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Antigen processing and presentation are the cornerstones of adaptive immunity. B cells cannot generate high-affinity antibodies without T cell help. CD4 + T cells, which provide such help, use antigen-specific receptors that recognize major histocompatibility complex (MHC) molecules in complex with peptide cargo. Similarly, eradication of virus-infected cells often depends on cytotoxic CD8 + T cells, which rely on the recognition of peptide-MHC complexes for their action. The two major classes of glycoproteins entrusted with antigen presentation are the MHC class I and class II molecules, which present antigenic peptides to CD8 + T cells and CD4 + T cells, respectively. This Review describes the essentials of antigen processing and presentation. These pathways are divided into six discrete steps that allow a comparison of the various means by which antigens destined for presentation are acquired and how the source proteins for these antigens are tagged for degradation, destroyed and ultimately displayed as peptides in complex with MHC molecules for T cell recognition., (© 2022. Springer Nature Limited.)

Human CD4(+) T cells process and present functional class II MHC-peptide complexes, but the endogenous peptide repertoire of these non-classical antigen presenting cells remains unknown. We eluted and sequenced HLA-DR-bound self-peptides presented by CD4(+) T cells in order to compare the T cell-derived peptide repertoire to sequences derived from genetically identical B cells. We identified several novel epitopes derived from the T cell-specific proteome, including fragments of CD4 and IL-2. While these data confirm that T cells can present peptides derived from the T-cell specific proteome, the vast majority of peptides sequenced after elution from MHC were derived from the common proteome. From this pool, we identified several identical peptide epitopes in the T and B cell repertoire derived from common endogenous proteins as well as novel endogenous epitopes with promiscuous binding. These findings indicate that the endogenous HLA-DR-bound peptide repertoire, regardless of APC type and across MHC isotype, is largely derived from the same pool of self-protein.

Although it has long been known that human CD4(+) T cells can express functional class II MHC molecules, the role of lysosomal proteases in the T cell class II MHC processing and presentation pathway is unknown. Using CD4(+) T cell clones that constitutively express class II MHC, we determined that cathepsin S is necessary for invariant chain proteolysis in T cells. CD4(+)HLA-DR(+) T cells down-regulated cathepsin S expression and activity 18 h after activation, thereby ceasing nascent class II MHC product formation. This blockade resulted in the loss of the invariant chain fragment CLIP from the cell surface, suggesting that-like professional APC-CD4(+) HLA-DR(+) cells modulate self-Ag presentation as a consequence of activation. Furthermore, cathepsin S expression and activity, and concordantly cell surface CLIP expression, was reduced in HLA-DR(+) CD4(+) T cells as compared with B cells both in vitro and ex vivo.

The principal components of both MHC class I and class II antigen processing and presentation pathways are well known. In dendritic cells, these pathways are tightly regulated by Toll-like-receptor signalling and include features, such as cross-presentation, that are not seen in other cell types. However, the exact mechanisms involved in the subcellular trafficking of antigens remain poorly understood and in some cases are controversial. Recent data suggest that diverse cellular machineries, including autophagy, participate in antigen processing and presentation, although their relative contributions remain to be fully elucidated. Here, we highlight some emerging themes of antigen processing and presentation that we think merit further attention.

We explored the role of antigen valency in B cell receptor (BCR) activation and rearrangement of intracellular MHC class II compartments as factors that contribute to the efficacy of antigen presentation. Using primary B cells that express a hen egg lysozyme (HEL)-specific BCR, we found that oligomeric HEL more efficiently promoted both BCR activation and internalization than did monovalent HEL, although monovalent HEL, unlike monovalent Fab fragments of anti-Ig, readily triggered the BCR. Nonetheless, oligovalent ligation positions the BCR in a membrane microdomain that is distinct from one engaged in the course of monovalent ligation, as judged by detergent extraction of the BCR. Furthermore, oligovalent HEL induced more pronounced rearrangement of MHC class II-containing antigen-processing compartments. Using videomicroscopy we observed in real time the rearrangement of MHC class II compartments as well as delivery of antigen in primary B cells. The observed increase in rearrangement of MHC class II-positive compartments and the disposition of antigen-bound BCRs therein correlates with improved presentation of a HEL-derived epitope. Although monomeric HEL efficiently engages the BCR, presentation of HEL-derived epitopes is impaired compared to oligovalent antigens. This trait may help explain the known ability of soluble, disaggregated antigen to induce a state of B cell tolerance.

