Despite extensive investigations demonstrating that immune responses are induced by many experimental DNA vaccines and that their character and magnitude can be readily manipulated, many of the processes noted above, related to DNA vaccines are still a “black box” with respect to the precise cell phenotypes, cell–cell interactions and

anatomical and temporal aspects of the initiation and maintenance of DNA vaccine immune responses. Studies such as these are difficult because of the paucity of tools necessary BMS-387032 price to investigate these low frequency events, but crucial for the rational design and application of DNA vaccines. We have therefore applied a variety of novel tools to address these questions directly in vivo for the first time. Following intramuscular injection, free and cell-associated pDNA has been found in muscle, peripheral blood [24], lymph nodes draining the injection site [19] and other sites including the bone marrow [25], minutes to months after injection [19], [26], [27] and [28]. Similar to others [19], we found labelled, cell-associated pDNA in the peripheral blood within 1 h of DNA injection and within cells of distal LNs, spleen and bone marrow by 24 h. We have not excluded the possibility that cells may be responsible for pDNA transport to the spleen and bone marrow, however our finding of pDNA in peripheral

blood within 1 h suggests that pDNA is carried as free DNA. Contrary to recent reports [29] we found no evidence for naïve CD4 T cell priming in the BM following pDNA injection. Our finding of pDNA-bearing Venetoclax mw cells in this site may have important consequences for both mobilisation of APC precursors from the BM into the periphery, as well as the maintenance of long-term memory following DNA vaccination. Our data suggests that CD11b+B220−MHCIIlow cells in the BM acquire pDNA. This phenotype is consistent with monocytes or neutrophils [30] which migrate from sites of inflammation to the BM and lead to antigen presentation directly or following engulfment by another APC [30]. Although it is understood that DNA vaccines

result in sustained Ag expression at the site of injection [31], in some cases more than 12 months [16], [31], [32], [33] and [34], the exact contribution of this Ag to initiating and maintaining immune responses is far from clear. all The cell types engaged in antigen production following intramuscular pDNA injection are predominantly myocytes, although direct transfection of, and antigen expression by, haematopoietic cells (including CD11b+ cells) at the injection site, has been reported [21], [35] and [36]. Although it is believed that somatic cells such as myocytes serve as Ag factories, that continue to “tickle” naïve and perhaps memory cells, precisely how and when Ag gets from these Ag depots to CD4 and CD8 T cells in secondary lymphoid tissue is not clear.