Although the percentage of GFP+ cells in the CD11clow/− population following 10 μg, 1 μg and 0.1 μg Ag doses appeared elevated compared to PBS/LPS control, particularly check details in draining CLN and BLN, these were not statistically significant. The proportion of CD11clow/− cells containing GFP
following 100 μg Ag, was higher in the local cervical and brachial LNs than in more distal inguinal and axial LNs (data not shown). Background correction, calculated by subtracting mean values for PBS control from dose values revealed that GFP+ cells could be detected at low Ag doses ( Fig. 2A and B, insets). The amount of cell-associated GFP from doses less than 100 μg may be below the level of sensitivity of GFP detection by flow cytometry. Lymphoid tissue autofluorescence also impacts on assay sensitivity. Analysis of cells displaying pMHC complexes (i.e. Y-Ae+) revealed that we could detect complexes in more than 20% of all CD11chigh cells in the draining CLNs (Fig. 2C) and BLNs (not shown) at the 100 μg dose. Decreasing amounts of Ag resulted in corresponding
decreases in the percentages of CD11c+Y-Ae+ cells, with the limit of detection of pMHC complexes between 1 μg and 100 ng of administered Ag. pMHC complex detection in CD 11clow/− selleck kinase inhibitor cells showed a similar trend. As was the case for detection of GFP+ cells, variability within the small group (n = 3), limited statistical significance. Both the CD11chigh and CD11clow/− populations also showed increased, although not statistically significant, Y-Ae mean fluorescence down to a dose of 100–10 ng Ag (data not shown). These results indicate that with controlled and careful detailed analyses, we can detect both Ag and cells displaying pMHC complexes following administration Dipeptidyl peptidase of about 1 μg–100 ng Ag, and this is the upper limit of Ag that we might expect to be produced following pDNA injection. The kinetics of Ag distribution and presentation is likely to vary depending on the route (e.g. subcutaneous vs. intramuscular) and the type of immunisation (e.g. protein vs. pDNA), and we wished to determine the kinetics of appearance of pMHC complexes for both protein and pDNA immunisation. The aim of this protein
injection study was to study the kinetics of Ag distribution in a widely studied situation such as subcutaneous injection. As has been shown for EαRFP previously , EαGFP+ cells, i.e. cell-associated EαGFP, can be found in the neck-draining CLNs and BLNs within 1 h of Ag injection in both CD11chigh (Fig. 3A) and CD11clow/− (Fig. 3B) cells. Fluorescence microscopy indicated that in addition to this cell-associated Ag, much of the injected Ag appeared to be extracellular (Fig. 1D). After this initial wave of antigen positive cells in the draining LNs, the number of cells carrying or associated with Ag decreased until 12–24 h when GFP+ cells reappeared in draining LNs. CD11c+GFP+ cells reappeared in the BLNs prior to their reappearance in the CLNs (Fig.