Towards a systems understanding of MHC class I and MHC class II ...

REVIEWS

Towards a systems understanding of MHC class I and MHC class II antigen presentation Jacques Neefjes*, Marlieke L. M. Jongsma*, Petra Paul* and Oddmund Bakke‡

Abstract | The molecular details of antigen processing and presentation by MHC class I and class II molecules have been studied extensively for almost three decades. Although the basic principles of these processes were laid out approximately 10 years ago, the recent years have revealed many details and provided new insights into their control and specificity. MHC molecules use various biochemical reactions to achieve successful presentation of antigenic fragments to the immune system. Here we present a timely evaluation of the biology of antigen presentation and a survey of issues that are considered unresolved. The continuing flow of new details into our understanding of the biology of MHC class I and class II antigen presentation builds a system involving several cell biological processes, which is discussed in this Review. Cross-presentation The ability of certain antigen-presenting cells to load peptides that are derived from exogenous antigens onto MHC class I molecules. This property is atypical, because most cells exclusively present peptides from their endogenous proteins on MHC class I molecules. Cross-presentation is essential for the initiation of immune responses to viruses that do not infect antigen-presenting cells.

*Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. ‡ Centre for Immune Regulation, Department of Molecular Biosciences, University of Oslo, Norway. Correspondence to J.N. e‑mail: [email protected] doi:10.1038/nri3084 Published online 11 November 2011

MHC class I and class II molecules are similar in func tion: they present peptides at the cell surface to CD8+ and CD4+ T cells, respectively. These peptides originate from different sources — intracellular for MHC class I mol ecules and exogenous for MHC class II molecules — and are obtained via different pathways1. An interesting link, termed cross-presentation, exists between the two pathways, whereby exogenous antigens are presented by MHC class I molecules2. In addition, cytosolic (endogenous) proteins can be presented by MHC class II molecules when these proteins are degraded through autophagy or other path ways3. Furthermore, the various mechanisms that patho gens have evolved to manipulate the MHC class I and class II pathways have provided new insights into the biol ogy of antigen presentation4; however, we will not discuss these topics further, as they have recently been reviewed2–4. In this Review, MHC class I antigen presentation is discussed, followed by MHC class II presentation. We start with a description of the basic pathways, and then consider the recent advances in the field to arrive at a systems description of antigen presentation by MHC molecules. Furthermore, we have asked colleagues in the antigen processing and presentation field to provide their opinions on unresolved issues.

The basics of MHC class I antigen presentation MHC class I molecules are expressed by all nucleated cells and present protein fragments of cytosolic and nuclear origin at the cell surface. It has been concluded from the work of many groups that antigens are degraded by

cytosolic and nuclear proteasomes. The resulting peptides are translocated into the endoplasmic reticulum (ER) by transporter associated with antigen presentation (TAP) to access MHC class I molecules. In the ER, the MHC class I heterodimer is assembled from a polymorphic heavy chain and a light chain called β2-microglobulin (β2m). A peptide is the third component required for stability, as it inserts itself deep into the MHC class I peptide-binding groove, which accommodates peptides of 8–9 amino acids (FIG. 1). Without peptides, MHC class I molecules are stabilized by ER chaperone proteins such as calreticulin, ERp57 (also known as PDIA3), protein disulphide isomerase (PDI) and the dedicated chaperone tapasin. Tapasin interacts with TAP, thereby coupling peptide translocation into the ER with peptide delivery to MHC class I molecules. When peptides bind to MHC class I molecules, the chaperones are released and fully assembled peptide–MHC class I complexes leave the ER for presentation at the cell surface1. Conversely, pep tides and MHC class I molecules that fail to associate in the ER are returned to the cytosol for degradation5,6.

The complexity of the MHC class I pathway Recent observations have highlighted the complexity of various steps in this basic pathway, as described below. Antigen processing. MHC class I molecules present antigens that have been generated by proteasomemediated degradation of proteins that are at the end of their functional lives. The half-lives of proteins vary

NATURE REVIEWS | IMMUNOLOGY

VOLUME 11 | DECEMBER 2011 | 823 © 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS

Autophagy Any process involving delivery of a portion of the cytoplasm to lysosomes that does not involve direct transport through the endocytic or vacuolar protein-sorting pathways.

DRiPs (Defective ribosomal products). Misfolded proteins that result from defective transcription or translation.

Cytotoxic T lymphocytes (CTLs). T cells that express the glycoprotein CD8 at the cell surface and that are capable of killing cells after recognizing peptides presented by MHC class I molecules.

Pulse-chase experiments A method to examine a cellular process that occurs over time by following a molecule of interest, which is labelled at time-point zero.

Mammalian target of rapamycin (mTOR). A conserved serine/ threonine protein kinase that regulates cell growth and metabolism, as well as cytokine and growth factor expression, in response to environmental cues. mTOR receives stimulatory signals from RAS and phosphoinositide 3‑kinase downstream of growth factors and nutrients (such as amino acids, glucose and oxygen).

microRNAs Small RNA molecules that regulate the expression of genes by binding to the 3ʹ-untranslated regions of specific mRNAs.

26S proteasome A giant multicatalytic protease that resides in the cytosol and the nucleus. The 20S core, which contains three distinct catalytic subunits, can be appended at either end by a 19S cap or an 11S cap. The binding of two 19S caps to the 20S core forms the 26S proteasome, which degrades polyubiquitylated proteins.

Thymic epithelial cells (TECs). Cortical TECs promote the survival of thymocytes that possess T cell receptors that can bind to self MHC molecules. Medullary TECs induce apoptosis in thymocytes specific for self antigens.

greatly, ranging from minutes to days, and therefore it would be expected that the time between the synthe sis of a protein and the presentation of its peptides by MHC class I molecules should be similar to the half-life of the protein. Such a notion, however, conflicts with observations that viral antigens are presented by MHC class I molecules much quicker than their natural halflives would allow. This contradiction was solved by the observation that a large fraction of proteins (varying from 30% to 70% of all proteins made) is immediately degraded after synthesis before forming functional proteins7,8. These so-called DRiPs (defective ribosomal products) are the result of defective transcription or translation9, alternative reading frame usage, failed assembly into larger protein complexes, the incor poration of wrong amino acids owing to mistakes by aminoacyl-tRNA synthetases or altered ubiquitin modifications9–13. DRiPs are immediately degraded to prevent the formation of protein aggregates, which would affect cell viability. Because of this translationcoupled degradation pathway, the translation of viral products is also directly coupled to MHC class I antigen presentation. DRiPs thus explain why influenza virus is recognized by T cells ~1.5 hours post-infection, rather than 8 hours later, when the first stable viral proteins are degraded in infected cells14. The study of MHC class I antigen presentation has revealed another surprise, namely the presentation of peptides that are not encoded in the genome. Proteases usually cleave off protein fragments, but they can also ligate two peptides, as in the case of protein splicing by the 20S core of the proteasome15,16. The resulting ligated peptides cannot be predicted on the basis of the genomic sequence and include neo-antigens that can be detected by tumour-specific cytotoxic T lymphocytes (CTLs)17. Pulse-chase experiments have indicated that a sub stantial fraction of MHC class I molecules never binds proper peptides18 and that these MHC molecules are ultimately degraded by the ER-associated protein degra dation (ERAD) system5. The concentration of peptides (which is related to the rate of protein synthesis) is thus rate limiting in the MHC class I antigen presentation response. The synthesis of proteins may be affected by various stimuli, such as interferon‑γ (IFNγ), ion izing radiation (via mammalian target of rapamycin (mTOR) activation)19 and the expression of particular microRNAs20–22. Under these conditions, a defined set of proteins is selectively produced at higher levels, and this results in alterations in the peptidome presented by MHC class I molecules. The presentation of new pep tides may alter MHC class I antigen presentation and CD8+ T cell responses, as illustrated by the enhanced antitumour responses by CD8+ T cells in response to ionizing radiation19,23. In summary, the MHC class I peptidome includes a substantial fraction of DRiPs, which are derived from proteins that have never been functional, and the lev els of DRiPs are controlled by external factors that can affect translation. In addition, imperfections in trans lation mechanisms together with peptide ligation can

%& 6EGNN

6%4 /*%ENCUU+

2GRVKFG βO

)QNIK '4 '4#&

6#2

2TQVGKPUKP VJGE[VQUQN QTPWENGWU

2TQVGCUQOG

2GRVKFGU

Figure 1 | The basic MHC class I antigen presentation pathway. The presentation of intracellular antigenic 0CVWTG4GXKGYU^+OOWPQNQI[ peptides by MHC class I molecules is the result of a series of reactions. First, antigens are degraded by the proteasome. Then, the resulting peptides are translocated via transporter associated with antigen presentation (TAP) into the endoplasmic reticulum (ER) lumen and loaded onto MHC class I molecules. Peptide–MHC class I complexes are released from the ER and transported via the Golgi to the plasma membrane for antigen presentation to CD8+ T cells. β2m, β2-microglobulin; ERAD, ER-associated protein degradation; TCR, T cell receptor.

generate new peptide fragments that are not encoded in the genome but are still immunogenic. Therefore, studies on the MHC class I-associated peptidome have expanded our knowledge about the peptide repertoire24 and have also uncovered cell biological processes that were previously unknown. The proteasome. The 26S proteasome is composed of a 20S core barrel that has protease activity 25 and two 19S caps. It generates the bulk of peptides for MHC class I molecules and defines the carboxyl terminus of these peptides26. The resulting peptides then only need amino‑terminal trimming. Two ‘alternative’ proteas omes have been described: the immunoproteasome, which is expressed by many immune cells; and the thymus-specific proteasome, which is expressed in thymic epithelial cells (TECs)27. Immune cell- or thymusspecific variants of the proteolytic subunits are incorpo rated into the 20S barrel, and this alters the degradation pattern of the proteasome28,29. Immunoproteasomes were thought to improve MHC class I antigen presen tation by selectively generating particular (immuno genic) peptides. Obviously, (immuno)proteasomes have to generate fragments for all of the different MHC class I alleles, which bind to different peptide sequences.

824 | DECEMBER 2011 | VOLUME 11

www.nature.com/reviews/immunol © 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS Therefore, it is difficult to envision how immuno proteasomes can improve the production of peptides for all MHC class I alleles, as the generation of one pep tide will always be at the cost of another one. Recently, an unexpected function of the immunoproteasome was revealed. Immune stress and IFNγ exposure lead to the production of reactive oxygen species (ROS), which can cause protein misfolding 30. The levels of these mis folded proteins exceed the capacity of the constitutive 26S proteasome, yielding protein aggregates that can result in pathology. However, immunoproteasomes are more active than 26S proteasomes under conditions of immune stress or IFNγ exposure, enabling them to handle the expanded pool of substrates and prevent the aggregation of proteins. Therefore, rather than being more selective in making peptides for MHC class I pres entation, immunoproteasomes may function to handle an expanded protein substrate pool, resulting in the production of more peptides. MHC class I molecules simply sample and present the results.

HLA-DM An MHC-like molecule that acts as a chaperone in MHC class II peptide loading.

