis is useful in evaluating the host response to periodontopathic antigens.

Enhanced prevalence of T cells expressing TCRBV8S2 and TCRBV8S3 in hearts of chronically Trypanosoma cruzi-infected mice

We have analysed the relative T cell receptor (TCR) BV gene usage in T cells from hearts and spleens of CBA/HJ mice chronically infected with the Tulahuén strain of Trypanosoma cruzi. During chronic infection, CBA/HJ mice recruit T cells at the major site of inflammation (i.e. the heart), with over-representation of certain TCRBV gene subfamilies (TCRBV8S2 and TCRBV8S3). In contrast, no signal or a very weak message from a limited number of T cells was recorded from one heart of the control group. No alteration of TCRBV distribution was recorded in spleens of chronically infected CBA/HJ. Our findings indicate that there is a preferential TCRBV gene usage in the T cell response in the hearts of chronically infected mice. Furthermore, the pattern of CDR3 lengths in inflammatory T cells was altered.

Clonal expansion of mycobacterial heat-shock protein-reactive T lymphocytes in the synovial fluid and blood of rheumatoid arthritis patients.

To examine the reactivity pattern and T cell receptor (TCR) characteristics of mycobacterial heat-shock protein 65 (hsp65)-reactive T cells generated from paired synovial fluid (SF) and peripheral blood (PB) samples obtained from rheumatoid arthritis (RA) patients and from healthy subjects. The reactivity pattern of hsp65-reactive T cell clones generated under limiting-dilution conditions was analyzed in 3H-thymidine incorporation assays. The TCR variable regions of these hsp65-reactive T cells were characterized by polymerase chain reaction with TCR AV- and BV-specific primers and by DNA sequence analysis of the third complementarity-determining region (CDR3). The hsp65-reactive T cells derived both from RA patients and controls preferentially recognized the 1-170 and 303-540 regions of hsp65 and did not cross-react with human hsp60. The hsp65-reactive T cell clones derived from RA patients displayed a restricted TCR AV and BV gene usage, which can be attributed to the limited clonal origin(s) of the independent T cell clones, as evidenced by CDR3 sequence analysis. These clonally expanded T cells were found in both PB and SF and in different inflamed joints of RA patients. Our study suggests that there is in vivo clonal activation and expansion of mycobacterial hsp65-reactive T cells in patients with RA.