Cell Receptor Vα Research Articles

Microfluidics sorting enables the isolation of an intact cellular pair complex of CD8+ T cells and antigen-presenting cells in a cognate antigen recognition-dependent manner.

Adaptive immune responses begin with cognate antigen presentation-dependent specific interaction between T cells and antigen-presenting cells. However, there have been limited reports on the isolation and analysis of these cellular complexes of T cell-antigen-presenting cell (T/APC). In this study, we successfully isolated intact antigen-specific cellular complexes of CD8+ T/APC by utilizing a microfluidics cell sorter. Using ovalbumin (OVA) model antigen and OT-I-derived OVA-specific CD8+ T cells, we analyzed the formation of antigen-specific and antigen-non-specific T/APC cellular complexes and revealed that the antigen-specific T/APC cellular complex was highly stable than the non-specific one, and that the intact antigen-specific T/APC complex can be retrieved as well as enriched using a microfluidics sorter, but not a conventional cell sorter. The single T/APC cellular complex obtained can be further analyzed for the sequences of T cell receptor Vα and Vβ genes as well as cognate antigen information simultaneously. These results suggested that this approach can be applied for other antigen and CD8+ T cells of mice and possibly those of humans. We believe that this microfluidics sorting method of the T/APC complex will provide useful information for future T cell immunology research.

Characterizations of distribution and clonality of T cell receptor Vα and T cell receptor Vβ subfamily T lymphocytes in diffuse large B cell lymphoma patients

Objective To investigate the situation of T cell receptor (TCR) Vα and TCR Vβ gene subfamily repertoire and clonal proliferation of T lymphocytes from peripheral blood of patients with diffuse large B-cell lymphoma (DLBCL). Methods April, May and July 2006, a total of three patients with DLBCL who were admitted in the First Affiliated Hospital of Guangzhou Medical University were collected in this study. Another of three healthy individuals who received medical examination in the same period were included into control group. The study protocol was approved by the Ethical Review Board of Investigation in Human Beings of the First Affiliated Hospital of Guangzhou Medical University. Informed consent was obtained from all participants. There were no significant differences of subjects' general clinical features between two groups (P>0.05). The blood specimens (10 mL) from the subjects were collected at the clinical laboratory. The complementarity determining region 3 (CDR3) of 29 TCR Vα and 24 TCR Vβ subfamily genes in peripheral blood T lymphocytes were amplified by real-time polymerase chain reaction (RT-PCR). CDR3 sequence and length of RT-PCR products were analyzed by gene scan. The clinical data of two groups were retrospectively analyzed: The expression of TCR Vα and Vβ subfamily genes in T lymphocytes of two groups were compared, and clonal proliferation of T lymphocytes in study group was analyzed. Results ① Different from the control group, 20-22 TCR Vα and 9-20 TCR Vβ subfamilies were identified in 3 DLBCL patients of study groups, respectively. ② Oligoclonality, biclonality and oligoclonal tendency of one or more TCR Vα and TCR Vβ subfamilies T lymphocytes were observed in all 3 DLBCL patients. TCR Vα subfamily genes expressed in clonal proliferative T lymphocytes were TCR Vα6, Vα8, Vα14, Vα15, Vα21, Vα22, Vα23 and Vα25. TCR Vβ subfamily genes expressed in clonal proliferative T lymphocytes were TCR Vβ1, Vβ3, Vβ13, Vβ15 and Vβ16. T lymphocytes predominately expressed TCR Vα6, Vα8, Vα14, Vα15, Vα23 and TCR Vβ13, Vβ15 subfamily genes were all in clonal proliferation. TCR Vα6, Vα23 and TCR Vβ3, Vβ13 subfamily T lymphocytes were obviously monoclonal proliferation, and their gene scan results were all closely monoclonaliity shape. Conclusions TCR Vα and TCR Vβ subfamilies T lymphocytes from peripheral blood of patients with DLBCL showed characteristics of advantage utilization and clonal proliferation. Key words: Lymphoma, large B-cell, diffuse; Genes, T-cell receptor alpha; Genes, T-cell receptor beta; T-lymphocytes