The MHC class I peptide-loading complex with some modifications. Once peptides have been generated, they have to gain access to MHC class I molecules that are waiting for peptides in the ER. The peptide transporter TAP pumps cytosolic peptides into the ER lumen. As TAP is not located at the nuclear face of the nuclear envelope, nuclear peptides must first diffuse into the cytosol to encounter TAP31. TAP also acts as a platform for the folding of MHC class I molecules by binding to one or more molecules of tapasin (the dedicated MHC class I chaperone)32. In the ER, MHC class I molecules are partially folded and stabilized by two additional common chaperones, calreticulin and ERp57, and possibly by the peptide-binding chaperone PDI33. Calreticulin is a lectin that binds N‑glycosylated MHC class I molecules, and this results in a more sta ble interaction between MHC class I molecules and tapasin34. The complex of TAP, tapasin, MHC class I, ERp57 and calreticulin is called the peptide-loading complex (PLC). The PLC ensures efficient peptide loading onto MHC class I molecules. After binding to a peptide of sufficient affinity, the MHC class I complex is released from the chaperones and can pass the ER quality control system for expression at the plasma membrane. Tapasin may also act as a peptide editor, with a role similar to that of HLA-DM in MHC class II presentation (see below). The presence of tapasin has been shown to result in the loading of a more diverse array of peptides, albeit one with a lower average affinity 35. However, the mechanism behind this remains puzzling. TAP translocates peptides of between 8 and 16 amino acids into the ER36. These peptides may require further trimming in the ER before binding to MHC class I molecules37. One ER aminopeptidase — termed ER aminopeptidase associated with antigen process ing (ERAAP) — has been identified in mice, and two have been found in humans and termed ER amino peptidase 1 (ERAP1) and ERAP2 (REFS 38–40) . As ERAAP is not part of the PLC, many peptides have to

be trimmed outside of the PLC in the ER lumen and then re-enter the PLC to bind to MHC class I mole cules (or find free MHC class I molecules outside of the PLC). ERAAP probably trims peptides to a mini mal length of 8 amino acids, which is still suitable for MHC class I loading 41. When these peptides fail to find MHC class I molecules, they are removed by the ERAD pathway and re-enter the cytosol for destruction6 or a new round of TAP translocation and MHC class I consideration42 (FIG. 2). Antigen presentation through neighbouring cells. The MHC class I pathway is designed to present antigenic peptides that are generated in an infected cell. This is meant to prevent collateral damage or innocent bystander killing. However, many cells are linked by gap junctions that allow the transfer of small cytosolic molecules or ions (such as ATP, Ca2+ and peptides) into the cytosol of the neighbouring cell. Peptides can then enter the antigen presentation pathway of neigh bouring cells43. Gap junctions remain stable during the initial stages of apoptosis (until membrane blebbing occurs), and peptides generated by caspases in dying cells can be transferred to non-apoptotic neighbouring cells for antigen presentation44. In lymph nodes, den dritic cells (DCs) can form gap junctions by contact ing other cells, and this may promote cross-priming. In mice, gap junctions also allow peptides to move from tumour cells into antigen-presenting cells (APCs) to induce strong antitumour responses45. Therefore, the MHC class I antigen presentation pathway appears to share information with its direct neighbours at the cost of some innocent bystander killing.

MHC class I polymorphism In most species, MHC class I heavy chains are encoded by three genes (HLA‑A, HLA‑B and HLA‑C in humans), and all three are polymorphic, constitut ing the most unique characteristic of MHC molecules. Polymorphism of the MHC proteins results in differ ent peptide-binding grooves that recognize unique peptides owing to variations in the anchor residues to which peptides dock46. In-depth analyses of the biology of the MHC class I locus and its alleles have revealed additional differences. Usually, HLA‑A and HLA‑B exhibit higher expression levels than HLA‑C. Human HLA‑C (and H2‑L in mice) is poorly expressed for several reasons, including transcriptional and/or posttranscriptional control by microRNAs18,20 and a more restricted peptide repertoire that limits assembly 47. Differences between the products of the three loci have been most extensively studied in humans. HLA‑A and HLA‑C bind efficiently to the PLC, whereas HLA‑B molecules do not 48. However, HLA‑B is loaded with peptide and transported to the plasma membrane more quickly than HLA‑A and HLA‑C18,49. This sug gests that association with the PLC does not promote faster peptide loading and that HLA‑A and HLA‑C may sample more peptides, as a result of editing by tapasin35, thus delaying the release of the peptide–MHC class I complex from the PLC.



REVIEWS %& 6EGNN

0GKIJDQWTKPIEGNN QT#2%

6%4

2GRVKFG

/*%ENCUU+

)CR LWPEVKQP

)QNIK

7DKSWKVKP

'4##2 '4NWOGP

%CNTGVKEWNKP

'4#& '4R 6CRCUKP

'PFQUQOG

sCC

.[UQUQOG

6#2

612 CC

/#4%*

#OKPQRGRVKFCUGU

622++

2GRVKFG RQQN

CC 1VJGTRGRVKFCUGU .KICVGF RGRVKFGU

2GRVKFGU

2GRVKFGU

2TQVGCUQOG

'PFQIGPQWU RTQVGKP

'PFQIGPQWU RTQVGKP

+ORGTHGEVRTQVGKP

&4K2U

6TCPUNCVKQP

0WENGWU

6TCPUETKRVKQP

Figure 2 | Complexity of the MHC class I antigen presentation pathway. The life of antigens starts with transcription and translation. Many proteins are imperfectly made, and these are termed defective ribosomal products (DRiPs). In a similar manner to functional endogenous proteins, DRiPs are degraded by the proteasome in the nucleus and cytosol. The proteasome can also generate new non-genetically encoded antigens by ligation. The resulting peptides are 0CVWTG4GXKGYU^+OOWPQNQI[ substrates for cytosolic aminopeptidases, such as thimet oligopeptidase (TOP) and tripeptidyl peptidase II (TPPII), which trim and destroy most peptides. A small fraction of peptides escape terminal destruction by translocation into the endoplasmic reticulum (ER) lumen via transporter associated with antigen presentation (TAP), which is part of the peptide loading complex (PLC; which also contains MHC class I molecules, ERp57, calreticulin and tapasin). Peptides may bind with MHC class I molecules directly or they may require further trimming by ER aminopeptidase associated with antigen processing (ERAAP) before they are considered suitable for MHC class I binding in or outside the PLC. Peptide–MHC class I complexes are then released from the PLC and the ER and are transported to the plasma membrane for antigen presentation to CD8+ T cells. Peptides and MHC class I molecules that fail to meet each other are transported back into the cytosol by the ER‑associated protein degradation (ERAD) system. Here, they are further trimmed or destroyed by peptidases and the proteasome. A fraction of peptides can enter the MHC class I pathway in neighbouring cells following translocation through gap junctions. Surface MHC class I molecules can be ubiquitylated by MARCH family proteins to promote their internalization and lysosomal degradation. A fraction of endocytosed MHC class I molecules is recycled after peptide exchange with endosomal peptides. APC, antigen-presenting cell.



REVIEWS The different polymorphic forms encoded within the MHC class I locus also behave differently. Some forms are almost always loaded with peptides (for example, most HLA‑B alleles in humans), whereas only a propor tion of other variants are loaded (30–70% for HLA‑A and HLA‑C)18. Possibly, MHC class I molecules that fail to acquire peptides under normal conditions are avail able for peptides that arise during viral infection or stress conditions. If so, such MHC class I alleles may present immunodominant peptides more readily, which may explain why certain HLA‑A and HLA‑C alleles cooper ate with HLA‑B alleles in protecting against HIV infec tion50. Often the linkage of particular MHC alleles to pathology is not understood; for example, HLA‑B27 is strongly linked to ankylosing spondylitis (a form of rheumatoid arthritis). Various explanations have been proposed, including the expression of an HLA‑B27 heavy chain dimer at the plasma membrane51 and the presentation of more stable peptides52. Recently, varia tions in the sequence of ERAP1 in HLA‑B27+ patients were reported to contribute to ankylosing spondylitis in a large patient cohort study53. Cells with ERAP1 mutants had a ~40% lower peptidase activity in vitro, suggesting that aberrant peptide processing for HLA‑B27 contrib uted to this autoimmune disease. Whereas the routes of antigen presentation by MHC class I molecules are understood in general terms, the consequences of poly morphisms in these molecules are only beginning to be appreciated but should explain the linkage to infection susceptibility and autoimmune diseases.

The MHC class I pathway in numbers For any functional reaction it is important that the sub strate, the enzymes and the product are in balance. For MHC class I presentation this is all about numbers. One cell contains approximately 2 × 109 molecules of pro tein, but only expresses 10,000–500,000 MHC class I molecules (depending on the cell type). As a result, the proteasome generates a 104–106-fold excess of peptides to MHC class I molecules, and so not all of these peptides can be presented54,55. Therefore, it could be assumed that when a viral protein appears, the new viral peptides are added to a long queue waiting to be loaded onto MHC class I molecules. However, the rapid rate of antigen presentation that is observed following viral infection implies the absence of such a queue. In fact, TAP and (most) MHC class I alleles can handle more peptides than are present under normal conditions18,56, suggest ing that peptides — rather than the TAP–MHC class I antigen presentation system — are rate-limiting in this system. How is this possible? Peptides have a half-life of 6–10 seconds in the cyto sol of living cells31. This suggests that the vast major ity are degraded before gaining access to TAP, thus preventing a peptide queue for presentation. Cytosolic peptides are degraded by an array of aminopeptidases. Tripeptidyl peptidase II (TPPII), as well as neurolysin, can cleave large peptide fragments57–60. Smaller pep tides of 8–15 amino acids are handled by thimet oligo peptidase (TOP), and even smaller ones by several other peptidases (TABLE 1). Mice deficient for these peptidases

Table 1 | Proteases in the MHC class I pathway Protease

Specificity

Proteasome

Cleaves after basic, hydrophobic or acidic residues; Lactacystin, MG132, epoxomycin, endoprotease activity on unfolded proteins and bortezomib (Velcade; Millennium peptides Pharmaceuticals)

Inhibitors

Generates most MHC class I peptides

TPPII

Aminopeptidase for long peptides (>15 amino acids); removes fragments of three or more amino acids after a lysine residue

Butabindide, AAF-CMK

Generates peptides for some MHC class I alleles57; generates an HIV Nef peptide59

Nardilysin

Aminopeptidase or endopeptidase; cleaves before basic amino acids60,146,147

Bestatin, ortho-phenanthroline

PRAME epitope generation for HLA‑A3

TOP

Aminopeptidase or endotrypticpeptidase for peptides of 8–15 amino acids148

Cpp-AAF-pAB

Generates MART1 epitopes for HLA‑A2 but destroys other epitopes148; downregulates MHC class I cell surface expression149

LAP

Aminopeptidase that removes hydrophobic or aromatic amino acids

Bestatin

MHC class I downregulation following LAP overexpression31; LAP deficiency has no effect on total antigen presentation150

PSA

Aminopeptidase for small peptides with broad specificity, including for poly‑Q peptides151

Puromycin

Generation of a VSV epitope for MHC class I152

Bleomycin hydrolase

Cysteine protease with aminopeptidase and endopeptidase activity for small peptides

AAF-CMK, E64

Generation of a VSV epitope for MHC class I152

IDE

Broad specificity protease

ortho-phenanthroline

Generation of a MAGE3 peptide153

ACE

Transmembrane cell surface carboxypeptidase that Captopril undergoes folding in the ER

Overexpression affects the MHC class I peptidome; HIV gp160 epitope generation154

ERAAP

ER-localized aminopeptidase for peptides over 8 amino acids (acts as a molecular ruler)155

Trims peptides for MHC class I in the ER; affects the MHC class I peptidome39,156,157

Aminopeptidase inhibitors such as amastatin

Function

AAF-CMK, Ala-Ala-Phe-chloromethylketone; ACE, angiotensin-converting enzyme; Cpp-AAF-pAB, carboxyphenylpropyl-Ala-Ala-Phe-aminobenzoate; ER, endoplasmic reticulum; ERAAP, ER‑associated aminopeptidase; IDE, insulin-degrading enzyme; LAP, leucine aminopeptidase; MAGE3, melanoma-associated antigen 3; MART1, melanoma antigen recognized by T cells 1; PRAME, a melanoma antigen; PSA, puromycin-sensitive aminopeptidase; TPPII, tripeptidyl peptidase II; TOP, thimet oligopeptidase; VSV, vesicular stomatitis virus.



