Chemical cross-linking of class I molecules on cells creates receptive peptide binding sites.

Class I heterodimers on the surface of cells are generally unreceptive to binding peptides in the absence of exogenous beta 2-microglobulin. Paraformaldehyde covalently cross-links beta 2-microglobulin to class I heavy chains in situ and stabilizes empty class I heterodimers. Functionally, this cross-linking creates receptive class I peptide binding sites by acting on beta 2-microglobulin-associated molecules. The presentation of preexisting peptide-class I complexes is also enhanced. These findings support a model whereby a structural alteration, the dissociation of beta 2-microglobulin, limits the existence of receptive class I molecules on normal cells and may control the half-life of active class I molecules.     

A single amino acid substitution in an MHC class I molecule allows heteroclitic recognition by lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes

Class I molecules of the MHC bind foreign and endogenous peptides allowing recognition by the TCR on CTL. The recognition and killing of cells infected with lymphocytic choriomeningitis virus (LCMV) depends on the recognition of LCMV peptides bound to class I MHC. Mutations in class I MHC molecules have enabled the delineation of regions in the class I molecule important for binding peptides and for interaction with the TCR. We have constructed a library of class I mutants using saturation mutagenesis and report a phenotypic change resulting from a single amino acid substitution that results in the heteroclitic (increased) killing of LCMV-infected cells. This amino acid change, asparagine to serine at position 30, is in a conserved region of the class I molecule contacting the alpha 3 domain. This mutation does not result in increased expression of the class I molecule on the cell surface, does not affect the binding of CD8, and does not affect allogeneic recognition. Cold target experiments show that this heteroclitic killing is due to increased recognition by CTL. These data point toward a critical function for this region of the class I molecule in the binding of peptides or their presentation to CTL.     

Thermolabile H-2Kb molecules expressed by transporter associated with antigen processing-deficient RMA-S cells are occupied by low-affinity peptides.

RMA-S cells do not express functional TAP, yet they express MHC class I molecules at the cell surface, especially at reduced temperatures (26 degrees C). It is generally assumed that such class I molecules are "empty," devoid of any associated peptide. A radiochemical approach was used to label class I-associated peptides and to determine the extent to which Kb molecules in RMA-S cells are associated with peptides. These studies revealed that at 26 degrees C Kb molecules in RMA-S cells are occupied with self-peptides. Such peptides stably associate with Kb at 26 degrees C but easily dissociate from them at 37 degrees C, suggesting low-affinity interactions between Kb and the associated peptides. At 26 degrees C, at least some of these Kb molecules are stably expressed in a peptide-receptive state on the cell surface, whereas at 37 degrees C they are short lived and are only transiently capable of binding and presenting exogenously supplied OVA 257-264 peptide for presentation to CD8+ Kb-restricted T lymphocytes. Thus contrary to current models of class I assembly in TAP-deficient RMA-S cells, the presumably "empty" molecules are in fact associated with peptides at 26 degrees C. Together, our data support the existence of an alternative mechanism of peptide binding and display by MHC class I molecules in TAP-deficient cells that could explain their ability to present Ag.     

Assembly and transport of MHC class II molecules.

At the surface of antigen-presenting cells MHC class I and class II molecules present peptides to respectively CD8+ and CD4+ T cells. MHC class I molecules acquire peptides right after synthesis in the endoplasmic reticulum. MHC class II molecules do not acquire peptides in the endoplasmic reticulum but instead associate with a third chain, the invariant chain which impedes peptide binding. Subsequently the invariant chain takes MHC class II molecules to the endosomal/lysosomal compartment thanks to a targeting signal retained in its cytoplasmic tail. It then dissociates from the MHC class II dimer to allow it to bind peptides.     

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.     

