Comparing Pooled Peptides with Intact Protein for Accessing Cross-presentation Pathways for Protective CD8+ and CD4+ T Cells

Abstract

To better understand the mechanisms of intracellular trafficking and presentation of exogenous peptides by antigen-presenting cells (APC), we compared the handling of overlapping 24-mer peptides from HIV Nef either mixed or covalently linked in tandem in one protein. Once internalized, peptides trafficked not only to endosomes but also to cytosol, and activated CD8 ؉ and CD4؉ T cells. In contrast, whole protein was found to traffic only to the endosomal compartments, and primarily activated CD4 ؉ T cells. Finally, with adjuvant, overlapping peptides were capable of protecting against lethal viral challenge, whereas the intact protein was less protective. These data suggest that overlapping long peptides are cross-presented through more varied intracellular routes and are more efficient in priming protective immunity than the whole protein.Despite much progress in vaccine development, there are still several challenges for design of subunit vaccines. Against intracellular pathogens, it is especially important to immunize protective CD4 ϩ and CD8 ϩ T cell responses. The former recognize epitopes presented by major histocompatibility complex (MHC) 3 class II molecules that are normally loaded mainly with peptides derived from exogenous antigens. Natural processing and loading are catalyzed by HLA-or H2-DM in acidic lysosomes (1).By contrast, CD8 ϩ T cell responses typically depend on epitope loading via the class I pathway. This "endogenous" route starts from the cytosol of any cells naturally infected by the relevant virus, which may not include professional antigen presenting cells (pAPCs). Once processed by cytosolic proteases, viral peptide fragments are transported into the endoplasmic reticulum, and loaded onto MHC class I molecules, which then traffic to the surface where they can present to CD8 ϩ T cells (2, 3). For exogenous antigens, "cross-presentation" by pAPCs is essential for priming CD8 ϩ as well as CD4 ϩ T cells.Although they must be crucial for most CD8 ϩ T cell vaccination strategies, the mechanisms underlying cross-presentation are not understood. First, internalized antigens are known to enter the cytoplasm, although the mechanisms remain unknown. Once in the cytoplasm, they can follow the conventional MHC class I pathway, i.e. processing by cytosolic proteasomes or proteases, transport into the endoplasmic reticulum, loading onto MHC class I molecules, and then trafficking to the surface for recognition by T cells (2, 3). Second, antigens internalized into endocytic compartments could be degraded by the local proteases into peptides and then loaded onto MHC class I molecules in the endocytic compartments that are recycled to and from the cell surface (4). Third, endosomes may fuse with the endoplasmic reticulum, allowing direct access of such peptides for loading onto nascent MHC class I molecules there before presentation at the cell surface (5); however, this theory remains controversial (6, 7).It has been hypothesized that endocytic and cytosolic proteases process antigens differently. If ...

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