Control of MHC class I traffic from the endoplasmic reticulum by cellular chaperones and viral anti-chaperones

MHC class I molecules assemble with peptides in the endoplasmic reticulum (ER). To ensure that only peptide-loaded MHC molecules leave the ER, empty molecules are retained by ER-resident chaperones, most notably the MHC-specific tapasin. ER exit of class I MHC is also controlled by viruses, but for the opposite purpose of preventing peptide presentation to T cells. Interestingly, some viral proteins are able to retain MHC class I molecules in the ER despite being transported. By contrast, other viral proteins exit the ER only upon binding to class I MHC, thereby rerouting newly synthesized class I molecules to intracellular sites of proteolysis. Thus, immune escape can be achieved by reversing, inhibiting or redirecting the chaperone-assisted MHC class I folding, assembly and intracellular transport.     

Activation of human T cell clones and Jurkat cells by cross-linking class I MHC molecules

Cross-linking class I MHC molecules on human T cell clones by reacting them with various mAb directed at either monomorphic or polymorphic determinants on class I MHC molecules followed by cross-linking with GaMIg stimulated a rise in intracellular free calcium concentration ([Ca2+]i), and induced proliferation and IL-2 production. T cell clones varied in the mean density of class I MHC molecules and the capacity to respond to mAb to class I MHC molecules. However, the functional responses of the clones did not correlate with class I MHC density or the CD4/CD8 phenotype. mAb to polymorphic class I MHC determinants were less able to induce an increase in [Ca2+]i and a functional response in the T cell clones. Additive stimulatory effects were noted when mAb against both HLA-A and HLA-B determinants were employed. Cross-linking class I MHC molecules on Jurkat cells induced a rise by [Ca2+]i and induced IL-2 production upon co-stimulation with PMA. Cross-linking class I MHC molecules on mutant Jurkat cells that expressed diminished levels of CD3 and were unable to produce IL-2 in response to anti-CD3 stimulation triggered both a rise in [Ca2+]i and IL-2 production with PMA co-stimulation. In contrast, cross-linking class I MHC molecules on mutant Jurkat cells that were CD3- stimulated neither a rise in [Ca2+]i nor IL-2 production. The combination of mAb to CD28 or ionomycin and PMA, however, was able to induce IL-2 production by CD3- Jurkat cells. The data demonstrate that cross-linking class I MHC molecules delivers a functionally important signal to T cell clones and Jurkat cells and indicate that class I MHC molecules may function to transduce activation signals to T cells. In addition, the data demonstrate that transmission of an activation signal via class I MHC molecules requires CD3 expression. The data, therefore, support a central role for CD3 in the transduction of activation signals to T cells via class I MHC molecules.     

Double labelling of major histocompatability complex molecules and lysosomal protein lamp-1 on human dendritic cells

Summary In this study, double labelling for major histocompatability complex (MHC) class I and class II molecules and for MHC molecules and the lysosomal membrane protein lamp-1 on ultrathin cryosections of dendritic cells isolated from human peripheral blood was performed. The plasma membrane proved to be positive for both MHC class I and MHC class II molecules and was labelled for only a very few lamp-1 molecules. MHC class I and MHC class II molecules did not co-localize intracellularly except in some peripherally located vesicles. However, many MHC class II-labelled vesicles were present in a juxtanuclear position but only some of them were co-labelled for lamp-1. These results indicate the presence of a separate, non-lysosomal compartment for class II molecules in dendritic cells.     

A glycosylated type I membrane protein becomes cytosolic when peptide: N-glycanase is compromised

The human cytomegalovirus-encoded glycoprotein US2 catalyzes proteasomal degradation of Class I major histocompatibility complex (MHC) heavy chains (HCs) through dislocation of the latter from the endoplasmic reticulum (ER) to the cytosol. During this process, the Class I MHC HCs are deglycosylated by an N-glycanase-type activity. siRNA molecules designed to inhibit the expression of the light chain, beta(2)-microglobulin, block the dislocation of Class I MHC molecules, which implies that US2-dependent dislocation utilizes correctly folded Class I MHC molecules as a substrate. Here we demonstrate it is peptide: N-glycanase (PNGase or PNG1) that deglycosylates dislocated Class I MHC HCs. Reduction of PNGase activity by siRNA expression in US2-expressing cells inhibits deglycosylation of Class I MHC HC molecules. In PNGase siRNA-treated cells, glycosylated HCs appear in the cytosol, providing the first evidence for the presence of an intact N-linked type I membrane glycoprotein in the cytosol. N-glycanase activity is therefore not required for dislocation of glycosylated Class I MHC molecules from the ER.     

