Autophagy and intracellular surveillance: Modulating MHC class II antigen presentation with stress

MHC class I and II proteins selectively acquire peptides from self and foreign antigens for display to T lymphocytes. The spectrum of peptides displayed by class I and II proteins is critical for self-tolerance as well as the development of immunity against invasive pathogens and tumors. Traditionally, immunologists held that class I and II molecules were restricted in surveying distinct subcellular domains for ligands. With this model, endogenous antigens processed in the cytosol give rise to peptides for MHC class I presentation to cytotoxic T cells, whereas engulfed exogenous antigens degraded in endosomes and lysosomes are destined for MHC class II presentation to CD4⁺ T lymphocytes. However, alternative pathways for delivering exogenous antigens to MHC class I molecules have now been characterized (1). Similarly, biochemical and functional studies indicate that MHC class II molecules display peptides from cytoplasmic proteins for CD4⁺ T cell recognition with potential relevance to viral immunity, tumor immunity, and autoimmunity (2). Therefore, defining the specific mechanism(s) by which MHC class II molecules in endosomal and lysosomal compartments access peptides derived from intracellular antigens within the cytoplasm and nucleus has become a priority. Recent studies using specific viral and bacterial antigens as well as an autoantigen point to three possible pathways: bulk autophagy (3, 4), chaperone-mediated autophagy (5), and a TAP-dependent pathway (6). In this issue of PNAS, Dengjel et al. (7) take a different approach to this same problem, examining how the induction of autophagy alters the complex spectrum of peptides displayed by MHC class II molecules. Using serum starvation of human B-lymphoblasts to induce autophagy for different time periods, the authors demonstrate notable changes in the peptides associated with class II molecules. Remarkably, these authors also demonstrate that the induction of autophagy by starvation alters the balance of active proteases within lysosomes.

Multiple mechanisms for protein degradation exist within a cell, including the ubiquitin/proteasome pathway and several distinct pathways of lysosomal proteolysis (Fig. 1). Lysosomal degradation pathways can also regulate the turnover of cytoplasmic and nuclear proteins as well as membrane organelles (8). In microautophagy, small portions of the cytosol are internalized via lysosomal invaginations, and proteins are continuously degraded in the lumen of this organelle even under resting conditions. During nutrient deprivation, macroautophagy is induced as a mechanism to salvage amino acids. In macroautophagy, the cytoplasm is sequestered into double-membraned structures known as autophagosomes, which fuse with endosomes and lysosomes. After fusion, the vacuolar material is degraded and recycled. In contrast with these bulk autophagy pathways, a third lysosomal degradation pathway, termed chaperone-mediated autophagy (CMA), does not require any vesicular trafficking (9). Rather, in CMA, specific cytosolic proteins are transported into lysosomes via a molecular chaperone/receptor complex. Heat shock cognate protein 70/73 (hsc70) serves as one chaperone for this pathway, and an isoform of the lysosome-associated membrane protein-2, lamp-2a, functions as the lysosomal receptor for CMA. Although short periods of starvation initiate macroautophagy, prolonged nutrient deprivation results in the cessation of this process and the induction of CMA. Thus, the cell utilizes multiple pathways of protein degradation under both resting and stress-induced conditions.

Fig. 1.

Multiple pathways of protein degradation in lysosomes. In microautophagy, portions of the cytosol are continuously internalized via lysosomal invaginations. In macroautophagy, the cytoplasm is sequestered into double-membraned structures, known as autophagosomes, which fuse with lysosomes. In CMA, specific cytosolic proteins are transported into lysosomes via a molecular chaperone/receptor complex composed of hsc70 and lamp-2. Cytoplasmic antigens may first be processed by cytosolic proteases before transport to lysosomes. These antigenic fragments intersect with MHC class II molecules in a mature endosomal compartment known as the MIIC before presentation to CD4⁺ T cells.

