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

tel-00827710, version 1 - 29 May 2013
Several key improvements have been reported since the initial description of multimer
technology [16,8,17]. First, as multimerization is the key to overcoming the relatively low
intrinsic affinity of TCR/MHC interaction, MHC-I multimers now exist as tetramers,
pentamers and dextramers (with the latter containing >10 MHC I / pep complexes). Monomer
production has also been substantially optimized. Most notable is the work of Schumacher
and colleagues, who demonstrated the possibility to generate high-throughput production of
MHC I / pep complexes using a photo-destructible peptide that permits an exchange reaction
with peptides of interest [18]. Additionally, the implementation of a dump channel, dual
tetramer labeling and multiplexing have all helped establish a robust foundation for
translating this technology into the clinics [18-21]. Nonetheless, there exist remaining
technical limitations: staining methods, analysis protocols, validation and data sharing have to
be standardized [22]; and the limit of detection for standard multimer assays is 10 -4 , which
does not allows for direct detection of rare antigen-specific populations such as naive ones
[23].
In order to improve the limit of detection, MHC multimer staining has recently been
combined with magnetic bead enrichment [24], a concept initially developed in mice for
assessment of CD4 + T and CD8 + T cells [25-27]. Following from these studies, our lab, as
well as others, developed a similar enrichment protocol for human CD8 + T cells (Figure 2)
[23,28]. Efforts have been made to standardize the procedure - herein described in details -
and to optimize any details in order to achieve sufficient sensitivity to allow detection of
naive antigen-specific T cells from human peripheral blood. This protocol permits up to 100-
fold increased detection of antigen-specific populations, allowing assessment of populations
with frequencies as low as 10 -6 . As such, it is now possible to characterize the naive T cell
repertoire, opening up new opportunities for defining how T cells are selected, as well as to
investigate aspects of their homeostasis [29]. These approaches may also serve as powerful
strategies for tracking rare antigen-experienced self-, tumor-, transplant- or microbe-specific
T cells, either in mice or in humans, in turn providing insight into parameters that shape
immune T-cell responses. This unit describes our method for labeling antigen-specific CD8 +
T cells obtained from mice or from human peripheral blood with MHC class I multimers, for
enriching and enumerating them, and eventually multiplexing the assay and/or coupling it to
intracellular cytokine staining (ICS) procedure. Future developments in cytometric systems
(e.g., mass spectroscopy-based cytometry) and gene expression studies (e.g., single cell PCR)
will further extend these approaches and provide an unprecedented look at the immune
repertoire [8,11].
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