Human cytomegalovirus immediate early glycoprotein US3 retains MHC class I molecules by transient association

Human cytomegalovirus (HCMV) interferes with major histocompatibility complex (MHC) class I antigen presentation by a sequential multistep process to escape T cell surveillance. During the immediate early phase of infection, the glycoprotein US3 prevents intracellular transport of MHC class I molecules. Interestingly, US3 displays a significantly shorter half-life than US3-retained MHC class I molecules. Here we show that US3 associates only transiently with MHC class I molecules, exits the ER, and is inefficiently retrieved from the Golgi. US3 was degraded in a post-Golgi compartment, most likely lysosomes, because: i) Brefeldin A treatment prolonged the half-life of US3; and ii) US3 co-localized with the lysosomal marker protein LAMP in chloroquine-treated cells. In contrast, MHC class I molecules remained stable in the ER. Upon inhibition of protein synthesis MHC class I molecules were released suggesting that a continuous supply of newly synthesized US3 molecules is required for inhibition of transport. Thus, US3 does not seem to retain MHC class I molecules by a retrieval mechanism. Instead, our observations are consistent with US3 preventing MHC class I trafficking by blocking forward transport.     

CD99 regulates the transport of MHC class I molecules from the Golgi complex to the cell surface

The down-regulation of surface expression of MHC class I molecules has recently been reported in the CD99-deficient lymphoblastoid B cell line displaying the characteristics of Hodgkin's and Reed-Sternberg phenotype. Here, we demonstrate that the reduction of MHC class I molecules on the cell surface is primarily due to a defect in the transport from the Golgi complex to the plasma membrane. Loss of CD99 did not affect the steady-state expression levels of mRNA and protein of MHC class I molecules. In addition, the assembly of MHC class I molecules and the transport from the endoplasmic reticulum to the cis-Golgi occurred normally in the CD99-deficient cells, and no difference was detected between the CD99-deficient and the control cells in the pattern and degree of endocytosis. Instead, the CD99-deficient cells displayed the delayed transport of newly synthesized MHC class I molecules to the plasma membrane, thus causing accumulation of the molecules within the cells. The accumulated MHC class I molecules in the CD99-deficient cells were colocalized with alpha-mannosidase II and gamma-adaptin in the Golgi compartment. These results suggest that CD99 may be associated with the post-Golgi trafficking machinery by regulating the transport to the plasma membrane rather than the endocytosis of surface MHC class I molecules, providing a novel mechanism of MHC class I down-regulation for immune escape.     

The oxidoreductase ERp57 efficiently reduces partially folded in preference to fully folded MHC class I molecules

The oxidoreductase ERp57 is an integral component of the peptide loading complex of major histocompatibility complex (MHC) class I molecules, formed during their chaperone-assisted assembly in the endoplasmic reticulum. Misfolded MHC class I molecules or those denied suitable peptides are retrotranslocated and degraded in the cytosol. The presence of ERp57 during class I assembly suggests it may be involved in the reduction of intrachain disulfides prior to retrotranslocation. We have studied the ability of ERp57 to reduce MHC class I molecules in vitro. Recombinant ERp57 specifically reduced partially folded MHC class I molecules, whereas it had little or no effect on folded and peptide-loaded MHC class I molecules. Reductase activity was associated with cysteines at positions 56 and 405 of ERp57, the N-terminal residues of the active CXXC motifs. Our data suggest that the reductase activity of ERp57 may be involved during the unfolding of MHC class I molecules, leading to targeting for degradation.     

