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

tel-00827710, version 1 - 29 May 2013
adjuvant. Specifically, we made an effort to use a relatively low dose of antigen (5x10 5
splenocytes injected/mouse), but the dose of adjuvant was quite high (100µg of poly
I:C/mouse). Poly I:C is known to be toxic at high concentrations and this dose could not be
injected into humans for this reason (Nicodemus and Berek, 2010). Further experiments
should be performed to titrate the immunostimulatory capacity of lower doses of adjuvant. In
parallel, the administration of antigen physically linked with adjuvant, which is known to
reduce toxicity and, involves lower doses of adjuvant, can also be examined.
3) Other recently developed techniques
Not only did tetramer-based enrichment allowed for this new approach to study T cell
responses, but we were able to combine this strategy with other established techniques such as
immunoscope or intracellular cytokine staining to gain a much more in-depth understanding
of the function and specificity of the antigen-specific T cells isolated by enrichment. The
combination of these techniques provides the opportunity to extend T cell analysis to the
study of rare populations and even further to single cell analysis, through the combination of
enrichment and single cell PCR. Several additional techniques have been recently developed
to permit an in-depth study of T cells.
(a) Transfer of single cell
The transfer of TCR-transgenic T cells has provided many details about antigen-specific T
cell responses, mainly because these populations could be followed over time upon transfer
using congenic surface markers. One of the main limitations of these types of studies was that
the data obtained were reflective of a bulk population, not a single cell. It is now well
established that an antigen-specific T cell population is composed of a diverse heterogeneous
mix of cells. To really gain insight into the molecular events of T cell priming and activation,
information at the single cell level is required. Stemberger et al. developed a specialized
injection system, which allowed them to transfer just a single cell. To do this, they purified
CD8 + OT-I T cells, diluted them, and applied them to a glass slide. Under the microscope,
they were able to aspirate a single cell into the tip of a glass microinjection needle. This single
cell was directly transferred intraperitoneally into a recipient mouse (Stemberger et al., 2007).
This work showed the feasibility of the approach and allowed them to demonstrate that a
single transferred T cell has polyfunctional potential and can develop into several effector and
memory subsets.
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