REVIEWS Immunoribosomes A subset of ribosomes that is thought to be responsible for the production of defective ribosomal products.

usually do not show obvious effects on total antigen presentation61. Possibly, various peptidases can com pensate for the activities of each other, and imprecise N‑terminal trimming might always be corrected by the ER aminopeptidase ERAAP62. However, cytosolic pepti dases are essential for the generation of particular MHC class I peptides, although they are not required for the bulk peptide pool (TABLE 1). Incorporating the destructive power of peptidases in the reaction scheme of MHC class I antigen presenta tion has some interesting consequences. For example, the proportion of cytosolic peptides that escape destruc tion and associate with MHC class I molecules must be less than 0.1% to explain why the number of input peptides — as made by the proteasome — is the limiting factor at the PLC. This implies that proteins expressed at a copy number of 1,000 or lower will not be recog nized by the immune system, as their peptides will not survive the collective peptidase activities unless they are unusually stable52,63. One way to selectively improve the presentation of pathogen-derived peptides would be to couple protein production and degradation to efficient entry into the MHC class I pathway 64. Proteasomes might localize close to TAP and deliver peptides directly, hence minimizing the effects of the peptidase pool (although there is cur rently no evidence to support this suggestion). In addi tion, antigens may be produced by immunoribosomes that favour presentation, but how immunoribosomes dis tinguish proteins that give rise to antigens from nor mal proteins is unclear. A recent study identified a ribosome-associated chaperone termed BAG6 that tracks mislocalized ER proteins during translation and couples this to degradation by the proteasome65. It is conceivable that there are additional systems coupling translation to degradation that will illustrate how these processes are linked and how they generate DRiPs for swift antigen presentation. The emerging picture is a stochastic model with an input of a large excess of pep tides and an output of only a few surviving peptides bound to MHC class I molecules. How the location of peptide generation, the total pool of peptidases and the possible action of chaperones skew the peptide pool for

MHC class I antigen presentation is unclear (TABLE 2). This step in the MHC class I pathway prevents absolute prediction of presented peptides, as prediction meth ods are currently based only on information about MHC class I anchor residues and TAP and proteasome specificities66,67.

The end of MHC class I life MHC class I complexes at the cell surface are relatively stable as a result of peptide editing and the ER quality control system. However, free MHC class I heavy chains are also present at the plasma membrane. They must result from dissociation of the MHC class I heterodimer, as free MHC class I heavy chains rarely leave the ER in β2m-deficient mice or cell lines68. Acidic pH69, lowaffinity peptides or other causes may be responsible for the dissociation, and this pool of MHC class I heavy chains can reassemble in the presence of exogenous peptides and β 2m (when β2m is present in serum at high quantities)70. Free MHC class I heavy chains have a shorter half-life on the cell surface than the full com plex, suggesting the existence of dedicated machinery that recognizes the different forms for degradation71. Studies on immune evasion by herpes simplex viruses have provided a potential mechanism for this degrada tion. These viruses encode two transmembrane proteins (K3 and K5) that contain a RING domain that ubiquity lates the cytoplasmic tail of target proteins to promote their internalization and lysosomal degradation72. These viral proteins mimic the human MARCH protein fam ily, and MARCH4 and MARCH9 have been shown to control the half-life of MHC class I molecules73. How MHC class I (or its free heavy chain) is recognized by these proteins is unclear. When MHC class I molecules are internalized into endosomes, they enter the classical MHC class II pres entation pathway. Subsequent acidification promotes the release of the associated peptides, which can be exchanged for new peptides generated by the endo cytic pathway. A fraction of MHC class I molecules is recycled, along with MHC class II molecules, to the cell surface for the presentation of endosomal antigen frag ments69,74. This pathway is not dominant under normal

Table 2 | Open issues on MHC class I antigen presentation Place

Percentage*

MHC class I‑related issue

1

42%

The rules for peptide generation and selection by cytosolic peptidases and the PLC

2

26%

The function of DRiPs

3

16%

The contribution of endosomes in MHC class I peptide loading

4

12%

The effects of MHC class I polymorphism

5

10%

The dedicated locations for antigen synthesis and degradation for presentation

6

10%

The alternative routes of antigen presentation in the absence of TAP or proteasomal activity

7

7%

The function of the immunoproteasome

8

7%

The fate and turnover of peptide-free MHC class I molecules at the plasma membrane

9

7%

The role and frequency of alternative and re-ligated peptides

DRiP, defective ribosomal product; PLC, peptide-loading complex; TAP, transporter associated with antigen presentation. *Data is based on the opinion of 43 experts in the field. Some experts mentioned more than one issue.



REVIEWS conditions, as only a few endosomal peptides have been extracted from MHC class I molecules, but it may be more prevalent under conditions of cross-presentation. The process of MHC class I antigen presentation has been resolved to a unique level of detail (FIG. 2). Many recent findings on the type of peptides that are pre sented and the function of various components of this system have deepened our insight into this process. The specificity of many steps has been resolved, most promi nently that of peptide binding to MHC class I molecules. However, although the collective knowledge of the differ ent steps in MHC class I antigen presentation allows us to determine which peptides are excluded from binding to MHC class I molecules, we cannot predict the actual peptides that are presented. How immunodominant peptides are generated and whether and how they differ from normal peptides is also not known with certainty. This indicates that gaps in our understanding have to be filled in order to move from descriptive to predictive biochemistry of MHC class I antigen presentation. %& 6EGNN

'ZQIGPQWU RTQVGKP

6%4

2GRVKFG

/*%ENCUU++

2TQVGCUG #2% 'CTN[ GPFQUQOG /++%

%.+2

)QNIK *.#&/

β

α

+K /*% ENCUU++

'4

Figure 3 | The basic MHC class II antigen presentation pathway. MHC class II α‑ and β‑chains assemble in the endoplasmic reticulum (ER) and form a complex with the 0CVWTG4GXKGYU^+OOWPQNQI[ invariant chain (Ii). The Ii–MHC class II heterotrimer is transported through the Golgi to the MHC class II compartment (MIIC), either directly and/or via the plasma membrane. Endocytosed proteins and Ii are degraded by resident proteases in the MIIC. The class II‑ associated Ii peptide (CLIP) fragment of Ii remains in the peptide-binding groove of the MHC class II dimer and is exchanged for an antigenic peptide with the help of the dedicated chaperone HLA-DM (known as H2‑M in mice). MHC class II molecules are then transported to the plasma membrane to present antigenic peptides to CD4+ T cells. APC, antigen-presenting cell; TCR, T cell receptor.

MHC class II versus MHC class I molecules One reason to discuss MHC class I and MHC class II molecules in a single review is that they overlap in a number of characteristics. Both classes have high levels of polymorphism; a similar three-dimensional struc ture (owing to the fact that they originate from one common founder gene by simple gene duplication); a genetic location within one locus; and a similar function in presenting peptides to the immune system. However, these molecules have different tissue distributions and differ in the types of antigenic peptides that they present owing to their use of different cell biological pathways. Here, we discuss how the MHC class II antigen presen tation pathway differs from the MHC class I pathway and thus presents protein fragments that are generated at different cellular locations. Like the MHC class I heavy chain, MHC class II molecules are encoded by three polymorphic genes (HLA-DR, HLA-DQ and HLA-DP in humans) that bind to different peptides. Many MHC class II alleles are the strongest genetic markers for several autoimmune dis eases, possibly owing to the peptides that they present 75. Although the different variants appear to associate dif ferently with the chaperone HLA-DM (see below)76, the effects of MHC class II polymorphism on the functions of MHC class II molecules are poorly studied when compared with MHC class I. The MHC class II pathway described below is mainly based on studies of HLA-DR and mouse MHC class II (I‑A and I‑E); the pathway may differ in details for other MHC class II molecules. The basics of MHC class II antigen presentation. Whereas MHC class I molecules are ubiquitously expressed, MHC class II molecules are primarily expressed by professional APCs, such as DCs, macrophages and B cells. It has been concluded from the work of many groups that the trans membrane α‑ and β‑chains of MHC class II are assem bled in the ER and associate with the invariant chain (Ii). The resulting Ii–MHC class II complex is transported to a late endosomal compartment termed the MHC class II compartment (MIIC). Here, Ii is digested, leav ing a residual class II‑associated Ii peptide (CLIP) in the peptide-binding groove of the MHC class II heterodimer. In the MIIC, MHC class II molecules require HLA-DM (H2‑DM in mice) to facilitate the exchange of the CLIP fragment for a specific peptide derived from a protein degraded in the endosomal pathway. MHC class II mol ecules are then transported to the plasma membrane to present their peptide cargo to CD4+ T cells (FIG. 3). In B cells, a modifier of HLA-DM is expressed called HLA-DO (H2‑O in mice), and this protein associates with HLA-DM and restricts HLA-DM activity to more acidic compartments, thus modulating peptide binding to MHC class II molecules77. Cross-presentation aside, MHC class I molecules present peptides of cytosolic origin, whereas MHC class II molecules bind to peptides that are derived from proteins degraded in the endocytic pathway. Their combined specificities cover antigens from almost all cellular compartments. However, essential differences in the pathways complicate this basic paradigm. In



REVIEWS addition, various issues are not well understood, and it is not currently possible to calculate the efficien cies of the reactions leading to MHC class II peptide loading, as no reports have provided the quantitative data required.

Crohn’s disease An inflammatory autoimmune disease of the gastrointestinal tract characterized by abdominal pain, vomiting and diarrhoea.

