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MHC class I (MHC-I) molecules present a blueprint of the intracellular proteome to T cells allowing them to control infection or malignant transformation. As a response, pathogens and tumor cells often downmodulate MHC-I mediated antigen presentation to escape from immune surveillance. Although the fundamental rules of antigen presentation are known in detail, the players in this system are not saturated and new modules of regulation have recently been uncovered. Here, we update the understanding of antigen presentation by MHC-I molecules and how this can be exploited by tumors to prevent exposure of the intracellular proteome. This knowledge can provide new ways to improve immune responses against tumors and pathogens.

Summary The endosomal system constitutes a highly dynamic vesicle network used to relay materials and signals between the cell and its environment.1 Once internalized, endosomes gradually mature into late acidic compartments and acquire a multivesicular body (MVB) organization through invagination of the limiting membrane (LM) to form intraluminal vesicles (ILVs).2 Cargoes sequestered into ILVs can either be delivered to lysosomes for degradation or secreted following fusion of the MVB with the plasma membrane.3 It has been speculated that commitment to ILVs is not a terminal event, and that a return pathway exists, allowing “back-fusion” or “retrofusion” of intraluminal membranes to the LM.4 The existence of retrofusion as a way to support membrane equilibrium within the MVB has been widely speculated in various cell biological contexts, including exosome uptake5 and major histocompatibility complex class II (MHC class II) antigen presentation.6, 7, 8, 9 Given the small physical scale, retrofusion of ILVs cannot be measured with conventional techniques. To circumvent this, we designed a chemically tunable cell-based system to monitor retrofusion in real time. Using this system, we demonstrate that retrofusion occurs as part of the natural MVB lifestyle, with attributes parallel to those of viral infection. Furthermore, we find that retrofusion and exocytosis coexist in an equilibrium, implying that ILVs inert to retrofusion comprise a significant fraction of exosomes destined for secretion. MVBs thus contain three types of ILVs: those committed to lysosomal degradation, those retrofusing ILVs, and those subject to secretion in the form of exosomes. Video abstract, Graphical abstract, Highlights • MVBs are complex organelles with intraluminal vesicles bound by the limiting membrane • Intraluminal membranes are in a dynamic equilibrium with the limiting membrane • Retrofusion of internal vesicles is controlled by processes used for viral fusion • Exosomes arise from internal MVB vesicles not participating in retrofusion, Multivesicular bodies (MVBs) are complex organelles harboring internal vesicles. Using a chemically controlled system, Perrin et al. visualize a part of these internal vesicles fusing back to the limiting membrane. This process of retrofusion exists in equilibrium with lysosomal degradation and exosome release and is inhibited by antiviral proteins.

HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CD8+ T cell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity.

SummaryHLA class I (HLA-I) drives immune responses by presenting antigen-derived peptides to cognate CD8+T cells. This process is often hijacked by tumors and pathogens for immune evasion. Since therapeutic options for restoring HLA-I antigen presentation are limited, we aimed to identify new HLA-I pathway targets. By iterative genome-wide screens we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augments B3GNT5 enzyme activity, resulting in upregulated levels of surface (neo)lacto-series GSLs. These GSLs sterically impede molecular interactions with HLA-I and diminish CD8+T cell activation. In accordance, a disturbed SPPL3-B3GNT5 pathway in glioma associates with decreased patient survival. Importantly, we show that this immunomodulatory effect can be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that functionally inhibits antigen presentation and represents a potential therapeutic target in cancer, infection and autoimmunity.

HLA class I (HLA-I) drives immune responses by presenting antigen-derived peptides to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Since therapeutic options for restoring HLA-I antigen presentation are limited, we aimed to identify new HLA-I pathway targets. By iterative genome-wide screens we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augments B3GNT5 enzyme activity, resulting in upregulated levels of surface (neo)lacto-series GSLs. These GSLs sterically impede molecular interactions with HLA-I and diminish CD8+ T cell activation. In accordance, a disturbed SPPL3-B3GNT5 pathway in glioma associates with decreased patient survival. Importantly, we show that this immunomodulatory effect can be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that functionally inhibits antigen presentation and represents a potential therapeutic target in cancer, infection and autoimmunity.

