T-cell Receptor Vα Research Articles

Genomic analysis and identification of a novel superantigen, SargEY, in Staphylococcus argenteus isolated from atopic dermatitis lesions.

Staphylococcus aureus is frequently isolated from active lesions of atopic dermatitis (AD) patients. We reported that 17%-22% of S. aureus isolated from AD patients produced a novel superantigen staphylococcal enterotoxin Y (SEY). Unlike many S. aureus superantigens that activate T cells via T-cell receptor (TCR) Vß, SEY activates T cells via TCR Vα and stimulates cytokine secretion. Staphylococcus argenteus was isolated from AD patients during the surveillance for S. aureus. Phylogenetic comparison of the genome indicated that the isolate was very similar to S. aureus causing skin infections. The isolate encoded a SEY-like protein, designated SargEY, which, like SEY, activated T cells via the TCR Vα. ST2250 is the only lineage positive for SargEY gene. ST2250 S. argenteus harboring a superantigen SargEY gene may be a novel staphylococcal clone that infects human skin and is involved in the exacerbation of AD.

Local chicken breeds exhibit abundant TCR-V segments but similar repertoire diversity

The thymus-derived lymphocytes of jawed vertebrates have four T-cell receptor (TCR) chains that play a significant role in immunity. As chickens have commercial value, their immune systems require a great deal of attention. Local chicken breeds are an essential part of poultry genetic resources in China. Here, we used high-throughput sequencing to analyze the TCRα and TCRβ repertoires and their relative expression levels in the native chicken breeds Baier Buff, Longyou Partridge, Xiaoshan, and Xianju. We found that TCR Vα and TCR Vβ were expressed and included 17, 19, 17, and six segments of the Vα2, Vα3, Vβ1, and Vβ2 subgroups, respectively. V-J pairing was biased; Jα11 was utilized by nearly all Vα segments and was the most commonly used. Breed-specific V segments and V-J pairings were detected as well. The results of the principal coordinate analysis (PCoA) as well as the V-J pairing and CDR3 diversity analyses suggested that the four local chicken breeds did not significantly differ in terms of TCR diversity. Hence, they expressed not significant differentiation, and they are rich genetic resources for the development and utilization of immune-related poultry breeding.

T-cell receptor variable region usage in Chagas disease: A systematic review of experimental and human studies.

T cells recognize their ligand, the peptide major histocompatibility complex (MHC), via the T-cell receptor (TCR), which is composed of covalently linked α and β or γ and δ chains. This recognition is critical for T-cell ontogeny and controls the selection, activation, and function of T lymphocytes. Specific TCR αβ variable regions have been associated with immunopathogenesis of Chagas disease. Here, we present a systematic review that compiles experimental in vivo and human data regarding the preferential expression of variable alpha (Vα) and variable beta (Vβ) chain regions in Trypanosoma cruzi infection. The original studies indexed in PubMed/Medline, Scopus, and Web of Science databases were screened according to the PRISMA strategy. The analysis showed that expression of TCR Vα subfamilies were evaluated in one human study, and, unlike TCR Vβ, TCR Vα presented a more restricted usage. Despite the great variability in the usage of TCR Vβ regions in human Chagas disease, a down-regulation of TCR Vβ5 expression by T cells from patients in the acute phase of the disease was shown. Opposingly, this TCR region was found overly expressed in CD4+ T cells from chronic Chagas patients. It was also demonstrated that murine Vβ9+ T cells derived from nonlymphoid organs of T. cruzi-infected animals had a modulatory profile, while splenic Vβ9+ T cells produced inflammatory cytokines, indicating that although they display the same TCR Vβ region usage, these cells are functionally distinct. Despite the limitations of few papers and year of publication of the studies, compiling the data derived from them reveals that further investigation of TCR usage will point to their potential role in protective or pathogenic responses, as biomarkers of disease progression, and in the search for dominant peptides potentially useful for the development of vaccines or therapies.

