EpsinR,a target for pyrenocine B,role in endogenous MHC‐II‐restricted antigen presentation

While the presentation mechanism of antigenic peptides derived from exogenous proteins by MHC class II molecules is well understood, relatively little is known about the presentation mechanism of endogenous MHC class II‐restricted antigens. We therefore screened a chemical library of 200 compounds derived from natural products to identify inhibitors of the presentation of endogenous MHC class II‐restricted antigens. We found that pyrenocine B, a compound derived from the fungus Pyrenochaeta terrestris, inhibits presentation of endogenous MHC class II‐restricted minor histocompatibility antigen IL‐4 inducible gene 1 (IL4I1) by primary dendritic cells (DCs). Phage display screening and surface plasmon resonance (SPR) analysis were used to investigate the mechanism of suppressive action by pyrenocine B. EpsinR, a target molecule for pyrenocine B, mediates endosomal trafficking through binding of soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs). Lentiviral‐mediated short hairpin (sh) RNA downregulation of EpsinR expression in DCs resulted in a decrease in the responsiveness of CD4⁺ T cells. Our data thus suggest that EpsinR plays a role in antigen presentation, which provides insight into the mechanism of presentation pathway of endogenous MHC class II‐restricted antigen.     

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.     

Carbon monoxide decreases endosome–lysosome fusion and inhibits soluble antigen presentation by dendritic cells to T cells.

Heme oxygenase‐1 (HO‐1) inhibits immune responses and inflammatory reactions via the catabolism of heme into carbon monoxide (CO), Fe²⁺, and biliverdin. We have previously shown that either induction of HO‐1 or treatment with exogenous CO inhibits LPS‐induced maturation of dendritic cells (DCs) and protects in vivo and in vitro antigen‐specific inflammation. Here, we evaluated the capacity of HO‐1 and CO to regulate antigen presentation on MHC class I and MHC class II molecules by LPS‐treated DCs. We observed that HO‐1 and CO treatment significantly inhibited the capacity of DCs to present soluble antigens to T cells. Inhibition was restricted to soluble OVA protein, as no inhibition was observed for antigenic OVA‐derived peptides, bead‐bound OVA protein, or OVA as an endogenous antigen. Inhibition of soluble antigen presentation was not due to reduced antigen uptake by DCs, as endocytosis remained functional after HO‐1 induction and CO treatment. On the contrary, CO significantly reduced the efficiency of fusion between late endosomes and lysosomes and not by phagosomes and lysosomes. These data suggest that HO‐1 and CO can inhibit the ability of LPS‐treated DCs to present exogenous soluble antigens to naïve T cells by blocking antigen trafficking at the level of late endosome–lysosome fusion.     

The exogenous pathway for antigen presentation on major histocompatibility complex class II and CD1 molecules

The endosomes and lysosomes of antigen-presenting cells host the processing and assembly reactions that result in the display of peptides on major histocompatibility complex (MHC) class II molecules and lipid-linked products on CD1 molecules. This environment is potentially hostile for T cell epitope and MHC class II survival, and the influence of regulators of protease activity and specialized chaperones that assist MHC class II assembly is crucial. At present, evidence indicates that individual proteases make both constructive and destructive contributions to antigen processing for MHC class II presentation to CD4 T cells. Some features of CD1 antigen capture within the endocytic pathway are also discussed.     

Processing of exogenous hepatitis B surface antigen particles for Ld-restricted epitope presentation depends on exogenous β2- microglobulin

Processing of exogenous hepatitis B surface antigen (HBsAg) particles in an endolysosomal compartment generates peptides that bind to the major histocompatibility complex (MHC) class I molecule L^d and are presented to CD8⁺ cytotoxic T lymphocytes. Surface-associated ‘empty’ MHC class I molecules associated neither with peptide, nor with β2-microglobulin (β2m) are involved in this alternative processing pathway of exogenous antigen for MHC class I-restricted peptide presentation. Here, we demonstrate that internalization of exogenous β2m is required for endolysosomal generation of presentation-competent, trimeric L^d molecules in cells pulsed with exogenous HBsAg. These data point to a role of endocytosed exogenous β2m in the endolysosomal assembly of MHC class I molecules that present peptides from endosomally processed, exogenous antigen.     

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.     

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.     