Relatively small genomes and high replication rates allow viruses and bacteria to accumulate mutations. This continuously presents the host immune system with new challenges. On the other side of the trenches, an increasingly well-adjusted host immune response, shaped by coevolutionary history, makes a pathogen's life a rather complicated endeavor. It is, therefore, no surprise that pathogens either escape detection or modulate the host immune response, often by redirecting normal cellular pathways to their advantage. For the purpose of this chapter, we focus mainly on the manipulation of the class I and class II major histocompatibility complex (MHC) antigen presentation pathways and the ubiquitin (Ub)-proteasome system by both viral and bacterial pathogens. First, we describe the general features of antigen presentation pathways and the Ub-proteasome system and then address how they are manipulated by pathogens. We discuss the many human cytomegalovirus (HCMV)-encoded immunomodulatory genes that interfere with antigen presentation (immunoevasins) and focus on the HCMV immunoevasins US2 and US11, which induce the degradation of class I MHC heavy chains by the proteasome by catalyzing their export from the endoplasmic reticulum (ER)-membrane into the cytosol, a process termed ER dislocation. US2- and US11-mediated subversion of ER dislocation ensures proteasomal degradation of class I MHC molecules and presumably allows HCMV to avoid recognition by cytotoxic T cells, whilst providing insight into general aspects of ER-associated degradation (ERAD) which is used by eukaryotic cells to purge their ER of defective proteins. We discuss the similarities and differences between the distinct pathways co-opted by US2 and US11 for dislocation and degradation of human class I MHC molecules and also a putatively distinct pathway utilized by the murine herpes virus (MHV)-68 mK3 immunoevasin for ER dislocation of murine class I MHC. We speculate on the implications of the three pathogen-exploited dislocation pathways to cellular ER quality control. Moreover, we discuss the ubiquitin (Ub)-proteasome system and its position at the core of antigen presentation as proteolysis and intracellular trafficking rely heavily on Ub-dependent processes. We add a few examples of manipulation of the Ub-proteasome system by pathogens in the context of the immune system and such diverse aspects of the host-pathogen relationship as virus budding, bacterial chromosome integration, and programmed cell death, to name a few. Finally, we speculate on newly found pathogen-encoded deubiquitinating enzymes (DUBs) and their putative roles in modulation of host-pathogen interactions.

Viruses that establish long-term infections in their hosts have evolved a number of methods to interfere with the activities of the innate and adaptive immune systems. Control of viral infections is achieved in part through the action of cytotoxic T lymphocytes (CTLs) that recognize cytosolically derived antigenic peptides in the context of class I major histocompatibility complex (MHC) molecules. Viral replication within host cells produces abundant proteinaceous fodder for proteasomal digestion and display by class I MHC products. Tactics that disrupt antigen-presentation pathways and prevent the display of peptides to CD8(+) CTLs have been favored during the course of host-virus co-evolution. Viral immunoevasins exploit diverse cellular processes to interfere with host antiviral functions. The study of such viral factors has uncovered novel host proteins that assist these viral factors in their task and that themselves perform important cellular functions. Here, we focus on viral immunoevasins that, together with their cellular targets, interfere with antigen-presentation pathways. In particular, we emphasize the intersection of the cellular quality-control machinery in the endoplasmic reticulum with the herpesvirus proteins that have co-opted it.

Class II MHC molecules survey the endocytic compartments of APCs and present antigenic peptides to CD4 T cells. In this context, lysosomal proteases are essential not only for the generation of antigenic peptides but also for proteolysis of the invariant chain to allow the maturation of class II MHC molecules. Recent studies with protease inhibitors have implicated the asparagine endopeptidase (AEP) in class II MHC-restricted Ag presentation. We now report that AEP-deficient mice show no differences in processing of the invariant chain or maturation of class II MHC products compared with wild-type mice. In the absence of AEP, presentation to primary T cells of OVA and myelin oligodendrocyte glycoprotein, two Ags that contain asparagine residues within or in proximity to the relevant epitopes was unimpaired. Cathepsin (Cat) L, a lysosomal cysteine protease essential for the development to CD4 and NK T cells, fails to be processed into its mature two-chain form in AEP-deficient cells. Despite this, the numbers of CD4 and NK T cells are normal, showing that the single-chain form of Cat L is sufficient for its function in vivo. We conclude that AEP is essential for processing of Cat L but not for class II MHC-restricted Ag presentation.

