Peptide selection by class I molecules of the major histocompatibility complex

Class I molecules of the major histocompatibility complex (MHC) bind peptides derived from cytoplasmic proteins. Comparison of over 100 such peptides reveals the importance of the carboxy-terminal residue in selective binding. Recent evidence implicates the proteases and transporters of the processing pathway in providing peptides with the correct residues at the carboxyl terminus.     

Human-specific evolution of killer cell immunoglobulin-like receptor recognition of major histocompatibility complex class I molecules

In placental mammals, natural killer (NK) cells are a population of lymphocytes that make unique contributions to immune defence and reproduction, functions essential for survival of individuals, populations and species. Modulating these functions are conserved and variable NK-cell receptors that recognize epitopes of major histocompatibility complex (MHC) class I molecules. In humans, for example, recognition of human leucocyte antigen (HLA)-E by the CD94:NKG2A receptor is conserved, whereas recognition of HLA-A, B and C by the killer cell immunoglobulin-like receptors (KIRs) is diversified. Competing demands of the immune and reproductive systems, and of T-cell and NK-cell immunity-combined with the segregation on different chromosomes of variable NK-cell receptors and their MHC class I ligands-drive an unusually rapid evolution that has resulted in unprecedented levels of species specificity, as first appreciated from comparison of mice and humans. Counterparts to human KIR are present only in simian primates. Observed in these species is the coevolution of KIR and the four MHC class I epitopes to which human KIR recognition is restricted. Unique to hominids is the emergence of the MHC-C locus as a supplier of specialized and superior ligands for KIR. This evolutionary trend is most highly elaborated in the chimpanzee. Unique to the human KIR locus are two groups of KIR haplotypes that are present in all human populations and subject to balancing selection. Group A KIR haplotypes resemble chimpanzee KIR haplotypes and are enriched for genes encoding KIR that bind HLA class I, whereas group B KIR haplotypes are enriched for genes encoding receptors with diminished capacity to bind HLA class I. Correlating with their balance in human populations, B haplotypes favour reproductive success, whereas A haplotypes favour successful immune defence. Evolution of the B KIR haplotypes is thus unique to the human species.     

Identification of class I major histocompatibility complex encoded molecules in the amphibian Xenopus

Class I-like molecules have been immunoprecipitated from Xenopus leukocytes and erythrocytes with alloantisera directed against major histocompatibility complex (MHC)-linked antigens. The heavy chains, depending on the allele examined, have molecular weights of 40 000–44 000 of which 3000 daltons are asparagine-linked carbohydrates, probably present as one N-linked glycan. The presumed analogue of ₂-microglobulin has a molecular weight of 13 000 and bears no asparagine-linked glycans. Family studies show that the heavy chains are encoded by genes residing in or closely linked to the MHC.Abbreviations used in this paper MHC major histocompatibility complex - CML cell-mediated lympholysis - MLR mixed leukocyte reaction - APBS amphibian phosphate-buffered saline - kd kilodalton - LG Xenopus laevis — Xenopus gilli species hybrids - IEF isoelectric focusing Founded and supported by F. Hoffmann-La Roche & Co., Limited Company, CH-4005 Basel, Switzerland.     

The lytic cycle of Epstein-Barr virus is associated with decreased expression of cell surface major histocompatibility complex class I and class II molecules

Human herpesviruses utilize an impressive range of strategies to evade the immune system during their lytic replicative cycle, including reducing the expression of cell surface major histocompatibility complex (MHC) and immunostimulatory molecules required for recognition and lysis by virus-specific cytotoxic T cells. Study of possible immune evasion strategies by Epstein-Barr virus (EBV) in lytically infected cells has been hampered by the lack of an appropriate permissive culture model. Using two-color immunofluorescence staining of cell surface antigens and EBV-encoded lytic cycle antigens, we examined EBV-transformed B-cell lines in which a small subpopulation of cells had spontaneously entered the lytic cycle. Cells in the lytic cycle showed a four- to fivefold decrease in cell surface expression of MHC class I molecules relative to that in latently infected cells. Expression of MHC class II molecules, CD40, and CD54 was reduced by 40 to 50% on cells in the lytic cycle, while no decrease was observed in cell surface expression of CD19, CD80, and CD86. Downregulation of MHC class I expression was found to be an early-lytic-cycle event, since it was observed when progress through late lytic cycle was blocked by treatment with acyclovir. The immediate-early transactivator of the EBV lytic cycle, BZLF1, did not directly affect expression of MHC class I molecules. However, BZLF1 completely inhibited the upregulation of MHC class I expression mediated by the EBV cell-transforming protein, LMP1. This novel function of BZLF1 elucidates the paradox of how MHC class I expression can be downregulated when LMP1, which upregulates MHC class I expression in latent infection, remains expressed in the lytic cycle.     

Calreticulin is expressed on the cell surface of activated human peripheral blood T lymphocytes in association with major histocompatibility complex class I molecules.