Rapid identification of tumor-reactive T-cell receptors by RNA preamplification-based single-cell sequencing

T-cell receptor (TCR)-transduced T (TCR-T) cell therapy has shown promising efficacy in the clinical treatment of malignant cancers. However, the populations covered by reported TCRs are still limited. Tumor infiltrating lymphocytes (TILs) are natural reservoirs of tumor-reactive T cells and TCRs. Approaches are required for the fast and cost-effective identification of tumor-reactive TCRs from TILs. The widely employed TCR identification approaches by the clonal expansion of TILs involve a TCR singularization process for the direct pairing of TCR Vα and the Vβ chain. However, the clonal expansion of T cells is well known to require extensive time and effort due to the involvement of T cell cultures. Several single-cell multiplexing PCR methods followed by Sanger sequencing have been developed, representing a cost-effective and fast approach for single-cell TCR identification. In this study, an RNA-based preamplification step was included in the single-cell TCR sequencing, which can reduce the multiplexing PCR amplification to one round. Moreover, the cDNA product of RNA preamplification is derived from the whole genome mRNA, instead of TCR mRNA only by multiplexing primers-based DNA preamplification, which is valuable for many other analyses (e.g., phenotypic analysis) of the tumor-reactive T cells that can be correlated with the identified TCRs. The feasibility for both single α chain and dual α chain TILs of this approach highlights its potential value as a rapid and cost-effective sequencing strategy for the development of TCR-T therapies for solid cancers.

T Cells Engaging the Conserved MHC Class Ib Molecule Qa-1b with TAP-Independent Peptides Are Semi-Invariant Lymphocytes.

The HLA-E homolog in the mouse (Qa-1b) is a conserved MHC class Ib molecule presenting monomorphic peptides to germline-encoded natural killer receptor CD94/NKG2A. Previously, we demonstrated the replacement of this canonical peptide by a diverse peptidome upon deficiency of the TAP peptide transporter. Analysis of this Qa-1b-restricted T cell repertoire against these non-mutated neoantigens revealed characteristics of conventional hypervariable CD8+ T cells, but also of invariant T cell receptor (TCR)αβ T cells. A shared TCR Vα chain was used by this subset in combination with a variety of Vβ chains. The TCRs target peptide ligands that are conserved between mouse and man, like the identified peptide derived from the transcriptional cofactor Med15. The thymus selection was studied in a TCR-transgenic mouse and emerging naïve CD8+ T cells displayed a slightly activated phenotype, as witnessed by higher CD122 and Ly6C expression. Moreover, the Qa-1b protein was dispensable for thymus selection. Importantly, no self-reactivity was observed as reported for other MHC class Ib-restricted subsets. Naïve Qa-1b restricted T cells expanded, contracted, and formed memory cells in vivo upon peptide vaccination in a similar manner as conventional CD8+ T cells. Based on these data, the Qa-1b restricted T cell subset might be positioned closest to conventional CD8+ T cells of all MHC class Ib populations.

Towards an Understanding of Protective Cellular Immunity - a TCR Transgenic Mouse Reactive with Chlamydia muridarum

Abstract A major limitation in understanding the development of protective T cell memory against Chlamydia is the difficulty in characterizing low frequencies of antigen-specific T cells. To better understand the kinetics and phenotype of specific CD4 T cells, we developed a C. muridarum-specific T-Cell Receptor (TCR) transgenic (Tg) mouse. Primed T cells from C57BL/6 mice immune to C. muridarum were stimulated with EB/RB for 5 days and fused with BW5147 cells. Specific clones producing IL-2 and IFN-γ were harvested and screened for TCR Vα and Vβ by qPCR. Cloned transgenes were extracted, purified, and co-microinjected into fertilized oocytes. PCR and FACS confirmed integration and expression. Tg T cells were analyzed ex vivo for activation markers and cytokine production after EB/RB stimulation. 90% of peripheral CD4+ T cells express TCR transgenes and exhibited enhanced proliferation and increased expression of IFN-γ, IL-2, and CD69, compared to controls. Naive T cells were adoptively transferred from CD45.2 Tg mice into CD45.1 mice one day before intravaginal inoculation with C. muridarum. Tg cells showed enhanced proliferation and TNF-α, IL-2, and IFN-γ production by day 5 compared to controls. Tg T cells collected from SLOs and oviducts on days 5, 8, 12, 22, 44, and 65 post-infection exhibited enhanced expression of memory and activation markers. Adoptive transfer of Tg or WT CD4 T cells to Rag1−/− mice rescued an otherwise lethal C. muridarum genital infection. WT and Tg CD4 T cells exhibited comparable chlamydial clearance from the genital tract. Adoptive transfer of Chlamydia-specific CD4+ T cells offers a powerful approach to characterize cellular activation, differentiation, and memory development.