The elimination of pathogens and pathogen-infected cells initially rests on the rapid deployment of innate immune defences. Should these defences fail, it is the lymphocytes--T cells and B cells--with their antigen-specific receptors that must rise to the task of providing adaptive immunity. Technological advances are now allowing immunologists to correlate data obtained in vitro with in vivo functions. A better understanding of T-cell activation in vivo could lead to more effective strategies for the treatment and prevention of infectious and autoimmmune diseases.

The initiation of most cytotoxic immune responses requires MHC class I-restricted presentation of internalized antigens to CD8(+) T lymphocytes, a process called cross-presentation. In dendritic cells (DC), the only antigen-presenting cells that activate naive T cells, cross-presentation is particularly efficient after internalization of opsonized antigens or immune complexes, which are cross-presented through a proteasome- and transporter associated with antigen processing (TAP)-dependent MHC class I antigen presentation pathway. We now show that FcgammaR-mediated cross-presentation is tightly regulated during DC maturation. Cross-presentation increases soon after activation by lipopolysaccharides, and it is then inhibited in fully mature cells. The initial induction of cross-presentation results from an increase of both antigen internalization and delivery to the cytosol, and from a slight rise in the activity of the proteasome and TAP. The subsequent block of cross-presentation in mature DC is a consequence of the selective down-modulation of antigen internalization and cytosolic delivery, while proteasome and TAP activities continue to rise. Therefore, FcgammaR-mediated cross-presentation is regulated during DC maturation by the selective control of antigen internalization and transport to the cytosol.

Cytotoxic T lymphocyte (CTL)-mediated immune responses rely on the efficiency of MHC class I ligand generation and presentation by antigen presenting cells (APCs). Whereas the abnormal expression of MHC molecules and transporters associated with antigen processing (TAPs) are commonly discussed as factors that modulate antigen presentation, much less is known about possible regulatory mechanisms at the level of proteolysis responsible for the generation of antigenic peptides. The ubiquitin-proteasome system is recognized as the major component responsible for this process in the cytosol and its activity can be regulated by cytokines, such as IFN-gamma. However, new evidence suggests the involvement of other proteases that can contribute to cytosolic proteolysis and therefore, to the quality and quantity of antigen production. Here, we review recent findings on an increasing number of proteolytic enzymes linked to antigen presentation, and we discuss how regulation of cytosolic protease activities might have implications for immune escape mechanisms that could be used by tumor cells and pathogens.

Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagosomes in live antigen-presenting cells. We find that an ingested particle sequentially encounters distinct protease activities during phagosomal maturation. Incorporation of active proteases into the phagosome of the macrophage cell line J774 indicates that phagosome maturation involves progressive fusion with early and late endocytic compartments. In contrast, phagosome biogenesis in bone marrow-derived dendritic cells (DCs) and macrophages preferentially involves endocytic compartments enriched in cathepsin S. Kinetics of phagosomal maturation is faster in macrophages than in DCs. Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide. This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation. Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte.

To address the role of different proteases in degradation of antigen destined for MHC class II-restricted presentation, we generated cathepsin-deficient mice carrying a transgenic B cell receptor (BCR) specific for hen egg lysozyme (HEL). We demonstrate that degradation of HEL in B lymphocytes is highly processive and does not result in discrete processing intermediates. Moreover, degradation of HEL does not require initial unlocking of the antigen by any of the cathepsins tested. Using mass spectrometry and microsequencing, we show that all major cathepsins (CatS, CatL, CatB, and CatD) digest HEL in vitro with considerable redundancy, although some preferential cleavages are evident. These observations have a functional correlate: when triggered by cathepsin S-deficient antigen-presenting cells, T cells that recognize different HEL epitopes fail to present two HEL-derived epitopes, while a third epitope is presented independently of the activity of cysteine proteases. We conclude that the proteolytic processing machinery is redundant, and that several proteases can substitute for each other to degrade a given antigen. However, a certain degree of proteolytic specificity is demonstrable for the generation of particular epitopes, notably by CatS.

Antigen presentation by MHC class II molecules relies on the action of endocytic proteases, which are differentially expressed in antigen-presenting cells and are regulated by different components of the immune system. Endocytic enzymes process and convert exogenous antigens into peptidic determinants capable of interaction with MHC class II molecules. Chemical and genetic tools have recently been developed to study the role of lysosomal proteases in antigen presentation.