Thymus‐specific serine protease regulates positive selection of a subset of CD4+ thymocytes

Thymus‐specific serine protease (TSSP) was initially reported as a putative protease specifically expressed in the endosomal compartment of cortical thymic epithelial cells (cTEC). As such, TSSP is potentially involved in the presentation of the self‐peptides that are bound to MHC class II molecules expressed at the cTEC surface and are involved in the positive selection of CD4⁺ thymocytes. We tested this hypothesis by generating mutant mice deprived of Prss16, the gene encoding TSSP. TSSP‐deficient mice produced normal numbers of T cells, despite a decrease in the percentage of cTEC expressing high surface levels of MHC class II. By using sensitive transgenic models expressing MHC class II‐restricted TCR transgenes (Marilyn and OT‐II), we showed that the absence of TSSP markedly impaired the selection of Marilyn and OT‐II CD4⁺ T cells. In contrast, selection of CD8⁺ T cells expressing an MHC class I‐restricted TCR transgene (OT‐I) was unaffected. Therefore, TSSP is involved in the positive selection of some CD4⁺ T lymphocytes and likely constitutes the first serine protease to play a function in the intrathymic presentation of self‐peptides bound to MHC class II complexes.     

Disruption of HLA class II antigen presentation in Burkitt lymphoma: implication of a 47 000 MW acid labile protein in CD4+ T‐cell recognition

While Burkitt lymphoma (BL) has a well‐known defect in HLA class I‐mediated antigen presentation, the exact role of BL‐associated HLA class II in generating a poor CD4⁺ T‐cell response remains unresolved. Here, we found that BL cells are deficient in their ability to optimally stimulate CD4⁺ T cells via the HLA class II pathway. This defect in CD4⁺ T‐cell recognition was not associated with low levels of co‐stimulatory molecules on BL cells, as addition of external co‐stimulation failed to elicit CD4⁺ T‐cell activation by BL. Further, the defect was not caused by faulty antigen/class II interaction, because antigenic peptides bound with measurable affinity to BL‐associated class II molecules. Interestingly, functional class II–peptide complexes were formed at acidic pH 5·5, which restored immune recognition. Acidic buffer (pH 5·5) eluate from BL cells contained molecules that impaired class II‐mediated antigen presentation and CD4⁺ T‐cell recognition. Biochemical analysis showed that these molecules were greater than 30 000 molecular weight in size, and proteinaceous in nature. In addition, BL was found to have decreased expression of a 47 000 molecular weight enolase‐like molecule that enhances class II‐mediated antigen presentation in B cells, macrophages and dendritic cells, but not in BL cells. These findings demonstrate that BL likely has multiple defects in HLA class II‐mediated antigen presentation and immune recognition, which may be exploited for future immunotherapies.     

Sculpting MHC class II–restricted self and non‐self peptidome by the class I Ag‐processing machinery and its impact on Th‐cell responses

It is generally assumed that the MHC class I antigen (Ag)‐processing (CAP) machinery — which supplies peptides for presentation by class I molecules — plays no role in class II–restricted presentation of cytoplasmic Ags. In striking contrast to this assumption, we previously reported that proteasome inhibition, TAP deficiency or ERAAP deficiency led to dramatically altered T helper (Th)‐cell responses to allograft (HY) and microbial (Listeria monocytogenes) Ags. Herein, we tested whether altered Ag processing and presentation, altered CD4⁺ T‐cell repertoire, or both underlay the above finding. We found that TAP deficiency and ERAAP deficiency dramatically altered the quality of class II‐associated self peptides suggesting that the CAP machinery impacts class II–restricted Ag processing and presentation. Consistent with altered self peptidomes, the CD4⁺ T‐cell receptor repertoire of mice deficient in the CAP machinery substantially differed from that of WT animals resulting in altered CD4⁺ T‐cell Ag recognition patterns. These data suggest that TAP and ERAAP sculpt the class II–restricted peptidome, impacting the CD4⁺ T‐cell repertoire, and ultimately altering Th‐cell responses. Together with our previous findings, these data suggest multiple CAP machinery components sequester or degrade MHC class II–restricted epitopes that would otherwise be capable of eliciting functional Th‐cell responses.     