Immunogenicity of HLA Class I and II Double Restricted Influenza A-Derived Peptides

The aim of the present study was to identify influenza A-derived peptides which bind to both HLA class I and -II molecules and by immunization lead to both HLA class I and class II restricted immune responses. Eight influenza A-derived 9-11mer peptides with simultaneous binding to both HLA-A*02:01 and HLA-DRB1*01:01 molecules were identified by bioinformatics and biochemical technology. Immunization of transgenic HLA-A*02:01/HLA-DRB1*01:01 mice with four of these double binding peptides gave rise to both HLA class I and class II restricted responses by CD8 and CD4 T cells, respectively, whereas four of the double binding peptides did result in HLA-A*02:01 restricted responses only. According to their cytokine profile, the CD4 T cell responses were of the Th2 type. In influenza infected mice, we were unable to detect natural processing in vivo of the double restricted peptides and in line with this, peptide vaccination did not decrease virus titres in the lungs of intranasally influenza challenged mice. Our data show that HLA class I and class II double binding peptides can be identified by bioinformatics and biochemical technology. By immunization, double binding peptides can give rise to both HLA class I and class I restricted responses, a quality which might be of potential interest for peptide-based vaccine development.     

Tapasin is a facilitator, not an editor, of class I MHC peptide binding

Tapasin has been proposed to function as a peptide editor to displace lower affinity peptides and/or to favor the binding of high affinity peptides. Consistent with this, cell surface HLA-B8 molecules in tapasin-deficient cells were less stable and the peptide repertoire was substantially altered. However, the binding affinities of peptides expressed in the absence of tapasin were unexpectedly higher, not lower. The peptide repertoire from cells expressing soluble tapasin was similar in both appearance and affinity to that presented in the presence of full-length tapasin, but the HLA-B8 molecules showed altered cell surface stability characteristics. Similarly, the binding affinities of HLA-A*0201-associated peptides from tapasin(+) and tapasin(-) cells were equivalent, although steady state HLA-A*0201 cell surface expression was decreased and the molecules demonstrated reduced cell surface stability on tapasin(-) cells. These data are inconsistent with a role for tapasin as a peptide editor. Instead, we propose that tapasin acts as a peptide facilitator. In this role, it stabilizes the peptide-free conformation of class I MHC molecules in the endoplasmic reticulum and thus increases the number and variety of peptides bound to class I MHC. Full-length tapasin then confers additional stability on class I MHC molecules that are already associated with peptides.     

Purification and Reconstitution of the Antigen Transport Complex TAP: A PREREQUISITE FOR DETERMINATION OF PEPTIDE STOICHIOMETRY AND ATP HYDROLYSIS*

The transporter associated with antigen processing (TAP) is an essential machine of the adaptive immune system that translocates antigenic peptides from the cytosol into the endoplasmic reticulum lumen for loading of major histocompatibility class I molecules. To examine this ABC transport complex in mechanistic detail, we have established, after extensive screening and optimization, the solubilization, purification, and reconstitution for TAP to preserve its function in each step. This allowed us to determine the substrate-binding stoichiometry of the TAP complex by fluorescence cross-correlation spectroscopy. In addition, the TAP complex shows strict coupling between peptide binding and ATP hydrolysis, revealing no basal ATPase activity in the absence of peptides. These results represent an optimal starting point for detailed mechanistic studies of the transport cycle of TAP by single molecule experiments to analyze single steps of peptide translocation and the stoichiometry between peptide transport and ATP hydrolysis.     

Class I-restricted presentation occurs without internalization or processing of exogenous antigenic peptides

Previous studies have shown that glutaraldehyde-fixed cells can present fragmented, but not native, Ag to class II-restricted T cells. This presumably occurs via direct binding of peptides to class II molecules at the cell surface. More recently, it has been shown that viable target cells can present peptides and endogenous, but not exogenous, protein Ag in association with class I MHC molecules to CTL. We have derived CTL specific for a chicken OVA peptide (OVA258-276) recognized in association with H-2Kb. These CTL recognize target cells that endogenously synthesize OVA and cells "loaded" with native OVA but fail to recognize target cells in the presence of exogenous native OVA. Thus, OVA must be intracellularly located to be processed and presented for CTL recognition. It remains unclear, however, whether exogenous peptides require internalization and further processing by target cells or are able to associate directly with class I molecules at the cell surface for CTL recognition. We provide evidence that glutaraldehyde-fixed cells can present synthetic peptides to H-2Kb- and H-2Db-restricted CTL and that such presentation does not require internalization or processing. The peptides used range in size from 16 to 48 amino acids in length. In contrast, glutaraldehyde-fixed cells are incapable of presenting Ag to CTL specific for influenza nucleoprotein and OVA if the cells are fixed within 1 h of viral influenza infection or loading with OVA. Thus, CTL recognition of antigenic peptides appears to occur via direct binding of peptides to class I molecules at the cell surface and does not require any intracellular processing events.     

