Rethinking peptide supply to MHC class I molecules

The notion that peptides bound to MHC class I molecules are derived mainly from newly synthesized proteins that are defective, and are therefore targeted for immediate degradation, has gained wide acceptance. This model, still entirely hypothetical, has strong intuitive appeal and is consistent with some experimental results, but it is strained by other findings, as well as by established and emerging concepts in protein quality control. While not discounting defectiveness as a driving force for the processing of some proteins, we propose that MHC-class-I-restricted epitopes are derived mainly from nascent proteins that are accessed by the degradation machinery prior to any assessment of fitness, and we outline one way in which this could be accomplished.     

MAPPP: MHC class I antigenic peptide processing prediction

MAPPP is a bioinformatics tool for the prediction of potential antigenic epitopes presented on the cell surface by major histocompatibility complex class I (MHC I) molecules to CD8 positive T lymphocytes. It combines existing predictions for proteasomal cleavage with peptide anchoring to MHC I molecules.     

The quality control of MHC class I peptide loading

The assembly of major histocompatibility complex (MHC) class I molecules is one of the more widely studied examples of protein folding in the endoplasmic reticulum (ER). It is also one of the most unusual cases of glycoprotein quality control involving the thiol oxidoreductase ERp57 and the lectin-like chaperones calnexin and calreticulin. The multistep assembly of MHC class I heavy chain with beta(2)-microglobulin and peptide is facilitated by these ER-resident proteins and further tailored by the involvement of a peptide transporter, aminopeptidases, and the chaperone-like molecule tapasin. Here we summarize recent progress in understanding the roles of these general and class I-specific ER proteins in facilitating the optimal assembly of MHC class I molecules with high affinity peptides for antigen presentation.     

Polymorphic peptide transporters in MHC class I monomorphic Syrian hamster

We have already shown that in species with highly polymorphic major histocompatibility complex (MHC) class I molecules (human, mouse) no functional polymorphism of the peptide transporters TaP1 and TAP2 is detectable (Lobigs and Müllbacher 1993).Investigating the antigen-presentation machinery of the class I MHC I expression via recombinant vaccinia viruses MHC class I expression via recombinant vaccinia viruses (VV) we found that six hamster cell lines fall into two phenotypic classes. Four cell lines (HaK, FF, MF-2, and HT-1) showed no defect in expressing four different H2 class I molecules (K^K, K^d, K^b, D^d) and the appropriate VV peptide recognized by mouse VV-immune cytotoxic T (Tc) cells on the cell surface. Two cell lines (BHK-21 and NIL-2) expressed D^d and K^b in association with VV peptides as recognized by VV-immune, H2-restricted Tc cells but not K^k and K^d. However, K^d was expressed on the cell surface, as shown by fluorescence-activated cell sorter (FACS) analysis and alloreactive Tc-cell recogniction. K^k is only surface-expressed in these two cell lines when superinfected with two VV recombinants encoding rat TAP1 (VV-mtp1) and TAP2 (VV-mtp2). Superinfection with VV-mtpl and VV-mtp2 rendered both cell lines, after infection with either VV-K^k or V-K^d, susceptible to lysis by either K^k-orK^d- restricted VV-immune Tc cells. Thus Syrian hamster cell lines express functionally polymorphic peptide transporters.The TAP2 gene from FF cells was cloned and sequenced; comparison with human, mouse, and rat TAP2 sequences show 78%, 88% and 87% similarity, respectively.     

Comparison of rat MHC class I antigens by peptide mapping

Monoclonal antibodies specific for the rat major histocompatibility complex (MHC) class I antigens RT1.Aⁿ, RT1.A^u, and RT1.E^u were used for immunoprecipitation of antigens biosynthetically radiolabeled with¹⁴C- or³H-labeled arginine, lysine, and tyrosine; with arginine or tyrosine alone; and with or without tunicamycin in the culture medium. Heavy chains of the glycosylated and unglycosylated antigens were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their tryptic and chymotryptic peptides were compared by high performance liquid chromatography. The antigens coded by the same locus in two different haplotypes (Aⁿ and A^u) differed by 30%, whereas the products of two different loci in the same haplotype (A^u and E^u) differed only by 1–3%. Comparative analysis of the data for samples labeled with single amino acids indicated that two amino acids in A^u have been substituted by an arginine and probably by a tyrosine residue, respectively, in E^u. The high degree of homology between the products of theA andE loci in the same haplotype accounts for the difficulty in detecting recombinational events within the MHC of the rat by classical serological approaches.We dedicate this publication to Professor Paul Doty on the occasion of his sixty-fifth birthday     

MHC class I multimers

T lymphocytes play a key role in the immune response to both foreign and self peptide antigens, which they recognize in combination with MHC molecules. In the past it has been difficult to analyse objectively the specificity, frequency and intensity of T cell responses. The recent application of fluorescent-labelled MHC class I multimers, however, has provided a powerful experimental approach to the direct visualisation of antigen-specific T cells. As a result, our perspective of how T cells respond to both viruses and other antigens in vivo has been greatly enhanced.     