Many persistent viruses have evolved the ability to subvert MHC class I antigen presentation. Indeed, human cytomegalovirus (HCMV) encodes at least four proteins that down-regulate cell-surface expression of class I. The HCMV unique short (US)2 glycoprotein binds newly synthesized class I molecules within the endoplasmic reticulum (ER) and subsequently targets them for proteasomal degradation. We report the crystal structure of US2 bound to the HLA-A2/Tax peptide complex. US2 associates with HLA-A2 at the junction of the peptide-binding region and the alpha3 domain, a novel binding surface on class I that allows US2 to bind independently of peptide sequence. Mutation of class I heavy chains confirms the importance of this binding site in vivo. Available data on class I-ER chaperone interactions indicate that chaperones would not impede US2 binding. Unexpectedly, the US2 ER-luminal domain forms an Ig-like fold. A US2 structure-based sequence alignment reveals that seven HCMV proteins, at least three of which function in immune evasion, share the same fold as US2. The structure allows design of further experiments to determine how US2 targets class I molecules for degradation.

The major histocompatibility complex (MHC) class II-associated invariant chain (Ii) regulates intracellular trafficking and peptide loading of MHC class II molecules. Such loading occurs after endosomal degradation of the invariant chain to a approximately 3-kD peptide termed CLIP (class II-associated invariant chain peptide). Cathepsins L and S have both been implicated in degradation of Ii to CLIP in thymus and peripheral lymphoid organs, respectively. However, macrophages from mice deficient in both cathepsins S and L can process Ii and load peptides onto MHC class II dimers normally. Both processes are blocked by a cysteine protease inhibitor, indicating the involvement of an additional Ii-processing enzyme(s). Comparison of cysteine proteases expressed by macrophages with those found in splenocytes and dendritic cells revealed two enzymes expressed exclusively in macrophages, cathepsins Z and F. Recombinant cathepsin Z did not generate CLIP from Ii-MHC class II complexes, whereas cathepsin F was as efficient as cathepsin S in CLIP generation. Inhibition of cathepsin F activity and MHC class II peptide loading by macrophages exhibited similar specificity and activity profiles. These experiments show that cathepsin F, in a subset of antigen presenting cells (APCs), can efficiently degrade Ii. Different APCs can thus use distinct proteases to mediate MHC class II maturation and peptide loading.

Antigen-presenting cells (APC) degrade proteins intracellularly to generate peptides, which are then bound by products of the major histocompatibility complex (MHC) and exposed on the surface of the APC for recognition by T cells. The supply of antigenic peptides and their association with MHC molecules requires the concerted action of a cohort of accessory molecules that includes chaperones, transporters of peptides, and the proteases that degrade the antigens.

Major histocompatibility complex class II antigen presentation requires the participation of lysosomal proteases in two convergent processes. First, the antigens endocytosed by the antigen-presenting cells must be broken down into antigenic peptides. Second, class II molecules are synthesized with their peptide-binding site blocked by invariant chain (Ii), and they acquire the capacity to bind antigens only after Ii has been degraded in the compartments where peptides reside. The study of genetically modified mice deficient in single lysosomal proteases has allowed us to determine their role in these processes. Cathepsins (Cat) B and D, previously considered major players in MHC class II antigen presentation, are dispensable for degradation of Ii and for generation of several antigenic determinants. By contrast, Cat S plays an essential role in removal of Ii in B cells and dendritic cells, whereas Cat L apparently does so in thymic epithelial cells. Accordingly, the absence of Cat S and L have major consequences for the onset of humoral immune responses and for T-cell selection, respectively. It is likely that other as yet uncharacterized lysosomal enzymes also play a role in Ii degradation and in generation of antigenic determinants. Experiments involving drugs that interfere with protein traffic suggest that more than one mechanism for Ii removal, probably involving different proteases, can co-exist in the same antigen-presenting cell. These findings may allow the development of protease inhibitors with possible therapeutic applications.

For a broader view of the role of H-2M as an accessory molecule in antigen presentation, we investigated the degree to which different MHC class II isotypes and alleles depend on H-2M to function in vivo. We generated H-2M-deficient animals expressing Ek/b or Ak molecules in addition to the Ab molecules already present in the mutant strain, and compared the ability of the different MHC class II molecules to present antigen at the cell surface for recognition by T cells, and contribute to positive selection of CD4+ T cells in the thymus. Biochemical analyses were performed to assess MHC class II maturation, and to determine the peptide content of the molecules. In the absence of H-2M, Ek/b molecules contained a more heterogeneous set of class II-associated invariant chain peptides (CLIP) than Ab did, which, unlike Ab-CLIP complexes, were not SDS-stable. Unlike Ab molecules, both Ek/b and Ak efficiently presented exogenously added peptides to T cells in the absence of H-2M. In addition, epitopes from some proteins, especially those known to be invariant chain independent, were presented by Ak molecules in the mutant animals. To our surprise, expression of Ek/b overcame the positive selection defect observed in H-2M-deficient mice expressing Ab alone. In contrast, Ak expression did not augment positive selection of CD4+ T cells in the mutant animals. Some of these findings in vivo contrast significantly with findings from in vitro studies on murine MHC class II molecules in human DM-deficient cell lines.