ER quality control in the biogenesis of MHC class I molecules

Class I molecules of the major histocompatibility complex play a vital role in cellular immunity, reporting on the presence of viral or tumor-associated antigens by binding peptide fragments of these proteins and presenting them to cytotoxic T cells at the cell surface. The folding and assembly of class I molecules is assisted by molecular chaperones and folding catalysts that comprise the general ER quality control system which also monitors the integrity of the process, disposing of misfolded class I molecules through ER associated degradation (ERAD). Interwoven with general ER quality control are class I-specific components such as the peptide transporter TAP and the tapasin-ERp57 chaperone complex that supply peptides and monitor their loading onto class I molecules. This ensures that at the cell surface class I molecules will possess mainly optimal peptides with a long half-life. In this review we discuss these processes as well as a number of strategies that viruses have evolved to subvert normal class I assembly within the ER and thereby evade immune recognition by cytotoxic T cells.     

Dissociation of beta 2-microglobulin leads to the accumulation of a substantial pool of inactive class I MHC heavy chains on the cell surface

A large pool of free class I heavy chains is detected in situ on the plasma membrane of living cells. These chains are present on cells of different MHC genotypes and appear to exist under physiological conditions in vivo. These molecules arise from the dissociation of previously assembled class I heterodimers at the cell surface. The ratio of intact to dissociated heterodimers is strongly affected by the occupancy of the peptide-binding site of the class I molecule. Upon dissociation of the heterodimer, the class I molecule is functionally inactive. These findings may help to explain why class I molecules on the cell surface are unreceptive to binding peptides yet readily associate with peptides in the presence of exogenous beta 2-microglobulin. These results have implications for understanding the distinct functions of class I versus class II molecules and how the immunological identity of cells is preserved.     

Large scale mass spectrometric profiling of peptides eluted from HLA molecules reveals N-terminal-extended peptide motifs

The majority of >2000 HLA class I molecules can be clustered according to overlapping peptide binding specificities or motifs recognized by CD8(+) T cells. HLA class I motifs are classified based on the specificity of residues located in the P2 and the C-terminal positions of the peptide. However, it has been suggested that other positions might be relevant for peptide binding to HLA class I molecules and therefore be used for further characterization of HLA class I motifs. In this study we performed large-scale sequencing of endogenous peptides eluted from K562 cells (HLA class I null) made to express a single HLA molecule from HLA-B*3501, -B*3502, -B*3503, -B*3504, -B*3506, or -B*3508. Using sequence data from >1,000 peptides, we characterized novel peptide motifs that include dominant anchor residues extending to all positions in the peptide. The length distribution of HLA-B35-bound peptides included peptides of up to 15 residues. Remarkably, we determined that some peptides longer than 11 residues represented N-terminal-extended peptides containing an appropriate HLA-B35 peptide motif. These results provide evidence for the occurrence of endogenous N-terminal-extended peptide-HLA class I configurations. In addition, these results expand the knowledge about the identity of anchor positions in HLA class I-associated peptides that can be used for characterization of HLA class I motifs.     