BackgroundCross-presentation of exogenous antigens in HLA-class I molecules by professional antigen presenting cells (APCs) is crucial for CD8+ T cell function. Recent murine studies show that several non-professional APCs, including cancer-associated fibroblasts (CAFs) also possess this capacity. Whether human CAFs are able to cross-present exogenous antigen, which molecular pathways are involved in this process and how this ultimately affects tumor-specific CD8+ T cell function is unknown.MethodsIn this study, we investigated the ability of human colorectal cancer (CRC)-derived CAFs to cross-present neoantigen-derived synthetic long peptides (SLPs), corresponding to tumor-derived mutant peptides, and how this affects tumor-specific T-cell function. Processing of the SLP was studied by targeting components of the cross-presentation machinery through CRISPR/Cas9 and siRNA-mediated genetic ablation to identify the key molecules involved in fibroblast-mediated cross-presentation. Multispectral flow cytometry and killing assays were performed to study the effect of fibroblast cross-presentation on T cell function.ResultsHere, we show that human CRC-derived CAFs display an enhanced capacity to cross-present neoantigen-derived SLPs when compared with normal colonic fibroblasts. Cross-presentation of antigens by fibroblasts involved the lysosomal protease cathepsin S. Cathepsin S expression by CAFs was detected in situ in human CRC tissue, was upregulated in ex vivo cultured CRC-derived CAFs and showed increased expression in normal fibroblasts after exposure to CRC-conditioned medium. Cognate interaction between CD8+ T cells and cross-presenting CAFs suppressed T cell function, reflected by decreased cytotoxicity, reduced activation (CD137) and increased exhaustion (TIM3, LAG3 and CD39) marker expression.ConclusionThese data indicate that CAFs may directly suppress tumor-specific T cell function in an antigen-dependent fashion in human CRC.

Invariant chain (Ii) is an important multifunctional player in the regulation of adaptive immune responses and is responsible for several cellular functions related to MHCI and MHCII antigen loading and antigen presentation. While regulating endosomal trafficking of MHCII and other proteins that bind to Ii, this molecule is able to influence the endosomal pathway delaying the maturation of endosomes to the late endosomal loading compartments. When expressed in cells Ii is found to increase endosomal size, but the mechanisms for this is not known. We used RNAi silencing to identify SNARE proteins controlling Ii induced increase of endosomal size and delay of the endosomal pathway. Ii was found to interact with the SNARE protein Vti1b. Vti1b localized at the contact sites of fusing Ii positive endosomes and a tailless Ii was able to relocate Vti1b to the plasma membrane. Furthermore, silencing Vti1b, abrogated the delay in endosomal maturation induced by Ii expression. In conclusion, Ii interacts with Vti1b and this interaction is fundamental for Ii-mediated alteration of the endosomal pathway. We propose that Ii, by interacting with SNAREs, in particular Vti1B in the biosynthetic pathway of antigen presenting cells, is able to assemble SNARE directed fusion partners in the early part of the endosomal pathway that lead to a slower endosomal maturation for efficient antigen processing and antigen loading.

Antigen-presenting cells (APCs) capture and present pathogens to T cells, thus arousing adaptive immune responses geared at the elimination of these invaders. In APCs, pathogens acquired from the extracellular space intersect with MHC class II (MHC-II) molecules in the endolysosomal system, where processing and loading of antigenic peptides occur. The resulting complexes can then be directed to the cell surface for recognition by T cells. To achieve this, the endosomal pathway of APCs must undergo dramatic rearrangements upon pathogen encounter. In this review we discuss recent strides in our understanding of how APCs modulate the organization and function of their endolysosomes to best suit different stages of antigen acquisition, processing and presentation cascade.

Dendritic cells (DCs) take up antigen in the periphery, migrate to secondary lymphoid organs, and present processed antigen fragments to adaptive immune cells and thus prime antigen-specific immunity. During local inflammation, recirculating monocytes are recruited from blood to the inflamed tissue, where they differentiate to macrophages and DCs. In this study, we found that monocytes showed high transporter associated with antigen processing (TAP)–dependent peptide compartmentalization and that after antigen pulsing, they were not able to efficiently stimulate antigen-specific T lymphocytes. Nevertheless, upon in vitro differentiation to monocyte-derived DCs, TAP-dependent peptide compartmentalization as well as surface major histocompatibility complex I turnover decreased and the cells efficiently restimulated T lymphocytes. Although TAP-dependent peptide compartmentalization decreased during DC differentiation, TAP expression levels increased. Furthermore, TAP relocated from early endosomes in monocytes to the endoplasmic reticulum (ER) and lysosomal compartments in DCs. Collectively, these data are compatible with the model that during monocyte-to-DC differentiation, the subcellular relocation of TAP and the regulation of its activity assure spatiotemporal separation of local antigen uptake and processing by monocytes and efficient T-lymphocyte stimulation by DCs.

Summary The peptide-loading complex is a bottleneck in antigen presentation by major histocompatibility complex (MHC) class I molecules. While the structures of its individual components were known, the recent report of the 7.2 A structure of the entire complex now fits them into their functional context, explaining this monumental step in antigen acquisition by MHC class I molecules.

The cytokine interferon‐γ (IFNγ) can induce expression of MHC class II (MHCII) on many different cell types, leading to antigen presentation to CD4(+) T cells and immune activation. This has also been linked to anti‐tumour immunity and graft‐versus‐host disease. The extent of MHCII upregulation by IFNγ is cell type‐dependent and under extensive control of epigenetic regulators and signalling pathways. Here, we identify novel genetic and chemical factors that control this form of MHCII expression. Loss of the oxidative stress sensor Keap1, autophagy adaptor p62/SQSTM1, ubiquitin E3‐ligase Cullin‐3 and chromatin remodeller BPTF impair IFNγ‐mediated MHCII expression. A similar phenotype is observed for arsenite, an oxidative stressor. Effects of the latter can be reversed by the inhibition of HDAC1/2, linking oxidative stress conditions to epigenetic control of MHCII expression. Furthermore, dimethyl fumarate, an antioxidant used for the treatment of several autoimmune diseases, impairs the IFNγ response by manipulating transcriptional control of MHCII. We describe novel pathways and drugs related to oxidative conditions in cells impacting on IFNγ‐mediated MHCII expression, which provide a molecular basis for the understanding of MHCII‐associated diseases.