Molecular cloning of cDNA that encode MHC class I molecules from a New World primate (Saguinus oedipus). Natural selection acts at positions that may affect peptide presentation to T cells

To investigate the evolutionary pressures that drive the generation of polymorphism in primate MHC class I molecules, three cDNA that encode MHC class I alleles from a New World monkey, the cotton-top tamarin (Saguinus oedipus), were cloned and sequenced. These tamarin MHC class I alleles contained amino acid substitutions not found in any of the previously sequenced human MHC class I alleles. Moreover, the majority of these unique amino acid substitutions was located in the Ag recognition site at positions that have been shown to be critical in the presentation of viral peptides to T cells in mice and humans. These data suggest that selective pressures on MHC class I molecules preferentially act on the Ag recognition site and that the peptide binding or presenting functions of these molecules may drive the generation of MHC class I polymorphism. The novel Ag recognition sites of the tamarin MHC class I molecules, in addition to their restricted polymorphism, might account for the unusual susceptibility of the cotton-top tamarin to human pathogens.     

Association of tapasin and COPI provides a mechanism for the retrograde transport of major histocompatibility complex (MHC) class I molecules from the Golgi complex to the endoplasmic reticulum

Tapasin is a subunit of the transporter associated with antigen processing (TAP). It associates with the major histocompatibility complex (MHC) class I. We show that tapasin interacts with beta- and gamma-subunits of COPI coatomer. COPI retrieves membrane proteins from the Golgi network back to the endoplasmic reticulum (ER). The COPI subunit-associated tapasin also interacts with MHC class I molecules suggesting that tapasin acts as the cargo receptor for packing MHC class I molecules as cargo proteins into COPI-coated vesicles. In tapasin mutant cells, neither TAP nor MHC class I are detected in association with the COPI coatomer. Interestingly, tapasin-associated MHC class I molecules are antigenic peptide-receptive and detected in both the ER and the Golgi. Our data suggest that tapasin is required for the COPI vesicle-mediated retrograde transport of immature MHC class I molecules from the Golgi network to the ER.     

Comparison of the capacity of murine and human class I MHC molecules to stimulate T cell activation.

The mechanism underlying the apparent differences in the capacity of murine and human class I MHC molecules to function as signal transducing structures in T cells was examined. Cross-linking murine class I MHC molecules on splenic T cells did not stimulate an increase in intracellular calcium ([Ca2+]i) and failed to induce proliferation in the presence of IL-2 or PMA. In contrast, modest proliferation was induced by cross-linking class I MHC molecules on murine peripheral blood T cells or human class I MHC molecules on murine transgenic spleen cells, but only when costimulated with PMA. Moreover, cross-linking murine class I MHC molecules or the human HLA-B27 molecule on T cell lines generated from transgenic murine splenic T cells stimulated only modest proliferation in the presence of PMA, but not IL-2. On the other hand, cross-linking murine class I MHC molecules expressed by the human T cell leukemic line, Jurkat, transfected with genes for these molecules, generated a prompt increase in [Ca2+]i, and stimulated IL-2 production in the presence of PMA. The results demonstrate that both murine and human class I MHC molecules have the capacity to function as signal transducing structures, but that murine T cells are much less responsive to this signal.     

Broad recognition of cytotoxic T cell epitopes from the HIV-1 envelope protein with multiple class I histocompatibility molecules.

