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Direct measurement of T-cell receptor repertoire diversity with AmpliCot.

ABSTRACT: Many studies require the measurement of nucleic acid sequence diversity. Here we describe a method, called AmpliCot, that measures the sequence diversity of PCR products on the basis of DNA hybridization kinetics, thereby avoiding the time, expense and biases associated with cloning and sequencing. SYBR Green dye is used to measure DNA hybridization kinetics in a homogeneous, automated fashion. PCR products are prepared in wholly double-stranded homoduplex form for a baseline measurement of DNA concentration. The DNA is melted and then reannealed under stringent conditions that allow only homoduplexes to form. The sequence diversity of a sample is proportional to the product of its concentration and the time required for it to anneal. After validating AmpliCot with a library of diverse sequences, we use it to measure the diversity of expressed rearrangements of the gene encoding the T-cell antigen receptor (TCR) beta chain. AmpliCot measurements are in good agreement with previous estimates of murine TCR repertoire diversity that required extensive cloning and sequencing.

SUBMITTER: Baum PD

PROVIDER: S-EPMC2366817 | biostudies-literature | 2006 Nov

REPOSITORIES: biostudies-literature

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Direct measurement of T-cell receptor repertoire diversity with AmpliCot.

Baum Paul D PD McCune Joseph M JM

Nature methods 20061101 11

Many studies require the measurement of nucleic acid sequence diversity. Here we describe a method, called AmpliCot, that measures the sequence diversity of PCR products on the basis of DNA hybridization kinetics, thereby avoiding the time, expense and biases associated with cloning and sequencing. SYBR Green dye is used to measure DNA hybridization kinetics in a homogeneous, automated fashion. PCR products are prepared in wholly double-stranded homoduplex form for a baseline measurement of DNA ...[more]

PMID: 17060913

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Project description:The tumor immune response is dependent on the interaction between tumor cells and the T-cell subset expressing the T-cell receptor (TCR) repertoire that infiltrates into the tumor microenvironment. The present study explored the diversity and shared TCR repertoires expressed on the surface of locoregional T cells and identified the T lymphocyte subsets infiltrating into esophageal squamous cell carcinoma (ESCC), in order to provide insight into the efficiency of immunotherapy and the development of a novel immune-oriented therapeutic strategy. A total of 53 patients with ESCC were enrolled in the present study, and immunohistochemical analysis of CD3, CD8, CD45RO, FOXP3, CD274, HLA class I and AE1/AE3 was performed. Digital pathological assessment was performed to evaluate the expression level of each marker. The clinicopathological significance of the immuno relation high (IR-Hi) group was assessed. Adaptor ligation PCR and next-generation sequencing were performed to explore the diversity of the TCR repertoire and to investigate the shared TCR repertoire in the IR-Hi group. Repertoire dissimilarity index (RDI) analysis was performed to assess the diversity of TCR, and the existence of shared TCRα and TCRβ was also investigated. Further stratification was performed according to the expression of markers of different T-cell subsets. Patients were stratified into IR-Hi and immuno relation low (IR-Lo) groups. Cancer-specific survival and recurrence-free survival rates were significantly improved in the IR-Hi group compared with in the IT-Lo group. The diversity of the TCR repertoire was significantly higher in the IR-Hi group. TCR repertoire analysis revealed 27 combinations of TCRα and 23 combinations of TCRβ VJ regions that were shared among the IR-Hi group. The IR-Hi group was divided into three clusters. Overall, the current findings revealed that the IR-Hi group maintained the diversity of TCR, and a portion of the IR-Hi cases held the T cells with shared TCR repertoires, implying recognition of shared antigens. The prognosis of patients with ESCC was affected by the existence of immune response cells and may possibly be stratified by the T-cell subsets.

Project description:BackgroundThe importance of CD4⁺ and CD8⁺ T cells in protection against tuberculosis (TB) is well known, however, the association between changes to the T cell repertoire and disease presentation has never been analyzed. Characterization of T-cells in TB patients in previous study only analyzed the TCR β chain and omitted analysis of the Vα family even though α chain also contribute to antigen recognition. Furthermore, limited information is available regarding the heterogeneity compartment and overall function of the T cells in TB patients as well as the common TCR structural features of Mtb antigen specific T cells among the vast numbers of TB patients.Methodology/principal findingsCDR3 spectratypes of CD4⁺ and CD8⁺ T cells were analyzed from 86 patients with TB exhibiting differing degrees of disease severity, and CDR3 spectratype complexity scoring system was used to characterize TCR repertoire diversity. TB patients with history of other chronic disease and other bacterial or viral infections were excluded for the study to decrease the likely contribution of TCRs specific to non-TB antigens as far as possible. Each patient was age-matched with a healthy donor group to control for age variability. Results showed that healthy controls had a normally diversified TCR repertoire while TB patients represented with restricted TCR repertoire. Patients with mild disease had the highest diversity of TCR repertoire while severely infected patients had the lowest, which suggest TCR repertoire diversity inversely correlates with disease severity. In addition, TB patients showed preferred usage of certain TCR types and have a bias in the usage of variable (V) and joining (J) gene segments and N nucleotide insertions.Conclusions/significanceResults from this study promote a better knowledge about the public characteristics of T cells among TB patients and provides new insight into the TCR repertoire associated with clinic presentation in TB patients.

Project description:T cell populations are regulated both by signals specific to the T-cell receptor (TCR) and by signals and resources, such as cytokines and space, that act independently of TCR specificity. Although it has been demonstrated that disruption of either of these pathways has a profound effect on T-cell development, we do not yet have an understanding of the dynamical interactions of these pathways in their joint shaping of the T cell repertoire. Complete DiGeorge Anomaly is a developmental abnormality that results in the failure of the thymus to develop, absence of T cells, and profound immune deficiency. After receiving thymic tissue grafts, patients suffering from DiGeorge anomaly develop T cells derived from their own precursors but matured in the donor tissue. We followed three DiGeorge patients after thymus transplantation to utilize the remarkable opportunity these subjects provide to elucidate human T-cell developmental regulation. Our goal is the determination of the respective roles of TCR-specific vs. TCR-nonspecific regulatory signals in the growth of these emerging T-cell populations. During the course of the study, we measured peripheral blood T-cell concentrations, TCRbeta V gene-segment usage and CDR3-length spectratypes over two years or more for each of the subjects. We find, through statistical analysis based on a novel stochastic population-dynamic T-cell model, that the carrying capacity corresponding to TCR-specific resources is approximately 1000-fold larger than that of TCR-nonspecific resources, implying that the size of the peripheral T-cell pool at steady state is determined almost entirely by TCR-nonspecific mechanisms. Nevertheless, the diversity of the TCR repertoire depends crucially on TCR-specific regulation. The estimated strength of this TCR-specific regulation is sufficient to ensure rapid establishment of TCR repertoire diversity in the early phase of T cell population growth, and to maintain TCR repertoire diversity in the face of substantial clonal expansion-induced perturbation from the steady state.

Dataset Information

Direct measurement of T-cell receptor repertoire diversity with AmpliCot.

Publications

Direct measurement of T-cell receptor repertoire diversity with AmpliCot.

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