C-type lectins are key players in immune regulation by driving distinct functions of antigen-presenting cells. The C-type lectin CLEC16A gene is located at 16p13, a susceptibility locus for several autoimmune diseases, including multiple sclerosis. However, the function of this gene and its potential contribution to these diseases in humans are poorly understood. In this study, we found a strong upregulation of CLEC16A expression in the white matter of multiple sclerosis patients (n = 14) compared to non-demented controls (n = 11), mainly in perivascular leukocyte infiltrates. Moreover, CLEC16A levels were significantly enhanced in peripheral blood mononuclear cells of multiple sclerosis patients (n = 69) versus healthy controls (n = 46). In peripheral blood mononuclear cells, CLEC16A was most abundant in monocyte-derived dendritic cells, in which it strongly co-localized with human leukocyte antigen class II. Treatment of these professional antigen-presenting cells with vitamin D, a key protective environmental factor in multiple sclerosis, downmodulated CLEC16A in parallel with human leukocyte antigen class II. Knockdown of CLEC16A in distinct types of model and primary antigen-presenting cells resulted in severely impaired cytoplasmic distribution and formation of human leucocyte antigen class II-positive late endosomes, as determined by immunofluorescence and electron microscopy. Mechanistically, CLEC16A participated in the molecular machinery of human leukocyte antigen class II-positive late endosome formation and trafficking to perinuclear regions, involving the dynein motor complex. By performing co-immunoprecipitations, we found that CLEC16A directly binds to two critical members of this complex, RILP and the HOPS complex. CLEC16A silencing in antigen-presenting cells disturbed RILP-mediated recruitment of human leukocyte antigen class II-positive late endosomes to perinuclear regions. Together, we identify CLEC16A as a pivotal gene in multiple sclerosis that serves as a direct regulator of the human leukocyte antigen class II pathway in antigen-presenting cells. These findings are a first step in coupling multiple sclerosis-associated genes to the regulation of the strongest genetic factor in multiple sclerosis, human leukocyte antigen class II.

Specific immune responses are critically dependent on protein degradation products in the form of peptides. These peptides are presented by major histocompatibility complexes (MHCs), and recognition of MHC-peptide complexes by the immune system determines successful pathogen elimination, transplant rejection, autoimmunity or death. Here we review the immune response from the peptide's perspective and discuss the fate of peptides in cells before presentation by MHC complexes. We then discuss how peptides are altered post-translationally to yield immune responses and how peptides can be engineered to achieve strong immune responses following vaccination. Although peptides are simple from a chemical perspective, they are complex in their immunological consequences.

Since the discovery of major histocompatibility complex (MHC) molecules, it took some 40 years to arrive at a coherent picture of how MHC class I and MHC class II molecules really work. This is a story of proteases and MHC-like chaperones that support the MHC class I and II molecules in presenting peptides to the immune system. We now understand that the MHC system shapes both the repertoire of presented peptides and the subsequent T cell responses, with important implications ranging from transplant rejection to tumor immunotherapies. Here we present an illustrated review on the ins and outs of MHC class I and MHC class II antigen presentation.

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.

MHC class II molecules (MHC-II) present antigenic fragments acquired in the endocytic route to the immune system for recognition and activation of CD4+ T cells. This ignites a series of immune responses. MHC-II strongly correlates to most autoimmune diseases. Understanding the biology of MHC-II is therefore expected to translate into novel means of autoimmunity control or immune response improvement. Although the basic cell biology of MHC-II antigen presentation is well understood, many novel aspects have been uncovered in recent years including means of antigen delivery, preparation for MHC-II loading, transport processes and vaccination strategies. We will discuss past, present and future of these insights into the biology of MHC-II.

SummaryMHC class II molecules (MHC-II) present peptides to T helper cells to facilitate immune responses and are strongly linked to autoimmune diseases. To unravel processes controlling MHC-II antigen presentation, we performed a genome-wide flow cytometry-based RNAi screen detecting MHC-II expression and peptide loading followed by additional high-throughput assays. All data sets were integrated to answer two fundamental questions: what regulates tissue-specific MHC-II transcription, and what controls MHC-II transport in dendritic cells? MHC-II transcription was controlled by nine regulators acting in feedback networks with higher-order control by signaling pathways, including TGFβ. MHC-II transport was controlled by the GTPase ARL14/ARF7, which recruits the motor myosin 1E via an effector protein ARF7EP. This complex controls movement of MHC-II vesicles along the actin cytoskeleton in human dendritic cells (DCs). These genome-wide systems analyses have thus identified factors and pathways controlling MHC-II transcription and transport, defining targets for manipulation of MHC-II antigen presentation in infection and autoimmunity.

