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tel-00827710, version 1 - 29 May 2013 (b) Barcoding technology An elegant strategy of cellular barcoding has recently been developed to analyze the kinship between different T cell populations (Schumacher et al., 2010). A retroviral plasmid library was generated in which each individual virus carried a unique molecular “barcode”. T cells were infected and, thus, labeled by retroviral transduction and then reintroduced into mice. After immunization and the resulting differentiation of transferred T cells, different cell subsets were sorted, DNA was isolated and the overlap of barcodes between different functional subsets was analyzed. Using this technique, lineage relationships could be analyzed between T cell subsets. The power of cellular barcoding has already been harnessed to answer two long-standing questions. First, it was used to determine whether populations of T cells found in a specific location or those that share a common functional activity, come from a common progenitor (Schepers et al., 2008). Following this study, van Heijst and colleagues made use of this approach to determine the number of precursors that are recruited to form a given effector T cell population depending on the conditions of immunization (van Heijst et al., 2009). (c) Combicolor approach The consistent improvement of flow cytometry technology has also provided new tools by which to study T cell responses. It is now possible to combine many fluorophores in the same experiment. While one tetramer labeled by one colour was used before to characterize a T cell response, it is now possible to simultaneously detect multiple different antigen-specific T cells with several tetramers within the same sample. In particular, Hadrup and colleagues developed a novel combinatorial method, in which each specific T cell is labeled with a mix of identical tetramers conjugated to different fluorophores. Each antigen-specificity is labeled and identified by a unique fluorophore combination. In this way, 15 specificities can be detected concurrently by using 4 different fluorophores (Hadrup et al., 2009). Depending on the frequency of the cells of interest, cells can be stained directly or upon tetramer enrichment. Although previous studies that characterized the T cell response were performed by examining cells at the population level, the trend is now to study rare populations and characterize the response at the single cell level by using the multiple new techniques described here, either alone or in combination, including tetramer-based enrichment. Indeed, 142

tel-00827710, version 1 - 29 May 2013 it has been demonstrated that the quality of the response at a cell-by-cell level is crucial to predict the outcome of a response. II. IMPACT OF THE ROUTE OF IMMUNIZATION ON CD8 + T CELL CROSS-PRIMING A. The route of immunization impacts the efficiency of CD8 + T cell cross-priming but not the diversity of antigen-specific T cells Using our model of cross-presentation we identified both common characteristics and differences between the CD8 + T cell responses generated after local versus systemic administration of cell-associated antigen. 1) Kinetic of the response Our first question was to examine whether the route of immunization affected the kinetics of establishing a productive T cell response. We initially observed a faster kinetic upon i.v. immunization as compared to i.d. immunization (Figure 21). This result was expected because antigen was delivered directly in the blood, which is screened for antigen in the spleen. In this way, the antigen should be rapidly taken up by APCs and presented to T cells. In contrast, antigen injected i.d. first must reach the draining lymph node, either by itself or after engulfment by migratory DCs prior to T cell activation. This need for more time to arrive at the location of optimal T cell priming could explain the difference in the kinetic of the immune response. 2) Quality of the response More surprisingly we demonstrated that while delayed, the local i.d. immunization leads to a more robust cross-priming, resulting in a higher percentage of IFNγ-producing cells, as well as multifunctional T cells, and a higher IFNγ production on a per cell basis (Figures 24 and 25). Based on prior patient studies and experimental models of HIV, Leishmania major and Mycobacterium tuberculosis, the T cell quality appears to be important for an efficient host response and eventual control of the infectious agent (Almeida et al., 2007; Darrah et al., 2007; Precopio et al., 2007). Therefore, we were able to conclude that local immunization leads to a better, overall response. Interestingly, the tetramer-based enrichment technique allowed us to perform this in-depth study and identify the small differences between the two conditions studied. Despite the enhanced CD8 + T cell cross-priming obtained with i.d. Page 143 of 256

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