| Class I MHC (MHC-I) complexes are displayed on nearly all nucleated cells and predominantly display peptides that are synthesized within the cell. However, professional antigen presenting cells can take up “exogenous” antigenic material and present peptide:MHC-I in a process termed the alternate MHC-I pathway. This can occur via cytosolic or vacuolar pathways and is important in priming naive T cells. Vacuolar alternate class I MHC Ag processing involves binding of exogenous peptides to MHC-I complexes in post-Golgi compartments. We investigated the source and nature of these peptide receptive MHC-1 complexes and the environment of peptide exchange.; We first examined the role of previously bound peptides on the generation of peptide-receptive MHC-I complexes. We found that incubation of macrophages from TAP1−/− mice with a Kb binding peptide could generate a cohort of MHC-I molecules that could subsequently influence presentation of a second “readout” peptide. Pre-incubation of macrophages with FAPGNYPAL, KVVRFDKL or RGYVYQGL peptide enhanced presentation of SIINFEKL peptide in soluble form and to a greater degree from E. coli bacteria expressing ovalbumin. These stabilizing peptides bound Kb complexes for subsequent dissociation and SIINFEKL binding. The high affinity SIINFEKL peptide decreased subsequent FAPGNYPAL presentation, indicating that relative peptide affinities influence Kb binding. Peptide exchange was encouraged at acidic pH values. Thus, peptide dissociation and exchange contributes to presentation of peptides through the alternate MHC-I pathway and this process is influenced by peptide affinity and pH.; Second, we examined the alternate MHC-I pathway in C57BL/6, TAP1−/− and tapasin−/− macrophages. We found that processing from bacterial antigen was diminished for TAP1−/− and tapasin−/− macrophages. Presentation of soluble peptide, however, was undiminished. We hypothesize that TAP1−/− and tapasin−/− macrophages lack MHC-I complexes with sufficient stability to function in the environment of the phagolysosome. Kb complexes on these cell types are less stable on the cell surface and more sensitive to acid pH, but can be stabilized at sub-physiological temperatures to present peptide from a bacterial source. We conclude that the functions of tapasin and TAP are required to produce stable MHC-I complexes that can enter phagolysosomes and acquire peptides derived from particulate sources. |