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

Distribution and Clonality of the Vα and Vβ T-Cell Receptor Repertoire of Regulatory T Cells in Leukemia Patients With and Without Graft Versus Host Disease

Graft versus host disease (GVHD) is the main complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Recent data indicated that regulatory T (Treg) cells might relate to GVHD, and such functions might be mediated by certain T-cell receptor (TCR) subfamily of Treg cells. Thus, we analyzed the distribution and clonality of the TCR Vα and Vβ repertoire of Treg cells from leukemia patients with and without GVHD after allo-HSCT. Numerous TCR Vα subfamilies, including Vα1, Vα9, Vα13, Vα16-19, and Vα24-29, were absent in Treg cells after allo-HSCT. The usage numbers for the TCR Vα and Vβ subfamilies in Treg cells from patients without GVHD appeared more widely. The expression frequencies of Vα10 or Vα20 between both groups were significantly different. Moreover, the expression frequency of TCR Vβ2 subfamily in patients without GVHD was significantly higher than that in patients with GVHD. Oligoclonally expanded TCR Vα and Vβ Treg cells were identified in a few samples in both groups. Restricted utilization of the Vα and Vβ subfamilies and the absence of some important TCR rearrangements in Treg cells may be related to GVHD due to a lower regulating function of Treg subfamilies.

T cell receptor (TCR) V gene usage in patients with systemic necrotizing vasculitis.

Wegener's granulomatosis (WG) and polyarteritis nodosa (PAN) are systemic necrotizing vasculitides of unknown etiology. These disorders run a fatal course if untreated. T lymphocytes are implicated in the pathogenesis of WG, since they have been found to infiltrate affected organs, and sIL-2R correlates with disease activity. To elucidate further the role of T cells in necrotizing vasculitis, we have used a panel of 12 TCR V-specific MoAbs to investigate the number of cells expressing certain V alpha and V beta gene segments in the CD4+ and CD8+ subsets of altogether 11 patients with WG or PAN. In the group of patients, we found abnormal expansions of T cells using particular TCR V alpha or V beta gene products. These T cell expansions were more numerous, of a dramatically higher magnitude, and frequently more often found in the CD4 subset, compared with T cell expansions identified in healthy individuals. In long-term studies of the T cell expansions for up to 18 months, a heterogeneous pattern was revealed, with no obvious correlation to clinical features such as disease activity or treatment. Studies of TCR V gene usage in this group of patients may help in understanding the pathogenesis of necrotizing vasculitis, and in the identification of unknown antigens, and may open the possibility to a highly selective immunotherapy by targeting disease-mediating T cells.

CTLA-4 controls the thymic development of both conventional and regulatory T cells through modulation of the TCR repertoire

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4; CD152) is of pivotal importance for self-tolerance, with deficiency or unfavorable polymorphisms leading to autoimmune disease. Tolerance to self-antigens is achieved through thymic deletion of highly autoreactive conventional T (Tconv) cells and generation of FoxP3(+) regulatory T (Treg) cells. The main costimulatory molecule, CD28, augments the negative selection of Tconv cells and promotes the generation of FoxP3(+) Treg cells. The role of its antagonistic homolog CTLA-4, however, remains a topic of debate. To address this topic, we investigated the thymic development of T cells in the presence and absence of CTLA-4 in a T-cell receptor (TCR) transgenic mouse model specific for the myelin basic protein peptide Ac1-9. We reveal that CTLA-4 is expressed in the corticomedullary region of the thymus. Its absence alters the response of CD4(+)CD8(-) thymocytes to self-antigen recognition, which affects the quantity of the Treg cells generated and broadens the repertoire of peripheral Tconv cells. T-cell repertoire alteration after deletion of CTLA-4 results from changes in TCR Vα and Jα segment selection as well as CDR3α composition in Tconv and Treg cells. CTLA-4, therefore, regulates the early development of self-reactive T cells in the thymus and plays a key role in central tolerance.

