MHC class I-restricted presentation of maleylated protein binding to scavenger receptors

Pathways for loading exogenous protein-derived peptides on MHC class I are thought to be present mainly in monocyte-lineage cells and to involve phagocytosis- or macropinocytosis-mediated antigenic leakage into either cytosol or extracellular milieu to give peptide access to MHC class I. We show that maleylation of OVA enhanced its presentation to an OVA-specific MHC class I-restricted T cell line by both macrophages and B cells. This enhanced presentation involved uptake through receptors of scavenger receptor (SR)-like ligand specificity, was TAP-1-independent, and was inhibited by low levels (2 mM) of ammonium chloride. No peptide loading of bystander APCs by maleylated (maleyl) OVA-pulsed macrophages was detected. Demaleylated maleyl-OVA showed enhanced MHC class I-restricted presentation through receptor-mediated uptake and remained highly sensitive to 2 mM ammonium chloride. However, if receptor binding of maleyl-OVA was inhibited by maleylated BSA, the residual presentation was relatively resistant to 2 mM ammonium chloride. Maleyl-OVA directly introduced into the cytosol via osmotic lysis of pinosomes was poorly presented, confirming that receptor-mediated presentation of exogenous maleyl-OVA was unlikely to involve a cytosolic pathway. Demaleylated maleyl-OVA was well presented as a cytosolic Ag, consistent with the dependence of cytosolic processing on protein ubiquitination. Thus, receptor-specific delivery of exogenous protein Ags to APCs can result in enhanced MHC class I-restricted presentation, suggesting that the exogenous pathway of peptide loading for MHC class I may be a constitutive property dependent mainly on the quantity of Ag taken up by APCs.     

The impact of misfolding versus targeted degradation on the efficiency of the MHC class I-restricted antigen processing

Evidence suggests that most epitopes presented by MHC class I molecules are derived from those newly synthesized proteins that are defective due to errors during manufacture. We examined epitope production from model cytosolic and exocytic proteins modified in various ways. Substrates containing a degradation targeting sequence demonstrated very rapid turnover and enhanced epitope production, as was the case for substrate retargeted from endoplasmic reticulum to cytosol. For less radical alterations, including point mutation and deletion and elimination of glycosylation sites, despite detectable changes in folding, half-life was only moderately decreased and there were no significant increases in epitope production. Puromycin, which causes premature termination of protein synthesis, also had no impact upon epitope production. It appears that most defective proteins are not rapidly dispensed with and the targeting of most nascent proteins for Ag processing is not tied to quality control.     

MHC class I-restricted presentation of exogenous antigen by thymic antigen-presenting cells in vitro and in vivo.

We have used a T-T hybridoma, RF33.70, to detect the MHC class I-restricted presentation of exogenous native OVA by thymic APC in vitro. Presentation of OVA with class I molecules by thymic APC requires intracellular processing. Phenotypic analyses indicated that low bouyant density, MHC class II+, FcR+ cells are capable of using this presentation pathway. In order to determine whether thymic APC have this function in vivo, thymic APC were isolated from mice after i.v. injection of native OVA. We find that OVA is presented in association with MHC class I, but not class II, molecules in the thymus. This is in contrast to splenic APC, which present exogenous OVA with both class I and class II molecules under these conditions. Our findings have implications for the repertoire of self-peptides that might be presented by thymic APC to developing T lymphocytes.     

Acidosis improves uptake of antigens and MHC class I-restricted presentation by dendritic cells

It is widely appreciated that inflammatory responses in peripheral tissues are usually associated to the development of acidic microenvironments. Despite this, there are few studies aimed to analyze the effect of extracellular pH on immune cell functions. We analyzed the impact of acidosis on the behavior of dendritic cells (DCs) derived from murine bone marrow. We found that extracellular acidosis (pH 6.5) markedly stimulated the uptake of FITC-OVA, FITC-dextran, and HRP by DCs. In fact, to reach similar levels of endocytosis, DCs cultured at pH 7.3 required concentrations of Ag in the extracellular medium almost 10-fold higher compared with DCs cultured at pH 6.5. Not only the endocytic capacity of DCs was up-regulated by extracellular acidosis, but also the expression of CD11c, MHC class II, CD40, and CD86 as well as the acquisition of extracellular Ags by DCs for MHC class I-restricted presentation. Importantly, DCs pulsed with Ag under acidosis showed an improved efficacy to induce both specific CD8(+) CTLs and specific Ab responses in vivo. Our results suggest that extracellular acidosis improves the Ag-presenting capacity of DCs.     

