Ultrastructural study of internalization and recycling of antigen by antigen presenting cells.

The evidence which suggests that a helper T (Th) cell recognizes a processed form of a soluble protein antigen in association with a class II MHC antigen on the surface of an antigen presenting cell (APC) has raised many questions and much controversy. A major question that remains unanswered is what is the cellular site(s) and mechanism(s) in which such an antigen is handled by an APC. The controversy relates to the issue of whether a protein antigen is required to be processed by an APC before it is presented to a Th cell. A currently favored hypothesis of antigen presentation, which stems mainly from analyses of T cell reactivity to peptides of protein antigens, is that such antigens are internalized by an APC, processed intracellularly, recycled to the cell surface and then presented to Th cells. As a first test of this hypothesis, we reasoned that although it is currently difficult to study the biochemistry of antigen processing, it is possible to study whether a protein antigen is internalized by an APC and recycled to its surface. In this report, colloidal gold conjugates of pork insulin (PI-Au), a tryptic peptide of pork insulin lacking the insulin receptor-binding portion of the molecule (TI-Au), myoglobin (MYO-Au), and apomyoglobin (APO-Au) were used to follow the pathway(s) and kinetics of antigen internalization and recycling in TA3 B hybridoma cells. Transmission electron micrographs of the routes followed by the PI-Au and TI-Au conjugates suggest that these antigens are internalized and recycled to the surface of an antigen presenting cell within 2-4 hr. In contrast, the patterns obtained for MYO-Au and APO-Au suggest that either the internalization of these antigens serves to channel them into a degradative pathway or that the kinetics of recycling of these antigens is slower. The two types of patterns observed may not be mutually exclusive and the purpose of internalization may vary, depending on the nature of the antigen. These data represent the first analysis of the kinetics and pathways of internalization and recycling of an antigen by an APC. It is impossible to formally prove that the pathway we have demonstrated is the one responsible for processing for antigen presentation. Nevertheless, these results support the notion that a protein antigen is handled intracellularly by an APC and recycled to the surface before it is presented to a Th cell.