The present paradigm is that primary B cells are nonphagocytosing cells. In this study, we demonstrate that human primary B cells are able to internalize bacteria when the bacteria are recognized by the BCR. BCR-mediated internalization of Salmonella typhimurium results in B cell differentiation and secretion of anti-Salmonella Ab by the Salmonella-specific B cells. In addition, BCR-mediated internalization leads to efficient Ag delivery to the MHC class II Ag-loading compartments, even though Salmonella remains vital intracellularly in primary B cells. Consequently, BCR-mediated bacterial uptake induces efficient CD4+ T cell help, which boosts Salmonella-specific Ab production. BCR-mediated internalization of Salmonella by B cells is superior over extracellular Ag extraction to induce rapid and specific humoral immune responses and efficiently combat infection.

TNF family member CD70 is the ligand of CD27, a costimulatory receptor that shapes effector and memory T cell pools. Tight control of CD70 expression is required to prevent lethal immunodeficiency. By selective transcription, CD70 is largely confined to activated lymphocytes and dendritic cells (DC). We show here that, in addition, specific intracellular routing controls its plasma membrane deposition. In professional antigen-presenting cells, such as DC, CD70 is sorted to late endocytic vesicles, defined as MHC class II compartments (MIIC). In cells lacking the machinery for antigen presentation by MHC class II, CD70 travels by default to the plasma membrane. Introduction of class II transactivator sufficed to reroute CD70 to MIIC. Vesicular trafficking of CD70 and MHC class II is coordinately regulated by the microtubule-associated dynein motor complex. We show that when maturing DC make contact with T cells in a cognate fashion, newly synthesized CD70 is specifically delivered via MIIC to the immunological synapse. Therefore, we propose that routing of CD70 to MIIC serves to coordinate delivery of the T cell costimulatory signal in time and space with antigen recognition.

MHC class I molecules present a variable but limited repertoire of antigenic peptides for T-cell recognition. Understanding how peptide selection is achieved requires mechanistic insights into the interactions between the MHC I and candidate peptides. We find that, at first encounter, MHC I H-2K(b) considers a wide range of peptides, including those with expanded N termini and unfitting anchor residues. Discrimination occurs in the second step, when noncanonical peptides dissociate with faster exchange rates. This second step exhibits remarkable temperature sensitivity, as illustrated by numerous noncanonical peptides presented by H-2K(b) in cells cultured at 26 °C relative to 37 °C. Crystallographic analyses of H-2K(b)-peptide complexes suggest that a conformational adaptation of H-2K(b) drives the decisive step in peptide selection. We propose that MHC class I molecules consider initially a large peptide pool, subsequently refined by a temperature-sensitive induced-fit mechanism to retain the canonical peptide repertoire.

The combination of genetically encoded fluorescent probes and advanced microscopic techniques has dramatically propelled the understanding of cell biology. Highly complex reactions can now be studied in detail in a relatively cost-effective and easy manner and, perhaps most importantly, in the context of a single living cell. In the past decade, numerous reports have uncovered the localization of key molecules in virtually all cellular processes. However, there remains a need for more accurate determination of genuine protein-protein interactions and quantification of highly dynamic processes, which has resulted in the revival of several biophysical techniques. Recent applications of these techniques have deepened understanding of processes involved in antigen presentation to the immune system.

Intracellular proteins are degraded by the proteasome, and resulting peptides surviving cytoplasmic peptidase activity can be presented by MHC class I molecules. Here, we show that intracellular aminopeptidases degrade peptides within seconds, almost irrespectively of amino acid sequence. N- but not C-terminal extension increases the half-life of peptides until they are 15 amino acids long. Beyond 15 amino acids, peptides are exclusively trimmed by the peptidase TPPII, which displays both exo- and endopeptidase activity. Surprisingly, most proteasomal degradation products are handled by TPPII before presentation by MHC class I molecules. We define three distinct proteolytic activities during antigen processing in vivo. Proteasome-generated peptides relevant for antigen presentation are mostly 15 amino acids or longer. These require TPPII activity for further trimming before becoming substrates for other peptidases and MHC class I. The heterogeneous pool of aminopeptidases will process TPPII products into MHC class I peptides and beyond.

MHC class I molecules bind short peptides and present them to CD8+ T cells. Contrary to textbook descriptions, the generation of MHC class-I-associated peptides from endogenous proteins is a highly dynamic and remarkably inefficient process. Here, we describe recent experiments that show how nascent and mature proteins are degraded into peptides that are trimmed, transported and trimmed again to enable presentation of a small portion of the generated peptides. By linking the failure rate of protein synthesis with antigen presentation, a rapid T-cell response is ensured, which is crucial in combating viral infections. Presentation on MHC class I molecules is achieved by less than 0.1% of the specific peptides that have survived intracellular destruction. The other peptides are converted into free amino acids that are used for recycling into new proteins.

