Since the discovery that T���cell immunity relies on ���denatured, unfolded, sequential determinants���1 of pro��� teins, whereas B���cell (that is, antibody) recognition of the same protein antigen is determined by its tertiary structure, immunologists have been fascinated with anti��� gen processing and presentation. Decades of work have elucidated the pathways that generate peptide���MHC complexes. As a result, we can now explain most of the fundamental differences between B��� and T���cell antigen recognition2,3 and such knowledge is useful for vaccine design and other immune���based interventions. Recognition of antigens in the peptide���binding groove of surface���expressed MHC class I and class II molecules by specific T���cell receptors is central to T���cell activation. To fulfil their physiological function, MHC proteins must first acquire peptide antigens, a function that is executed differently by the two main classes of MHC molecules. For MHC class I molecules, the goal is to report on intracellular events (such as viral infection, the presence of intracellular bacteria or cellular transformation) to CD8+ T cells4. MHC class I molecules are composed of heavy chains and an invari��� ant light chain, known as ��2���microglobulin. The events of the biosynthesis of MHC class I molecules can be summarized in six steps: one, acquisition of antigenic peptides two, tagging of the antigenic peptide for destruction by ubiquitylation three, proteolysis four, delivery of peptides to the endoplasmic reticulum (ER) five, binding of peptides to MHC class I molecules and six, display of peptide���MHC class I complexes on the cell surface (FIG.��1). For MHC class II molecules, the goal is to sample the extracellular milieu and present antigens to CD4+ T cells4. Similar to MHC class I mole��� cules, the ����� and �����chain of MHC class II molecules are synthesized in the ER and associate with the invari��� ant chain (Ii also known as CD74) for proper folding, trafficking and protection of the antigen���binding groove5. Newly assembled MHC class II molecules are then delivered by vesicular transport to endolysosomal�� compartments that supply peptide antigens. Following peptide loading, peptide���MHC class II complexes are delivered to the cell surface. Despite the involvement of different molecules and cellular compartments, the gen��� eration of peptide���MHC class II complexes can be strat��� ified into the same six steps as those for peptide���MHC class I complexes. The molecular expression of MHC class II molecules is mostly restricted to professional antigen���presenting cells (APCs), including macrophages and dendritic cells (DCs). DCs possess many unique features of antigen processing and presentation not seen in other cell types. Immature DCs reside in the tissue (for example, in the skin, lungs and gastrointestinal tract) and undergo remarkable transformation following exposure to pathogens. Pathogen-associated��molecular��patterns and their vertebrate receptors, including Toll���like receptors (TLRs)6,7, influence the dynamics of antigen acquisition, cytoskeletal rearrangements and regulation of MHC bio��� synthesis, all of which affect both MHC class I and class II antigen processing and presentation8,9. Likewise, the machinery of protein translation and degradation, which is required for generating antigenic peptides for presenta��� tion, is carefully regulated following DC activation10. DC activation by TLR ligands is also required for the formation *Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA. ���Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA. ��Division of Infectious Diseases, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114, USA. ||Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. Correspondence to H.L.P. e���mail: ploegh@wi.mit.edu doi:10.1038/nri2368 Published��online��18��July��2008 Endolysosomal compartments Endosomes��that��have��fused�� with��lysosomes.��This��acidic�� environment��allows�� degradation��of��antigens.�� The known unknowns of antigen processing and presentation Jatin M. Vyas*�����, Annemarthe G. Van der Veen* and Hidde L. Ploegh*|| Abstract | The principal components of both MHC class I and class II antigen processing and presentation pathways are well known. In dendritic cells, these pathways are tightly regulated by Toll-like-receptor signalling and include features, such as cross-presentation, that are not seen in other cell types. However, the exact mechanisms involved in the subcellular trafficking of antigens remain poorly understood and in some cases are controversial. Recent data suggest that diverse cellular machineries, including autophagy, participate in antigen processing and presentation, although their relative contributions remain to be fully elucidated. Here, we highlight some emerging themes of antigen processing and presentation that we think merit further attention. REVIEWS NATuRE REvIEwS | immunology voLuME 8 | AuguST 2008 | 607 �� 2008 Macmillan Publishers Limited. All rights reserved.

