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: 2.25). 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 Followers
 · 
266 Views
  • 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 05/2014; 9(5):e97335. DOI:10.1371/journal.pone.0097335 · 3.53 Impact Factor
  • 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; 78(2). DOI:10.1111/sji.12079 · 1.88 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: During infection and autoimmune disease, activation and expansion of T cells take place. Consequently, the TCR repertoire contains information about ongoing and past diseases. Analysis and interpretation of the human TCR repertoire are hampered by its size and stochastic variation and by the diversity of Ags and Ag-presenting molecules encoded by the MHC, but are highly desirable and would greatly impact fundamental and clinical immunology. A subset of the TCR repertoire is formed by invariant T cells. Invariant T cells express interdonor-conserved TCRs and recognize a limited set of Ags, presented by nonpolymorphic Ag-presenting molecules. Discovery of the three known invariant T cell populations has been a tedious and slow process, identifying them one by one. Because conservation of the TCR α-chain of invariant T cells is much higher than the β-chain, and because the TCR α-chain V gene segment TRAV1-2 is used by two of the three known invariant TCRs, we employed next-generation sequencing of TCR α-chains that contain the TRAV1-2 gene segment to identify 16 invariant TCRs shared among many blood donors. Frequency analysis of individual clones indicates these T cells are expanded in many donors, implying an important role in human immunity. This approach extends the number of known interdonor-conserved TCRs and suggests that many more exist and that these TCR patterns can be used to systematically evaluate human Ag exposure.

Full-text (4 Sources)

Download
56 Downloads
Available from
May 26, 2014