A few cases have been described of antigenic determinants that are broadly presented by multiple class II MHC molecules, especially murine I-E or human DR, in which polymorphism is limited to the beta chain, and the alpha chain is conserved. However, no similar cases have been studied for presentation by class I MHC molecules. Because both domains of the MHC peptide binding site are polymorphic in class I molecules, exploring permissiveness in class I presentation would be of interest, and also such broadly presented antigenic determinants would clearly be useful for vaccine development. We had defined an immunodominant determinant, P18, of the HIV-1 gp160 envelope protein recognized by human and murine CTL. To determine the range of class I MHC molecules that could present this peptide and to determine whether two HIV-1 gp160 Th cell determinants, T1 and HP53, could also be presented by class I MHC molecules, we attempted to generate CTL specific for these three peptides in 10 strains of B10 congenic mice, representing 10 MHC types, and BALB/c mice. P18 was presented by at least four different class I MHC molecules from independent haplotypes (H-2d, p, u, and q to CD8+ CTL. In H-2d and H-2q the presentation was mapped to the D-end class I molecule, and for Dd, a requirement for both the alpha 1 and alpha 2 domains of Dd, not Ld, was found. HP53 was also presented by the same four different class I MHC molecules to CD8+ CTL although at higher concentrations. T1 was presented by class I molecules in three different strains of distinct MHC types (B10.M, H-2f; B10.A, H-2a; and B10, H-2b) to CTL. The CTL specific for P18 and HP53 were shown to be CD8+ and CD4- and to kill targets expressing endogenously synthesized whole gp160 as well as targets pulsed with the corresponding peptide. To compare the site within each peptide presented by the different class I molecules, we used overlapping and substituted peptides and found that the critical regions of each peptide are the similar for all four MHC molecules. Thus, antigenic sites are broadly or permissively presented by class I MHC molecules even without a nonpolymorphic domain as found in DR and I-E, and these sequences may be of broad usefulness in a synthetic vaccine.     

Macrophages present pinocytosed exogenous antigen via MHC class I whereas antigen ingested by receptor-mediated endocytosis is presented via MHC class II

Macrophages present exogenous Ag either via MHC class I or MHC class II molecules. We investigated whether the mode of hemagglutinin (HA) uptake influences the class of MHC molecule by which this Ag is presented. Normally, HA is ingested by receptor-mediated endocytosis, but this may be switched to macropinocytosis and pinocytosis by adding phorbol esters to the cells. This switch resulted in altered intracellular routing of ingested Ag and a transition from Ag presentation via MHC class II molecules to presentation via MHC class I molecules. Similarly, inhibition of receptor-mediated HA endocytosis, by treating the cells with the HA receptor destroying enzyme neuraminidase, abrogated Ag presentation via MHC class II molecules and induced presentation via MHC class I molecules. If, however, under these conditions, receptor-mediated uptake of HA was restored, by virtue of HA/anti-HA Ab interaction and subsequent uptake of HA via the Fc receptor, presentation via MHC class II was restored as well, whereas presentation of HA via MHC class I molecules was no longer detectable. We conclude that in macrophages the mode of Ag uptake is decisive in determining via which class of MHC molecules Ag is presented: pinocytosis and macropinocytosis produce exclusive presentation of exogenous Ag via MHC class I molecules whereas receptor-mediated endocytosis leads exclusively to presentation via class II molecules.     

Human cytomegalovirus UL18 utilizes US6 for evading the NK and T-cell responses

Human cytomegalovirus (HCMV) US6 glycoprotein inhibits TAP function, resulting in down-regulation of MHC class I molecules at the cell surface. Cells lacking MHC class I molecules are susceptible to NK cell lysis. HCMV expresses UL18, a MHC class I homolog that functions as a surrogate to prevent host cell lysis. Despite a high level of sequence and structural homology between UL18 and MHC class I molecules, surface expression of MHC class I, but not UL18, is down regulated by US6. Here, we describe a mechanism of action by which HCMV UL18 avoids attack by the self-derived TAP inhibitor US6. UL18 abrogates US6 inhibition of ATP binding by TAP and, thereby, restores TAP-mediated peptide translocation. In addition, UL18 together with US6 interferes with the physical association between MHC class I molecules and TAP that is required for optimal peptide loading. Thus, regardless of the recovery of TAP function, surface expression of MHC class I molecules remains decreased. UL18 represents a unique immune evasion protein that has evolved to evade both the NK and the T cell immune responses.     

