Autophagy promotes MHC class II presentation of peptides from intracellular source proteins

Abstract

MHC-peptide complexes mediate key functions in adaptive immunity. In a classical view, MHC-I molecules present peptides from intracellular source proteins, whereas MHC-II molecules present antigenic peptides from exogenous and membrane proteins. Nevertheless, substantial crosstalk between these two pathways has been observed. We investigated the influence of autophagy on the MHC-II ligandome and demonstrated that peptide presentation is altered considerably upon induction of autophagy. The presentation of peptides from intracellular and lysosomal source proteins was strongly increased on MHC-II in contrast with peptides from membrane and secreted proteins. In addition, autophagy influenced the MHC-II antigen-processing machinery. Our study illustrates a profound influence of autophagy on the class II peptide repertoire and suggests that this finding has implications for the regulation of CD4(+) T cell-mediated processes.

Fig. 1.

Starvation enhances the level of autophagic vacuoles. Autophagic vacuoles were stained with the specific dye MDC (13) and analyzed by fluorescence microscopy or fluorescence spectroscopy. Awells cells were incubated for 24 h in DMEM (control cells) (A), 6 h HBSS (B), or 24 h HBSS (starved cells) (C), subsequently, for 10 min with MDC, washed, and immediately analyzed by fluorescence microscopy. Autophagic vacuoles are marked with an arrow. (D) Intracellular MDC measurement by fluorescence spectroscopy; unstained cells were used as negative control.

Fig. 2.

Altered peptide presentation on HLA-DR under starvation. Displayed are the relative intensity ratios of HLA-DR-eluted peptides from starved cells (6 and 24 h) and control cells as assessed by LC-MS. Peptides were quantified by their relative peak heights in mass spectra and grouped according to the cellular localization of their source proteins: membrane plus secreted proteins and intracellular plus lysosomal proteins. Data of serial LC-MS runs were normalized to the abundant peptide LSSWTAADTAAQITQR, which showed only marginal differences in presentation levels (Table 1). Horizontal bars indicate the mean intensity ratios for each group. Marked in a box are the four peptides that showed the highest presentation levels after 24 h of starvation. Their source proteins are localized in the nucleus and in lysosomes.

Fig. 3.

Changes of lysosomal protease composition during autophagy. (A) Affinity labeling of active cathepsins. Endocytic extracts were generated from control cells, cells after 6 h and 24 h of starvation, and human peripheral blood monoyctes, respectively, by differential centrifugation as reported in refs. and . Five-microgram total endocytic protein (1.5 μg in monocytes) were either directly incubated with the active site-restricted biotinylated affinity label DCG-0N as described (lanes: 2, control cells; 3, 6 h of starvation; 4, 24 h of starvation; and 9, monocytes) or were subjected to 95°C as negative control (lane 1). In addition, control cells were incubated with the CatS-inhibitor LHVS (25 nM), the CatB-inhibitor Ca074 (1 μM), the pan-cysteine protease inhibitors leupeptin (1 mM), or E64 (25 μM) (lanes 5–8) for 45 min at 37°C before labeling as further controls. Active cathepsins were visualized after resolution by SDS/PAGE by streptavidin-HRP blot: CatZ, CatB, CatH, and CatS at 36, 33, 30, and 28 kDa, respectively. (B) Cathepsin polypeptides probed by Western blot. Identical amounts of total cellular protein from control cells (lane 1) and cells undergoing autophagy (6 and 24 h of starvation, respectively; lane 2 and lane 3) were probed for CatS, CatC, CatD, CatH, β-actin, and LAMP-1 by Western blot.

Fig. 4.

MBP_83–99 digestion with lysosomal extracts from control cells and cells undergoing autophagy. (A) Preferential detected cleavage sites. MBP_83–99 was incubated at pH 5.4 with lysosomal extracts from control cells and cells undergoing 24 h autophagy for 3 h. Breakdown products were subsequently separated by RP-HLPC and analyzed by MALDI-MS and Edman microsequencing. (B) RP-HLPC chromatogram of control cell MBP breakdown products at 214 nm. The annotated peaks correspond to the major breakdown products of MBP_83–99 as identified by MALDI MS and Edman microsequencing. The corresponding chromatogram of autophagic cells is not shown.