Chaperones and folding of MHC class I molecules in the endoplasmic reticulum

In this review we discuss the influence of chaperones on the general phenomena of folding as well as on the specific folding of an individual protein, MHC class I. MHC class I maturation is a highly sophisticated process in which the folding machinery of the endoplasmic reticulum (ER) is heavily involved. Understanding the MHC class I maturation per se is important since peptides loaded onto MHC class I molecules are the base for antigen presentation generating immune responses against virus, intracellular bacteria as well as tumours. This review discusses the early stages of MHC class I maturation regarding BiP and calnexin association, and differences in MHC class I heavy chain (HC) interaction with calnexin and calreticulin are highlighted. Late stage MHC class I maturation with focus on the dedicated chaperone tapasin is also discussed.     

An immunodominant MHC class II-restricted tumor antigen is conformation dependent and binds to the endoplasmic reticulum chaperone, calreticulin.

There is accumulating evidence that CD4(+) T cell responses are important in antitumor immunity. Accordingly, we generated CD4(+) T cells against the murine CT26 colon cancer. Three of three independent CT26-specific CD4(+) hybridomas were found to recognize the high m.w. precursor of the env gene product gp90. The CD4(+) response was completely tumor specific in that the same glycoprotein expressed by other tumors was not recognized by the CT26-specific hybridomas. The recognition of gp90 by the hybridomas was strictly dependent on the conformation of gp90. Different procedures that disrupted the conformation of the glycoprotein, such as disulfide bond reduction and thermal denaturation, completely abrogated recognition of gp90 by all three hybridomas. In CT26 cells, but not in other tumor cells tested, a large proportion of gp90 was retained in the endoplasmic reticulum, mostly bound to the endoplasmic reticulum chaperone, calreticulin. Although calreticulin was not essential for the stimulation of the gp90-specific hybridomas, most of the antigenic form of gp90 was bound to it. The antigenicity of gp90 correlated well with calreticulin binding, reflecting the fact that specificity of binding of calreticulin to its substrate required posttranslational modifications that were also necessary for the generation of this tumor-specific CD4(+) epitope.     

Towards a systems understanding 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-restricted presentation of intracellular antigen.

An endogenously produced immunoglobulin light chain (lambda 2(315] is processed and presented to T cells in association with major histocompatibility complex (MHC) class II molecules. Using transfectants producing variant forms of lambda 2(315) that are neither expressed on the cell surface nor secreted, we demonstrate that intracellular lambda 2(315), which has never been exported outside of the cell, is the source of processed lambda 2(315) idiotype. This challenges the currently accepted paradigm that endogenous antigens are only presented by MHC class I molecules. Variants of lambda 2(315) protein that are retained in the endoplasmic recticulum (ER) are also presented. Variants that are expressed in the cytosol as well as those that are transported into the nucleus rather than the ER are not presented. Thus, the ER is likely to be the processing compartment.     

Anti-peptide antibody blocks peptide binding to MHC class I molecules in the endoplasmic reticulum

The finding that MHC class I molecules are physically associated with the TAP transporter has suggested that peptides may be directly transported into the binding groove of the class I molecules rather than into the lumen of the endoplasmic reticulum (ER) where they subsequently would encounter class I molecules by diffusion. Such a mechanism would protect peptides from peptidases in the ER and/or escaping back into the cytoplasm. However, we find that an anti-peptide Ab that is cotranslationally transported into the ER prevents TAP-transported peptides from being presented on class I molecules. The Ab only blocks the binding of its cognate peptide (SIINFEKL) but not other peptides (KVVRFKDL, ASNENMETM, and FAPGNYPAL). Therefore, most TAP-transported peptides must diffuse through the lumen of the ER before binding stably to MHC class I molecules.     

ATGs help MHC class II,but inhibit MHC class I antigen presentation

We have recently shown that the LC3/Atg8 lipidation machinery of macroautophagy is involved in the internalization of MHC class I molecules. Decreased internalization in the absence of ATG5 or ATG7 leads to MHC class I surface stabilization on dendritic cells and macrophages, resulting in elevated CD8⁺ T cell responses during viral infections and improved immune control. Here, we discuss how the autophagic machinery supports MHC class II restricted antigen presentation, while compromising MHC class I presentation via internalization and degradation.     