A new method for quantitative analysis of the T cell receptor V region repertoires in healthy common marmosets by microplate hybridization assay

The common marmoset, Callithrix jacchus, is one of the smallest primates and is increasingly used for an experimental nonhuman primate model in many research fields. Analysis of T cell receptor (TCR) repertoires is a powerful tool to investigate T cell immunity in terms of antigen specificity and variability of TCR expression. However, monoclonal antibodies specific for many TCR Vα or Vβ chains have not been created. We have recently identified a large number of TCRα chain variable (TRAV) and TCRβ chain variable (TRBV) sequences from a cDNA library of common marmosets. The purpose of this study is to develop a new method for analysis of TCR repertoires in the common marmoset using this sequence information. This method is based on a microplate hybridization technique using 32 TRAV-specific and 32 TRBV-specific oligoprobes following an adaptor-ligation PCR. This enables the easy quantitation of the respective TRAV and TRBV expression levels. No cross-hybridization among specific-oligoprobes and very low variances in repeated measures of the same samples was found, demonstrating high specificity and reproducibility. Furthermore, this method was validated by an antihuman Vβ23 antibody which specifically bound to marmoset Vβ23. Using this method, we analyzed TCR repertoires from various tissue samples (PBMCs, spleen, lymph node and thymus) and isolated T cell subpopulations (CD4+CD8+, CD4+CD8− and CD4−CD8+) from the thymus of 10 common marmosets. Neither tissue-specific nor T cell subpopulation-specific differences was found in TRAV and TRBV repertoires. These results suggest that, unlike mice, TCR repertoires in the common marmoset are not affected by endogenous superantigens and are conserved among individuals, among tissues, and among T cell subpopulations. Thus, TCR repertoire analysis with high specificity and reproducibility is a very useful technique, with the potential to replace flow cytometric analysis using a panel of TRV-specific antibodies, many of which remain unavailable.

An anti-human ovarian carcinoma and CD3 bispecific single-chain antibody mediates CDR3 spectratype drift of T cell receptor alpha and beta chains

To analyze the drift of T cell receptor (TCR) Vα and Vβ gene family CDR3 spectratype in response to ovarian carcinoma cells mediated by an anti-human ovarian carcinoma/CD3 bispecific single-chain antibody (BHL-1), and explore the mechanism of the bispecific single-chain antibody-mediated T cell immune response. Immunoscopic spectratyping technique was used to analyze the TCR repertoire diversity (CDR3 spectratype distribution) of the T cells from 6 healthy donors before and after stimulation of the cells with human ovarian carcinoma in the presence of BHL-1. The predominant usage of TCR α and Vβ chain CDR3 was analyzed after the stimulation, and sequence analysis was performed for the CDR3 region of the monoclonal T cells. The spectratypes of Vα and Vβ gene family TCR CDR3 region showed a Gaussian distribution before stimulation of the T cells from the 6 donors. After stimulation of the T cells, CDR3 spectratype drift occurred in the T cells, and some TCR Vα and Vβ families showed an anomalous and oligoclonal expansion. Different CDR3 sequences of the Vα and Vβ gene family TCR were found in the monoclonal T cells stimulated with BHL-1. CDR3 spectratype drift occurs in TCR α and Vβ chains of T cells after stimulation with human ovarian carcinoma cells and BHL-1, indicating that the predominant usage of TCR Vα and Vβ families is associated with the specific T cell immune response mediated by BHL-1.

Biomolecular Characteristics of Specific Regulartory T Cells of Graft-Versus-Disease Based on T-Cell Receptor Analysis,