MHC class I multimers

T lymphocytes play a key role in the immune response to both foreign and self peptide antigens, which they recognize in combination with MHC molecules. In the past it has been difficult to analyse objectively the specificity, frequency and intensity of T cell responses. The recent application of fluorescent-labelled MHC class I multimers, however, has provided a powerful experimental approach to the direct visualisation of antigen-specific T cells. As a result, our perspective of how T cells respond to both viruses and other antigens in vivo has been greatly enhanced.     

The nonclassical MHC class I molecule Qa-1 forms unstable peptide complexes

The MHC class Ib molecule Qa-1 is the primary ligand for mouse CD94/NKG2A inhibitory receptors expressed on NK cells, in addition to presenting Ags to a subpopulation of T cells. CD94/NKG2A receptors specifically recognize Qa-1 bound to the MHC class Ia leader sequence-derived peptide Qdm. Qdm is the dominant peptide loaded onto Qa-1 under physiological conditions and this peptide has an optimal sequence for binding to Qa-1. Peptide dissociation experiments demonstrated that Qdm dissociates from soluble or cell surface Qa-1(b) molecules with a t(1/2) of approximately 1.5 h at 37 degrees C. In comparison, complexes of an optimal peptide (SIINFEKL) bound to the MHC class Ia molecule H-2K(b) dissociated with a t(1/2) in the range from 11 to 31 h. In contrast to K(b), the stability of cell surface Qa-1(b) molecules was independent of bound peptides, and several observations suggested that empty cell surface Qa-1(b) molecules might be unusually stable. Consistent with the rapid dissociation rate of Qdm from Qa-1(b), cells become susceptible to lysis by CD94/NKG2A(+) NK cells under conditions in which new Qa-1(b)/Qdm complexes cannot be continuously generated at the cell surface. These results support the hypothesis that Qa-1 has been selected as a specialized MHC molecule that is unable to form highly stable peptide complexes. We propose that the CD94/NKG2A-Qa-1/Qdm recognition system has evolved as a rapid sensor of the integrity of the MHC class I biosynthesis and Ag presentation pathway.     

Functional screening of a retroviral peptide library for MHC class I presentation

We have used retroviral vector technology to develop a method for functional screening of combinatorial peptide libraries expressed inside mammalian cells with the ultimate goal of identifying new drug targets. The method was validated in a library screening experiment based on antigen presentation of small peptides. A library encoding SIXNXEKX-peptides, where X designates randomised positions corresponding to major histocompatibility (MHC) class I anchor residues, was generated in a retroviral vector. The library was transduced into a population of antigen presenting cells (APCs) known to mediate MHC class I restricted presentation of the SIINFEKL peptide. The cellular library was screened by using an antigen presentation assay in which a T cell hybridoma recognising the MHC class I/SIINFEKL peptide complex was employed. Using this experimental model, we identified two positive cellular clones both encoding SIINFEKL peptides with identical codon usage. This number corresponded well to the expected frequency of SIINFEKL in the library. The lack of identification of other peptides capable of activating the T-hybridoma supports previous findings of a high degree of specificity at the level of peptide-loading of MHC-molecules. The result further demonstrates the potential of using combinatorial libraries for functional screening and selection of effector peptides stably expressed in mammalian cells.     

Conserved MHC class I peptide binding motif between humans and rhesus macaques

Since the onset of the HIV pandemic, the use of nonhuman primate models of infection has increasingly become important. An excellent model to study HIV infection and immunological responses, in particular cell-mediated immune responses, is SIV infection of rhesus macaques. CTL epitopes have been mapped using SIV-infected rhesus macaques, but, to date, a peptide binding motif has been described for only one rhesus class I MHC molecule, Mamu-A*01. Herein, we have established peptide-live cell binding assays for four rhesus MHC class I molecules: Mamu-A*11, -B*03, -B*04, and -B*17. Using such assays, peptide binding motifs have been established for all four of these rhesus MHC class I molecules. With respect to the nature and spacing of crucial anchor positions, the motifs defined for Mamu-B*04 and -B*17 present unique features not previously observed for other primate species. The motifs identified for Mamu-A*11 and -B*03 are very similar to the peptide binding motifs previously described for human HLA-B*44 and -B*27, respectively. Accordingly, naturally processed peptides derived from HLA-B*44 and HLA-B*27 specifically bind Mamu-A*11 and Mamu-B*03, respectively, indicating that conserved MHC class I binding capabilities exist between rhesus macaques and humans. The definition of four rhesus MHC class I-specific motifs expands our ability to accurately detect and quantitate immune responses to MHC class I-restricted epitopes in rhesus macaques and to rationally design peptide epitope-based model vaccine constructs destined for use in nonhuman primates.     