Major histocompatibility complex (MHC) class I molecules usually present endogenous peptides at the cell surface. This is the result of a cascade of events involving various dedicated proteins like the peptide transporter associated with antigen processing (TAP) and the ER chaperone tapasin. However, alternative ways for class I peptide loading exist which may be highly relevant in a process called cross-priming. Both pathways are described here in detail. One major difference between these pathways is that the proteases involved in the generation of peptides are different. How proteases and peptidases influence peptide generation and degradation will be discussed. These processes determine the amount of peptides available for TAP translocation and class I binding and ultimately the immune response.

Meeting abstracts Anti-tumor immune responses are often hampered by an excess of coinhibition of T cells, but also by a lack of MHC antigen presentation and costimulation. The latter are not yet successfully exploited as targets for immunotherapy, while augmenting MHC and costimulatory molecules

MHC class I and MHC class II molecules present peptides to the immune system to drive proper T cell responses. Pharmacological modulation of T-cell responses can offer treatment options for a range of immune-related diseases. Pharmacological downregulation of MHC molecules may find application in treatment of auto-immunity and transplantation rejection while pharmacological activation of antigen presentation would support immune responses to infection and cancer. Since the cell biology of MHC class I and MHC class II antigen presentation is understood in great detail, many potential targets for manipulation have been defined over the years. Here, we discuss how antigen presentation by MHC molecules can be modulated by pharmacological agents and how chemistry can further support the study of antigen presentation in general. The chemical biology of antigen presentation by MHC molecules shows surprising options for immune modulation and the development of future therapies.

The 20S proteasome is involved in the processing of MHC class I-presented Ags. A number of epitopes is known to be generated as precursor peptides requiring trimming either before or after translocation into the endoplasmic reticulum (ER). In this study, we have followed the proteasomal processing and TAP-dependent ER translocation of the immunodominant epitope of the murine CMV immediate early protein pp89. For the first time, we experimentally linked peptide generation by the proteasome system and TAP-dependent ER translocation. Our experiments show that the proteasome generates both an N-terminally extended 11-mer precursor peptide as well as the correct H2-Ld 9-mer epitope, a process that is accelerated in the presence of PA28. Our direct peptide translocation assays, however, demonstrate that only the 11-mer precursor peptide is transported into the ER by TAPs, whereas the epitope itself is not translocated. In consequence, our combined proteasome/TAP assays show that the 11-mer precursor is the immunorelevant peptide product that requires N-terminal trimming in the ER for MHC class I binding.

Antigenic peptide binding to MHC class II molecules in the endocytic pathway occurs via a multifactorial process that requires the support of a specialized lysosomal chaperone called HLA-DM. DM shows both in primary amino acid sequence and quaternary structure a high homology to both MHC class I and class II molecules. Like the peptide presenting class II molecules, DM is expressed in all professional antigen presenting cells. DM catalyzes the dissociation of peptides that do not bind stably to the class II peptide-binding groove, thereby leading to the preferential presentation of stably binding antigenic peptides. The recently discovered HLA-DO molecule is mainly expressed in B cells and associates with DM, thereby markedly affecting DM function. Like DM, the genes encoding the HLA-DO heterodimer lie within the MHC class II region and exhibit strong homology to classical class II molecules. This review evaluates the unique effects of DO on DM-mediated antigen presentation by MHC class II molecules and discusses the possible physiological relevance for the B cell-specific expression of DO and its function.

Heat shock proteins (HSPs) like glycoprotein (gp)96 (glucose-regulated protein 94 [grp94]) are able to induce specific cytotoxic T lymphocyte (CTL) responses against cells from which they originate. Here, we demonstrate that for CTL activation by gp96-chaperoned peptides, specific receptor-mediated uptake of gp96 by antigen-presenting cells (APCs) is required. Moreover, we show that in both humans and mice, only professional APCs like dendritic cells (DCs), macrophages, and B cells, but not T cells, are able to bind gp96. The binding is saturable and can be inhibited using unlabeled gp96 molecules. Receptor binding by APCs leads to a rapid internalization of gp96, which colocalizes with endocytosed major histocompatibility complex (MHC) class I and class II molecules in endosomal compartments. Incubation of gp96 molecules isolated from cells expressing an adenovirus type 5 E1B epitope with the DC line D1 results in the activation of E1B-specific CTLs. This CTL activation can be specifically inhibited by the addition of irrelevant gp96 molecules not associated with E1B peptides. Our results demonstrate that only receptor-mediated endocytosis of gp96 molecules leads to MHC class I–restricted re-presentation of gp96-associated peptides and CTL activation; non–receptor-mediated, nonspecific endocytosis is not able to do so. Thus, we provide evidence on the mechanisms by which gp96 is participating in the cross-presentation of antigens from cellular origin.

Major histocompatibility complex class II molecules present peptides to CD4+ T cells. New findings indicate that conventional and plasmacytoid dendritic cells handle these molecules differently after activation.