Abstract 4067Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an important therapetic option for a number of malignant and refractory haematological diseases. Graft versus host disease (GVHD) is the main complications following allo-HSCT. Recent data indicated that regulartory T (Treg) cells might participate in mediating GVHD and graft-versus-leukemia (GVL) effect after allo-HSCT. However, the distribution and clonality of T cell receptor (TCR) repertoire of specific Treg cells in GVHD remains unclear. To further characterize this feature, we analyzed the distribution and clonality of TCR repertoire of Treg cells at GVHD onset to further understand the biological characteristics of specific Treg cells subsets of GVHD.The complementarity-determining region 3 (CDR3) sizes of TCR repertoire (TCR Vα, Vβ, Vδ and Vγ subfamilies) were analyzed in the Treg cells of recipients at GVHD onset, using RT-PCR and genescan technique. To determine the expression levels and expression pattern of TCR Vγ I-III subfamily genes, the expression levels and expression pattern of CD3 γ, δ, ε, ζ genes, and the expression levels of regulatory genes of Treg cells (FOXP3 and GATA-3 genes), we performed quantitative analysis by real-time PCR.The frequency of TCR Vβ subfamilies of Treg cells at GVHD onset was significant lower than that in the Treg cells without GVHD. Oligoclonality were detected in TCR Vα 15, Vα 23, Vβ 14 and Vδ 3 subfamilies of the Treg cells from GVHD patients, and oligoclonality were detected in TCR Vα 4, Vα 21, Vβ 16 and Vβ 19 subfamilies of the Treg cells from patients without GVHD. The TCR Vγ I-III subfamilies expression levels in Treg cells were decreased significantly after allo-HSCT. The expression pattern of TCR Vγ subfamilies of Treg cells at GVHD onset was TCR Vγ III>TCR Vγ I>TCR Vγ II, and different with the expression pattern of Treg cells without GVHD (TCR Vγ I>TCR Vγ II>TCR Vγ III). The expression pattern of CD3 γ, δ, ε, ζ genes of Treg cells at GVHD onset was also changed, and GVHD might mainly influence the expression levels of CD3 δ and CD3 ε genes of Treg cells. The expression levels of GATA-3 gene had a positive correlation with the expression levels of FOXP3 gene in Treg cells with GVHD. The expression levels of GATA-3 gene in Treg cells at GVHD onset were significant higher than that in the Treg cells without GVHD, but the expression levels of FOXP3 gene in Treg cells at GVHD onset were significant lower than that in the Treg cells without GVHD.In conclusion, oligoclonality of TCR Vα 15, Vα 23, Vβ 14 and Vδ 3 subfamilies of Treg cells might related with GVHD. The expression pattern of high TCR Vγ III gene expression and low TCR Vγ II gene expression in Treg cells might correlate with GVHD. GVHD might mainly influence the expression levels of CD3 δ and CD3 ε genes in TCR signal transduction of Treg cells. The high expression levels of GATA-3 gene in Treg cells might play a role in mediating GVHD, and GATA-3 could be a novel treatment target of GVHD immunotherapy. Disclosures:Wu:Guangdong Natural Science Foundation (No.10451051501005778), Science and Technology Planning Project of Guangdong Province of China (No.2009A030200007) and China Postdoctoral Science Foundation (No.200902332, No.20080440776): Research Funding. Liu:Guangdong Natural Science Foundation (No.10451051501005778), Science and Technology Planning Project of Guangdong Province of China (No.2009A030200007) and China Postdoctoral Science Foundation (No.200902332, No.20080440776): Research Funding.

Immune pathomechanism of drug hypersensitivity reactions

Drug hypersensitivity research has progressed enormously in recent years, and a greater understanding of mechanisms has contributed to improved drug safety. Progress has been made in genetics, enabling personalized medicine for certain drugs, and in understanding drug interactions with the immune system. In a recent meeting in Rome, the clinical, chemical, pharmacologic, immunologic, and genetic aspects of drug hypersensitivity were discussed, and certain aspects are briefly summarized here. Small chemicals, including drugs, can induce immune reactions by binding as a hapten to a carrier protein. Park (Liverpool, England) demonstrated (1) that drug haptens bind to protein in patients in a highly restricted manner and (2) that irreversibly modified carrier proteins are able to stimulate CD4(+) and CD8(+) T cells from hypersensitive patients. Drug haptens might also stimulate cells of the innate immune system, in particular dendritic cells, and thus give rise to a complex and complete immune reaction. Many drugs do not have hapten-like characteristics but might gain them on metabolism (so-called prohaptens). The group of Naisbitt found that the stimulation of dendritic cells and T cells can occur as a consequence of the transformation of a prohapten to a hapten in antigen-presenting cells and as such explain the immune-stimulatory capacity of prohaptens. The striking association between HLA-B alleles and the development of certain drug reactions was discussed in detail. Mallal (Perth, Australia) elegantly described a highly restricted HLA-B∗5701-specific T-cell response in abacavir-hypersensitive patients and healthy volunteers expressing HLA-B∗5701 but not closely related alleles. Expression of HLA-B∗1502 is a marker known to be necessary but not sufficient to predict carbamazepine-induced Stevens-Johnson syndrome/toxic epidermal necrolysis in Han Chinese. The group of Chen and Hong (Taiwan) described the possible "missing link" because they showed that the presence of certain T-cell receptor (TCR) clonotypes was necessary to elicit T-cell responses to carbamazepine. The role of TCRs in drug binding was also emphasized by Pichler (Bern, Switzerland). Following up on their "pharmacological interactions of drugs with immune receptors" concept (p-i concept), namely that drugs can bind directly to TCRs, MHC molecules, or both and thereby stimulate T cells, they looked for drug-binding sites for the drug sulfamethoxazole in drug-specific TCRs: modeling revealed up to 7 binding sites on the CDR3 and CDR2 regions of TCR Vα and Vβ. Among many other presentations, the important role ofregulatory T cells in drug hypersensitivity was addressed.