Normalization of obesity-associated insulin resistance through immunotherapy

Progressive obesity and its associated metabolic syndromes represent a globally growing challenge, yet mechanistic understanding and current therapeutics are unsatisfactory. We discovered that CD4+ T-lymphocytes, resident in visceral adipose tissue (VAT), control insulin-resistance in diet-induced obese (DIO) mice and likely humans. DIO VAT-associated T cells display biased TCR-Vα repertoires suggesting antigen-specific expansion. CD4+ T-lymphocyte control of glucose homeostasis is compromised in DIO when VAT accumulates pathogenic IFNγ-secreting Th1 cells, overwhelming static numbers of Th2 (CD4+GATA-3+) and regulatory Foxp3+ T cells. CD4+ T cell transfer into DIO, lymphocyte-free RAGnull mice reversed weight gain and insulin resistance predominately through Th2 cells. Brief systemic treatment with αCD3 antibody or its F(ab′)2 fragment, restores the Th1/Foxp3+ balance and reverses insulin resistance for months, despite continuing high-fat diet. The progression of obesity-associated metabolic abnormalities is physiologically under CD4+ T cell control, with expansion of adipose tissue-resident T cells that can be manipulated by immunotherapy.

No restriction of intrathyroidal T cell receptor V alpha families in the thyroid of Graves' disease.

Recently it has been reported that the intrathyroidal T cells in Graves' disease display restriction in V alpha T cell receptor (TcR) gene family usage, although this is not found with TcR V beta gene families in the same individuals. We have performed a qualitative analysis of TcR V alpha family usage in 12 patients with Graves' disease by reverse transcription and polymerase chain reaction (PCR) amplification of RNA extracted from isolated, unstimulated intrathyroidal lymphocytes and from snap-frozen whole thyroid specimens. No restriction was observed, with 10-15 V alpha gene families being amplified in all cases. The pattern of usage was similar to that in peripheral blood lymphocytes derived from normal subjects (n = 3) and from patients with Graves' disease (n = 3), as well as that present in the thyroids of patients with non-autoimmune toxic multinodular goitre (n = 4). These results indicated that there is no marked restriction of the unselected intrathyroidal T cell population in patients with Graves' disease who have been treated with antithyroid drugs.

Localization of CD4+ T cell epitope hotspots to exposed strands of HIV envelope glycoprotein suggests structural influences on antigen processing.

The spectrum of immunogenic epitopes presented by the H2-IA(b) MHC class II molecule to CD4(+) T cells has been defined for two different (clade B and clade D) HIV envelope (gp140) glycoproteins. Hybridoma T cell lines were generated from mice immunized by a sequential prime and boost regime with DNA, recombinant vaccinia viruses, and protein. The epitopes recognized by reactive T cell hybridomas then were characterized with overlapping peptides synthesized to span the entire gp140 sequence. Evidence of clonality also was assessed with antibodies to T cell receptor Valpha and Vbeta chains. A total of 80 unique clonotypes were characterized from six individual mice. Immunogenic peptides were identified within only four regions of the HIV envelope. These epitope hotspots comprised relatively short sequences ( approximately 20-80 aa in length) that were generally bordered by regions of heavy glycosylation. Analysis in the context of the gp120 crystal structure showed a pattern of uniform distribution to exposed, nonhelical strands of the protein. A likely explanation is that the physical location of the peptide within the native protein leads to differential antigen processing and consequent epitope selection.

Comparison of the T Cell Patterns in Leprous and Cutaneous Sarcoid Granulomas: Presence of Vα24-Invariant Natural Killer T Cells in T-Cell-Reactive Leprosy Together with a Highly Biased T Cell Receptor Vα Repertoire

The T-cell-reactive (eg, tuberculoid and reversal) forms of leprosy represent a well-defined granulomatous reaction pattern against an invading pathogen. The immune response in cutaneous sarcoidosis is a granulomatous condition that pathologically is very similar to T-cell reactive leprosy. However, it lacks a defined causative agent. In view of the role of NKT cells in murine granulomas induced by mycobacterial cell walls, we have searched for the presence of NKT cells in the cutaneous lesions of both leprosy and sarcoidosis. These cells were present in T-cell-reactive leprosy but were undetectable in cutaneous sarcoidosis. We have also studied the TCR Valpha repertoire in the two diseases. In addition to Valpha24(+) NKT cells, all patients with T-cell-reactive leprosy showed a very restricted T-cell-reactive Valpha repertoire with a strong bias toward the use of the Valpha6 and Valpha14 segments. Valpha6 and Valpha14(+) T cells were polyclonal in terms of CDR3 length and Jalpha usage. In contrast, most sarcoidosis patients showed a diverse usage of Valpha chains associated with clonal or oligoclonal expansions reminiscent of antigen-driven activation of conventional T cells. Thus the origin and perpetuation of the two kinds of granulomatous lesions appear to depend on altogether distinct T-cell recruiting mechanisms.

