Article

Primer sets for cloning the human repertoire of T cell Receptor Variable regions.

Department of Medical Sciences and Interdisciplinary Research Centre for Autoimmune Diseases, via Solaroli 17, 28100, Novara, Italy.
BMC Immunology (Impact Factor: 2.61). 09/2008; 9:50. DOI: 10.1186/1471-2172-9-50
Source: PubMed

ABSTRACT Amplification and cloning of naïve T cell Receptor (TR) repertoires or antigen-specific TR is crucial to shape immune response and to develop immuno-based therapies. TR variable (V) regions are encoded by several genes that recombine during T cell development. The cloning of expressed genes as large diverse libraries from natural sources relies upon the availability of primers able to amplify as many V genes as possible.
Here, we present a list of primers computationally designed on all functional TR V and J genes listed in the IMGT, the ImMunoGeneTics information system. The list consists of unambiguous or degenerate primers suitable to theoretically amplify and clone the entire TR repertoire. We show that it is possible to selectively amplify and clone expressed TR V genes in one single RT-PCR step and from as little as 1000 cells.
This new primer set will facilitate the creation of more diverse TR libraries than has been possible using currently available primer sets.

0 Bookmarks
 · 
161 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Sjögren's syndrome (SjS), an autoimmune disease characterized by exocrine gland dysfunction leading to dry mouth and dry eye diseases, is typified by progressive leukocyte infiltrations of the salivary and lacrimal glands. Histologically, these leukocyte infiltrations generally establish peri-ductal aggregates, referred to as lymphocytic foci (LF), that occassionally appear as germinal center-like structures. The formation and organization of these LF suggest an important and dynamic role for helper T cells (TH), specifically TH1, TH2 and the recently discovered TH17, in development and onset of clinical SjS, considered a B cell-mediated hypersensitivity type 2 disease. Despite an ever-increasing focus on identifying the underlying etiology of SjS, defining factors that initiate this autoimmune disease remains a mystery. Thus, determining interactions between infiltrating TH cells and exocrine gland tissue (auto-)antigens represents a fertile research endeavor. This review discusses pathological functions of TH cells in SjS, the current status of TH cell receptor gene rearrangements associated with human and mouse models of SjS, and potential future prospects for identifying receptor-autoantigen interactions. This article is protected by copyright. All rights reserved.
    Scandinavian Journal of Immunology 05/2013; · 2.20 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Human induced pluripotent stem cells (hiPSCs) have enormous potential for the treatment of inherited and acquired disorders. Recently, antigen-specific T lymphocytes derived from hiPSCs have been reported. However, T lymphocyte populations with broad T cell receptor (TCR) diversity have not been generated. We report that hiPSCs derived from skin biopsy are capable of producing T lymphocyte populations with a broad TCR repertoire. In vitro T cell differentiation follows a similar developmental program as observed in vivo, indicated by sequential expression of CD7, intracellular CD3 and surface CD3. The γδ TCR locus is rearranged first and is followed by rearrangement of the αβ locus. Both γδ and αβ T cells display a diverse TCR repertoire. Upon activation, the cells express CD25, CD69, cytokines (TNF-α, IFN-γ, IL-2) and cytolytic proteins (Perforin and Granzyme-B). These results suggest that most, if not all, mechanisms required to generate functional T cells with a broad TCR repertoire are intact in our in vitro differentiation protocol. These data provide a foundation for production of patient-specific T cells for the treatment of acquired or inherited immune disorders and for cancer immunotherapy.
    PLoS ONE 01/2014; 9(5):e97335. · 3.73 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: T-cells are central players in the immune response against both pathogens and cancer. Their specificity is solely dictated by the T-cell receptor (TCR) they clonally express. As such, the genetic modification of T lymphocytes using pathogen- or cancer-specific TCRs represents an appealing strategy to generate a desired immune response from peripheral blood lymphocytes. Moreover, notable objective clinical responses were observed in terminally ill cancer patients treated with TCR-gene modified cells in several clinical trials conducted recently. Nevertheless, several key aspects of this approach are the object of intensive research aimed at improving the reliability and efficacy of this strategy. Herein, we will survey recent studies in the field of TCR-gene transfer dealing with the improvement of this approach and its application for the treatment of malignant, autoimmune, and infectious diseases.
    Frontiers in Immunology 01/2012; 3:186.

Full-text (3 Sources)

View
33 Downloads
Available from
May 26, 2014