8A) No such increase was observed in the pCIneo group This incr

8A). No such increase was observed in the pCIneo group. This increase in the %Tg preceded cell division as no CFSE dye dilution was observed by d3 (data not shown). We speculate that this is indicative of retention of Eα-specific T cells or inhibition of T cell egress from the lymphoid tissues, due to stable APC-T cell interactions as we [22], and others [23] have noted in other T cell priming regimes. There was no corresponding increase in the percentage of non-Tg CD4+ T cells in draining LNs (Fig. 8A), distal peripheral LNs or spleen (data not shown), suggesting that the TEa GDC-0973 ic50 accumulation we

observed was Ag-driven. Concomitantly, we observed significant blastogenesis of Eα-specific T cells, in all tissues of pCI-EαRFP and pCI-EαGFP-immunised mice (Fig. 8A). No TEa blasts mTOR inhibitor therapy were found in pCIneo-immunised groups. These results are strongly suggestive of presentation Eα peptide to Eα-specific CD4+ T cells at d3 following plasmid vaccination and that T cells in the draining, and distal LNs and spleen have seen Ag by this time. In order to determine if there were any differences in the kinetics of T cell activation in these anatomically distinct lymphoid tissues, we analysed cell

division history using adoptive transfer of CFSE-labelled TEa T cells. By d5 we observed Eα-specific T cell division in draining lymph nodes, but little division in more distal peripheral LNs and the spleen (Fig. 8B and C). However by d10 we found TEa division in all lymphoid tissues examined, with the highest proportion of divided cells being found in the spleen. Thus although the T cell response to pDNA-encoded Ag appears to commence in the local draining lymph nodes, this is superceded by responses in the spleen. We also examined intermediate timepoints, and have never observed

the multiple division peaks, typically found when using CFSE for T cell proliferation, suggesting that the Eα-specific T cells had divided in a different location and ADP ribosylation factor once divided had migrated to the tissues examined, or that very few naïve re-circulating T cells synchronously enter cell division, presumably due to limiting amounts of Ag. Only when they have divided more than 6 times have they accumulated sufficiently for us to detect cell division. We were unable to find evidence for Ag presentation at timepoints other than d3. These results correlate with the appearance of pMHC complexes in draining lymph nodes, hence from our data it appears that Ag presentation peaks 3 days after DNA immunisation.

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