Distribution of Productive Antigen‐Processing Activity for MHC class II Presentation in Macrophages

Abstract

We demonstrated that an epitope from the recombinant protective antigen (rPA) of Bacillus anthracis was presented by mature major histocompatibility complex class II (MHC-II) molecules, whereas an epitope from the recombinant virulent (rV) antigen of Yersinia pestis was presented by newly synthesized MHC-II. We addressed which endosomal compartments were involved in the antigen processing of each epitope. Bone-marrow-derived macrophages were subjected to subcellular fractionation; fractions were analysed for the expression of endosomal markers and used as a source of enzyme activity for the processing of rPA and rV antigens. The rPA epitope was productively processed by dense lysosomal fractions and light membrane fractions expressing early endosomal markers Rab5 and early endosomal antigen-1 as well as markers of antigenpresenting compartments (MHC-II, DM, DO and Ii chain). In contrast, the rV epitope was productively processed only by dense fractions with lysosomal activity. No productive antigen-processing activity was associated with fractions of intermediate density expressing Rab7 and Rab9, characteristic of late endosomes. The data suggest that endosomal compartments expressing Rab5 guanosine triphosphatase can productively process protein antigens for presentation by mature MHC class II molecules.

Major Histocompatibility Complex Class II-Peptide Complexes Internalize Using a Clathrin- and Dynamin-independent Endocytosis Pathway

“…In fact, we have found that the kinetics of cell surface pMHC-II arrival on CD63 ϩ antigen processing compartments is significantly delayed relative to that of cell surface MHC-II-Ii complexes. 4 We have also found that internalized MHC-II from the cell surface can traffic back to multivesicular bodies and be secreted on exosomes from B cells (47), demonstrated that the endocytosis pathway identified here leads to MHC-II delivery to multivesicular antigen processing compartments. Given the large number of pMHC-II on the cell surface at any given time, clathrin-and dynamin-independent endocytosis of Ii-free pMHC-II to both early endosomal and lysosomelike antigen processing compartments could be a major source of MHC-II for peptide loading (as discussed below).…”

“…In agreement with this, we find that mutation of the target lysine of MHC-II ubiquitination has no effect on the kinetics of pMHC-II endocytosis in HeLa-CIITA cells. 4 S22N mutant (panel B). The distribution of each endosomal protein (green) and internalized pMHC-II (red) was analyzed by confocal microscopy as described above.…”

“…earlier endosomal compartments and are, in fact, destroyed in late, more acidic lysosomes (2)(3)(4)(5). Therefore MHC-II must follow a trafficking pathway that takes them to lysosome-like antigen processing compartments but still allows access to early endosomes.…”

Phagosomes Induced by Cytokines Function as anti-Listeria Vaccines

“…The STAT-1-signaling route is connected to the induction of MHC II expression and transport to antigen-loading compartments (MIIC) (23). MIIC vesicles contain MHC II molecules loaded with peptides (26), and SDS-stable ␣␤ MHC II dimers provide a valid measurement of peptide-loaded MHC II molecules (27)(28)(29). We found that the phagosomes (P-IFN, P-IL-6, and P-NT lanes in Fig.…”

LIMP-2 Links Late Phagosomal Trafficking with the Onset of the Innate Immune Response to Listeria monocytogenes

“…We based our hypothesis on two observations: (i) different MIIC and phagocytic vesicles contain LAMP-1 and LIMP-2 as characteristic markers (11,17,19,20,(23)(24)(25)(26), and (ii) both proteins appear as key regulators of late trafficking events (19,(27)(28)(29). Therefore, they were good candidates for the molecules that connect the late trafficking processes with innate immunity to LM.…”