The complexity of the MHC class II pathway MHC class II expression. Unlike MHC class I expres sion, the expression of MHC class II molecules is restricted to APCs. However, MHC class II expression can be induced by IFNγ and other stimuli in non-APCs, including mesenchymal stromal cells78, fibroblasts and endothelial cells79, as well as in epithelial cells and enteric glial cells during Crohn’s disease80,81 and eosinophilic oesophagitis82. In addition, dermatoses (such as psoria sis83) can induce MHC class II expression by keratino cytes84. Non-APCs can express MHC class II molecules in the absence of co-stimulatory molecules to maintain peripheral tolerance158. But how is the expression of MHC class II controlled in APCs and non-APCs? The master regulator of MHC class II expres sion is MHC class II transactivator (CIITA). CIITA is recruited to the X1, X2 and Y box elements in the MHC class II locus by the MHC class II enhanceosome (which contains cAMP-responsive-element-binding protein (CREB), nuclear transcription factor Y (NFY) and the regulatory factor X (RFX) complex) (reviewed in REF. 85). CIITA expression is regulated in a more complex manner, yielding CIITA isoforms I, III and IV 86,87, which are expressed in different cell types. Transcriptional control of the MHC class II locus in DCs involves an additional layer of regulation. In imma ture DCs, the type I CIITA promoter is bound by four factors, namely PU.1, interferon-regulatory factor 8 (IRF8), nuclear factor-κB (NF‑κB) and SP1. This leads to high levels of CIITA transcription and, as a result, high levels of MHC class II transcription. During DC maturation, this complex is replaced by a complex con taining PR domain zinc finger protein 1 (PRDM1; also known as BLIMP1) that inhibits CIITA transcription88 (FIG. 4b) . In addition, CIITA requires phosphoryla tion89,90 and monoubiquitylation91,92 before being active as the MHC class II transcription factor in APCs. By combining the results of a genome-wide small interfering RNA (siRNA) screen with those of quan titative PCR assays, five upstream regulators of CIITA were recently identified. These were cell division cycleassociated protein 3 (CDCA3), RMND5B, CCR4‑NOT transcription complex subunit 1 (CNOT1), mitogenactivated protein kinase 1 (MAPK1) and pleckstrin homology domain-containing family A member 4 (PLEKHA4). By determining how these factors con trolled the expression of each other, a complex feed back mechanism in control of CIITA and MHC class II transcription was uncovered93 (FIG. 4a). In fact, a com plex transcriptional feedback mechanism is the only possible mechanism to explain how a master regulator of transcription (in this case, CIITA) is controlled by the next factor that is controlled by the next and so on. However, the factors that constitute the feedback mechanism must also be controlled. Further systems

biology analyses showed that feedback control of CIITA expression is determined by the combined activ ities of transforming growth factor‑β (TGFβ) signalling and chromatin modifications that lead to MHC class II transcription in APCs93. Tissue-specific regulation of MHC class II expression is thus the consequence of two general inputs: chromatin modifications (includ ing epigenetics) and signalling by external factors. The latter was noticed earlier, as some cell types only express MHC class II molecules under inflammatory conditions (see above). In summary, transcription of the MHC class II locus is controlled by the master regulator CIITA, which in turn is regulated by post-translational modifications and factors that are mainly, but not exclusively, active in immune cells. Under defined conditions of signalling and chromatin modifications, CIITA and MHC class II molecules can be expressed in non-immune cells, often in response to infections or inflammation. MHC class II transport from the ER to the MIIC. Although both MHC class I and MHC class II mol ecules are assembled in the ER, MHC class I molecules need to be loaded with peptide to leave the ER, whereas MHC class II molecules associate with Ii94. Four differ ent splice variants of Ii exist, with variation in the cyto plasmic tail (the p33 and p35 variants) or inclusion of an additional exon encoding a protease inhibitor of the cystatin family (the p43 and p45 variants)95,96. Whereas the individual α- and β-chains of MHC class II are trapped in the ER, the assembled MHC class II αβ heterodimer starts to slowly leave the ER, and this is further accelerated by the binding of Ii. It is believed that the CLIP region of Ii blocks the MHC class II peptide-binding groove, thus preventing the binding of other peptides in the ER. Indeed, the levels of endog enous antigen presentation are higher in Ii-deficient mice97, but biochemical analyses of the same mice sug gest that this is not an efficient process, as hardly any MHC class II molecules are in a stable peptide-loaded form in the absence of Ii98,99. Particular antigens can access MHC class II molecules after TAP-dependent translocation into the ER100, but the vast majority of peptides fail to bind to MHC class II molecules in the ER owing to the presence of Ii. The cytoplasmic tail of Ii contains two classical dileucine sorting motifs that direct MHC class II mol ecules to endosomal compartments96 (FIG. 4c). These sort ing motifs are recognized by the sorting adaptors AP1 (a trans-Golgi network adaptor) and AP2 (a plasma mem brane adaptor)101. Thus, Ii may direct MHC class II mol ecules to traffic to the MIIC directly from the trans-Golgi network or by endocytosis from the plasma membrane. Endocytosis (which is AP2 dependent) is preferred in human cervical carcinoma cells (HeLa cells) and imma ture DCs102,103, whereas direct sorting (which is AP1 dependent) may be dominant in mature DCs104. In sum mary, Ii is essential for various steps in the life of MHC class II molecules, but it may take different routes to its final destination in an endosomal compartment; here, Ii is degraded and MHC class II finally acquires its peptide.



REVIEWS H %& 6EGNN

'ZQIGPQWU RTQVGKP

+OOCVWTG&%

/CVWTG&%

+.

%& /#4%*

6%4

/#4%*

2GRVKFG /*%ENCUU++

E

7DKSWKVKP $NQEMQH GPFQE[VQUKU

&GITCFCVKQP

G $KPFKPIVQCEVKP

#2

/#4%*

/[QUKP'

! #EVKP

%NCVJTKP

2 (' #4 #4.

/[QUKP++

F /++% 2TQVGCUG

/++% 6TCPURQTVCNQPIOKETQVWDWNGU

-KPGUKP

%.+2 !

.[UQUQOG

'5%46 4GVTQ HWUKQP!

!

)QNIK

&[PGKP

!

4#$

0WENGWU

*.#&/

4+.2

%++6#

/++%

/61%

'4

D

C

+OOCVWTG&%

5KIPCNNKPICPFEJTQOCVKPOQFKȮECVKQPU

/CVWTG&%

27 52 +4( 0(κ$ %&%#

4/0&$

%016

/#2-

2.'-*#

%++6#

%++6#

%++6#

%++6# ! 4(: %4'$ 0(;

24&/

/*%ENCUU++

! 4(: %4'$ 0(; /*%ENCUU++IGPGU

Figure 4 | Complexity of the MHC class II antigen presentation pathway. Insights into the various steps of the MHC class II pathway are shown in different boxes projected on the basic pathway of MHC class II antigen presentation. a | Transcription of the MHC class II genes is controlled by the master regulator MHC class II transactivator (CIITA), ensuring tissue-specific expression. The expression of CIITA is controlled by a feedback loop of factors that are subsequently controlled by two general processes: signalling and chromatin modifications. b | CIITA expression is controlled in immature dendritic cells (DCs) by an activating transcriptional complex that contains PU.1, SP1, IRF8 and NF‑κB, whereas it is inhibited in mature DCs by PR domain zinc finger protein 1 (PRDM1). Consequently, CIITA (with other factors) induces transcription of the MHC class II genes in immature DCs but not in mature DCs. c | The adaptor protein AP2 drives the internalization of Ii–MHC class II complexes into clathrin-coated vesicles at the plasma membrane for transport to the MHC class II compartment (MIIC). d | In the MIIC, Ii is degraded and MHC class II molecules interact with HLA-DM. Most HLA-DM and MHC class II molecules are located and interact in the internal structures formed by the cytosolic endosomal sorting complex required for transport (ESCRT) machinery. Whether ‘retrofusion’ of internal HLA-DM and MHC

/*%ENCUU++IGPGU

class II molecules to the outer membrane of the MIIC occurs is still unknown. e | The MIIC or its tubular extensions are transported to the cell membrane 0CVWTG4GXKGYU^+OOWPQNQI[ by the microtubule-based motor proteins dynein and kinesin. These proteins have MIIC-localized receptors, such as RAB7-interacting lysosomal protein (RILP) for the dynein motor. The final step involves actin-based myosin motors that interact with the MIIC either via Ii (which binds to myosin II) or via the GTPase ADP-ribosylation factor-like protein 14 (ARL14), which recruits an effector protein, ARF7EP, that acts as a receptor for the motor protein myosin 1E. This latter mechanism controls MIIC secretion in immature DCs. f | In immature DCs, internalization of MHC class II molecules from the plasma membrane may require the ubiquitin ligase MARCH1, which is controlled by interleukin‑10 (IL‑10). CD83 on mature DCs prevents this ubiquitylation of MHC class II molecules and thus stabilizes MHC class II molecules on the cell surface. CDCA3, cell division cycle-associated protein 3; CNOT1, CCR4‑NOT transcription complex subunit 1; CREB, cAMP-responsiveelement-binding protein; IRF8, interferon-regulatory factor 8; MAPK1, mitogen-activated protein kinase 1; NF‑κB, nuclear factor-κB; NFY, nuclear transcription factor Y; PLEKHA4, pleckstrin homology domain-containing family A member 4; RFX, regulatory factor X; TCR, T cell receptor.



REVIEWS

Tetraspanin A member of a family of proteins that contain four transmembrane domains. Some tetraspanins are highly restricted to specific tissues, whereas others are widely distributed. Members of this family have been implicated in cell activation, proliferation, adhesion, motility, differentiation and cancer.

Endosomal sorting complex required for transport (ESCRT). A complex of proteins required for the recognition and sorting of ubiquitin-modified proteins into the luminal vesicles of multivesicular bodies.

Exosomes Small vesicles that are released from activated cells. They are bounded by a lipid bilayer that is derived either from the plasma membrane or from the membrane of internal vesicles of the MIIC.

The MHC class II peptide-loading compartment. MHC class I molecules bind to peptides while stabilized in a partially folded state by ER chaperones, but this is prob ably different for MHC class II molecules, as endosomes are not known to contribute to folding. The location of MHC class II peptide loading has been a matter of debate since the MIIC was visualized by electron microscopy in 1990 (REF. 105). At that time, the MIIC was shown to contain MHC class II molecules and Ii, to have a multilamellar morphology, to be acidic and to contain lysosomal proteases and CD63, which defined it as a late endosomal compartment 105. Other structures were subsequently identified and a revised definition for the MIIC was required. The MHC class II chaperone HLA-DM was found to localize in late endosomes106, where it stabilizes MHC class II molecules that are either bound to or devoid of CLIP, thus preventing the aggregation or degradation of MHC class II molecules prior to the binding of high-affinity peptides107 (FIG. 4d). Following these findings, further studies identified the late endo somal tetraspanin proteins 108 (which interact with HLA-DM and MHC class II molecules and probably induce the formation of a proteinaceous network) and the proteases cathepsin S and cathepsin L, which degrade Ii109. An in vitro reconstitution experiment defined the molecules minimally required for the MIIC as MHC class II, HLA-DM and cathepsins110, and the combined data suggest that a late endosomal structure with at least these three factors would fulfil the criteria for the MIIC. A complicating factor is that the MIIC is not homo geneous but exists in multiple morphologies (multi vesicular, mixed and multilamellar) that may represent different maturation states. MHC class II, HLA-DM and other molecules are located mainly in the inter nal structures of the MIIC; to reach these sites they have to be ubiquitylated and sorted by the endosomal sorting complex required for transport (ESCRT) machin ery on the outer membrane of the MIIC111. Moreover, fluorescence resonance energy transfer (FRET) stud ies have suggested that HLA-DM interacts with MHC class II on the internal vesicles of the MIIC and not on its outer membrane112. Therefore, it is thought that the internal vesicles carrying MHC class II mol ecules and HLA-DM fuse back to the outer membrane of the MIIC so that they can be embedded in the plasma membrane rather than secreted in the form of exosomes. However, this process of ‘retrofusion’ has not yet been defined at the molecular level. Another model proposes that peptide-loaded MHC class II molecules that appear on the plasma membrane in DCs origi nate from the MIIC outer membrane and that MHC class II molecules on internal vesicles are destined for degradation113. However, as most MHC class II mol ecules are found on internal vesicles, a major loss of MHC class II would be expected to occur if they were destined for degradation, and this was not observed in biochemical experiments114. The molecular mech anisms of retrofusion (if any) need to be defined to resolve this issue.

