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

tel-00827710, version 1 - 29 May 2013
To test these hypothesis, Ova tetramer-positive CD8 + T cells were sorted by FACS, followed
by TCR gene amplification and characterization of the distribution of Vβ-Jβ CDR3 lengths
present. This method, called immunoscope, accurately evaluates TCR diversity (Pannetier et
al., 1993) (Figure 28). Specifically, antigen-specific T cells were sorted based on tetramer
staining upon enrichment. RNA was extracted and cDNA was synthetized. The different Vβ
germline genes can be clustered in 24 families according to their level of homology. PCR
reactions using specific primers for each Vβ family and one for Cβ segment were performed.
This gave the frequency of the different Vβ families in the T cell pool. Then, a second step
was performed to obtain further information: a nested primer specific for the constant region
was used for an elongation step. These products were then analyzed to obtain an
immunoscope profile with the length of the CDR3 region.
Figure 28. Immunoscope allows the determination of the Vβ family diversity as well as the
length of the CDR3 region.
In our experiment, 5x10 3 cells per mouse, isolated from 5 mice per group, were pooled for the
analysis. As a control, we purified twenty-five thousand bulk CD3 + CD8 + T cells from a non-
immunized animal. 22 Vβ families were detected in both the non-immunized and immunized
animals. Data are represented as a profile of the Vβ-Jβ products obtained, plotted in arbitrary
intensity units as a function of the size of the DNA fragment (Pannetier et al., 1993). As
expected, analysis of the expanded antigen-specific cells in immunized animals showed a
non-Gaussian distribution of the peaks as compared to the naïve bulk CD8 + population
(Figure 29A). Notably, the Vβ 12.1 and 13.1 families were highly represented in the
immunized animals, consistent with prior reports (Dillon et al., 1994). (Please note the change
in nomenclature – the populations found here correspond with Vβ 5 and Vβ 8 respectively).
To determine the diversity of the T cell responses the number of distinct peaks detected in all
immunoscope profiles were determined (Figure 29B). As shown, the number of peaks was
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