MHC class II molecules usually present exogenous antigens, but peptidome analyses have also identified many antigens from cytosolic or nuclear sources. In this issue of Immunity, Schmid et al. (2007) show that MHC class II molecules can present these through autophagosomes.

Proteasomes are proteolytic complexes involved in non-lysosomal degradation which are localized in both the cytoplasm and the nucleus. The dynamics of proteasomes in living cells is unclear, as is their targeting to proteins destined for degradation. To investigate the intracellular distribution and mobility of proteasomes in vivo, we generated a fusion protein of the proteasome subunit LMP2 and the green fluorescent protein (GFP). The LMP2-GFP chimera was quantitatively incorporated into catalytically active proteasomes. The GFP-tagged proteasomes were located within both the cytoplasm and the nucleus. Within these two compartments, proteasomes diffused rapidly, and bleaching experiments demonstrated that proteasomes were transported slowly and unidirectionally from the cytoplasm into the nucleus. During mitosis, when the nuclear envelope has disintegrated, proteasomes diffused rapidly throughout the dividing cell without encountering a selective barrier. Immediately after cell division, the restored nuclear envelope formed a new barrier for the diffusing proteasomes. Thus, proteasomes can be transported unidirectionally over the nuclear membrane, but can also enter the nucleus upon reassembly during cell division. Since proteasomes diffuse rapidly in the cytoplasm and nucleus, they may perform quality control by continuous collision with intracellular proteins, and degrading those proteins that are properly tagged or misfolded.

During biosynthesis, MHC class II molecules travel through the endocytic pathway and interact with antigenic peptides before their stable insertion in the plasma membrane. The process of class II association with these peptides and their final deposition at the cell surface are essential steps in boosting specific antibody responses. Therefore, the study of class II molecules is important in understanding how cell-biological events can direct an immune response.

Multidrug resistance (MDR) to anti-cancer drugs has been associated with the overexpression of P-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP), both being members of the ATP-binding cassette (ABC) superfamily of transporters. We investigated whether in addition to P-gp and MRP, another ABC transporter, the transporter associated with antigen processing (TAP), is associated with MDR. TAP plays a major role in MHC class I-restricted antigen presentation by mediating peptide translocation over the endoplasmic reticulum membrane. TAP1 and P-gp share a significant degree of homology among their transmembrane domains, which are thought to be the primary determinants of substrate specificity, and both can apparently mediate the translocation of peptides. Using immunocytochemistry and Western blot, TAP was overexpressed in parallel with MHC class I in several MDR human cancer cell lines. TAP was overexpressed more frequently in MRP-positive MDR cell lines (three out of three) than in P-gp positive MDR cells (two out of five). Reversal of resistance resulted in a decrease in TAP levels. Transfection of the TAP genes into TAP-deficient lymphoblastoid T2 cells conferred mild resistance to etoposide, vincristine and doxorubicin (2- to 2.5-fold). Furthermore, etoposide and vincristine inhibited TAP-dependent peptide translocation to the endoplasmic reticulum. Collectively, our results suggest that TAP may modestly contribute to the MDR phenotype, in particular in MRP- overexpressing MDR cells. Further insight into the role of TAP in MDR will require the study of other transfectants, as well as the investigation of TAP expression in P-gp and MRP-negative MDR cancer cell lines. Images Figure 1 Figure 2

Like many other antigen presenting cells, macrophages present antigenic fragments in the context of MHC class I or class II molecules. Here we will summarize our contributions to the more cell biological aspects of the process of antigen presentation. We will therefore follow the MHC molecules from their assembly or construction to the site of action (the plasma membrane).

The major histocompatibility complex (MHC)-encoded transporters associated with antigen processing (TAP) translocate peptides from the cytosol into the lumen of the endoplasmic reticulum (ER) where they associate with MHC class I molecules. The length of class I-binding peptides is usually 8-11 amino acids, but examples of significantly longer peptides have been described. The preferred lengths and upper and lower size limits for peptides translocated by TAP have not been determined in detail because in the currently used test systems, peptides are subject to proteolytic degradation. In the present study, three sets of individual peptides or partially randomized peptide libraries ranging between 6 and 40 residues were used that contained a radiolabeled tyrosine and a consensus sequence for ER-specific N-glycosylation at opposite ends, thus ensuring that only nondegraded peptides were monitored in the transport/glycosylation assay. For three different transporters, rat TAP1/2a, rat TAP1/2u and hTAP, the most efficient ATP-dependent transport was observed for peptides with 8-12 amino acids. Hexamers and longer peptides of up to 40 amino acids were also translocated, albeit less efficiently. For two of the three sets of peptides analyzed, rat TAP1/2a showed a less stringent length selection than rat TAP1/2u and human TAP. The superior transport of the decamer of the TNKT.. Y series was not due to faster degradation or less efficient glycosylation of shorter or longer length variants. A binding assay with TAP-containing microsomes revealed a high affinity for the radiolabeled decamer (KD = 580 nM), while other length variants were clearly inferior in their binding affinities. Thus, TAP binds and preferentially translocates peptides with a length suitable for binding to MHC class I molecules, but peptides that are considerably longer may also be substrates. About 10(5) peptide binding sites per cell equivalent of microsomes were determined, providing an estimate for the number of TAP complexes in the ER membrane.