Although the intracellular location for the pep tide loading of MHC class II molecules seems to be in the MIIC, many issues have yet to be resolved. These include the mechanism of MHC class II entry into the MIIC and the functional role of trimeric Ii in mediat ing the fusion of early endosomes115 and in regulating intracellular transport of MHC class II molecules116. MHC class II molecules will probably present differ ent peptides when they are loaded in these different parts of the endosomal pathway, with different levels of help from HLA-DM117. Finally, degradation of antigens is delayed in immature DCs, possibly as a mechanism to store antigens for presentation over long periods of time118. Whereas the minimal MIIC has been defined, the consequences of differences in MIIC morphology, different proteolytic activities, controlled acidification during DC maturation, retrofusion and other pro cesses need to be defined for a more complete under standing of the intracellular process of MHC class II antigen loading. MHC class II transport from the MIIC to the plasma membrane. Late endosomal compartments such as the MIIC are not typical recycling structures; how ever, MHC class II molecules, HLA-DM, tetraspanins and other molecules are transported from the MIIC to the plasma membrane. The contents of the MIIC, including MHC class II molecules, are released from the MIIC after a specific time period. This release is controlled by factors such as cholesterol, cytosolic pH, kinases and GTPases. Fast transport of the MIIC and other vesicles is driven by microtubule-based motors, namely dynein (for inward transport) and the kinesin family (for outward transport), whereas slow vesicle transport involves the actin-based myosin motor family. Motor proteins require vesicle receptors, the activity of which is controlled by other processes. The molecular basis for this part of MHC class II biology is largely undefined, with a few exceptions. Inward transport of the MIIC along microtubules by the dynein motor is controlled by RAB7-interacting lysosomal protein (RILP) on the MIIC (FIG. 4e), and RILP in turn is controlled by the cho lesterol sensor OSBP-related protein 1L (ORP1L) and the ER‑resident protein VAMP-associated protein A (VAPA)119. The ER thus controls the transport of the MIIC, and this explains the effect of cholesterol on MHC class II antigen presentation120. DCs may be unique in that the transport of MHC class II-containing vesicles from the MIIC to the plasma membrane is regulated by maturation signals, which induce higher levels of surface expression of MHC class II molecules at the cost of the intracellular pool of MHC class II molecules121,122. In DCs, lipopolysaccharide triggers the formation of tubules that originate from the MIIC, generating a complex network of moving vesi cles and tubules that may all fuse to the plasma mem brane123–125. What controls MHC class II transport in DCs? Two actin-based motors have been implicated. The common actin-based motor myosin II may interact with Ii to control MHC class II transport in DCs126 (FIG. 4e).



REVIEWS Toll-like receptor A member of a group of receptors that recognize components derived from a wide range of pathogens and switch on gene transcription that leads to cell activation and cytokine secretion.

Another pathway controlling MHC class II transport in DCs was identified using an integrated siRNA and cell biology screen. First, siRNAs affecting MHC class II expression were defined, then the target genes that were downregulated following DC maturation were identi fied, and finally the remaining candidates were silenced in immature DCs. The silencing of some of these genes induced the redistribution of MHC class II molecules in a manner that was similar to what was observed in mature DCs, although the cells remained immature with respect to other activation markers93. These candidates included the gene encoding GTPase ADP-ribosylation factor-like protein 14 (ARL14; also known as ARF7), which is located on the MIIC and recruits the effector protein ARF7EP (previously known as C11ORF46), which acts as a receptor for the motor protein myo sin 1E93. This pathway controls MHC class II export in DCs (FIG. 4e). How maturation signals by lipopoly saccharide control these pathways is unclear, but they may show some resemblance to the induced secretion of other lysosome-related organelles, such as cytolytic granules, melanosomes and Weibel–Palade bodies127. The end of an MHC class II molecule. Similarly to MHC class I molecules, MHC class II molecules do not have an infinite life. However, MHC class II complexes are relatively stable (having already survived late endo somal conditions) and do not dissociate at the plasma membrane. In addition, the half-life of MHC class II molecules greatly increases following DC matura tion121,122. So how are these molecules finally degraded? MHC class II (like MHC class I) can be ubiquitylated by MARCH1 (REF. 128). Because the expression levels of MARCH1 — and therefore the ubiquitylation of MHC class II molecules — decrease when DCs mature, ubiq uitylation was proposed to control MHC class II halflife129. Interleukin‑10 downregulates surface expression of MHC class II molecules and controls the expression of MARCH1 (REFS 130,131). In addition, the co-stimulatory molecule CD83 is highly expressed by mature DCs and inhibits the interaction between MARCH1 and MHC class II molecules, thereby preventing MHC class II ubiquitylation132. These observations suggest a causal link between ubiquitylation and MHC class II half-life (FIG. 4d).

However, this link has recently been challenged. Mice engineered to express MHC class II molecules with amino acid substitutions that prevent their ubiq uitylation still show normal levels of antigen presenta tion by MHC class II molecules, although MHC class II expression at the plasma membrane was slightly ele vated133. It is possible that MHC class II ubiquitylation is involved in another process, namely sorting within the endosomal pathway, rather than in endocytosis and degradation118,134. In summary, MHC class II molecules are extraordi narily stable but still display cell type-specific half-lives. The mechanisms that control MHC class II degradation have not been established but could involve ubiquityla tion133. It is most likely that MHC class II molecules are degraded in a similar way to any other lysosomal pro tein, by lysosomal proteolysis, but the exact mechanism distinguishing old MHC class II molecules from new ones is unresolved. Outside-in signalling by MHC class II. MHC class II molecules mediate inside-out signalling when present ing peptides to T cells, but recent data suggest that MHC class II molecules also function as signalling receptors, resulting in outside-in signalling (reviewed in REF. 135). This can lead to the apoptosis of activated APCs and results in the termination of immune responses136. By contrast, engagement of MHC class II molecules on melanoma cells by the ligand lymphocyte activation gene 3 (LAG3) expressed by infiltrating lymphocytes can prevent cell death by activating survival pathways137. As MHC class II molecules have short cytoplasmic tails without detectable signalling motifs, adaptor molecules must be involved to transduce the outside-in signals135. Toll-like receptor (TLR) activation induces the association of intracellular MHC class II molecules with CD40 and Bruton’s tyrosine kinase (BTK), resulting in prolonged BTK activation and TLR signalling-specific gene tran scription138. In addition to CD40, the B cell receptor complex components CD79a and CD79b139, the IgE receptor 140 and CD19 (REF. 141) have been reported to be involved in MHC class II‑associated signal transduction. Signalling through MHC class II molecules is a new con cept, and the consequences of this have to be revealed in the future.

Table 3 | Open issues on MHC class II antigen presentation Place

Percentage*

MHC class II‑related issue

1

21%

The mechanism and role of HLA-DM (and HLA-DO) in optimizing MHC class II peptide loading

2

16%

How do pathogens and TLRs control MHC class II?

3

16%

The mechanism by which MHC class II molecules present cytosolic antigens

4

16%

Are MIICs specialized immuno-endosomes or general late endosomes?

5

14%

The role and mechanism of MHC class II antigen presentation in non-professional APCs

6

9%

The mechanism that controls MHC class II transport to the plasma membrane

7

9%

The exact role of MHC class II ubiquitylation

8

7%

The function of MHC class II molecules on exosomes

APC, antigen-presenting cell; MIIC, MHC class II compartment; TLR, Toll-like receptor. *Data is based on the opinions of 43 experts in the field. Some experts mentioned more than one issue.



REVIEWS Conclusions and perspectives The biology of the MHC class I and MHC class II anti gen presentation pathways has been studied extensively owing to their fundamental role in controlling immune responses and their involvement in transplantation, infection, vaccination and autoimmunity. Although antigen presentation is understood in a high level of detail, this in fact represents still only sketches of the total system. For a deeper understanding, modern technologies such as siRNA screens allow genomewide consideration of relevant molecular relationships. This can yield comprehensive lists of new molecules involved in any process. An integration of siRNA data with flow cytometry or microscopy data and with tran scriptional information from quantitative PCR and microarrays has allowed the identification of vari ous novel pathways, placing new GTPases and motor proteins in control of MHC class II transport 93. Such experimental data sets can be integrated with others derived from siRNA or genetic screens and expres sion or protein–protein interaction databases to build pathways in silico. These pathways then have to be

1.

2. 3. 4.

5.

6.

7.

8. 9. 10.

11.

12. 13.

14.

15.

Vyas, J. M., Van der Veen, A. G. & Ploegh, H. L. The known unknowns of antigen processing and presentation. Nature Rev. Immunol. 8, 607–618 (2008). Kurts, C., Robinson, B. W. & Knolle, P. A. Crosspriming in health and disease. Nature Rev. Immunol. 10, 403–414 (2010). Crotzer, V. L. & Blum, J. S. Autophagy and adaptive immunity. Immunology 131, 9–17 (2010). Horst, D., Verweij, M. C., Davison, A. J., Ressing, M. E. & Wiertz, E. J. Viral evasion of T cell immunity: ancient mechanisms offering new applications. Curr. Opin. Immunol. 23, 96–103 (2011). Hughes, E. A., Hammond, C. & Cresswell, P. Misfolded major histocompatibility complex class I heavy chains are translocated into the cytoplasm and degraded by the proteasome. Proc. Natl Acad. Sci. USA 94, 1896–1901 (1997). Koopmann, J. O. et al. Export of antigenic peptides from the endoplasmic reticulum intersects with retrograde protein translocation through the Sec61p channel. Immunity 13, 117–127 (2000). Reits, E. A., Vos, J. C., Gromme, M. & Neefjes, J. The major substrates for TAP in vivo are derived from newly synthesized proteins. Nature 404, 774–778 (2000). Schubert, U. et al. Rapid degradation of a large fraction of newly synthesized proteins by proteasomes. Nature 404, 770–774 (2000). Li, M. et al. Widespread RNA and DNA sequence differences in the human transcriptome. Science 333, 53–58 (2011). Yewdell, J. W. & Hickman, H. D. New lane in the information highway: alternative reading frame peptides elicit T cells with potent antiretrovirus activity. J. Exp. Med. 204, 2501–2504 (2007). Berglund, P., Finzi, D., Bennink, J. R. & Yewdell, J. W. Viral alteration of cellular translational machinery increases defective ribosomal products. J. Virol. 81, 7220–7229 (2007). Netzer, N. et al. Innate immune and chemically triggered oxidative stress modifies translational fidelity. Nature 462, 522–526 (2009). Dolan, B. P. et al. Distinct pathways generate peptides from defective ribosomal products for CD8+ T cell immunosurveillance. J. Immunol. 186, 2065–2072 (2011). Khan, S. et al. Cutting edge: neosynthesis is required for the presentation of a T cell epitope from a longlived viral protein. J. Immunol. 167, 4801–4804 (2001). Vigneron, N. et al. An antigenic peptide produced by peptide splicing in the proteasome. Science 304, 587–590 (2004).

experimentally validated to avoid noise in our under standing of the MHC class I and MHC class II antigen presentation pathways. In addition, our understanding of MHC class I and MHC class II antigen presentation can be — and in fact already is — translated into treatment options142–144. A deeper understanding of antigen presentation by these molecules should result in additional targets for therapeutic manipulation of the immune system. Many groups have recently uncovered new steps in the antigen processing and presentation system. However, many unknowns and controversies remain, which are summarized in TABLE 2 and TABLE 3 based on the opinions from experts in the fields of MHC class I and MHC class II biology. Whether immuno dominance of peptides can be predicted and why particular MHC class I or MHC class II alleles are associated with autoimmune diseases is mostly unclear (except for the known link between gluten, HLA‑DQ2 and HLA‑DQ8 and coeliac disease145), but we hope that these questions will be resolved in the coming years.

