MHC class II signaling in antigen-presenting cells

The MHC class II molecules have been recognized as signaling receptors for more than a decade, and recent work has revealed the importance of their signaling for the immune response. Today, we know that the function of MHC class II molecules on antigen-presenting cells (APCs) is not limited to their role as antigen-presenting structures; they are flexible receptors that, by triggering a variety of signaling pathways, can regulate APC activities from proliferation and maturation to apoptosis. Recent advances have provided insights into how these molecules might accommodate such regulation.     

High efficiency of endogenous antigen presentation by MHC class II molecules.

MHC class II molecules are involved in the presentation of both exogenous and endogenous antigens to CD4 T cells. Using the trans-membrane hemagglutinin (HA) from measles virus and the secreted hen egg lysozyme (HEL) as antigen models, we have compared the efficiency of MHC class II presentation by naive antigen presenting cells (APCs) pulsed with exogenous antigen with that of their transfected counterparts synthesizing endogenous antigen. B cells expressing even a very low amount of trans-membrane HA were found to present endogenous HA to I-Ed restricted T cell hybridomas with a high efficiency whereas their naive counterparts required to be pulsed with a comparatively high amount of exogenous HA. Similarly, MHC class II presentation of endogenous secreted HEL was found to be much more efficient when compared with that of exogenous HEL. Biochemical studies did not reveal any enhanced intracellular degradation of endogenous HEL. As expected, HEL was released in the surrounding medium within < 1 h. MHC class II presentation of endogenous HEL could not be explained by re-uptake by bystander APCs of HEL secreted in the surrounding medium. No sensitization of naive APCs could be observed either when co-cultured with HEL secreting cells or when cultured for 10 days with a sub-threshold amount of exogenous HEL. At the cell surface, I-Ed molecules immunoprecipitated from HEL secreting cells were found to be slightly enriched in SDS-resistant forms. These data raised the question of how peptides derived from endogenous transmembrane and secreted antigens can so efficiently reach an MHC class II loading compartment.     

Diversity in MHC class II antigen presentation

Processing exogenous and endogenous proteins for presentation by major histocompatibility complex (MHC) molecules to T cells is the defining function of antigen-presenting cells (APC) as major regulatory cells in the acquired immune response. MHC class II-restricted antigen presentation to CD4 T cells is achieved by an essentially common pathway that is subject to variation with regard to the location and extent of degradation of protein antigens and the site of peptide binding to MHC class II molecules. These subtle variations reveal a surprising flexibility in the ways a diverse peptide repertoire is displayed on the APC surface. This diversity may have profound consequences for the induction of immunity to infection and tumours, as well as autoimmunity and tolerance.     

Trafficking of MHC class II molecules in the late secretory pathway.

Antigen presenting cells (APCs) alert the immune system to attack by extracellular organisms; APCs achieve this via internalization, degradation, and display of antigenic fragments on the cell surface by MHC class II molecules. These class II molecules bind to an accessory protein, termed the invariant chain, that ensures proper folding of the molecules. Invariant-chain binding also directs class II molecules to lysosomes, which are probably the most important sites for antigen loading. Endosomes are intermediates in the transport of class-II-invariant chain complexes to antigen-processing compartments, whereas trafficking of class II-peptide complexes to the membrane (and beyond) is less-well understood. Unlike other APCs, dendritic cells alter their capacity to present peptides via MHC class II molecules during differentiation, revealing a complex level of regulated antigen-presentation by this APC subtype.     

Concentration of MHC class II molecules in lipid rafts facilitates antigen presentation

The plasma membranes of eukaryotic cells are not uniform and possess distinct cholesterol- and sphingolipid-rich raft microdomains that are enriched in proteins known to be essential for cellular function. Lipid raft microdomains are important for T cell receptor (TCR)-mediated activation of T cells. However, the importance of lipid rafts on antigen presenting cells (APCs) and their role in major histocompatibility (MHC) class II-restricted antigen presentation has not been examined. MHC class II molecules were found to be constitutively present in plasma membrane lipid rafts in B cells. Disruption of these microdomains dramatically inhibited antigen presentation at limiting concentrations of antigen. The inhibitory effect of raft disruption on antigen presentation could be overcome by loading the APCs with exceptionally high doses of antigen, showing that raft association concentrates MHC class II molecules into microdomains that allow efficient antigen presentation at low ligand densities.     

Association of MHC class II-peptide complexes with plasma membrane lipid microdomains

Activation of CD4(+) T cells requires the interaction of multiple T-cell receptors with MHC class II-peptide complexes on the surface of antigen-presenting cells (APCs). Recent studies have shown that MHC class II complexes are clustered in APC plasma membrane microdomains, thereby providing a mechanism for localized concentration of MHC class II-peptide complexes. The integrity of one type of APC membrane microdomain, the lipid raft, is important for antigen presentation to T cells. We propose a model in which the coordinated processes of MHC class II peptide loading and intracellular trafficking enhance T-cell activation by loading specific MHC class II-peptide complexes in discrete lipid raft microdomains.     

