Our finding Obeticholic Acid in vivo that Eα-specific T cells accumulate in peripheral

LNs and spleen, 3 days after injection of Eα-expressing plasmids, suggests that these cells are involved in Ag-specific interactions with Ag-bearing APCs. This is unlikely to be simply the result of LN shut down [48], [49] and [50] as the proportion of non-Tg CD4 T cells was unaltered at this timepoint (Fig. 8A). We routinely observe enlarged, hypercellular peripheral LNs between 24 and 48 h after immunisation with all plasmids, including pCIneo (data not shown), presumably due to CpG-driven non-specific inflammation, however we believe that the accumulation and/or inhibition of egress at d3 is Ag-driven and is indicative of ongoing Ag presentation. We also observed Eα-specific TEa blastogenesis at d3 and cell division by d4/d5, which is further indicative of Ag presentation occurring by d3. We were unable to find pMHC+ cells in the spleen, but

the fact that we observed concomitant T cell accumulation and blastogenesis in draining LNs, distal LNs and spleen indicates that these things are happening at diverse locations simultaneously. T cell division in the draining LNs preceded that in the distal LNs and spleen which suggest that although T cells appear to be activated at sites distal to the tissue injection site, perhaps they do not receive sufficient stimulus, Ag dose or inflammation-driven co-stimulation LGK 974 at these earliest time points, to enter cell cycle. While further experiments are required to conclusively determine that cells are dividing at these sites in situ, and have not just migrated, the fact that pDNA reaches lymph nodes distal to the injection site and the spleen, is suggestive of Ag presentation in situ. We cannot rule out Ag presentation, after d3, but we were unable to find pMHC+ cells after this timepoint. Increasing

the sensitivity of the Y-Ae detection method may reveal a longer duration of presentation. The duration oxyclozanide of antigenic stimulus determines the fate of naïve and effector cells, in terms of whether T cells will be activated or deleted. We know that Ag persists in the injection site and potentially the draining lymph node for many weeks, and therefore it is possible that naïve, re-circulating Ag-specific T cells may be subsequently exposed to Ag upon passage through Ag-containing lymphoid tissues, although this will depend on their precursor frequency. Whether or not these subsequently activated cells contribute to the effector or memory response is unclear. Recent evidence suggests that naïve CD4+ T cells that enter the immune response after the peak response, i.e. when Ag is limiting, divide less on primary response, but are better at responding upon subsequent Ag challenge, and acquire a long-lived central memory phenotype [44].