Background: Glycoproteins encoded by the major histocompatibility complex class I region (MHC class I) present peptide antigens to cytotoxic T cells (CTLs). Peptides are delivered to the site of MHC class I assembly by the transporter associated with antigen processing (TAP), and cell lines that lack this transporter are unable to present endogenous antigens to CTLs. Although it has been shown that a fraction of newly synthesized class I molecules are in physical association with TAP, it is not known whether this interaction is functionally relevant, or where on the class I molecule the TAP binding site might be. Results C1R cells transfected with a mutant HLA-A2.1 heavy chain (HC), where threonine at position 134 in the α2 domain is changed to lysine (T134K), are unable to present endogenous antigens to CTLs. We have studied the biochemistry of this mutant in C1R cells, and found that a large pool of unstable empty class I HC– β 2 m ( β -2 microglobulin) heterodimers exist that are rapidly transported to the cell surface. The T134K mutant seemed to bind peptide antigens and assemble with β 2 m as efficiently as wild-type HLA-A2.1. However, we show here that the inefficiency with which T134K presents intracellular antigen is associated with its inability to interact with the TAP heterodimer. Conclusion These experiments establish that the class I–TAP interaction is obligatory for the presentation of peptide epitopes delivered to the endoplasmic reticulum (ER) by TAP. Wild-type HLA-A2.1 molecules in TAP-deficient cells are retained in the ER, whereas T134K is rapidly released to the cell surface, but is unstable, suggesting a role for the TAP complex as an intracellular checkpoint that only affects the release of class I molecules with stably bound peptide ligands.

The assembly of major histocompatibility complex (MHC) class I and class II molecules, and their association with antigenic peptides, is analogous to a production line. Each molecule in the pathway has a specific job to do before it hands over its partially processed product to the next cog in the cellular machine. This tireless molecular workforce has been quietly generating functional MHC class I and class II complexes for millennia, yet it has only been in the past two decades that we have begun to understand how some of the many components work. MHC class I and class II molecules use different intracellular pathways and, consequently, present different peptides (with some exceptions).

MHC class II molecules (MHCII) are critical for presenting antigens to CD4(+) T-cells. They control ignition of CD4(+) T cells and are as such involved in most auto-immune diseases. To define proteins and pathways controlling MHCII antigen presentation and expression, we performed a genome-wide flow cytometry based RNAi screen. Hits were subsequently classified by two screens that monitored the intracellular distribution and transcription of MHCII. This multi-dimensional approach allowed subclassification of hits into functional groups as a first step to defining new pathways controlling MHCII antigen presentation. The datasets from this screen are used as a template for several follow-up studies. This overview focuses on how data from genome-wide screens can be used for target-lead finding, data mining, systems biology and systematic cell biology.

We studied the functional consequences of targeting class II molecules to either the cell surface or to endocytic structures by expressing HLA-DR1 in human kidney cells in the presence or absence of different forms of the invariant chain (Ii). Transfectants expressing class II molecules in the absence of Ii present influenza virus efficiently and co-expression of full length Ii does not further increase antigen presentation. Chimeric Ii containing the cytoplasmic domain of the transferrin receptor (Tfr-Ii) delivers class II molecules associated with Tfr-Ii to endosomal compartments, but this does not result in efficient antigen presentation. When class II molecules are targeted to the cell surface by Ii lacking either 15 (delta 15Ii) or 23 (delta 23Ii) amino acids from the cytoplasmic domain, a fraction of free class II molecules is also observed. Whereas delta 15Ii did not affect antigen presentation by class II molecules, delta 23Ii inhibited, but did not abrogate, the response. We show that class II molecules expressed in the presence of delta 23Ii can be internalized, followed by degradation of delta 23Ii and return of free class II alpha beta heterodimers to the cell surface. A fraction of the resulting free class II molecules is sodium dodecyl sulfate stable, indicating that internalization and reappearance of class II molecules at the cell surface can be an alternative route for antigen presentation. In all transfectants, class II molecules were found in endocytic compartments that labeled for CD63 and resembled the multilaminar MIIC compartments found in B cell lines. Ii is not required for endosomal targeting of class II molecules. The number of class II molecules observed in the multilaminar compartments correlates with the efficiency of antigen presentation.

With the discovery of MHC-encoded peptide transporters (TAP) came the identification of a new class of molecules within the immune system. TAP belongs to a large family of ATP-binding, multimembrane-spanning transporters that are expressed in a diversity of cells, from prokaryotic to mammalian, and show specificity for a variety of different substrates. TAP represents the solution to a major topological problem in immunology, namely the translocation of peptides, generated by cytosolic degradation of antigens, into the lumen of the endoplasmic reticulum where they associate with newly synthesized MHC class I molecules. A novel assay allows us to determine the requirements for the TAP-mediated peptide transport. First results indicate that TAP preselects peptides according to sequence and length in a way that is compatible with the characteristics of peptides isolated from class I molecules.