Compartmentalization of class II antigen presentation: contribution of cytoplasmic and endosomal processing

Summary: During antigen processing, peptides are generated and displayed in the context of major histocompatibility complex (MHC) class II molecules on the surface of antigen‐presenting cells (APCs) to modulate immune responses to foreign antigens and guide self‐tolerance. Exogenous and cytoplasmic antigens are processed by distinct routes within APCs to yield class II ligands. Exogenous antigens are internalized, processed, and bound to class II molecules within endosomal and lysosomal compartments of APCs. Studies reviewed here demonstrate the importance of reduction in regulating exogenous antigen presentation. The differential expression of a γ‐interferon‐inducible lysosomal thiol reductase in professional APCs and melanomas is discussed in the context of tumor immune evasion. Cytoplasmic autoantigens, by contrast, are degraded by the proteasome and other enzymes in the cytosol, with the resulting peptides translocating to endosomal and lysosomal compartments for intersection with class II molecules. Processing and editing of these antigenic peptides within endosomes and lysosomes may be critical in regulating their display via class II proteins. Multiple pathways may regulate the transit of cytosolic peptides to class II molecules. The role of lysosome‐associated membrane protein‐2a and heat‐shock cognate protein 70 in promoting cytoplasmic peptide presentation by MHC class II molecules is discussed.     

Exogenous CLIP localizes into endocytic compartment of cells upon internalization: implications for antigen presentation by MHC class II molecules

We have previously shown that exogenous CLIP (class II associated invariant chain peptide) downregulated MHC class II expression on antigen presenting cells (APC) and modulated T cell mediated immune responses. The present study was undertaken to investigate the mechanism of uptake of exogenously added CLIP peptide by APC. We found that exogenous CLIP is rapidly internalized by APC and it co-localize with MHC class II in intracellular compartments including early-, late-endosomes and lysosomes. We suggest that exogenous CLIP acts as an in vivo regulator of immune response by internalization and passage through the intracellular compartments where it interferes in peptide loading and recycling of MHC class II molecules to the APC surface. Therefore, exogenous CLIP regulates immune responses by modulation of antigen presentation by the APC.     

Vaccinia virus A35R inhibits MHC class II antigen presentation

The Vaccinia virus gene A35R (Copenhagen designation) is highly conserved in mammalian-tropic poxviruses and is an important virulence factor, but its function was unknown. We show herein that A35 does not affect viral infectivity, apoptosis induction, or replication; however, we found that A35 significantly inhibited MHC class II-restricted antigen presentation, immune priming of T lymphocytes, and subsequent chemokine and cytokine synthesis. A35 localized to endosomes and reduced the amount of a model antigenic peptide displayed in the cleft of class II MHC. In addition, A35 decreased VV specific T cell responses in vivo. Thus, this is the first report identifying a function for the A35 protein in virulence as well as the first report identifying a VV gene that inhibits peptide antigen presentation.     

Vesicles bearing MHC class II molecules mediate transfer of antigen from antigen-presenting cells to CD4+ T cells

Previous studies have provided evidence that myelin basic protein (MBP)-specific rat T cells acquire antigen via transfer of preformed peptide/MHC class II complexes from splenic antigen-presenting cells (APC). The purpose of the present study was to determine how T cells acquire peptide/MHC class II complexes from APC in vitro. Our results show that a MHC class II+ T cell line, R1-trans, released MHC class II-bearing vesicles that directly stimulated MBP-specific CD4+ T cells. Vesicles expressing complexes of MHC class II and MBP were also specifically cytotoxic to MBP-specific T cells. Surviving T cells acquired MHC class II/antigen complexes from these vesicles by a mechanism that did not require protein synthesis but depended on specific TCR interactions with peptide/self MHC complexes. Furthermore, MBP/MHC class II-bearing vesicles enabled T cells to present MBP to other T cell responders. These studies provide evidence that APC release vesicles expressing preformed peptide/MHC class II complexes that interact with clonotypic TCR, allowing MHC class II acquisition by T cells. Vesicular transport of antigen/MHC class II complexes from professional APC to T cells may represent an important mechanism of communication among cells of the immune system.     