16. Hanada, K., Yewdell, J. W. & Yang, J. C. Immune recognition of a human renal cancer antigen through post-translational protein splicing. Nature 427, 252–256 (2004). 17. Dalet, A. et al. An antigenic peptide produced by reverse splicing and double asparagine deamidation. Proc. Natl Acad. Sci. USA 108, e323–e331 (2011). References 9, 11 and 15–17 describe various examples of the generation of non-genetically encoded antigens that can be presented by MHC class I molecules. 18. Neefjes, J. J. & Ploegh, H. L. Allele and locus-specific differences in cell surface expression and the association of HLA class I heavy chain with β2‑microglobulin: differential effects of inhibition of glycosylation on class I subunit association. Eur. J. Immunol. 18, 801–810 (1988). 19. Reits, E. A. et al. Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy. J. Exp. Med. 203, 1259–1271 (2006). 20. Kulkarni, S. et al. Differential microRNA regulation of HLA‑C expression and its association with HIV control. Nature 472, 495–498 (2011). 21. Apcher, S. et al. Major source of antigenic peptides for the MHC class I pathway is produced during the pioneer round of mRNA translation. Proc. Natl Acad. Sci. USA 108, 11572–11577 (2011). 22. Gu, W. et al. Both treated and untreated tumors are eliminated by short hairpin RNA-based induction of target-specific immune responses. Proc. Natl Acad. Sci. USA 106, 8314–8319 (2009). 23. Ferrara, T. A., Hodge, J. W. & Gulley, J. L. Combining radiation and immunotherapy for synergistic antitumor therapy. Curr. Opin. Mol. Ther. 11, 37–42 (2009). 24. Mester, G., Hoffmann, V. & Stevanovic, S. Insights into MHC class I antigen processing gained from largescale analysis of class I ligands. Cell. Mol. Life Sci. 68, 1521–1532 (2011). 25. Sauer, R. T. & Baker, T. A. AAA+ proteases: ATPfueled machines of protein destruction. Annu. Rev. Biochem. 80, 587–612 (2011). 26. Cascio, P., Hilton, C., Kisselev, A. F., Rock, K. L. & Goldberg, A. L. 26S proteasomes and immunoproteasomes produce mainly N‑extended versions of an antigenic peptide. EMBO J. 20, 2357–2366 (2001). 27. Sijts, E. J. & Kloetzel, P. M. The role of the proteasome in the generation of MHC class I ligands and immune responses. Cell. Mol. Life Sci. 68, 1491–1502 (2011). 28. Toes, R. E. et al. Discrete cleavage motifs of constitutive and immunoproteasomes revealed by quantitative analysis of cleavage products. J. Exp. Med. 194, 1–12 (2001).


29. Nitta, T. et al. Thymoproteasome shapes immunocompetent repertoire of CD8+ T cells. Immunity 32, 29–40 (2010). 30. Seifert, U. et al. Immunoproteasomes preserve protein homeostasis upon interferon-induced oxidative stress. Cell 142, 613–624 (2010). This paper shows how immunological stress induces protein aggregation and pathology. Immunoproteasomes are more active than constitutive proteasomes and prevent aggregation and pathology. 31. Reits, E. et al. Peptide diffusion, protection, and degradation in nuclear and cytoplasmic compartments before antigen presentation by MHC class I. Immunity 18, 97–108 (2003). 32. Wearsch, P. A. & Cresswell, P. The quality control of MHC class I peptide loading. Curr. Opin. Cell Biol. 20, 624–631 (2008). 33. Park, B. et al. Redox regulation facilitates optimal peptide selection by MHC class I during antigen processing. Cell 127, 369–382 (2006). 34. Wearsch, P. A., Peaper, D. R. & Cresswell, P. Essential glycan-dependent interactions optimize MHC class I peptide loading. Proc. Natl Acad. Sci. USA 108, 4950–4955 (2011). 35. Zarling, A. L. et al. Tapasin is a facilitator, not an editor, of class I MHC peptide binding. J. Immunol. 171, 5287–5295 (2003). 36. Parcej, D. & Tampe, R. ABC proteins in antigen translocation and viral inhibition. Nature Chem. Biol. 6, 572–580 (2010). 37. Blanchard, N. et al. Endoplasmic reticulum aminopeptidase associated with antigen processing defines the composition and structure of MHC class I peptide repertoire in normal and virus-infected cells. J. Immunol. 184, 3033–3042 (2010). 38. Serwold, T., Gonzalez, F., Kim, J., Jacob, R. & Shastri, N. ERAAP customizes peptides for MHC class I molecules in the endoplasmic reticulum. Nature 419, 480–483 (2002). 39. Saveanu, L. et al. Concerted peptide trimming by human ERAP1 and ERAP2 aminopeptidase complexes in the endoplasmic reticulum. Nature Immunol. 6, 689–697 (2005). 40. Saric, T. et al. An IFN‑γ‑induced aminopeptidase in the ER, ERAP1, trims precursors to MHC class I‑presented peptides. Nature Immunol. 3, 1169–1176 (2002). 41. York, I. A. et al. The ER aminopeptidase ERAP1 enhances or limits antigen presentation by trimming epitopes to 8–9 residues. Nature Immunol. 3, 1177–1184 (2002). 42. Roelse, J., Gromme, M., Momburg, F., Hammerling, G. & Neefjes, J. Trimming of TAP-translocated peptides in the endoplasmic reticulum and in the cytosol during recycling. J. Exp. Med. 180, 1591–1597 (1994).


REVIEWS 43. Neijssen, J. et al. Cross-presentation by intercellular peptide transfer through gap junctions. Nature 434, 83–88 (2005). 44. Pang, B. et al. Direct antigen presentation and gap junction mediated cross-presentation during apoptosis. J. Immunol. 183, 1083–1090 (2009). 45. Saccheri, F. et al. Bacteria-induced gap junctions in tumors favor antigen cross-presentation and antitumor immunity. Sci. Transl. Med. 2, 44ra57 (2010). This study shows that gap junctions are induced by S. Typhimurium infection and are essential for generating a strong antitumour response with a S. Typhimurium-based antitumour vaccine. 46. Trowsdale, J. HLA genomics in the third millennium. Curr. Opin. Immunol. 17, 498–504 (2005). 47. Neisig, A., Melief, C. J. & Neefjes, J. Reduced cell surface expression of HLA‑C molecules correlates with restricted peptide binding and stable TAP interaction. J. Immunol. 160, 171–179 (1998). 48. Neisig, A., Wubbolts, R., Zang, X., Melief, C. & Neefjes, J. Allele-specific differences in the interaction of MHC class I molecules with transporters associated with antigen processing. J. Immunol. 156, 3196–3206 (1996). 49. Peh, C. A. et al. HLA‑B27‑restricted antigen presentation in the absence of tapasin reveals polymorphism in mechanisms of HLA class I peptide loading. Immunity 8, 531–542 (1998). 50. Leslie, A. et al. Additive contribution of HLA class I alleles in the immune control of HIV‑1 infection. J. Virol. 84, 9879–9888 (2010). 51. Malik, P., Klimovitsky, P., Deng, L. W., Boyson, J. E. & Strominger, J. L. Uniquely conformed peptidecontaining β2‑microglobulin‑free heavy chains of HLA‑B2705 on the cell surface. J. Immunol. 169, 4379–4387 (2002). 52. Herberts, C. A. et al. Cutting edge: HLA‑B27 acquires many N‑terminal dibasic peptides: coupling cytosolic peptide stability to antigen presentation. J. Immunol. 176, 2697–2701 (2006). 53. Evans, D. M. et al. Interaction between ERAP1 and HLA‑B27 in ankylosing spondylitis implicates peptide handling in the mechanism for HLA‑B27 in disease susceptibility. Nature Genet. 43, 761–767 (2011). 54. Princiotta, M. F. et al. Quantitating protein synthesis, degradation, and endogenous antigen processing. Immunity 18, 343–354 (2003). 55. Yewdell, J. W., Reits, E. & Neefjes, J. Making sense of mass destruction: quantitating MHC class I antigen presentation. Nature Rev. Immunol. 3, 952–961 (2003). 56. Reits, E. A., Vos, J. C., Gromme, M. & Neefjes, J. The major substrates for TAP in vivo are derived from newly synthesized proteins. Nature 404, 774–778 (2000). 57. Reits, E. et al. A major role for TPPII in trimming proteasomal degradation products for MHC class I antigen presentation. Immunity 20, 495–506 (2004). 58. York, I. A., Bhutani, N., Zendzian, S., Goldberg, A. L. & Rock, K. L. Tripeptidyl peptidase II is the major peptidase needed to trim long antigenic precursors, but is not required for most MHC class I antigen presentation. J. Immunol. 177, 1434–1443 (2006). 59. Kloetzel, P. M. Generation of major histocompatibility complex class I antigens: functional interplay between proteasomes and TPPII. Nature Immunol. 5, 661–669 (2004). 60. Kessler, J. H. et al. Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes. Nature Immunol. 12, 45–53 (2011). 61. Kawahara, M., York, I. A., Hearn, A., Farfan, D. & Rock, K. L. Analysis of the role of tripeptidyl peptidase II in MHC class I antigen presentation in vivo. J. Immunol. 183, 6069–6077 (2009). 62. Saveanu, L., Carroll, O., Hassainya, Y. & van Endert, P. Complexity, contradictions, and conundrums: studying post-proteasomal proteolysis in HLA class I antigen presentation. Immunol. Rev. 207, 42–59 (2005). 63. Lev, A. et al. The exception that reinforces the rule: crosspriming by cytosolic peptides that escape degradation. Immunity 28, 787–798 (2008). 64. Lev, A. et al. Compartmentalized MHC class I antigen processing enhances immunosurveillance by circumventing the law of mass action. Proc. Natl Acad. Sci. USA 107, 6964–6969 (2010). 65. Hessa, T. et al. Protein targeting and degradation are coupled for elimination of mislocalized proteins. Nature 475, 394–397 (2011).