Endogenous antigen presentation by major histocompatibility complex class II molecules can be understood if class II alpha beta heterodimers bind peptide in the endoplasmic reticulum (ER) before they associate with the invariant chain (Ii). We have studied the assembly of class II molecules from the free alpha, beta and Ii subunits to examine the existence of a class II alpha beta heterodimer as an intermediate in the assembly of class II alpha beta Ii heterotrimers in the ER. In human kidney cell transfectants, the free class II alpha and beta subunits and the class II alpha beta heterodimer are retained in the ER by association with the chaperonin immunoglobulin binding protein (BiP) and Ii is retained through its cytoplasmic tail. Co-expression of Ii results in release of BiP from class II alpha beta complexes and exit of class II alpha beta Ii heterotrimers from the ER. We show that the cytoplasmic tail and the transmembrane region of the class II alpha and beta chain is not essential for proper assembly of the class II alpha beta heterodimer. We followed assembly of the class II alpha beta Ii heterotrimers in wild-type cells. The class II subunits assemble post-translationally. No class II alpha beta heterodimers could be isolated as intermediates in the formation of class II alpha beta Ii heterotrimers, suggesting that peptide binding by class II molecules in the ER is necessarily inefficient.

Cytotoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C terminus of these CTL epitopes is considered to be produced by the proteasome. Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity. Nardilysin and TOP were required, either together or alone, for the generation of a tumor-specific CTL epitope from PRAME, an immunodominant CTL epitope from Epstein-Barr virus protein EBNA3C, and a clinically important epitope from the melanoma protein MART-1. TOP functioned as C-terminal trimming peptidase in antigen processing, and nardilysin contributed to both the C-terminal and N-terminal generation of CTL epitopes. By broadening the antigenic peptide repertoire, nardilysin and TOP strengthen the immune defense against intracellular pathogens and cancer.

BACKGROUND: The eradication of facultative intracellular bacterial pathogens, like Salmonella typhi, requires the concerted action of both the humoral immune response and the cytotoxic CD8(+) T cell response. Dendritic cells (DCs) are considered to orchestrate the cytotoxic CD8(+) T cell response via cross-presentation of bacterial antigens onto MHC class I molecules. Cross-presentation of Salmonella by DCs however, is accompanied by the induction of apoptosis in the DCs. Besides antibody production, B cells are required to clear Salmonella infection for other unknown reasons. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that Salmonella-specific B cells that phagocytose Salmonella upon BCR-ligation reactivate human memory CD8(+) T cells via cross-presentation yielding a Salmonella-specific cytotoxic T cell response. The reactivation of CD8(+) T cells is dependent on CD4(+) T cell help. Unlike the DCs, B cell-mediated cross-presentation of Salmonella does not coincide with apoptosis. CONCLUSIONS/SIGNIFICANCE: B cells form a new player in the activation of the cytotoxic effector arm of the immune response and the generation of effective adaptive immunity in Salmonella infection.

Antigen cross-presentation is a critical step in the elicitation of cell-mediated immune responses. Much research has been aimed at manipulating antigen cross-presentation to improve tumor immunotherapy and vaccination. In this issue of Science Translational Medicine, Saccheri et al. describe a mechanism for spurring successful antitumor responses by enhancing the transfer, to antigen-presenting cells, of tumor-specific antigens that leave the cancer cells via gap junctions induced by Salmonella infection of the melanoma tumor. Salmonella turns from foe to friend by promoting cross-presentation for strong antitumor immunity and tumor eradication.

CD70 is a TNF-related transmembrane molecule expressed by mature dendritic cells (DCs), which present antigens to T cells via major histocompatibility complex (MHC) molecules. In DCs, CD70 localizes with MHC class II molecules in late endosomal vesicles, known as MHC class II compartments (MIICs). MIICs are transported to the immune synapse when a DC contacts an antigen-specific CD4+ T cell. Consequently, MHC class II and CD70 are simultaneously exposed to the T cell. Thereby, T-cell activation via the antigen receptor and CD70-mediated co-stimulation are synchronized, apparently to optimize the proliferative response. We report here that the invariant chain (Ii), a chaperone known to transport MHC class II to MIICs, performs a similar function for CD70. CD70 was found to travel by default to the plasma membrane, whereas Ii coexpression directed it to late endosomes and/or lysosomes. In cells containing the MHC class II presentation pathway, CD70 localized to MIICs. This localization relied on Ii, since transport of CD70 from the Golgi to MIICs was impeded in Ii-deficient DCs. Biophysical and biochemical studies revealed that CD70 and Ii participate in an MHC-class-II-independent complex. Thus, Ii supports transport of both MHC class II and CD70 to MIICs and thereby coordinates their delivery to CD4+ T cells.

This article presents an integral model of TAP function that visualizes how peptide and ATP drive the conformational changes required for pore opening and peptide transfer into the major histocompatibility complex (MHC) class I loading complex.