Linkage of bacterial protein synthesis and presentation of MHC class I-restricted Listeria monocytogenes-derived antigenic peptides

The processing and MHC class I-restricted presentation of antigenic peptides derived from the p60 protein of the facultative intracellular bacterium Listeria monocytogenes is tightly linked to bacterial protein synthesis. We used non-linear regression analysis to fit a mathematical model of bacterial antigen processing to a published experimental data set showing the accumulation and decay of p60-derived antigenic peptides in L. monocytogenes-infected cells. Two alternative models equally describe the experimental data. The simulation accounting for a stable and a hypothetical rapidly degraded form of antigen predicts that the antigenic peptides p60 217-225 and p60 449-457 are derived from a putative instable form of p60 with an average intracellular half-life of approximately 3 minutes accounting for approximately 31% of all p60 molecules synthesized. The alternative model predicts that both antigenic peptides are processed from p60 degraded intracellularly with a half-life of 109 min and that antigen processing only occurs as long as bacterial protein synthesis is not inhibited. In order to decide between both models the intracellular accumulation of p60 in infected cells was studied experimentally and compared with model predictions. Inhibition of p60 degradation by the proteasome inhibitor epoxomicin revealed that during the first 3 h post infection approximately 30% of synthesized p60 molecules were degraded. This value is significantly lower than the approximately 50% degradation of p60 that would be expected in the presence of the predicted putative short-lived state of p60 and also fits precisely with the predictions of the alternative model, indicating that the tight connection of bacterial protein biosynthesis and antigen processing and presentation of L. monocyctogenes-derived antigenic peptides is not caused by the presence of a highly instable antigenic substrate.     

Exogenous peptides delivered by ricin require processing by signal peptidase for transporter associated with antigen processing-independent MHC class I-restricted presentation

In this study we demonstrate that a disarmed version of the cytotoxin ricin can deliver exogenous CD8(+) T cell epitopes into the MHC class I-restricted pathway by a TAP-independent, signal peptidase-dependent pathway. Defined viral peptide epitopes genetically fused to the N terminus of an attenuated ricin A subunit (RTA) that was reassociated with its partner B subunit were able to reach the early secretory pathway of sensitive cells, including TAP-deficient cells. Successful processing and presentation by MHC class I proteins was not dependent on proteasome activity or on recycling of MHC class I proteins, but rather on a functional secretory pathway. Our results demonstrated a role for signal peptidase in the generation of peptide epitopes associated at the amino terminus of RTA. We showed, first, that potential signal peptide cleavage sites located toward the N terminus of RTA can be posttranslationally cleaved by signal peptidase and, second, that mutation of one of these sites led to a loss of peptide presentation. These results identify a novel MHC class I presentation pathway that exploits the ability of toxins to reach the lumen of the endoplasmic reticulum by retrograde transport, and suggest a role for endoplasmic reticulum signal peptidase in the processing and presentation of MHC class I peptides. Because TAP-negative cells can be sensitized for CTL killing following retrograde transport of toxin-linked peptides, application of these results has direct implications for the development of novel vaccination strategies.     

Expression of a membrane protease enhances presentation of endogenous antigens to MHC class I-restricted T lymphocytes.

We find that expression of the membrane dipeptidyl carboxypeptidase angiotensin-converting enzyme (ACE) enhances presentation of certain endogenously synthesized peptides to major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes. ACE appears to function only in an intracellular secretory compartment of antigen-presenting cells. ACE-enhanced antigen presentation requires the expression of the putative antigenic peptide transporters, TAP1 and TAP2. These findings demonstrate that a protease can influence the processing of endogenously synthesized antigens and strongly suggest that longer peptides can be transported from the cytosol to a secretory compartment where trimming of antigenic peptides to the lengths preferred by MHC class I molecules can occur if the appropriate protease is present.     

MHC class II invariant chains in antigen processing and presentation

Most protein antigens cannot elicit a T-cell response unless they are processed to peptides, which are then presented to T lymphocytes by surface MHC class II molecules. Recent evidence supports an essential role of the invariant chain associated with class II MHC polypeptides in antigen processing.     

Disruption of MHC class II-restricted antigen presentation by vaccinia virus

Vaccinia virus (VV), currently used in humans as a live vaccine for smallpox, can interfere with host immunity via several discrete mechanisms. In this study, the effect of VV on MHC class II-mediated Ag presentation was investigated. Following VV infection, the ability of professional and nonprofessional APC to present Ag and peptides to CD4+ T cells was impaired. Viral inhibition of class II Ag presentation could be detected within 1 h, with diminished T cell responses dependent upon the duration of APC infection and virus titer. Exposure of APC to replication-deficient virus also diminished class II Ag presentation. Virus infection of APC perturbed Ag presentation by newly synthesized and recycling class II molecules, with disruptions in both exogenous and cytoplasmic Ag presentation. Virus-driven expression of an endogenous Ag, failed to restore T cell responsiveness specific for this Ag in the context of MHC class II molecules. Yet, both class II protein steady-state and cell surface expression were not altered by VV. Biochemical and functional analysis revealed that VV infection directly interfered with ligand binding to class II molecules. Together, these observations suggest that disruption of MHC class II-mediated Ag presentation may be one of multiple strategies VV has evolved to escape host immune surveillance.     

