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

tel-00827710, version 1 - 29 May 2013
tag. This approach allows you to work with the same multimer labeled in different colors, thus
improving specificity of the assay (see Note 14) and permits multiplexing different specificities in
the same tube (see Notes 16 and 22). Note that regardless of the source, high-quality monomers are
important, with monomer purity impacting multimerization. Moreover, the choice of streptavidin
reagent is critical, and it is recommended to purchase high-quality streptavidin conjugated to bright
fluorochromes.
7. When using MHC multimers, it is recommended to choose an appropriate method to validate the
specificity of tetramer-stained cells. Positive controls will be multimers targeting abundant
populations of CD8 T cells. For human, EBV BMLF1 280-288 or Influenza A-M1 58-66 can be used as a
positive control. For mice, the strategy will be to stain splenocytes from a TCR-transgenic mouse
with the corresponding multimer (CD8 OT-I T cells that are specific for H2-K b -SIINFEKL
complexes stained with H2-K b -SIINFEKL multimer for instance). It will help you to establish the
assay and ensure that enrichment is sufficient for detection of rare cells. Concerning naïve cells in
humans, MART1 26-35(Leu27) is a good choice, as it will also be a useful reference for establishing
gating parameters for naïve vs. memory populations [30]. Negative control tetramers can be
employed to help establish the assay, although there is the potential to observe CD8 + T cells with
the capacity to bind any MHCI, including self-antigens. An alternative option is the evaluation of
background staining based on the non-specific labeling of CD4 + T cells. That said, recent work has
suggested that even this interaction might be of physiologic relevance [28]. More definitive
controls are also important, such as assessment of TCR CDR3-variable region usage skewing,
peptide-induced TCR downregulation and, after cell sorting, TCR sequences analysis or TCR genes
transfer into immortalized cell lines to show that the specificity can be reconstituted [8].
8. Biotinylated monomers may be stored for months at -80°C. Stability testing is recommended. In
contrast, multimers are less stable, should be stored at 4 o C and ideally should be used within 4
weeks. Best is even to multimerize the amount you will need for each experiment one day before.
9. The use of a « Dump channel » is essential as it excludes cells that bind non-selectively to the MHC
multimer reagents. Its composition has to be reviewed in the context of the experimental aims. For
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