Targeting antigen in mature dendritic cells for simultaneous stimulation of CD4(+) and CD8(+) T cells

Due to their potent immunostimulatory capacity, dendritie cells (DQ have be come the centerpiece of many vaccine regimens. Immature DC (DCimm) capture, process, and present Ags to CD4(+) lymphocytes, which reciprocally activat e DCimm through CD40, and the resulting mature DC (DCmat) loose phagocytic capacity, but acquire the ability to efficiently stimulate CD8(+) lymphocyt es. Recombinant vaccinia viruses (rVV) provide a rapid, easy, and efficient method to introduce Ags into DC, but we observed that rVV infection of DCi mm results in blockade of DC maturation in response to all activation signa ls, including CD40L, monocyte-conditioned medium, LPS, TNF-alpha, and poly( I:C), and failure to induce a CD8(+) response. By contrast, DCmat can be in fected with rVV and induce a CD8(+) response, but, having lost phagocytic a ctivity, fail to process the Ag via the exogenous class 11 pathway. To over come these limitations, we used the CMV protein pp65 as a model Ag and desi gned a gene containing the lysosomal-associated membrane protein I targetin g sequence (Sig-pp65-LAMP1) to target pp65 to the class 11 compartment. DCm at infected with rVV-Sig-pp65-LAMP1 induced proliferation of pp65-specific CD4(+) clones and efficiently induced a pp65-specific CD4(+) response, sugg esting that after DC maturation the intracellular processing machinery for class I I remains intact for at least 16 h. Moreover, infection of DCmat wi th rVV-Sig-pp65-LAMP1 resulted in at least equivalent presentation to CD8() cells as infection with rVV-pp65. These results demonstrate that despite rVV interference with DCimm maturation, a single targeting vector can deliv er Ags to DCmat for the effective simultaneous stimulation of both CD4(+) a nd CD8(+) cells.