Endogenous TCR Recombination in TCR Tg Single RAG-Deficient Mice Uncovered by Robust In Vivo T Cell Activation and Selection

Recombination activating gene (RAG)-deficient TCR (T Cell Receptor) Tg (transgenic) mice are routinely used as sources of monoclonal T cells. We found that after the transfer of T cells from a RAG-2-deficient 5CC7 TCR Tg mice into allogeneic hosts we recovered a population of T cells expressing diverse αβ-TCRs. In fact, in the thymus and spleen of the 5CC7 RAG-2-deficient donor mice, we detected rare T cells expressing non-Tg TCR chains. Similar observations were obtained using T cells from two other TCR transgenic strains, namely RAG-2-deficient aHY and RAG-1-deficient OT-1 mice. The sequences of the endogenous TCR transcripts suggested that gene recombination could occur, albeit quite inefficiently, in the RAG-deficient mice we used. In agreement, we evidenced rare TCR Vα and Vβ-chain transcripts in non-Tg RAG-2-deficient mice. Since in these non-Tg RAG-deficient mice no mature T cells could ever be found, our findings suggested a role for the TCR Tg in rescuing rare recombined endogenous chains. Robust T-cell activation by the allogeneic environment favored the selection and expansion of the rare cells expressing endogenous TCRs. Potential mechanisms involved in the recombination of the endogenous TCR chains in the different strains of RAG-deficient mice used, and in particular the possibility of RAG-1 hypomorphism due to an incomplete knocking out procedure, are discussed. Our findings have important experimental implications for studies using TCR-Tg RAG-deficient cells as monoclonal T cell populations.

Evolution of T-cell clonality in a patient with Ph-negative acute lymphocytic leukemia occurring after interferon and imatinib therapy for Ph-positive chronic myeloid leukemia

IntroductionThe development of Philadelphia chromosome (Ph) negative acute leukemia/myelodysplastic syndrome (MDS) in patients with Ph-positive chronic myeloid leukemia (CML) is very rare. The features of restrictive usage and absence of partial T cell clones have been found in patients with CML. However, the T-cell clonal evolution of Ph-negative malignancies during treatment for CML is still unknown.ObjectiveTo investigate the dynamic change of clonal proliferation of T cell receptor (TCR) Vα and Vβ subfamilies in one CML patient who developed Ph-negative acute lymphoblastic leukemia (ALL) after interferon and imatinib therapy.MethodsThe peripheral blood mononuclear cells (PBMC) samples were collected at the 3 time points (diagnosis of Ph-positive chronic phase (CP) CML, developing Ph-negative ALL and post inductive chemotherapy (CT) for Ph-negative ALL, respectively). The CDR3 size of TCR Vα and Vβ repertoire were detected by RT-PCR. The PCR products were further analyzed by genescan to identify T cell clonality.ResultsThe CML patient who achieved complete cytogenetic remission (CCR) after 5 years of IFN-α therapy suddenly developed Ph-negative ALL 6 months following switch to imatinib therapy. The expression pattern and clonality of TCR Vα/Vβ T cells changed in different disease stages. The restrictive expression of Vα/Vβ subfamilies could be found in all three stages, and partial subfamily of T cells showed clonal proliferation. Additionally, there have been obvious differences in Vα/Vβ subfamily of T cells between the stages of Ph-positive CML-CP and Ph-negative ALL. The Vα10 and Vβ3 T cells evolved from oligoclonality to polyclonality, the Vβ13 T cells changed from bioclonality to polyclonality, when Ph-negative ALL developed.ConclusionsRestrictive usage and clonal proliferation of different Vα/Vβ subfamily T cells between the stages of Ph-positive CP and Ph-negative ALL were detected in one patient. These changes may play a role in Ph- negative leukemogenesis.