MHC presentation via autophagy and how viruses escape from it

T cells detect infected and transformed cells via antigen presentation by major histocompatibility complex (MHC) molecules on the cell surface. For T cell stimulation, these MHC molecules present fragments of proteins that are expressed or taken up by the cell. These fragments are generated by distinct proteolytic mechanisms for presentation on MHC class I molecules to cytotoxic CD8⁺ and on MHC class II molecules to helper CD4⁺ T cells. Proteasomes are primarily involved in MHC class I ligand and lysosomes, in MHC class II ligand generation. Autophagy delivers cytoplasmic material to lysosomes and, therefore, contributes to cytoplasmic antigen presentation by MHC class II molecules. In addition, it has been recently realized that this process also supports extracellular antigen processing for MHC class II presentation and cross-presentation on MHC class I molecules. Although the exact mechanisms for the regulation of these antigen processing pathways by autophagy are still unknown, recent studies, summarized in this review, suggest that they contribute to immune responses against infections and to maintain tolerance. Moreover, they are targeted by viruses for immune escape and could maybe be harnessed for immunotherapy.     

Ultrastructural analysis of thymic nurse cell epithelium

Thymic nurse cells (TNC), a paradigmatic cell type of cortical epithelium, are large lymphoid-epithelial cell complexes of thymocytes enclosed within vacuoles lined by the epithelial cell membrane. TNC express major histocompatibility complex (MHC) class I and class II molecules on their surface and vacuole-lining membranes at high density and it was suggested that TNC provide an optimal microenvironment for positive selection of T cells. In this report we present electron microscopical data demonstrating that chicken TNC display morphological structures of exocytosis previously shown for hormone-secreting cells. In TNC, however, exocytosis is restricted to the capillary cleft between the epithelial cell and engulfed thymocytes. Thus, besides physical contact between the epithelial cell and enclosed thymocytes, TNC may additionally influence the development of thymocytes through release of soluble factors in a restricted microenvironment. By employing the 3-(2,4-dinitroanilino)-3-amino-N-methyl-propylamine technique which at the ultrastructural level detects acidic organelles involved in processing of antigens presented by MHC class II molecules, we also show that TNC contain acidic compartments similar to classical antigen-presenting cells, i.e. early and late endosomes and lysosomes, albeit in a lower amount than in thymic dendritic cells. This fact provides evidence that TNC not only are capable of antigen presentation but also possess the intracellular machinery for antigen processing.     

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.     

Autophagy in innate and adaptive immunity against intracellular pathogens

Autophagy delivers cytoplasmic constituents for lysosomal degradation. Recent studies have demonstrated that this pathway mediates resistance to pathogens and is targeted for immune evasion by viruses and bacteria. Lysosomal degradation products, including pathogenic determinants, are then surveyed by the adaptive immune system to elicit antigen-specific T cell responses. CD4⁺ T helper cells have been shown to recognize nuclear and cytosolic antigens via presentation by major histocompatibility complex (MHC) class II molecules after autophagy. Furthermore, some sources of natural MHC class II ligands display characteristics of autophagy substrates, and autophagosomes fuse with late endosomes, in which MHC class II loading is thought to occur. Although MHC class II antigen processing via autophagy has so far mainly been described for professional antigen-presenting cells like B cells, macrophages, and dendritic cells, it might be even more important for cells with less endocytic potential, like epithelial cells, when these express MHC class II at sites of inflammation. Therefore, autophagy might contribute to immune surveillance of intracellular pathogens via MHC class II presentation of intracellular pathogen-derived peptides.     

Hsp90–peptide complexes stimulate antigen presentation through the class II pathway after binding scavenger receptor SREC-I

Molecular chaperones such as heat shock protein 90 (Hsp90) have been shown to form complexes with tumor antigens and can be used to prepare anticancer vaccines largely due to this property. Earlier studies had suggested that mice immunized with a molecular chaperone-based vaccine derived from tumors became immune to further vaccination and that both CD8⁺ and CD4⁺ T cells were activated by the chaperone vaccine in a manner dependent on scavenger receptor SREC-I. Here we have investigated mechanisms whereby SREC-I might facilitate uptake of Hsp90-conjugated peptides by APC into the MHC class II pathway for presentation to CD4⁺ T cells. Our studies showed that antigenic peptides associated with Hsp90 were taken up into the class II pathway by a mechanism dependent on SREC-I binding and internalization and presented to CD4⁺ T cells. In addition our studies showed that SREC-I could associate with MHC class II molecules on the cell surface and in intracellular endosomes, suggesting a mechanism involving facilitated uptake of peptides into the MHC class II pathway. These studies in addition to our earlier findings showed SREC-I to play a primary role in chaperone-associated antigen uptake both through cross priming of MHC class I molecules and entry into the class II pathway.     