Traffic of proteins and peptides across membranes for immunosurveillance by CD8(+) T lymphocytes: a topological challenge

Cytotoxic CD8(+) T lymphocytes kill infected cells that display major histocompatibility complex (MHC) class I molecules presenting peptides processed from pathogen proteins. In general, the peptides are proteolytically processed from newly made endogenous antigens in the cytosol and require translocation to the endoplasmic reticulum (ER) for MHC class I loading. This last task is performed by the transporters associated with antigen processing (TAP). Sampling of suspicious pathogen-derived proteins reaches beyond the cytosol, and MHC class I loading can occur in other secretory or endosomal compartments besides the ER. Peptides processed from exogenous antigens can also be presented by MHC class I molecules to CD8(+) T lymphocytes, in this case requiring delivery from the extracellular medium to the processing and MHC class I loading compartments. The endogenous or exogenous antigen can be processed before or after its transport to the site of MHC class I loading. Therefore, mechanisms that allow the full-length protein or processed peptides to cross several subcellular membranes are essential. This review deals with the different intracellular pathways that allow the traffic of antigens to compartments proficient in processing and loading of MHC class I molecules for presentation to CD8(+) T lymphocytes and highlights the need to molecularly identify the transporters involved.     

Exogenous peptides presented by transporter associated with antigen processing (TAP)-deficient and TAP-competent cells: intracellular loading and kinetics of presentation

This study investigates the differential capacity of TAP-deficient T2 cells, TAP-competent EBV cells, and immature and mature dendritic cells to present peptides to preformed CTL lines. It demonstrates that presentation of exogenous peptides involves peptide uptake and loading onto newly synthesized MHC class I molecules. This mechanism was best demonstrated for low affinity peptides in the presence of irrelevant peptides competing for HLA binding sites. Under these circumstances, inhibition of protein synthesis with cycloheximide or vesicular trafficking with brefeldin A significantly reduced the presentation of low affinity peptides. This was not restored by adding exogenous beta(2)-microglobulin to stabilize the MHC complex on the cell surface. In contrast, presentation of high affinity peptides was not sensitive to cycloheximide or brefeldin A, which suggests that different mechanisms may operate for presentation of high and low affinity peptides by TAP-competent cells. High affinity peptides can apparently compete with peptides in preloaded MHC class I molecules at the cell surface, whereas low affinity peptides require empty MHC molecules within cells. Accordingly, very high concentrations of exogenous low affinity peptides in conjunction with active MHC class I metabolism were required to allow successful presentation against a background of competing intracellular high affinity peptides in TAP-competent cells. These findings have implications for the design of peptide and protein-based vaccines.     

Molecular mechanism and species specificity of TAP inhibition by herpes simplex virus ICP47.

The immediate early protein ICP47 of herpes simplex virus (HSV) inhibits the transporter for antigen processing (TAP)-mediated translocation of antigen-derived peptides across the endoplasmic reticulum (ER) membrane. This interference prevents assembly of peptides with class I MHC molecules in the ER and ultimately recognition of HSV-infected cells by cytotoxic T-lymphocytes, potentially leading to immune evasion of the virus. Here, we demonstrate that recombinant, purified ICP47 containing a hexahistidine tag inhibits peptide import into microsomes of insect cells expressing human TAP, whereas inhibition of peptide transport by murine TAP was much less effective. This finding indicates an intrinsic species-specificity of ICP47 and suggests that no additional proteins interacting specifically with either ICP47 or TAP are required for inhibition of peptide transport. Since neither purified nor induced ICP47 inhibited photocrosslinking of 8-azido-ATP to TAP1 and TAP2 it seems that ICP47 does not prevent ATP from binding to TAP. By contrast, peptide binding was completely blocked by ICP47 as shown both by photoaffinity crosslinking of peptides to TAP and peptide binding to microsomes from TAP-transfected insect cells. Competition experiments indicated that ICP47 binds to human TAP with a higher affinity (50 nM) than peptides whereas the affinity to murine TAP was 100-fold lower. Our data suggest that ICP47 prevents peptides from being translocated by blocking their binding to the substrate-binding site of TAP.     