Characterization of conserved CDR3 sequence of TCR α- and β-chain genes in peripheral blood T-cells from patients with diffuse large B-cell lymphoma

T-cell immunodeficiency is a common feature in cancer patients, but clonally expanded T cells which were considered to have the specific anti-tumor cytotoxicity can be identified in most cancer patients, including those with hematological malignancies. We previously reported that there were skewed usages of T-cell receptor (TCR) Vα and TCR Vβ subfamily in clonally expanded T cells in the peripheral blood (PB) of patients with diffuse large B-cell lymphoma (DLBCL). In order to investigate the characteristics of the complementarity determining region 3 (CDR3) of TCR α- and β-chain genes in clonally expanded T cells, the sequences of TCR-CDR3 region of clonally expanded TCR Vα and TCR Vβ subfamily T cells were analyzed. Conserved amino acid motifs of TCR-CDR3 region were identified in different clonally expanded T cells. RSxYNTDKLI, SxGG, GGS, and DSxY motifs were found in the TCR Vα subfamily T-cell clones, while SxGTG, SRG, and GTxD motifs existed in the TCR Vβ subfamily T-cell clones. Moreover, the TCR harboring conserved motif in CDR3 region of some T-cell clones had high homology in the three-dimensional spatial structures. These findings revealed the mono/oligoclonally expanded T cells were derived from the stimulation of some antigens in the PB of patients with DLBCL, and may be recognizing an identical antigenic epitope.

Identification of Idiotype TCR Vα6, Vα10 and Vβ23 Relate to APL-Associated Antigen

Differentiation therapy with all-trans retinoic acid in combination with chemotherapy has significantly improved survival in patient with acute promyelocytic leukaemia (APL). However, relapse remains a major problem. In recent years, specific adoptive immunotherapy with tumor-specific T-cells was considered as a new approach to malignancy therapy. Recently, researches are focus on searching the generation of effective tumor antigen specific T-cells. The tumor antigen specific T cells could be determined by analysis of the T-cell receptor (TCR) Vα or Vβ repertoire, which display monoclonal or oligoclonal pattern. In order to develop the specific cellular immunotherapy in APL, in this present study, we analysis the clonality and the pattern of CDR3 sequence of TCR Vα and Vβ repertoire in peripheral blood T cells by RT-PCR and genescan technique, which was expected to define the APL associated TCR Vα or Vβ subfamily T cells. Oligoclonal T cells expressed TCR Vα6, Vα10 or Vβ23 genes were identified from one APL patient. The molecular characteristics of the CDR3 of Vα and Vβ genes rearrangement were Vα6-N-Jα17, Vα10-N-Jα35 and Vβ23-N-Dβ1-NJβ2.7. And the amino acid sequence of CDR3 region in TCR Vα6, Vα10 and Vβ23 subfamilies were CAMRENSAGNK, YLCAGDELLWECA or CASSSKWAGGTYEQY, respectively. These sequences were accepted by GenBank (Accession Number EU544946, EU544947 and EU647219). In conclusions, three idiotype Vα and Vβ sequences were identified in a case with APL, which was thought that may mediate the host specific immunity response for leukemia cells in APL. Farther investigation will be performed on construction the vector containing the antigen specific TCRs for gene transfer to establish TCR modified-T cells for specific immunotherapy of APL.