Constitutive internalization of murine MHC class I molecules

The total number of cell surface glycoprotein molecules at the plasma membrane results from a balance between their constitutive internalization and their egress to the cell surface from intracellular pools and/or biosynthetic pathway. Constitutive internalization is net result of constitutive endocytosis and endocytic recycling. In this study we have compared spontaneous internalization of murine major histocompatibility complex (MHC) class I molecules (K(d), D(d), full L(d), and empty L(d)) after depletion of their egress to the cell surface (Cycloheximide [CHX], brefeldin A [BFA]) and internalization after external binding of monoclonal antibody (mAb). MHC class I alleles differ regarding their cell surface stability, kinetics, and in the way of internalization and degradation. K(d) and D(d) molecules are more stable at the cell surface than L(d) molecules and, thus, constitutively internalized more slowly. Although the binding of mAbs to cell surface MHC class I molecules results in faster internalization than depletion of their egress, it is still slow and, thereby, can serve as a model for tracking of MHC class I endocytosis. Internalization of fully conformed MHC class I molecules (K(d), D(d), and L(d)) was neither inhibited by chlorpromazine (CP) (inhibitor of clathrin endocytosis), nor with filipin (inhibitor of lipid raft dependent endocytosis), indicating that fully conformed MHC class I molecules are internalized via the bulk pathway. In contrast, internalization of empty L(d) molecules was inhibited by filipin, indicating that non-conformed MHC class I molecules require intact cholesterol-rich membrane microdomains for their constitutive internalization. Thus, conformed and non-conformed MHC class I molecules use different endocytic pathways for constitutive internalization.     

Redox-regulated Export of the Major Histocompatibility Complex Class I-Peptide Complexes from the Endoplasmic Reticulum

In contrast to the fairly well-characterized mechanism of assembly of MHC class I-peptide complexes, the disassembly mechanism by which peptide-loaded MHC class I molecules are released from the peptide-loading complex and exit the endoplasmic reticulum (ER) is poorly understood. Optimal peptide binding by MHC class I molecules is assumed to be sufficient for triggering exit of peptide-filled MHC class I molecules from the ER. We now show that protein disulfide isomerase (PDI) controls MHC class I disassembly by regulating dissociation of the tapasin-ERp57 disulfide conjugate. PDI acts as a peptide-dependent molecular switch; in the peptide-bound state, it binds to tapasin and ERp57 and induces dissociation of the tapasin-ERp57 conjugate. In the peptide-free state, PDI is incompetent to bind to tapasin or ERp57 and fails to dissociate the tapasin-ERp57 conjugates, resulting in ER retention of MHC class I molecules. Thus, our results indicate that even after optimal peptide loading, MHC class I disassembly does not occur by default but, rather, is a regulated process involving PDI-mediated interactions within the peptide-loading complex.     

The MHC class I homolog of human cytomegalovirus is resistant to down-regulation mediated by the unique short region protein (US)2, US3, US6, and US11 gene products

Human CMV encodes four unique short region proteins (US), US2, US3, US6, and US11, each independently sufficient for causing the down-regulation of MHC class I molecules on the cell surface. This down-regulation allows infected cells to evade recognition by cytotoxic T cells but leaves them susceptible to NK cells, which lyse cells that lack class I molecules. Another human CMV-encoded protein, unique long region protein 18 (UL18), is an MHC class I homolog that might provide a mechanism for inhibiting the NK cell response. The sequence similarities between MHC class I molecules and UL18 along with the ability of UL18 to form trimeric complexes with beta(2)-microglobulin and peptides led to the hypothesis that if the US and UL18 gene products coexist temporally during infection, the US proteins might down-regulate UL18 molecules, similar to their action on MHC class I molecules. We show here that temporal expression of US and UL18 genes partially overlaps during infection. However, unlike MHC class I molecules, the MHC class I homolog, UL18, is fully resistant to the down-regulation associated with the US2, US3, US6, and US11 gene products. The specific effect of US proteins on MHC class I molecules, but not on UL18, represents another example of how viral proteins have evolved to evade immune surveillance, avoiding fratricide by specifically targeting host proteins.     