Major histocompatibility complex class II-associated peptides determine the binding of the superantigen toxic shock syndrome toxin-1

Superantigens bind to major histocompatibility complex (MHC) class II proteins and interact with variable parts of the T cell antigen receptor (TCR) β-chain. Cross-linking the TCR with MHC class II molecules on the antigen-presenting cell by the superantigen leads to T cell activation that plays an essential role in pathogenesis. Recent crystallographic data have resolved the structure of the complexes between HLA-DR1 and staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 (TSST-1), respectively. For TSST-1, these studies have revealed possible contact sites between the superantigen and the HLA-DR1-bound peptide. Here, we show that TSST-1 binding is dependent on the MHC-II-associated peptides by employing variants of T2 mutant cells deficient in loading of peptides to MHC class II molecules as superantigen-presenting cells. On HLA-DR3-transfected T2 cells, presentation of TSST-1, but not SEB, was dependent on HLA-DR3-associated peptides. Thus, although these superantigens can be recognized in the context of multiple MHC class II alleles and isotypes, they clearly bind to specific subsets of MHC molecules displaying appropriate peptides.     

Molecules that modify antigen recognition

MHC class II molecules assemble in the presence of invariant chains. These probably serve not only to protect the peptide-binding site on MHC class II molecules from endogenous peptides, but also to sort MHC class II molecules from the Golgi complex to endosomes and there to retain the class II polypeptides to allow binding of peptides generated from exogenous antigens.     

Sustained cross‐presentation capacity of murine splenic dendritic cell subsets in vivo

An exclusive feature of dendritic cells (DCs) is their ability to cross‐present exogenous antigens in MHC class I molecules. We analyzed the fate of protein antigen in antigen presenting cell (APC) subsets after uptake of naturally formed antigen‐antibody complexes in vivo. We observed that murine splenic DC subsets were able to present antigen in vivo for at least a week. After ex vivo isolation of four APC subsets, the presence of antigen in the storage compartments was visualized by confocal microscopy. Although all APC subsets stored antigen for many days, their ability and kinetics in antigen presentation was remarkably different. CD8α⁺ DCs showed sustained MHC class I‐peptide specific CD8⁺ T‐cell activation for more than 4 days. CD8α^? DCs also presented antigenic peptides in MHC class I but presentation decreased after 48 h. In contrast, only the CD8α^? DCs were able to present antigen in MHC class II to specific CD4⁺ T cells. Plasmacytoid DCs and macrophages were unable to activate any of the two T‐cell types despite detectable antigen uptake. These results indicate that naturally occurring DC subsets have functional antigen storage capacity for prolonged T‐cell activation and have distinct roles in antigen presentation to specific T cells in vivo.     

Conformational heterogeneity of MHC class II induced upon binding to different peptides is a key regulator in antigen presentation and epitope selection

T cells bearing αβ receptors recognize antigenic peptides bound to class I and class II glycoproteins encoded in the major histocompatibility complex (MHC). Cytotoxic and helper T cells respond respectively to peptide antigens derived from endogenous sources presented by MHC class I, and exogenous sources presented by MHC II, on antigen presenting cells. Differences in the MHC class I and class II structures and their maturation pathways have evolved to optimize antigen presentation to their respective T cells. A main focus of our laboratory is on efforts to understand molecular events in processing of antigen for presentation by MHC class II. The different stages of MHC class II—interactions with molecular chaperons involved in folding and traffic from the ER through the antigen-loading compartments, peptide exchange, and transport to the cell surface have been investigated. Through intense research on biophysical and biochemical properties of MHC class II molecules, we have learned that the conformational heterogeneity of MHC class II induced upon binding to different peptides is a key regulator in antigen presentation and epitope selection, and a determinant of the ability of MHC class II to participate in peptide association or dissociation and interaction with the peptide editor HLA-DM.     

Failure of trafficking and antigen presentation by CD1 in AP-3-deficient cells

Endocytosed microbial antigens are primarily delivered to lysosomal compartments where antigen binding to MHC and CD1 molecules occurs in an acidic and proteolytically active environment. Signal-dependent delivery to lysosomes has been suggested for these antigen-presenting molecules, but molecular interactions with vesicular coat proteins and adaptors that direct their lysosomal sorting are poorly understood. Here CD1b but not other CD1 isoforms bound the AP-3 adaptor protein complex. In AP-3-deficient cells derived from patients with Hermansky-Pudlak syndrome type 2 (HPS-2), CD1b failed to efficiently gain access to lysosomes, resulting in a profound defect in antigen presentation. Since MHC class II traffics normally in AP-3-deficient cells, defects in CD1b antigen presentation may account for recurrent bacterial infections in HPS-2 patients.