Targeting Antigen in Mature Dendritic Cells for Simultaneous Stimulation of CD4+ and CD8+ T Cells

Abstract

Due to their potent immunostimulatory capacity, dendritic cells (DC) have become the centerpiece of many vaccine regimens. Immature DC (DCimm) capture, process, and present Ags to CD4+ lymphocytes, which reciprocally activate DCimm through CD40, and the resulting mature DC (DCmat) loose phagocytic capacity, but acquire the ability to efficiently stimulate CD8+ lymphocytes. Recombinant vaccinia viruses (rVV) provide a rapid, easy, and efficient method to introduce Ags into DC, but we observed that rVV infection of DCimm results in blockade of DC maturation in response to all activation signals, including CD40L, monocyte-conditioned medium, LPS, TNF-α, and poly(I:C), and failure to induce a CD8+ response. By contrast, DCmat can be infected with rVV and induce a CD8+ response, but, having lost phagocytic activity, fail to process the Ag via the exogenous class II pathway. To overcome these limitations, we used the CMV protein pp65 as a model Ag and designed a gene containing the lysosomal-associated membrane protein 1 targeting sequence (Sig-pp65-LAMP1) to target pp65 to the class II compartment. DCmat infected with rVV-Sig-pp65-LAMP1 induced proliferation of pp65-specific CD4+ clones and efficiently induced a pp65-specific CD4+ response, suggesting that after DC maturation the intracellular processing machinery for class II remains intact for at least 16 h. Moreover, infection of DCmat with 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 deliver Ags to DCmat for the effective simultaneous stimulation of both CD4+ and CD8+ cells.

HLA class I-restricted responses to vaccinia recognize a broad array of proteins mainly involved in virulence and viral gene regulation

“…Although the immunogenicity of virion structural proteins might be expected to be enhanced by their relatively high levels of expression and delivery to professional antigen presentation cells during viral penetration (25,31), we found that only one of 30 structural proteins was immunogenic (0.8%, P ϭ 0.0020) ( Table 8). By contrast, 12 of 50 proteins involved in genome replication (24%) and five of 21 (23.8%) virulence factors were immunogenic.…”

“…In fact, considering that 35 different ORFs were recognized and that a total of 258 potential ORFs were studied, it can be concluded that class I restricted responses can recognize at least 13.6% of the proteins encoded by the vaccinia virus genome. The 35 ORFs recognized were analyzed with respect to stage of expression (early͞intermediate vs. late) (21,(25)(26)(27)(28)(29) and function (structural protein, virulence factor, and regulation). We also analyzed the percent homology of the vaccinia epitopes with the comparable peptides in the genomes of variola major and MVA (Tables 8 and 9; see also Table 18, which is published as supporting information on the PNAS web site).…”

Efficient Generation of Antigen-Specific Cytotoxic T Cells Using Retrovirally Transduced CD40-Activated B Cells

“…For example, infusion of CMVspecific CD8 ϩ T cell clones to hemopoietic stem cell recipients resulted in the restoration of CMV-specific immunity (12,19). Various combinations of APC and CMV Ags have been developed for the generation of CMV-specific T cell responses including CMV-infected fibroblasts (20); CMV-infected MoDC (21,22); MoDC pulsed with pp65 peptides (23,24); MoDC pulsed with recombinant pp65 protein (25); adenovirus vector-infected MoDC (26); vaccinia virus vector-infected MoDC (27); LCL transfected with pp65 gene (28); and retrovirus vector-infected LCL (29). All of the methods described to date have limitations.…”

Induction of Influenza Matrix Protein 1 and MelanA-specific T lymphocytes in vitro using mRNA-electroporated dendritic cells

“…Incorporation of ubiquitin will result in an enhanced formation of peptides for MHC class I presentation, [17][18][19] whereas targeting sequences from the invariant chain (Ii) or lysosome-associated membrane protein (Lamp1) will lead to presentation of the antigen in the context of both MHC class I and class II, thus providing antigen-specific help. [20][21][22][23] Several strategies have been explored to deliver genes into DC. They can be divided into two groups: viral and nonviral strategies.…”