Lamp-2a facilitates MHC class II presentation of cytoplasmic antigens

Extracellular antigens are internalized and processed before binding MHC class II molecules within endosomal and lysosomal compartments of professional antigen presenting cells (APC) for subsequent presentation to T cells. Yet select cytoplasmic peptides derived from autoantigens also intersect and bind class II molecules via an unknown mechanism. In human B lymphoblasts, inhibition of the peptide transporter associated with antigen processing (TAP) failed to alter class II-restricted cytoplasmic epitope presentation. By contrast, decreased display of cytoplasmic epitopes via class II molecules was observed in cells with diminished expression of the lysosome-associated membrane protein-2 (Lamp-2). Overexpression of Lamp-2 isoform A (Lamp-2a), an established component of chaperone-mediated autophagy, enhanced cytoplasmic autoantigen presentation. Manipulating APC expression of heat shock cognate protein 70 (hsc70), a cofactor for Lamp-2a, also altered cytoplasmic class II peptide presentation. These results demonstrate a novel role for the lysosomal Lamp-2a-hsc70 complex in promoting immunological recognition and antigen presentation.     

Unraveling features of the natural MHC class II peptidome of skin-migrated dendritic cells

Dendritic cells (DCs) migrating from peripheral tissues at steady state are considered the most efficient antigen-presenting cells (APCs) involved in the induction of peripheral T-cell tolerance via self-antigen presentation on MHC class II molecules. However, difficulties in obtaining sufficient numbers of such DCs have precluded previous analyses of their natural MHC class II peptidome in laboratory animals or humans. Here, we overcome this difficulty by collecting the large quantities of sheep DCs that migrate from the skin via the afferent lymphatics at steady state to the draining lymph node. We compared the repertoire of MHC class II-bound peptides from afferent lymph DCs with autologous APCs derived from peripheral blood. A large fraction of the MHC class II peptidome from skin DCs was derived from membrane-recycling proteins (59%) and from proteins of the antigen presentation machinery (50%), whereas these types of peptides constituted a more limited fraction in blood APCs (21 and 11%, respectively). One sheep cytokeratin peptide was identified in the skin DC peptidome indicating active processing of epithelium-derived antigens. Conversely, peptides derived from cytosolic and soluble antigens of the extracellular milieu were more represented in blood APCs than skin DCs. The biased peptidome of skin-migrated DCs indicates that these cells express a peptide repertoire for the generation of self-reactive and/or regulatory T cells mainly directed toward DC molecules from internal and external membranes and to a lesser extent toward antigens of the extracellular milieu, including some tissue-specific peptides.     

Antigen-independent acquisition of MHC class II molecules by human T lymphocytes

We report here that human T lymphocytes have the capacity of acquiring large amounts of MHC class II molecules from various types of antigen-presenting cells (APC) in an antigen-independent manner. The transfer of MHC class II molecules from APC to T cell required direct cell-to-cell contact and appeared to involve the interaction of numerous adhesion molecules between these cells. Depletion of cholesterol from the plasma membrane reduced the amount of MHC class II transferred onto the T cells. Most significantly, the newly acquired MHC class II molecules were capable of efficiently presenting antigen to T helper cells. These results suggest that T cells are able to interact with other T cells to regulate immune responses by presenting MHC peptide complexes that have been snatched away from nearby APC.     

Differential glycosylation of MHC class II molecules on gastric epithelial cells: implications in local immune responses

Class II major histocompatibility complex (MHC) expression is a hallmark of antigen presenting cells (APC). Human gastric epithelial cells (GEC) express class II MHC and this expression increases during infection with Helicobacter pylori as does the number of CD4 T cells found adjacent or in between epithelial cells. These observations suggested that human GEC act as APCs. To characterize and compare class II MHC complexes with those present in conventional APC, immunoprecipitated class II MHC from GEC and B cells, as prototypic APC, were separated by two-dimensional electrophoresis. Although the composition of class II MHC from both cell phenotypes was similar, their electrophoretic mobility differed. Methodical elimination of carbohydrates, either enzymatically with endoglycosidase-H or blocking with tunicamycin, revealed that the deviations were due to differences in glycosylation in both cell phenotypes. When deglycosylated class II MHC alpha chains, beta chains, and the invariant chain from both cell phenotypes were mixed and run in the same gel, the core proteins had identical migration patterns. Because differences in glycosylation of class II MHC proteins may affect peptide selection and/or recognition by T cells, the noted differences in glycosylation of class II MHC expressed by GEC could be important in considering their potential role as APC locally.     