Activation of human T4 cells by cross-linking class I MHC molecules

These studies examined whether cross-linking class I MHC molecules results in functional or biochemical responses in human T4 cells. The initial studies demonstrated that cross-linking class I MHC molecules either by culturing highly purified T4 cells with immobilized mAb to class I MHC Ag or reacting the T4 cells with mAb to class I MHC Ag and then cross-linking the mAb with goat antimouse Ig (GaMIg) enhanced T4 cell proliferation induced by an immobilized mAb to CD3, OKT3. More-over, immobilized but not soluble mAb to class I MHC Ag enhanced T4 cell proliferation induced by the combination of two mAb to CD2, OKT11, and D66.2. Finally, T4 cells reacted with mAb to CD3 and class I MHC Ag proliferated in the presence of IL-2 when cross-linked with GaMIg more vigorously than T4 cells reacted with either mAb alone. Cross-linking class I MHC molecules was also found to stimulate T4 cells directly. T4 cells reacted with mAb to class I MHC Ag or beta 2 microglobulin and cross-linked with GaMIg proliferated vigorously in the presence of IL-2 or PMA. In addition, it was demonstrated that cross-linking class I MHC molecules by culturing T4 cells with immobilized mAb to class I MHC Ag induced T4 cell proliferation in the presence of IL-2. T4 cell proliferation in the presence of IL-2 and PMA could also be induced by reacting the cells with specific mAb to polymorphic determinants on class I MHC molecules and cross-linking with GaMIg. Cross-linking mAb to CD4 or CD11a did not have a similar functional effect on T4 cells. Finally it was demonstrated that adding GaMIg to T4 cells reacted with mAb to class I MHC Ag but not CD11a resulted in an increase in intracellular calcium concentration. The data demonstrate that cross-linking class I MHC molecules results in the generation of at least one activation signal, a rise in intracellular calcium concentration, and, thereby, stimulates human T4 cells.     

MHC class Ib molecules bridge innate and acquired immunity

Our understanding of the classical MHC class I molecules (MHC class Ia molecules) has long focused on their extreme polymorphism. These molecules present peptides to T cells and are central to discrimination between self and non-self. By contrast, the functions of the non-polymorphic MHC class I molecules (MHC class Ib molecules) have been elusive, but emerging evidence reveals that, in addition to antigen presentation, MHC class Ib molecules are involved in immunoregulation. As we discuss here, the subset of MHC class Ib molecules that presents peptides to T cells bridges innate and acquired immunity, and this provides insights into the origins of acquired immunity.     

MHC class I manipulation on cell surfaces by gene transfer of anti-MHC class I intrabodies--a tool for decreased immunogenicity of allogeneic tissue and cell transplants

Intrabodies (IB) are suitable tools to down-regulate the expression of cell surface molecules in general. In this work, the appearance of major histocompatibility (MHC) class I molecules on the cell surface could be prevented by the expression of intracellularly localized anti-MHC class I antibodies. The expression of MHC antigens presenting intracellularly synthetised peptides on the cell surface is the predominant reason for immunologic detection and rejection of allogeneic cell and tissue transplants. Allogeneic keratinocyte sheets might be a suitable tool for skin grafting. Within this study primary rat keratinocytes have been transfected with anti-MHC I-IB. Strong IB-expressing cells showed a MHC I "knockout" phenotype. The cells did not exhibit any significant alterations compared to non-transfected cells: the cell growth and the expression of other surface molecules were unaltered. Merely an enhanced intracellular accumulation of MHC I molecules could be detected. Notably, IB-expressing keratinocytes displayed a reduced susceptibility to allogeneic cytotoxic T cells in vitro compared to unmodified cells with a normal level of MHC I surface expression. These MHC I-deficient keratinocytes might be utilized in tissue-engineered allogeneic non-immunogeneic skin transplants. The principle of MHC class I manipulation in general can be used for other allogeneic cell and tissue-engineered transplants as well.     

Presentation of antigenic peptides by MHC class I molecules

The basis for the immune response against intracellular pathogens is the recognition by cytotoxic T lymphocytes of antigenic peptides derived from cytosolic proteins, which are presented on the cell surface by major histocompatibility complex (MHC) class I molecules. The understanding of MHC class I-restricted peptide presentation has recently improved dramatically with the elucidation of the structural basis for the specificity of peptide binding to MHC class I molecules and the identification of proteins encoded in the class II region of the MHC that are putatively involved in the production of peptides and their transport into the endoplasmic reticulum, where they assemble with class I molecules.