Voie d'immunisation et séquence d'administration de l ... - TEL

tel-00827710, version 1 - 29 May 2013
1) Current techniques
(a) Limiting dilutions analysis
For many years, limiting dilution analysis was the standard method used to determine the
frequency of antigen-specific T cells in a particular model. The main limitations of this
approach were the requirement for exogenous stimulation and expansion, which introduced
potential bias and significant inter-assay variability.
(b) Tetramer staining
The generation of MHC-I tetrameric complexes, originally described by Altman and Davis,
represents what has been proven to be a major technical advance for the study of the antigen-
specific T cell responses (Altman et al., 1996). MHC tetramers are reagents that carry 4 MHC
class I-peptide complexes and, thus, have the ability to interact with multiple TCRs at the
surface of a single CD8 + T cell. Fluorescent labeling of tetramers has allowed the
identification of antigen-specific T lymphocytes based on the avidity of their TCR. By
combining this method with other antibody-based staining protocols (surface markers or
intracellular cytokines), it is now possible to phenotypically and functionally characterize the
antigen-specific T cell response. Nevertheless, there remains one important technical
limitation: the limit of detection is relatively high – for tetramer staining, only one cell in 10 4
can be observed, which does not permit the direct detection of rare antigen-specific
populations such as circulating naïve antigen-specific T cells (Figure 13).
(c) ELISPOT and intracellular staining
Both ELISPOT and intracellular staining techniques allow the functional characterization of
the T cell populations of interest. These assays are based on the ability of antigen-specific T
cells to secrete cytokines upon short, in vitro restimulation with cognate peptide. Such
approaches are capable of distinguishing lymphocytes possessing the capacity to secrete a
given cytokine at the time of the assay, indicating the extend of prior T cell priming; however
this response corresponds only to a fraction of the antigen-specific population.
72