Colocalization of FcγRI-Targeted Antigen With Class I MHC: Implications for Antigen Processing

The high-affinity receptor for IgG (CD64 or FcγRI) is constitutively expressed exclusively on professional APCs (monocytes, macrophages, and dendritic cells). When Ag is targeted specifically to FcγRI, Ag presentation is markedly enhanced, although the mechanism of this enhancement is unknown. In an effort to elucidate the pathways involved in FcγRI targeting, we developed a model targeted Ag using enhanced green fluorescent protein (eGFP). This molecule, wH22xeGFP, consists of the entire humanized anti-FcγRI mAb H22 with eGFP genetically fused to the C-terminal end of each CH3 domain. wH22xeGFP binds within the ligand-binding region by its Fc end, as well as outside the ligand-binding region by its Fab ends, thereby cross-linking FcγRI. Confocal microscopy studies revealed that wH22xeGFP was rapidly internalized by the high-FcγRI-expressing cell line U937 10.6, but did not associate with intracellular proteins Rab4, Rab5a, or Lamp-1, suggesting that the targeted fusion protein was not localized in early endosomes, recycling vesicles, or lysosomes. Interestingly, wH22xeGFP was found colocalized with intracellular MHC class I, suggesting that FcγRI-targeted Ags may converge upon a class I processing pathway. These data are in agreement with studies in the mouse showing that FcγRI targeting can lead to Ag-specific activation of cytotoxic T cells. Data obtained from these studies should lead to a better understanding of how Ags targeted to FcγRI are processed and under what conditions they lead to presentation of antigenic peptides in MHC class I, as a foundation for the use of FcγRI-targeted Ags as vaccines.