Macroautophagy has been proposed to play an essential role during development and oncogenesis as well as in response to physiological stress, but its role in immunological processes, in particular MHC class II-restricted antigen presentation, has only been recently tested. An inhibitor of bulk autophagy, 3-methyladenine (3-MA), was used to determine whether macroautophagy played a role in the MHC class II-mediated presentation of intracellular antigens to CD4⁺ T lymphocytes (3, 4, 10–12). These studies revealed conflicting results, perhaps dependent on the nature of the antigen, its subcellular distribution, its half-life, and the cultivation of the antigen-presenting cells (APC). In the work described here, Dengjel et al. (7) chose a distinct approach: to induce autophagy by depriving cells of nutrients followed by analysis of the peptides displayed by MHC class II molecules. Using mass spectrometry to sequence and quantify the peptides associated with the class II molecule HLADR4, the authors observe that, although the majority of the ligands are derived from membrane proteins, a significant portion of these MHC class II-associated peptides also originate from intracellular proteins. After cells are starved for 6 h to induce autophagy, the HLADR4-restricted presentation of peptides from intracellular and lysosomal proteins increases almost 30%, and, with 24 h of nutrient deprivation, the increase is >50%. Additionally, the authors examine the gene expression profiles in cells after autophagy induction. Their results suggest that cells modify their gene expression to survive nutrient deprivation by up-regulating genes important in lysosomal trafficking and amino acid transport and metabolism while down-regulating genes critical for nucleic acid and protein synthesis. Thus, autophagy may induce multiple mechanisms, including changes in gene expression, to alter the peptides displayed via class II molecules and, as a consequence, influence the activation of specific CD4⁺ T lymphocytes.

Lysosomal acidic proteases were shown to be required for MHC class II-restricted presentation of a cytoplasmic antigen that was also 3-MA-sensitive (3), suggesting a possible link between autophagy and lysosomal antigen processing. Furthermore, Dengjel et al. (7) observe that the MHC class II-mediated presentation of certain peptides derived from the same intracellular protein are differentially affected by serum starvation, leading the authors to hypothesize that the induction of autophagy alters the function of lysosomal proteases. They demonstrate a time-dependent loss of cathepsin activity and expression after serum starvation. Consistent with this finding, the authors observe changes in the in vitro degradation of a model antigen using lysosomes extracted from normal or serum-starved cells. Interestingly, serum starvation is known to enhance the level of lysosomal lamp-2a by blocking the degradation of this transmembrane receptor for CMA (13). Thus, the reduction in the activity of lysosomal proteases may partially account for the stabilization of lamp-2a expression and, subsequently, the induction of CMA upon nutrient deprivation. Together, these results indicate that starvation-induced autophagy may alter the antigen-processing machinery for MHC class II-mediated peptide presentation.

Dengjel et al.'s studies suggest a role for autophagy in the MHC class II-restricted presentation of peptides from intracellular proteins.

During the initial hours of starvation, the induction of macroautophagy results in the uniform loss of cellular protein content. However, after longer periods of nutrient deprivation, the selective degradation of certain protein substrates via CMA ensures the removal of nonessential cellular proteins while maintaining those proteins critical for survival during long-term starvation. Interestingly, the induction of CMA in response to nutrient deprivation differs among tissues (8). For example, in the heart, initiation of this pathway occurs during the first day of starvation, whereas, in the liver, CMA is maximally activated only after 2–3 days of starvation. Studies with human B-lymphoblasts suggest that both macroautophagy and CMA can be detected in cells cultivated under nutrient-rich conditions (4, 5, 7). However, precisely how immune cell stress or differentiation modulates the balance between these pathways remains undefined. Here, Dengjel et al. (7) report that the accumulation of autophagic vesicles, as measured by monodansylcadaverine (MDC) in serum-starved cells, plateaus at 6 h after starvation, whereas the MHC class II-associated presentation of peptides from intracellular proteins is more pronounced at 24 h of nutrient deprivation. Although these results could simply be explained by the time-dependent decrease in lysosomal protease activity due to macroautophagy, other factors, such as the induction of CMA after extended periods of physiological stress, may also be involved in the MHC class II-mediated presentation of cytoplasmic antigens. CMA may be particularly important in MHC class II-mediated presentation of those cytosolic antigens dependent on the activity of cytoplasmic proteases, such as the proteasome or calpain, for their processing and those that are insensitive to bulk autophagy inhibitors such as 3-MA (10, 11). Lastly, chaperones such as hsc70 play critical roles in CMA, and, interestingly, the authors observe an increase in the mRNA expression of this cognate heat shock protein as well as the MHC class II-mediated presentation of a peptide derived from this chaperone (7). Thus, these studies suggest a role for autophagy in the MHC class II-restricted presentation of peptides from intracellular proteins in APC; however, more research is required to clearly elucidate the role of the various lysosomal degradation pathways in this process. Equally important will be in vivo investigations to dissect the role of macroautophagy as well as CMA in regulating host immunity to autoantigens, tumors, and pathogens.