Nature Reviews | Immunology 3 4 5 6 Vesicular transport Endoplasmic reticulum Golgi complex 2 Ubiquitin 1 Peptide epitope Peptide Ribosome mRNA Error Peptidases Amino- peptidase(s) Proteasome TAP Tapasin MHC class I ��2m Synthesis and assembly of MHC class I molecules Peptide���MHC class I complex Plasma membrane High error rate in translation Peptide-loading complex Calreticulin ERp57 Pathogen-associated molecular patterns Molecular��patterns��that���� are��found��in��pathogens��but���� not��in��mammalian��cells.�� Examples��include��terminally�� mannosylated��and�� polymannosylated��compounds,�� which��bind��the��mannose�� receptor,��and��various��microbial�� products,��such��as��bacterial�� lipopolysaccharides,�� hypomethylated��DNA,��flagellin�� and��double-stranded��RNA,�� which��bind��Toll-like��receptors. Endolysosomal tubules Highly��dynamic��subcellular�� structures��that��eminate��from�� late��endocytic���lysosomal���� and/or��phagolysosomal�� compartments.��They��are���� known��to��contain��MHC��class��II�� molecules,��CD63,��CD82��and�� lysosome-associated�� membrane��protein��1��(LAMP1),�� and��require��microtubules��for�� movement. Endocytic pathway A��trafficking��pathway��used��by�� all��cells��for��the��internalization�� of��molecules��from��the��plasma�� membrane��to��the�� endolysosomes. Cross-presentation The��ability��of��certain��antigen- presenting��cells��to��load�� peptides��that��are��derived��from�� exogenous��antigens��onto��MHC�� class��I��molecules.��Cross- presentation��is��important��for�� the��initiation��of��immune�� responses��to��viruses��that��do�� not��infect��antigen-presenting�� cells. Autophagy An��evolutionarily��conserved�� process��in��which��acidic��double- membrane��vacuoles,��known��as�� autophagosomes,��sequester�� intracellular��contents��(such��as�� damaged��organelles��and�� macromolecules)��and��target�� them��for��degradation,��through�� fusion��to��lysosomes.��This�� process��does��not��involve���� direct��transport��through��the�� endocytic��or��vacuolar��protein�� sorting��pathways. of endolysosomal��tubules, which contain numerous pro��� teins including MHC class II molecules, and deliver these proteins to the cell surface, where they are available to CD4+ T cells for potential activation11���13. DCs have a central role in the activation of naive T cells and therefore direct the adaptive immune response against invading microorganisms. But how do DCs participate in the immune response to intracellular microorganisms that do not directly infect APCs? First, whole microorganisms can transiently exist in the extra��� cellular space and be taken up by DCs into the endocytic�� pathway, where relevant antigens are loaded onto MHC class II molecules in endolysosomes. In addition, DCs possess the capacity to take up these antigens and trans��� fer them to the MHC class I pathway through a process referred to as cross-presentation, but the details of this process remain controversial. But, how do antigens from the extracellular environ��� ment gain access to the MHC class I pathway, which is normally restricted to the presentation of intracellular anti��� gens? Here, we review the evidence for the hypotheses that include the involvement of the ER dislocation machinery and channel���independent pathways in this process. The generation of peptides for both MHC class I and class II pathways have previously been viewed as the exclusive domain of the proteasome and lyso��� somal���associated proteases, respectively. Recent data indicate that additional pathways can participate in this process. For example, a role for autophagy, which is a ubiquitous process by which cells remove damaged organelles, in the generation of peptides for MHC molecules has been proposed. The pathways of antigen processing and presentation have recently been exten��� sively reviewed4,14 and, therefore, in this Review we focus on aspects of antigen processing and presentation that are less well understood or that remain controversial. MHC class II processing and presentation Focused on the extracellular environment, the MHC class II antigen presentation pathway intersects with the endocytic pathway to sample antigens. Extracellular anti��� gen is taken up by APCs and placed into a membrane��� delimited compartment, known as the phagosome. This phagosomal compartment undergoes a series of modifi��� cations that are in part dictated by its content and finally fuses with lysosomes to form the phagolysosome, in which the contents of the phagosome can interact with MHC class II molecules (FIG.��2). Peptide���loaded MHC class II molecules are then transported to the cell surface where they engage antigen���specific CD4+ T cells. Despite the apparent simplicity of this pathway, important questions remain, including the nature of the modifications made to the phagosome, the modes of delivery of MHC class II molecules to the cell surface and the contribution of autophagy to the MHC class II pathway. Phagosome maturation. Phagocytosis serves as an important mechanism for antigen acquisition and is restricted to professional APCs, which are responsible for the uptake of various particles, including microorganisms Figure 1 | Six steps for loading and trafficking of mHC class i molecules to the cell surface. Antigen processing and presentation by MHC class I molecules can be divided into six discrete steps. Step 1: acquisition of antigens from proteins with errors (for example, due to premature termination or misincorporation). Step 2: misfolded proteins are tagged with ubiquitin for degradation. Step 3: the proteasome degrades these ubiquitylated proteins into peptides. Step 4: the peptides are delivered to the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) complex. Step 5: peptide is loaded onto nascent MHC class I molecules this process is facilitated by members of the peptide-loading complex, such as tapasin and two housekeeping ER proteins, known as calreticulin and ERp57. Step 6: peptide-loaded MHC class I molecules are transported via the Golgi complex to the cell surface. The steps for peptide���MHC class II loading conceptually follow this same path. ��2m, ��2-microglobulin. REVIEWS 608 | AuguST 2008 | voLuME 8 www.nature.com/reviews/immunol �� 2008 Macmillan Publishers Limited. All rights reserved.