Restricted T cell receptor Vα gene rearrangements in T cells recognizing an immunodominant peptide of myelin basic protein in DR2 patients with multiple sclerosis

Restricted T cell receptor Vα gene rearrangements in T cells recognizing an immunodominant peptide of myelin basic protein in DR2 patients with multiple sclerosis

A Shannon entropy analysis of immunoglobulin and T cell receptor

In 1970, before any antigen-bound immunoglobulin structure had been solved, Elvin Kabat proposed that regions of high amino acid diversity would be the antigen binding sites of immunoglobulin (Kabat, 1970). Conversely, sites of low variability were proposed to be structural, framework regions. This variability was defined by Wu and Kabat as the number of different amino acids found at a site divided by the relative frequency of the most common amino acid at that site (Wu and Kabat, 1970). Several groups have subsequently devised improvements of Kabat-Wu variability analysis (Litwin and Jores, 1992). While these methods are somewhat better than Kabat Wu, they still suffer from Kabat-Wu's basic limitation: they account for only the most common one or two amino acids in estimating diversity. This leads to underestimates of low diversities and exaggerations of high diversities. Shannon information analysis eliminates serious bias and is more stable than Kabat-Wu and second generation measures of diversity (Jores et al. 1990; Wu and Kabat 1970). Statistical reliability can be measured using Shannon analysis, and Shannon measurements can be provided with error estimates. Here we use Shannon's method to analyze the amino acid diversity at each site of T cell receptor Vα and Vβ to identify complementarity determining regions and framework sites. Our results reveal that the T cell receptor is significantly more diverse than immunoglobulin-suggesting T cell receptor has more than the previously-discovered four complementarity determining regions. These new complementarity determining regions may represent a larger antigen combining site, additional combining sites, or an evolutionary strategy to avoid inappropriate interaction with other molecules

T cell receptor Vα and Vβ gene usage by tumour-infiltrating lymphocytes in oral squamous cell carcinoma

T cell receptor Vα and Vβ gene usage by tumour-infiltrating lymphocytes in oral squamous cell carcinoma

Dual conformations of a T cell receptor Vα homodimer: implications for variability in VαVβ domain association

The crystal structure of a mutant T cell receptor (TCR) Vα domain containing a grafted third complementarity-determining region (CDR3) from a different Vα was determined at 2.3Å resolution by molecular replacement using the wild-type Vα structure as a search model. Like the wild-type Vα domain, the mutant crystallized as a homodimer very similar to TCR VαVβ and antibody VL VH heterodimers, with the CDR loops disposed to form part of the antigen-binding site. However, the relative orientation of the two chains in the mutant Vα homodimer differs from that in the wild-type by a rotation of 14° such that the buried surface area in the dimer interface of the mutant is 140 Å2 less than in the wild-type. While the residues forming the interface are essentially the same in the two structures, there are only four pairs of interface hydrogen bonds in the case of the mutant compared with eight for the wild-type. These results suggest that multiple relative orientations of the Vα and Vβ domains of TCRs may be possible, providing a significant contribution to TCR combining site diversity.

Limited heterogeneity of rearranged T cell receptor V alpha and V beta transcripts in synovial fluid T cells in early stages of rheumatoid arthritis.