MHC class II molecules on the move for successful antigen presentation

Major histocompatibility complex class II (MHC II) molecules are targeted to endocytic compartments, known as MIIC, by the invariant chain (Ii) that is degraded upon arrival in these compartments. MHC II acquire antigenic fragments from endocytosed proteins for presentation at the cell surface. In a unique and complex series of reactions, MHC II succeed in exchanging a remaining fragment of Ii for other protein fragments in subdomains of MIIC before transport to the cell surface. Here, the mechanisms regulating loading and intracellular trafficking of MHC II are discussed.     

Structure-based selection of small molecules to alter allele-specific MHC class II antigen presentation

Class II major histocompatibility molecules are the primary susceptibility locus for many autoimmune disorders, including type 1 diabetes. Human DQ8 and I-A(g7), in the NOD mouse model of spontaneous autoimmune diabetes, confers diabetes risk by modulating presentation of specific islet peptides in the thymus and periphery. We used an in silico molecular docking program to screen a large "druglike" chemical library to define small molecules capable of occupying specific structural pockets along the I-A(g7) binding groove, with the objective of influencing presentation to T cells of the autoantigen insulin B chain peptide consisting of amino acids 9-23. In this study we show, using both murine and human cells, that small molecules can enhance or inhibit specific TCR signaling in the presence of cognate target peptides, based upon the structural pocket targeted. The influence of compounds on the TCR response was pocket dependent, with pocket 1 and 6 compounds inhibiting responses and molecules directed at pocket 9 enhancing responses to peptide. At nanomolar concentrations, the inhibitory molecules block the insulin B chain peptide consisting of amino acids 9-23, endogenous insulin, and islet-stimulated T cell responses. Glyphosine, a pocket 9 compound, enhances insulin peptide presentation to T cells at concentrations as low as 10 nM, upregulates IL-10 secretion, and prevents diabetes in NOD mice. These studies present a novel method for identifying small molecules capable of both stimulating and inhibiting T cell responses, with potentially therapeutic applications.     

MHC class II expression and antigen presentation by human endometrial cells

It has been demonstrated previously that mixed cell suspensions from the female reproductive tract consisting of human epithelial and stromal cells were capable of presenting foreign antigen to autologous T cells. There have been, however, no reported studies examining antigen presentation by isolated epithelial cells from the human female reproductive tract. It is now shown that freshly isolated epithelial cells from the uterine endometrium constitutively express MHC class II antigen and that class II was upregulated on cultured epithelium by interferon gamma (IFNγ). Using a highly purified preparation, it was demonstrated that these epithelial cells were able to process and present tetanus toxoid recall antigen driving autologous T cell proliferation. Cells isolated from the basolateral sub-epithelium stroma were also potent antigen presenting cells in this model system. Thus, isolated endometrial epithelial cells were able to directly process and present antigen to T cells and may be responsible for the transcytosis and delivery of antigen to professional antigen presenting cells found in the sub-epithelial stroma.     

Cowpox virus exploits the endoplasmic reticulum retention pathway to inhibit MHC class I transport to the cell surface

Major histocompatibility complex (MHC) class I molecules assemble with peptides in the ER lumen and are transported via Golgi to the plasma membrane for recognition by T cells. Inhibiting MHC assembly, transport, and surface expression are common viral strategies of evading immune recognition. Cowpox virus, a clinically relevant orthopoxvirus, downregulates MHC class I expression on infected cells. However, the viral protein(s) and mechanisms responsible are unknown. We identify CPXV203 as a cowpox virus protein that associates with fully assembled MHC class I molecules and blocks their transport through the Golgi. A C-terminal KTEL motif in CPXV203 closely resembles the canonical ER retention motif KDEL and is required for CPXV203 function, indicating that a physiologic pathway is exploited to retain MHC class I in the ER. This viral mechanism for MHC class I downregulation may explain virulence differences between clinical isolates of orthopoxviruses.     

MHC class II molecules: transport pathways for antigen presentation

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.