A long N-terminal-extended nested set of abundant and antigenic major histocompatibility complex class I natural ligands from HIV envelope protein

Viral antigens complexed with major histocompatibility complex (MHC) class I molecules are recognized by cytotoxic T lymphocytes on infected cells. Assays with synthetic peptides identify optimal MHC class I ligands often used for vaccines. However, when natural peptides are analyzed, more complex mixtures including long peptides bulging in the middle of the binding site or with carboxyl extensions are found, reflecting lack of exposure to carboxypeptidases in the antigen processing pathway. In contrast, precursor peptides are exposed to extensive cytosolic aminopeptidase activity, and fewer than 1% survive, only to be further trimmed in the endoplasmic reticulum. We show here a striking example of a nested set of at least three highly antigenic and similarly abundant natural MHC class I ligands, 15, 10, and 9 amino acids in length, derived from a single human immunodeficiency virus gp160 epitope. Antigen processing, thus, gives rise to a rich pool of possible ligands from which MHC class I molecules can choose. The natural peptide set includes a 15-residue-long peptide with unprecedented 6 N-terminal residues that most likely extend out of the MHC class I binding groove. This 15-mer is the longest natural peptide known recognized by cytotoxic T lymphocytes and is surprisingly protected from aminopeptidase trimming in living cells.     

Direct binding of peptide to empty MHC class I molecules on intact cells and in vitro

MHC class I molecules devoid of peptide are expressed on the cell surface of the mouse mutant lymphoma cell line RMA-S upon culture at reduced temperature. Empty class I molecules are thermolabile at the cell surface and in detergent lysates, but can be stabilized by the addition of presentable peptide; peptide binding appears to be a rapid process. Furthermore, class I molecules on the surface of RMA-S (H-2b haplotype) cells cultured at 26 degrees C can efficiently and specifically bind iodinated peptide presented by H-2Kb. Binding of iodinated peptide is also observed at a lower level for nonmutant cells (RMA) cultured at 26 degrees C. These experiments underscore the role for peptide in maintenance of the structure of class I molecules and, more importantly, provide two assay systems to study the interactions of peptides with MHC class I molecules independent of the availability of T cells that recognize a particular peptide-MHC class I complex.     

The human cytomegalovirus gene product US6 inhibits ATP binding by TAP

Human cytomegalovirus (HCMV) encodes several genes that disrupt the major histocompatibility complex (MHC) class I antigen presentation pathway. We recently described the HCMV-encoded US6 gene product, a 23 kDa endoplasmic reticulum (ER)-resident type I integral membrane protein that binds to the transporter associated with antigen processing (TAP), inhibits peptide translocation and prevents MHC class I assembly. The functional consequence of this inhibition is to prevent the cell surface expression of class I bound viral peptides and their recognition by HCMV-specific cytotoxic T cells. Here we describe a novel mechanism of action for US6. We demonstrate that US6 inhibits the binding of ATP by TAP1. This is a conformational effect, as the ER lumenal domain of US6 is sufficient to inhibit ATP binding by the cytosolic nucleotide binding domain of TAP1. US6 also stabilizes TAP at 37 degrees C and prevents conformational rearrangements induced by peptide binding. Our findings suggest that the association of US6 with TAP stabilizes a conformation in TAP1 that prevents ATP binding and subsequent peptide translocation.     

What to do with HLA-DO?

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

Endoplasmic reticulum aminopeptidase associated with antigen processing regulates quality of processed peptides presented by MHC class I molecules

Effective immune surveillance by CD8 T cells depends on the presentation of diverse peptides by MHC class I (pMHC I) molecules on the cell surface. The pMHC I repertoire is shaped in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with Ag processing (ERAAP). The ERAAP activity is required for producing peptides of appropriate length for generating optimal pMHC I. Paradoxically, ERAAP also inhibits generation of certain peptides such as the SVL9 (SSVVGVWYL) peptide encoded by the H13(a) histocompatibility gene and presented by D(b) MHC by an unknown mechanism. In this study, we show that the presentation of the SVL9-D(b) complex is inhibited when other peptides compete for binding D(b). Conversely, improving the binding of SVL9 peptide to D(b) suppresses the inhibition. Interestingly, the inhibitory effect of competitor peptides is observed only when ERAAP is expressed in the same cells. Thus, ERAAP, in concert with MHC I molecules, regulates the quality of processed peptides presented on the cell surface.