MHC class II molecules display antigenic peptides on cell surfaces for recognition by CD4(+) T cells. Proteolysis is required in this process both for degradation of invariant chain (Ii) from class II-Ii complexes to allow subsequent binding of peptides, and for generation of the antigenic peptides. The cysteine endoprotease, cathepsin S, mediates Ii degradation in human and mouse antigen-presenting cells. Studies described here examine the functional significance of cathepsin S inhibition on antigen presentation and immunity. Specific inhibition of cathepsin S in A20 cells markedly impaired presentation of an ovalbumin epitope by interfering with class II-peptide binding, not by obstructing generation of the antigen. Administration of a cathepsin S inhibitor to mice in vivo selectively inhibited activity of cathepsin S in splenocytes, resulting in accumulation of a class II-associated Ii breakdown product, attenuation of class II-peptide complex formation, and inhibition of antigen presentation. Mice treated with inhibitor had an attenuated antibody response when immunized with ovalbumin but not the T cell-independent antigen TNP-Ficoll. In a mouse model of pulmonary hypersensitivity, treatment with the inhibitor also abrogated a rise in IgE titers and profoundly blocked eosinophilic infiltration in the lung. Thus, inhibition of cathepsin S in vivo alters Ii processing, antigen presentation, and immunity. These data identify selective inhibition of cysteine proteases as a potential therapeutic strategy for asthma and autoimmune disease processes.

Antigen presentation by major histocompatibility complex (MHC) class II molecules requires the participation of different proteases in the endocytic route to degrade endocytosed antigens as well as the MHC class II-associated invariant chain (Ii). Thus far, only the cysteine protease cathepsin (Cat) S appears essential for complete destruction of Ii. The enzymes involved in degradation of the antigens themselves remain to be identified. Degradation of antigens in vitro and experiments using protease inhibitors have suggested that Cat B and Cat D, two major aspartyl and cysteine proteases, respectively, are involved in antigen degradation. We have analyzed the antigen-presenting properties of cells derived from mice deficient in either Cat B or Cat D. Although the absence of these proteases provoked a modest shift in the efficiency of presentation of some antigenic determinants, the overall capacity of Cat B-/- or Cat D-/- antigen-presenting cells was unaffected. Degradation of Ii proceeded normally in Cat B-/- splenocytes, as it did in Cat D-/- cells. We conclude that neither Cat B nor Cat D are essential for MHC class II-mediated antigen presentation.

Protein degradation plays an important role in the control and regulation of many crucial biological functions, ranging from cell cycle progression to presentation of viral antigens for scrutiny by cells of the immune system. At the heart of many of these catabolic events is the multicatalytic proteinase complex known as the proteasome. This large barrel-shaped protein complex executes a remarkable set of functions ranging from the complete destruction of abnormal and misfolded proteins to the specific proteolytic activation of crucial signaling molecules. Inhibitors of this proteolytic complex have thus been extremely useful for perturbing its function and deciphering its role in these diverse biological processes. Inhibitors of the proteasome consist mainly of peptides that are modified at the predicted site of hydrolysis with a reactive functional group capable of modifying the attacking nucleophile, either reversibly or irreversibly. Many of these inhibitors can be used in living cells and have proved to be invaluable tools for the study of proteasome function.

Major histocompatibility complex (MHC) class I molecules associate with peptides that are delivered from the cytosol to the lumen of the endoplasmic reticulum by the transporter associated with antigen processing (TAP). Liver microsomes of SHR and Lewis rats, which express different alleles of TAP (cim(b) and cim(a), respectively), accumulate different sets of peptides. Use of MHC congenic rats assigned this difference to the MHC, independent of the class I products expressed. Both the cim(a) and cim(b) TAP complexes translocate peptides with a hydrophobic carboxyl terminus, but translocation of peptides with a carboxyl-terminal His, Lys, or Arg residue is unique to cim(a). Thus, the specificity of the TAP peptide translocator restricts the peptides available for antigen presentation.