Targeting dendritic cell mannose receptors by mannosylating antigens represents a promising vaccination strategy. Using the model antigen ovalbumin (OVA) expressed recombinantly in bacterial and yeast vectors, we have previously demonstrated fungal mannosylation enhances antigen immunogenicity in the context of CD4(+) T cell responses. However, because protection against many tumors and pathogens is thought to require MHC class I-restricted T cell responses, the capacity of differentially mannosylated OVA antigens to induce antigen-specific CD8(+) T cell proliferation was determined. We found that mannosylated yeast-derived OVA antigens were more potent than their unmannosylated counterparts at inducing antigen-specific T cell proliferation. However, the type of mannosylation was critical as addition of extensive O-linked mannosylation increased lymphoproliferative responses while the presence of N-linked mannosylation was associated with decreased responses. Mannosylated OVA failed to stimulate TNF-alpha and IL-12 production from dendritic cells. These data suggest that vaccines incorporating mannosylation must take into account how the mannose groups are linked to the core antigen and may need to include an adjuvant to stimulate cytokine production.
The CCAAT displacement protein (CDP-cut/CUTL1/cux) performs a key proliferation-related function as the DNA binding subunit of the cell cycle controlled HiNF-D complex. HiNF-D interacts with all five classes (H1, H2A, H2B, H3, and H4) of the cell-cycle dependent histone genes, which are transcriptionally and coordinately activated at the G(1)/S phase transition independent of E2F. The tumor suppressor pRB/p105 is an intrinsic component of the HiNF-D complex. However, the molecular interactions that enable CDP and pRB to form a complex and thus convey cell growth regulatory information onto histone gene promoters must be further defined. Using transient transfections, we show that CDP represses the H4 gene promoter and that pRB functions with CDP as a co-repressor. Direct physical interaction between CDP and pRB was observed in glutathione-S-transferase (GST) pull-down assays. Furthermore, interactions between these proteins were established by yeast and mammalian two-hybrid experiments and co-immunoprecipitation assays. Confocal microscopy shows that subsets of each protein are co-localized in situ. Using a series of pRB mutants, we find that the CDP/pRB interaction, similar to the E2F/pRB interaction, utilizes the A/B large pocket (LP) of pRB. Thus, several converging lines of evidence indicate that complexes between CDP and pRB repress cell cycle regulated histone gene promoters.
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