Redox regulation facilitates optimal peptide selection by MHC class I during antigen processing

Activated CD8(+) T cells discriminate infected and tumor cells from normal self by recognizing MHC class I-bound peptides on the surface of antigen-presenting cells. The mechanism by which MHC class I molecules select optimal peptides against a background of prevailing suboptimal peptides and in a considerably proteolytic ER environment remained unknown. Here, we identify protein disulfide isomerase (PDI), an enzyme critical to the formation of correct disulfide bonds in proteins, as a component of the peptide-loading complex. We show that PDI stabilizes a peptide-receptive site by regulating the oxidation state of the disulfide bond in the MHC peptide-binding groove, a function that is essential for selecting optimal peptides. Furthermore, we demonstrate that human cytomegalovirus US3 protein inhibits CD8(+) T cell recognition by mediating PDI degradation, verifying the functional relevance of PDI-catalyzed peptide editing in controlling intracellular pathogens. These results establish a link between thiol-based redox regulation and antigen processing.     

Distinct functions of tapasin revealed by polymorphism in MHC class I peptide loading

Peptide assembly with class I molecules is orchestrated by multiple chaperones including tapasin, which bridges class I molecules with the TAP and is critical for efficient Ag presentation. In this paper, we show that, although constitutive levels of endogenous murine tapasin apparently are sufficient to form stable and long-lived complexes between the human HLA-B*4402 (B*4402) and mouse TAP proteins, this does not result in normal peptide loading and surface expression of B*4402 molecules on mouse APC. However, increased expression of murine tapasin, but not of the human TAP proteins, does restore normal cell surface expression of B*4402 and efficient presentation of viral Ags to CTL. High levels of soluble murine tapasin, which do not bridge TAP and class I molecules, still restore normal surface expression of B*4402 in the tapasin-deficient human cell line 721.220. These findings indicate distinct roles for tapasin in class I peptide loading. First, tapasin-mediated bridging of TAP-class I complexes, which despite being conserved across the human-mouse species barrier, is not necessarily sufficient for peptide loading. Second, tapasin mediates a function which probably involves stabilization of empty class I molecules and which is sensitive to structural compatibility of components within the loading complex. These discrete functions of tapasin predict limitations to the study of HLA molecules across some polymorphic and species barriers.     

Oligocarbamates as MHC class I ligands

Summary New ligands for major histocompatibility complex (MHC) class I molecules were prepared using a flexible automated synthesis of oligocarbamates. An efficient solution-phase synthesis was found for Fmoc-amino alcohols which are required as building blocks. The biological activity of the oligomeric peptidomimetics was demonstrated in a stabilizing assay with MHC class I presenting cells.     

Oligocarbamates as MHC class I ligands

New ligands for major histocompatibility complex (MHC) class I molecules were prepared using a flexible automated synthesis of oligocarbamates. An efficient solution-phase synthesis was found for Fmoc-amino alcohols which are required as building blocks. The biological activity of the oligomeric peptidomimetics was demonstrated in a stabilizing assay with MHC class I presenting cells.     

NMR study on the interaction between MHC class I protein and its antigen peptide

A major histcompatibility complex (MHC) class I protein H-2K(b) was expressed in a large scale as a fusion protein with thioredoxin and hexahistidine at the N-terminus to analyze the interaction with the antigen peptide SIYRYYGL. NMR spectra of the peptide in the mixture solution with the protein showed very broad signals, indicating the obviously clear existence of the dynamic interaction between the class I protein and the antigen peptide. The interaction of the protein and peptide was discussed as well as the surrounding atmosphere of the peptide in the complex.     

Technologies for MHC class I immunoproteomics

T cell epitopes are peptides, for instance derived from foreign, mutated or overexpressed proteins, that are displayed by MHC molecules on the cell surface and that are recognized by T lymphocytes. Knowledge of the identity of epitopes displayed by MHC molecules is of high value for diagnostic purposes and for the development of prophylactic and therapeutic immunotherapy regimens. Here we review key techniques in MHC class I epitope definition and we discuss recent developments in epitope discovery and their implications. Developments in epitope discovery strategies should ultimately lead to the definition of the MHC-associated peptidome.     

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.