We observed small clusters of GFP+ cells in draining popliteal LN

We observed small clusters of GFP+ cells in draining popliteal LNs at 24 h post-injection, however amplification of the GFP signal using anti-GFP Ig was required to visualise these rare cells (Fig. 7C). These results suggest pDNA-encoded Ag is in the tissue draining lymph node as early

as 24 h post-injection. As previously described for the EαGFP system, we could detect Y-Ae+ EαRFP+ cells in the subcapsular sinus (Fig. 7D) and paracortical areas of draining LNs, 24 h after EαRFP injection. However many Y-Ae+ cells in the T cell areas were EαRFP negative, suggesting that Ag had already been processed and hence no longer fluorescent, or that these cells contained levels of EαRFP below the limits of detection by immunofluorescence microscopy. We observed cells of a similar phenotype, Y-Ae+EαRFP−, in mice immunised with pCI-EαRFP. Three days after plasmid injection, we detected rare, sparsely distributed Y-Ae+EαRFP− PLX 4720 cells in the subcapsular sinus JAK assay of draining inguinal lymph nodes (Fig. 7E and F). No staining was observed in pCIneo-immunised mice or using the isotype control, mIgG2b (data not shown). We were unable to conclusively demonstrate pMHC+ cells in the T cell areas of peripheral lymph nodes or spleen, presumably because the level of pMHC complex on these very rare cells was below the sensitivity of detection of the immunofluorescence staining protocol. Others

have shown previously that Ag dose has consequences for both the number of pMHC complexes generated and T cell activation in vivo and hence we were interested to know if the apparently low level pMHC we observed on CD11c+ cells was Histamine H2 receptor sufficient for T cell activation and whether the pMHC complex staining we observed 3 days after DNA injection correlated temporally with the activation of Eα-specific CD4+ T cells. We also wanted to establish the precise anatomical localisation and kinetics of CD4+ T cell activation and proliferation following

intramuscular DNA injection and hence determine the relationship between pDNA distribution, pMHC+ cells and T cell activation. Therefore we used adoptive transfer of Eα-specific TEa T cells and kinetic analysis of activation and cell division following injection with Eα-expressing plasmids, to readout antigen presentation in vivo. The TEa TcR recognises the same pMHC complex as the Y-Ae mAb [12] and thus the initial activation/blastogenesis of these cells should be a good indication of the first time these cells see Ag, i.e. the precise timing of Ag presentation. At early timepoints (e.g. 12 h), we observed a transient upregulation of surface CD69 in both non-Tg and Tg CD4 T cells in pCI-EαRFP- and pCIneo-immunised mice, indicative of DNA-induced non-specific activation (data not shown). However by 24 h surface CD69 had returned to control levels (data not shown).

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