Analysis of the interindividual conservation of T cell receptor α- and β-chain variable regions gene in the peripheral blood of patients with systemic lupus erythematosus

The aim of this study was to find conserved motifs in specific T cell receptor (TCR) alpha- and beta-chains, and to analyse the association between complementarity determining region 3 (CDR3) spectratype and systemic lupus erythematosus (SLE) activity. TCR alpha-and beta-chain CDR3 spectratypes were analysed in 20 SLE patients. The CDR3 spectratypes of three patients were monitored over time, and the CDR3 regions of clonally expanded T cells were sequenced. CDR3 spectratype analysis showed prominent usage of TCR AV8, AV14, AV23, AV30, AV31, BV2, BV8, BV11, BV14, BV16, BV19 and BV24 families in SLE patients. The CDR3 spectratype showed dynamic change correlating with SLE activity. The sequence of the CDR3 region in clonally expanded T cells suggested a conserved GGX amino acid motif in both alpha- and beta-chains. The Ja34 and Jb2s1 region genes were found in high frequency. Both TCR Valpha and Vbeta gene usage is highly restricted in SLE, suggesting that the TCRs recognize a limited number of antigenic epitopes. The conserved motifs and limited use of joining region genes may indicate the recognition of similar antigenic epitopes in multiple individuals.

The Molecular Characteristics in CDR3 of TCR Vα and Vβ Genes Associated with cGVHD in Patients after Allogeneic Hematopoietic Stem Cell Transplantation.

Successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) requires reconstitution of normal T-cell immunity. Chronic graft-versus-host disease (cGVHD) is one of the major complications following allo-HSCT. The poor reconstitution of T-cell immunity (including the reconstitution of recent thymic output function and T-cell receptor (TCR) repertoire) was associated with cGVHD. In the previous study, we found that cGVHD predicted low TCR rearrangement excision circles (TRECs) levels and slow naïve T-cell recovery. Because GVHD displayed as clonal proliferation of special T-cells clones, which was triggered by donor T cells to recognize the host's allogene antigen, in the present study we analyzed the TCR Vα and Vβ repertoire and cloanlity in patients with cGVHD, in order to find the special T-cell clones associated with cGVHD and evaluate the molecular characteristics of the CDR3 of TCR Vα and Vβ repertoire of GVHD-associated T-cell clones. Peripheral blood mononuclear cells (PBMNCs) were obtained from 5 leukemia patients with cGVHD after allo-HSCT. The expression and cloanlity analysis of TCR Vα and Vβ repertoire were detected by RT-PCR and genescan technique. Six donors served as controls. Almost all of TCR Vα and Vβ repertoire with polyclonal pattern were identified in normal controls. However, the skew expression pattern of TCR Vα and Vβ repertoire could be detected in patients with cGVHD even more than 4 year after allo-HSCT. Among 29 Vα and 24 Vβ subfamilies, there were only 4∼12 Vα and 4∼11 Vβ subfamilies expressed in patients with cGVHD. Oligoclonal or monoclonal expanded T cells were identified in TCR Vα 2, 3, 6, 10, 12, 14, 15, 25, 26 and TCR Vβ 1, 3, 7∼9, 13, 17, 19, 20 subfamilies respectively. The CDR3 sequences were further analyzed and all the sequences were blasted by internet (http://www.ncbi.nlm.nih.gov) and confirmed that it belonged to specific TCR Vα or Vβ gene rearrangement. The lengths of CDR3 were ranged from 12 to 15 amino acids. The molecular characteristics of the CDR3 of TCR Vα and Vβ genes rearrangement were TCRVα 3 (new name: Vα 17*01)-N-Jα 48*01-Cα (motif: CATEVDFGNEKLIF), TCRVα 2 (new name: Vα 12–2*01)-N-Jα 20*01-Cα (motif: CAVNLNDYKLIF), TCRVβ 1 (new name: Vβ 9*01)-N-Dβ 2*01-N-Jβ 2–1*01-Cβ 2 (motif: CASSDPPETYNEQFF), TCRVβ 7 (new name: Vβ 4–3*01)-N-Dβ 1*01-Jβ 1–1*01-Cβ 1 (motif: CASSHESGNTEAFF). Some TCR subfamily genes shared similarity in CDR3 amino acid motif. The role of specific sequences of CDR3 of TCR Vα and Vβ repertoire and T-cell clones will be confirmed in vivo by animal models.