The role of tetraspanin CD63 in antigen presentation via MHC class II

Interactions between MHC class II (MHC II)-positive APCs and CD4(+) T cells are central to adaptive immune responses. Using an Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell line (LCL) as MHC II-positive APCs and CD4(+) T-cell clones specific for two endogenously expressed EBV antigens, we found that shRNA knockdown of the tetraspanin protein CD63 in LCL cells consistently led to increased CD4(+) T-cell recognition. This effect was not due to enhanced antigen processing nor to changes in MHC II expression since CD63 knockdown did not influence the amount or dimerization of MHC II in LCL cells. We therefore investigated the possible involvement of exosomes, small MHC II- and tetraspanin-abundant vesicles which are secreted by LCL cells and which we found could themselves activate the CD4(+) T-cell clones in an MHC II-dependent manner. While equal loadings of exosomes purified from the control and CD63(low) LCLs stimulated T cells to a comparable degree, we found that exosome production significantly increased following CD63-knockdown, suggesting that this may underlie the greater T-cell stimulatory capacity of the CD63(low) LCLs. Taken together, our data reveal a new insight into the mechanisms by which tetraspanins are involved in the regulation of MHC II-dependent T-cell stimulation.     

Presentation of endogenous viral proteins in association with major histocompatibility complex class II: On the role of intracellular compartmentalization,invariant chain and the TAP transporter system

Major histocompatibility complex (MHC) class II-associated antigen presentation is mainly linked to processing of exogenous antigens upon cellular uptake by endocytosis, but has also been observed for endogenously sythesized antigens. We have studied the MHC class II-associated presentation of the endogenously synthesized membrane associated glycoprotein (GP) and the cytosolic nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) in professional antigen presenting cells (APC) of mice. Since LCMV is a noncytopathic virus and minimally affects cellular protein synthesis. it is a convenient virus for the study of antigen presentation. In contrast, most other studies assessing class II-associated presentation of endogeneously synthesized viral antigens used cytolytic viruses such as vaccinia, measles and influenza virus, which drastically interfere with host cell functions. In addition, most studies were performed using non-professional APC. We found that class II-associated presentation of endogenously synthesized membrane associated LCMV-GP was efficient and could not be inhibited by chloroquine or leupeptin. Neither the transporter associated with processing (TAP) system nor the invariant chain (Ii) were significantly involved in this process. In contrast, MHC class II-associated presentation of endogenously synthesized cytosolic LCMV-NP was not observed even in Ii-deficient APC. Thus, MHC class II loading of endogenously synthesized LCMV-GP apparently does not require processing in acidie endosomal compartments as defined by chloroquine and leupeptin insensitivity. Furthermore, although the TAP molecules transport peptides of up to 15 amino acids in length, which potentially could bind to MHC class II molecules in the endoplasmic reticulum, such a process apparently does not occur for either the glycoprotein or the nucleoprotein. Therefore, the subcellular localization of an endogenously synthesized protein influences crucially whether or not MHC class II loading can occur independently of the acidic compartments usually involved in MHC class II loading.     

The lysosomal cysteine proteases in MHC class II antigen presentation

Summary: The endosomal pathway of antigen presentation leads to the display of peptides on major histocompatibility complex (MHC) class II molecules at the cell surface of antigen‐presenting cells (APCs). The pathway involves two major steps, invariant chain degradation and antigen processing, which take place in the late endosomes/lysosomes. So far, of the known lysosomal proteases, only cathepsin L and cathepsin S have been shown to have a non‐redundant role in endosomal presentation in vivo. Besides being engaged in the degradation of invariant chain, these enzymes also mediate the processing of antigens in distinct cell types. Surprisingly, these enzymes are active in different types of APCs, and this defined expression pattern seems to be enforced by regulatory mechanisms acting on multiple levels.     

Achieving stability through editing and chaperoning: regulation of MHC class II peptide binding and expression

Summary: In antigen‐presenting cells (APCs), loading of major histocompatibility complex class II (MHC II) molecules with peptides is regulated by invariant chain (Ii), which blocks MHC II antigen‐binding sites in pre‐endosomal compartments. Several molecules then act upon MHC II molecules in endosomes to facilitate peptide loading: Ii‐degrading proteases, the peptide exchange factor, human leukocyte antigen‐DM (HLA‐DM), and its modulator, HLA‐DO (DO). Here, we review our findings arguing that DM stabilizes a globally altered conformation of the antigen‐binding groove by binding to a lateral surface of the MHC II molecule. Our data imply changes in the interactions between specificity pockets and peptide side chains, complementing data from others that suggest DM affects hydrogen bonds. Selective weakening of peptide/MHC interactions allows DM to alter the peptide repertoire. We also review our studies in cells that highlight the ability of several factors to modulate surface expression of MHC II molecules via post‐Golgi mechanisms; these factors include MHC class II‐associated Ii peptides (CLIP), DM, and microbial products that modulate MHC II traffic from endosomes to the plasma membrane. In this context, we discuss possible mechanisms by which the association of some MHC II alleles with autoimmune diseases may be linked to their low CLIP affinity.