66. Tenzer, S. et al. Modeling the MHC class I pathway by combining predictions of proteasomal cleavage, TAP transport and MHC class I binding. Cell. Mol. Life Sci. 62, 1025–1037 (2005). 67. Lundegaard, C., Lund, O., Buus, S. & Nielsen, M. Major histocompatibility complex class I binding predictions as a tool in epitope discovery. Immunology 130, 309–318 (2010). 68. Martayan, A. et al. Class I HLA folding and antigen presentation in β2‑microglobulin‑defective Daudi cells. J. Immunol. 182, 3609–3617 (2009). 69. Gromme, M. et al. Recycling MHC class I molecules and endosomal peptide loading. Proc. Natl Acad. Sci. USA 96, 10326–10331 (1999). 70. Rocca, A. et al. Localization of the conformational alteration of MHC molecules induced by the association of mouse class I heavy chain with a xenogeneic β2‑microglobulin. Mol. Immunol. 29, 481–487 (1992). 71. Neefjes, J. J., Smit, L., Gehrmann, M. & Ploegh, H. L. The fate of the three subunits of major histocompatibility complex class I molecules. Eur. J. Immunol. 22, 1609–1614 (1992). 72. Boname, J. M. et al. Efficient internalization of MHC I requires lysine‑11 and lysine‑63 mixed linkage polyubiquitin chains. Traffic 11, 210–220 (2010). 73. Bartee, E., Mansouri, M., Hovey Nerenberg, B. T., Gouveia, K. & Fruh, K. Downregulation of major histocompatibility complex class I by human ubiquitin ligases related to viral immune evasion proteins. J. Virol. 78, 1109–1120 (2004). 74. Howe, C. et al. Calreticulin-dependent recycling in the early secretory pathway mediates optimal peptide loading of MHC class I molecules. EMBO J. 28, 3730–3744 (2009). 75. Fernando, M. M. et al. Defining the role of the MHC in autoimmunity: a review and pooled analysis. PLoS Genet. 4, e1000024 (2008). 76. Anders, A. K. et al. HLA-DM captures partially empty HLA-DR molecules for catalyzed removal of peptide. Nature Immunol. 12, 54–61 (2011). 77. Denzin, L. K., Fallas, J. L., Prendes, M. & Yi, W. Right place, right time, right peptide: DO keeps DM focused. Immunol. Rev. 207, 279–292 (2005). 78. Romieu-Mourez, R., Francois, M., Boivin, M. N., Stagg, J. & Galipeau, J. Regulation of MHC class II expression and antigen processing in murine and human mesenchymal stromal cells by IFN-γ, TGF-β, and cell density. J. Immunol. 179, 1549–1558 (2007). 79. Geppert, T. D. & Lipsky, P. E. Antigen presentation by interferon-γ‑treated endothelial cells and fibroblasts: differential ability to function as antigen-presenting cells despite comparable Ia expression. J. Immunol. 135, 3750–3762 (1985). 80. Bland, P. MHC class II expression by the gut epithelium. Immunol. Today 9, 174–178 (1988). 81. Koretz, K., Leman, J., Brandt, I. & Moller, P. Metachromasia of 3‑amino‑9‑ethylcarbazole (AEC) and its prevention in immunoperoxidase techniques. Histochemistry 86, 471–478 (1987). 82. Mulder, D. J. et al. Antigen presentation and MHC class II expression by human esophageal epithelial cells: role in eosinophilic esophagitis. Am. J. Pathol. 178, 744–753 (2011). 83. Schonefuss, A. et al. Upregulation of cathepsin S in psoriatic keratinocytes. Exp. Dermatol. 19, e80–e88 (2010). 84. Tjernlund, U. M., Scheynius, A., Kabelitz, D. & Klareskog, L. Anti‑Ia‑reactive cells in mycosis fungoides: a study of skin biopsies, single epidermal cells and circulating T lymphocytes. Acta Derm. Venereol. 61, 291–301 (1981). 85. Choi, N. M., Majumder, P. & Boss, J. M. Regulation of major histocompatibility complex class II genes. Curr. Opin. Immunol. 23, 81–87 (2011). 86. Muhlethaler-Mottet, A., Otten, L. A., Steimle, V. & Mach, B. Expression of MHC class II molecules in different cellular and functional compartments is controlled by differential usage of multiple promoters of the transactivator CIITA. EMBO J. 16, 2851–2860 (1997). 87. Reith, W., LeibundGut-Landmann, S. & Waldburger, J. M. Regulation of MHC class II gene expression by the class II transactivator. Nature Rev. Immunol. 5, 793–806 (2005). 88. Smith, M. A. et al. Positive regulatory domain I (PRDM1) and IRF8/Pu.1 counter-regulate MHC class II transactivator (CIITA) expression during dendritic cell maturation. J. Biol. Chem. 286, 7893–7904 (2011).


A study of the transcriptional regulation of MHC class II expression in immature and mature DCs. 89. Sisk, T. J., Nickerson, K., Kwok, R. P. & Chang, C. H. Phosphorylation of class II transactivator regulates its interaction ability and transactivation function. Int. Immunol. 15, 1195–1205 (2003). 90. Greer, S. F. et al. Serine residues 286, 288, and 293 within the CIITA: a mechanism for down-regulating CIITA activity through phosphorylation. J. Immunol. 173, 376–383 (2004). 91. Bhat, K. P., Truax, A. D. & Greer, S. F. Phosphorylation and ubiquitination of degron proximal residues are essential for class II transactivator (CIITA) transactivation and major histocompatibility class II expression. J. Biol. Chem. 285, 25893–25903 (2010). 92. Greer, S. F., Zika, E., Conti, B., Zhu, X. S. & Ting, J. P. Enhancement of CIITA transcriptional function by ubiquitin. Nature Immunol. 4, 1074–1082 (2003). 93. Paul, P. et al. A genome-wide multidimensional RNAi screen reveals pathways controlling MHC class II antigen presentation. Cell 145, 268–283 (2011). A genome-wide siRNA screen for factors that control expression and peptide loading of MHC class II molecules. Many unknown factors are identified and additional screens are presented to reveal new pathways controlling MHC class II expression and transport in immature DCs. 94. Busch, R., Doebele, R. C., Patil, N. S., Pashine, A. & Mellins, E. D. Accessory molecules for MHC class II peptide loading. Curr. Opin. Immunol. 12, 99–106 (2000). 95. Bertolino, P. & Rabourdin-Combe, C. The MHC class II‑ associated invariant chain: a molecule with multiple roles in MHC class II biosynthesis and antigen presentation to CD4+ T cells. Crit. Rev. Immunol. 16, 359–379 (1996). 96. Landsverk, O. J., Bakke, O. & Gregers, T. F. MHC II and the endocytic pathway: regulation by invariant chain. Scand. J. Immunol. 70, 184–193 (2009). 97. Bodmer, H., Viville, S., Benoist, C. & Mathis, D. Diversity of endogenous epitopes bound to MHC class II molecules limited by invariant chain. Science 263, 1284–1286 (1994). 98. Viville, S. et al. Mice lacking the MHC class II‑associated invariant chain. Cell 72, 635–648 (1993). 99. Bikoff, E. K. et al. Defective major histocompatibility complex class II assembly, transport, peptide acquisition, and CD4+ T cell selection in mice lacking invariant chain expression. J. Exp. Med. 177, 1699–1712 (1993). 100. Tewari, M. K., Sinnathamby, G., Rajagopal, D. & Eisenlohr, L. C. A cytosolic pathway for MHC class II‑ restricted antigen processing that is proteasome and TAP dependent. Nature Immunol. 6, 287–294 (2005). 101. Hofmann, M. W. et al. The leucine-based sorting motifs in the cytoplasmic domain of the invariant chain are recognized by the clathrin adaptors AP1 and AP2 and their medium chains. J. Biol. Chem. 274, 36153–36158 (1999). 102. Dugast, M., Toussaint, H., Dousset, C. & Benaroch, P. AP2 clathrin adaptor complex, but not AP1, controls the access of the major histocompatibility complex (MHC) class II to endosomes. J. Biol. Chem. 280, 19656–19664 (2005). 103. McCormick, P. J., Martina, J. A. & Bonifacino, J. S. Involvement of clathrin and AP‑2 in the trafficking of MHC class II molecules to antigen-processing compartments. Proc. Natl Acad. Sci. USA 102, 7910–7915 (2005). 104. Santambrogio, L. et al. Involvement of caspasecleaved and intact adaptor protein 1 complex in endosomal remodeling in maturing dendritic cells. Nature Immunol. 6, 1020–1028 (2005). 105. Peters, P. J., Neefjes, J. J., Oorschot, V., Ploegh, H. L. & Geuze, H. J. Segregation of MHC class II molecules from MHC class I molecules in the Golgi complex for transport to lysosomal compartments. Nature 349, 669–676 (1991). 106. Sanderson, F. et al. Accumulation of HLA-DM, a regulator of antigen presentation, in MHC class II compartments. Science 266, 1566–1569 (1994). 107. Kropshofer, H. et al. Editing of the HLA‑DR‑peptide repertoire by HLA-DM. EMBO J. 15, 6144–6154 (1996). 108. Engering, A. & Pieters, J. Association of distinct tetraspanins with MHC class II molecules at different subcellular locations in human immature dendritic cells. Int. Immunol. 13, 127–134 (2001). 109. Hsing, L. C. & Rudensky, A. Y. The lysosomal cysteine proteases in MHC class II antigen presentation. Immunol. Rev. 207, 229–241 (2005).