The identification of activated T cells in synovial fluid and synovium, and the association of rheumatoid arthritis (RA) with specific HLA-DR restriction elements, strongly suggest that these T cells play a critical role in the etiology and pathogenesis of RA. Analysis of the T cell receptor (TCR) repertoire in the early stages of RA might be an approach to identify those T cells involved in the initiation and/or perpetuation of the disease. TCR V alpha and V beta transcripts of synovial T cells, sampled at the early stages of RA, were amplified by reverse transcriptase-polymerase chain reaction. HLA-DR subtyping was determined by serologic analysis and dot-blot hybridization of polymerase chain reaction amplification products using digoxigenin-labeled, sequence-specific oligonucleotide probes. Our findings showed a limited heterogeneity of V alpha and V beta TCRs in synovial fluid T cells, and a preferential usage of TCR V alpha 17 in early RA. In contrast, in the later stages of RA, a more polyclonal TCR V alpha and V beta gene usage was observed. Our results support the view that induction of RA is driven by an oligoclonal immune response to an unknown antigen. These findings also suggest a pathogenetic role for V alpha 17 T cells in the early stages of RA.

VH shuffling can be used to convert an Fv fragment of anti-hen egg lysozyme specificity to one that recognizes a T cell receptor Vα

This study describes the isolation and characterization of Fv fragments that recognize a T cell receptor Vα (Vα 1934.4). A VH gene repertoire from an immunized mouse was recombined with the anti-hen egg lysozyme (HEL) VκD1.3 gene as single chain (sc)Fvs, and an Fv with reasonable affinity for binding to Vα1934.4 isolated. The Fv (VH14/VκD1.3) does not bind to HEL, indicating that the heavy chain shuffling has converted an anti-HEL specificity to one that recognizes the unrelated Vα1934.4. The association constant for the Fv-Vα1934.4 interaction has been determined using surface plasmon resonance (SPR) and is 1.2 × 10 7 M −1. Recombinant antibodies of reasonable affinity can therefore be generated by combining a VH library with a ‘fixed’ Vκ. To improve the affinity further, light chain shuffling has been used to generate an Fv (VH14/Vκ9) that has a 30-fold higher affinity for binding to Vα1934.4 than the parent (VH14/VκD1.3) Fv, and SPR measurements demonstrate that the affinity improvement is due to an increase in on-rate. Unexpectedly, Vκ9 differs from VκD1.3 by only two amino acids at positions 30 and 91 and, consistent with the change in binding affinity, both of these residues are located in CDRs.

T cell receptor V alpha bias can be determined by TCR-contact residues within an MHC-bound peptide.

Many antigen-specific T cell responses show profound V-region biases in one or both chains of the TCR alpha beta heterodimer. We have examined how changes in residues within an MHC-bound peptide can influence V-region selection. Single-chain TCR transgenic mice were derived using a beta-chain specific for the Kb-restricted peptide from ovalbumin, OVA257-264. Transgenic T cells were stimulated in vitro using OVA257-264 or a single residue variant having a lysine to aspartic acid substitution at determinant position 7 (7D). Recent crystallographic analyses have shown that this variant residue is involved in direct contact with the TCR. Sequence analysis of the T cell populations showed that the majority OVA257-264-specific T cells used V alpha 10-positive TCR while the majority of the 7D-specific TCR expressed the V alpha 4 element. In both peptide responses we found that some cell lines appeared to be made up of more than one clone expressing the same V alpha sequence but having limited variation at the V-J junction. These results show that the TCR contact residues within the target peptide can influence V-region bias and suggest that the first and second hypervariable regions of the TCR are directly or indirectly involved in determining peptide specificity.

Limited T cell receptor Vα and Vβ chain usage in HTLV-I specific cytotoxic T lymphocytes in HAM/TSP patients

Limited T cell receptor Vα and Vβ chain usage in HTLV-I specific cytotoxic T lymphocytes in HAM/TSP patients

Analysis of the T cell receptor Vα and Vβ repertoire in psoriasis

Analysis of the T cell receptor Vα and Vβ repertoire in psoriasis

T Cell Receptor Vα Gene Usage in Human T Cells Stimulated with SEE and SED

V alpha gene usage in human T cells stimulated with SEE and SED was investigated by using polymerase chain reaction with specific primers. V beta 8 T cells from normal blood donors PBMC were sorted at day 5 after stimulation with SEE and analysed for TCR-V alpha gene usage. Whole T cells stimulated with anti-CD3 MoAb or SED were also analysed and compared at different time points after stimulation. There was no biased V alpha gene usage found as a response to either of the two superantigens. These results show that V alpha gene usage of human T cells stimulated with SEE or SED is normal.