REVIEWS 110. Hartman, I. Z. et al. A reductionist cell-free major histocompatibility complex class II antigen processing system identifies immunodominant epitopes. Nature Med. 16, 1333–1340 (2010). 111. Raiborg, C. & Stenmark, H. The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins. Nature 458, 445–452 (2009). 112. Zwart, W. et al. Spatial separation of HLA-DM/HLA-DR interactions within MIIC and phagosome-induced immune escape. Immunity 22, 221–233 (2005). 113. ten Broeke, T., van Niel, G., Wauben, M. H., Wubbolts, R. & Stoorvogel, W. Endosomally stored MHC class II does not contribute to antigen presentation by dendritic cells at inflammatory conditions. Traffic 12, 1025–1036 (2011). 114. Neefjes, J. J., Stollorz, V., Peters, P. J., Geuze, H. J. & Ploegh, H. L. The biosynthetic pathway of MHC class II but not class I molecules intersects the endocytic route. Cell 61, 171–183 (1990). 115. Nordeng, T. W. et al. The cytoplasmic tail of invariant chain regulates endosome fusion and morphology. Mol. Biol. Cell 13, 1846–1856 (2002). 116. Landsverk, O. J., Barois, N., Gregers, T. F. & Bakke, O. Invariant chain increases the half-life of MHC II by delaying endosomal maturation. Immunol. Cell Biol. 89, 619–629 (2011). 117. Strong, B. S. & Unanue, E. R. Presentation of type B peptide–MHC complexes from hen egg white lysozyme by TLR ligands and type I IFNs independent of H2‑DM regulation. J. Immunol. 187, 2193–2201 (2011). 118. Trombetta, E. S., Ebersold, M., Garrett, W., Pypaert, M. & Mellman, I. Activation of lysosomal function during dendritic cell maturation. Science 299, 1400–1403 (2003). 119. Rocha, N. et al. Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7–RILP–p150Glued and late endosome positioning. J. Cell Biol. 185, 1209–1225 (2009). This study reveals the complex effects of motor regulation on the MIIC and other late endosomes. Cholesterol in late endosomes and/or the MIIC controls interactions with the ER protein VAPA, which removes the dynein motor from its receptor RILP, resulting in vesicle relocation. 120. Kuipers, H. F. et al. Statins affect cell-surface expression of major histocompatibility complex class II molecules by disrupting cholesterol-containing microdomains. Hum. Immunol. 66, 653–665 (2005). 121. Cella, M., Engering, A., Pinet, V., Pieters, J. & Lanzavecchia, A. Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells. Nature 388, 782–787 (1997). 122. Pierre, P. et al. Developmental regulation of MHC class II transport in mouse dendritic cells. Nature 388, 787–792 (1997). 123. Boes, M. et al. T‑cell engagement of dendritic cells rapidly rearranges MHC class II transport. Nature 418, 983–988 (2002). 124. Wubbolts, R. et al. Direct vesicular transport of MHC class II molecules from lysosomal structures to the cell surface. J. Cell Biol. 135, 611–622 (1996). 125. Kleijmeer, M. et al. Reorganization of multivesicular bodies regulates MHC class II antigen presentation by dendritic cells. J. Cell Biol. 155, 53–63 (2001). 126. Vascotto, F. et al. The actin-based motor protein myosin II regulates MHC class II trafficking and BCRdriven antigen presentation. J. Cell Biol. 176, 1007–1019 (2007). 127. Saftig, P. & Klumperman, J. Lysosome biogenesis and lysosomal membrane proteins: trafficking meets function. Nature Rev. Mol. Cell Biol. 10, 623–635 (2009). 128. de Gassart, A. et al. MHC class II stabilization at the surface of human dendritic cells is the result of maturation-dependent MARCH I down-regulation. Proc. Natl Acad. Sci. USA 105, 3491–3496 (2008).

129. Shin, J. S. et al. Surface expression of MHC class II in dendritic cells is controlled by regulated ubiquitination. Nature 444, 115–118 (2006). 130. Thibodeau, J. et al. Interleukin-10‑induced MARCH1 mediates intracellular sequestration of MHC class II in monocytes. Eur. J. Immunol. 38, 1225–1230 (2008). This study explains the effects of IL‑10 on MHC class II expression in human monocytes. IL‑10 controls MARCH1 expression, which in turn controls the half-life of MHC class II on the cell surface. 131. Koppelman, B., Neefjes, J. J., de Vries, J. E. & Waal Malefyt, R. Interleukin‑10 down-regulates MHC class II αβ peptide complexes at the plasma membrane of monocytes by affecting arrival and recycling. Immunity 7, 861–871 (1997). 132. Tze, L. E. et al. CD83 increases MHC II and CD86 on dendritic cells by opposing IL‑10‑driven MARCH1‑mediated ubiquitination and degradation. J. Exp. Med. 208, 149–165 (2011). This study shows how CD83 inhibits MHC class II ubiquitylation by MARCH1. 133. McGehee, A. M. et al. Ubiquitin-dependent control of class II MHC localization is dispensable for antigen presentation and antibody production. PLoS ONE 6, e18817 (2011). 134. Walseng, E. et al. Ubiquitination regulates MHC class II–peptide complex retention and degradation in dendritic cells. Proc. Natl Acad. Sci. USA 107, 20465–20470 (2010). This article shows how ubiquitylation regulates the degradation of internalized MHC class II molecules but not the endocytosis of MHC class II. 135. Al Daccak, R., Mooney, N. & Charron, D. MHC class II signaling in antigen-presenting cells. Curr. Opin. Immunol. 16, 108–113 (2004). 136. Drenou, B. et al. A caspase-independent pathway of MHC class II antigen-mediated apoptosis of human B lymphocytes. J. Immunol. 163, 4115–4124 (1999). 137. Hemon, P. et al. MHC class II engagement by its ligand LAG‑3 (CD223) contributes to melanoma resistance to apoptosis. J. Immunol. 186, 5173–5183 (2011). 138. Liu, X. et al. Intracellular MHC class II molecules promote TLR-triggered innate immune responses by maintaining activation of the kinase Btk. Nature Immunol. 12, 416–424 (2011). This comprehensive study reveals crosstalk between TLRs, MHC class II and CD40. A full mechanism is presented to describe how MHC class II molecules are involved in outside-in signalling. 139. Lang, P. et al. TCR-induced transmembrane signaling by peptide/MHC class II via associated Ig-α/β dimers. Science 291, 1537–1540 (2001). 140. Bonnefoy, J. Y. et al. The low-affinity receptor for IgE (CD23) on B lymphocytes is spatially associated with HLA-DR antigens. J. Exp. Med. 167, 57–72 (1988). 141. Bradbury, L. E., Kansas, G. S., Levy, S., Evans, R. L. & Tedder, T. F. The CD19/CD21 signal transducing complex of human B lymphocytes includes the target of antiproliferative antibody‑1 and Leu‑13 molecules. J. Immunol. 149, 2841–2850 (1992). 142. van der Burg, S. H. & Melief, C. J. Therapeutic vaccination against human papilloma virus induced malignancies. Curr. Opin. Immunol. 23, 252–257 (2011). 143. Mitea, C. et al. A universal approach to eliminate antigenic properties of α-gliadin peptides in celiac disease. PLoS ONE 5, e15637 (2010). 144. Baugh, M. et al. Therapeutic dosing of an orally active, selective cathepsin S inhibitor suppresses disease in models of autoimmunity. J. Autoimmun. 36, 201–209 (2011). 145. Fallang, L. E. et al. Differences in the risk of celiac disease associated with HLA‑DQ2.5 or HLA‑DQ2.2 are related to sustained gluten antigen presentation. Nature Immunol. 10, 1096–1101 (2009).


146. Chow, K. M. et al. Studies on the subsite specificity of rat nardilysin (N-arginine dibasic convertase). J. Biol. Chem. 275, 19545–19551 (2000). 147. Chow, K. M. et al. Nardilysin cleaves peptides at monobasic sites. Biochemistry 42, 2239–2244 (2003). 148. York, I. A. et al. The cytosolic endopeptidase, thimet oligopeptidase, destroys antigenic peptides and limits the extent of MHC class I antigen presentation. Immunity 18, 429–440 (2003). 149. Kim, S. I., Pabon, A., Swanson, T. A. & Glucksman, M. J. Regulation of cell-surface major histocompatibility complex class I expression by the endopeptidase EC3.4.24.15 (thimet oligopeptidase). Biochem. J. 375, 111–120 (2003). 150. Rock, K. L., York, I. A., Saric, T. & Goldberg, A. L. Protein degradation and the generation of MHC class I‑presented peptides. Adv. Immunol. 80, 1–70 (2002). 151. Bhutani, N., Venkatraman, P. & Goldberg, A. L. Puromycin-sensitive aminopeptidase is the major peptidase responsible for digesting polyglutamine sequences released by proteasomes during protein degradation. EMBO J. 26, 1385–1396 (2007). 152. Stoltze, L. et al. Two new proteases in the MHC class I processing pathway. Nature Immunol. 1, 413–418 (2000). 153. Parmentier, N. et al. Production of an antigenic peptide by insulin-degrading enzyme. Nature Immunol. 11, 449–454 (2010). 154. Shen, X. Z., Lukacher, A. E., Billet, S., Williams, I. R. & Bernstein, K. E. Expression of angiotensin-converting enzyme changes major histocompatibility complex class I peptide presentation by modifying C termini of peptide precursors. J. Biol. Chem. 283, 9957–9965 (2008). 155. Chang, S. C., Momburg, F., Bhutani, N. & Goldberg, A. L. The ER aminopeptidase, ERAP1, trims precursors to lengths of MHC class I peptides by a “molecular ruler” mechanism. Proc. Natl Acad. Sci. USA 102, 17107–17112 (2005). 156. Hammer, G. E., Gonzalez, F., James, E., Nolla, H. & Shastri, N. In the absence of aminopeptidase ERAAP, MHC class I molecules present many unstable and highly immunogenic peptides. Nature Immunol. 8, 101–108 (2007). 157. Nguyen, T. T. et al. Structural basis for antigenic peptide precursor processing by the endoplasmic reticulum aminopeptidase ERAP1. Nature Struct. Mol. Biol. 18, 604–613 (2011). References 39 and 155–157 show how ERAAP acts as a molecular ruler for MHC class I peptides and skews the peptide repertoire. 158. Kreisel, D. et al. Cutting edge: MHC class II expression by pulmonary nonhematopoietic cells plays a critical role in controlling local inflammatory responses. J. Immunol. 185, 3809–3813 (2010).

Acknowledgements

We thank our colleagues for their input in the controversial items section and I. Berlin, S. van Kasteren, O. Landsverk and A. Lammerts van Beuren-Brandt for critical reading. We apologize to our colleagues for not citing every relevant paper owing to length limitations. This work was supported by European Research Council (ERC) and Netherlands Organization for Scientific Research (NWO) grants to J.N. and an NWO visiting grant to O.B.

Competing interests statement

The authors declare no competing financial interests.

FURTHER INFORMATION Authors’ homepage: http://www.neefjeslab.nl ALL LINKS ARE ACTIVE IN THE ONLINE PDF


Towards a systems understanding of MHC class I and MHC class II ...

Towards a systems understanding of MHC class I and MHC class II ...

Suggest Documents

(MHC) Class II Molecules

CD11b MHC class II I-Ab CD11c MHC class II I-Ab - PLOS

Expression of MHC class I, MHC class II, and cancer germline ...

Erratum: MHC class I and MHC class II DRB gene ... - Springer Link

SPECIALIZED FUNCTIONS OF MHC CLASS I ... - BioMedSearch

Molecular Basis of MHC Class I

MHC class II antigens in alopecia areata

Presentation via Class II MHC Vaccination ...

Magnitude of Alloresponses to MHC Class I/II ... - Semantic Scholar

Noncognate Interaction with MHC Class II ...

Individual MHC class I and MHC class IIB ... - Wiley Online Library

Specific MHC class I supertype associated with

Mechanical Stress Downregulates MHC Class I

Peptidase II Involvement Tripeptidyl 1 MHC Class I Epitopes Requires ...

Engineering and characterization of a murine MHC class II ...

Thermodynamic and Kinetic Analysis of a PeptideâClass I MHC ...

and a Mode of MHC Class I Dimerization Peptide-Induced ...

Distinct Modes of Dual MHC Class I and Class II Res - The Journal of ...

7he MHC Class II Transactivator (CIITA): A Physiologic Inhibitor of

Expression of MHC class II molecules in different cellular and ...

MHC Class II Isotype-and Allele-Specific Attenuation of Experimental ...

Association with BiP and aggregation of class II MHC molecules ...

HLA-DRB1*11 and variants of the MHC class II

Generating MHC Class II+/Ii- phenotype after adenoviral ... - Nature

Towards a systems understanding of MHC class I and MHC class II ...