Calreticulin is an endoplasmic reticulum resident molecule known to be involved in the folding and assembly of major histocompatibility complex (MHC) class I molecules. In the present study, expression of calreticulin was analyzed in human peripheral blood T lymphocytes. Pulse-chase experiments in [35S]methionine-labeled T cell blasts showed that calreticulin was associated with several proteins in the endoplasmic reticulum and suggested that it was expressed at the cell surface. Indeed, the 60-kDa calreticulin was labeled by cell surface biotinylation and precipitated from the surface of activated T cells together with a protein with an apparent molecular mass of 46 kDa. Cell surface expression of calreticulin by activated T lymphocytes was further confirmed by immunofluorescence and flow cytometry, studies that showed that both CD8+ and CD4+ T cells expressed calreticulin in the plasma membrane. Low amounts of cell surface calreticulin were detected in resting T lymphocytes. By sequential immunoprecipitation using the conformation independent monoclonal antibody HC-10, we provided evidence that the cell surface 46-kDa protein co-precipitated with calreticulin is unfolded MHC I. These results show for the first time that after T cell activation, significant amounts of calreticulin are expressed on the T cell surface, where they are found in physical association with a pool of beta2-free MHC class I molecules.     

Misfolded major histocompatibility complex class I molecules accumulate in an expanded ER-Golgi intermediate compartment

Misfolded membrane proteins are rapidly degraded, often shortly after their synthesis and insertion in the endoplasmic reticulum (ER), but the exact location and mechanisms of breakdown remain unclear. We have exploited the requirement of MHC class I molecules for peptide to achieve their correct conformation: peptide can be withheld by introducing a null mutation for the MHC-encoded peptide transporter, TAP. By withholding TAP-dependent peptides, the vast majority of newly synthesized class I molecules fails to leave the endoplasmic reticulum and is degraded. We used mice transgenic for HLA-B27 on a TAP1- deficient background to allow visualization by immunoelectron microscopy of misfolded HLA-B27 molecules in thymic epithelial cells. In such HLA transgenic animals, the TAP mutation can be considered a genetic switch that allows control over the extent of folding of the protein of interest, HLA-B27, while the rate of synthesis of the constituent subunits remains unaltered. In TAP1-deficient, HLA-B27 transgenic animals, HLA-B27 molecules fail to assemble correctly, and do not undergo carbohydrate modifications associated with the Golgi apparatus, such as conversion to Endoglycosidase H resistance, and acquisition of sialic acids. We show that such molecules accumulate in an expanded network of tubular and fenestrated membranes. This compartment has its counterpart in normal thymic epithelial cells, and is identified as an ER-Golgi intermediate. We detect the presence of ubiquitin and ubiquitin-conjugating enzymes in association with this compartment, suggesting a nonlysosomal mode of degradation of its contents.     

Generation of major histocompatibility complex class I antigens

Presentation of antigenic peptides by major histocompatibility complex (MHC) class I molecules on the surface of antigen-presenting cells is an effective extracellular representation of the intracellular antigen content. The intracellular proteasome-dependent proteolytic machinery is required for generating MHC class I-presented peptides. These peptides appear to be derived mainly from newly synthesized defective ribosomal products, ensuring a rapid cytotoxic T lymphocyte-mediated immune response against infectious pathogens. Here we discuss the generation of MHC class I antigens on the basis of the currently understood molecular, biochemical and cellular mechanisms.     

Cell surface expression of major histocompatibility complex class I molecules is reduced in hepatitis C virus subgenomic replicon-expressing cells

The hepatitis C virus (HCV) causes chronic hepatitis in most infected individuals by evading host immune defenses. In this investigation, we show that HCV-infected cells may go undetected in the immune system by suppressing major histocompatibility complex (MHC) class I antigen presentation to cytotoxic T lymphocytes. Cells expressing HCV subgenomic replicons have lower MHC class I cell surface expression. This is due to reduced levels of properly folded MHC class I molecules. HCV replicons induce endoplasmic reticulum (ER) stress (K. Tardif, K. Mori, and A. Siddiqui, J. Virol. 76:7453-7459, 2002), which results from a decline in protein glycosylation. Decreasing protein glycosylation can disrupt protein folding, preventing the assembly of MHC class I molecules. This results in the accumulation of unfolded MHC class I. Therefore, the persistence and pathogenesis of HCV may depend upon the ER stress-mediated interference of MHC class I assembly and cell surface expression.     

Functions of ERp57 in the folding and assembly of major histocompatibility complex class I molecules

ERp57 is a thiol oxidoreductase of the endoplasmic reticulum that appears to be recruited to substrates indirectly through its association with the molecular chaperones calnexin and calreticulin. However, its functions in living cells have been difficult to demonstrate. During the biogenesis of class I histocompatibility molecules, ERp57 has been detected in association with free class I heavy chains and, at a later stage, with a large complex termed the peptide loading complex. This implicates ERp57 in heavy chain disulfide formation, isomerization, or reduction as well as in the loading of peptides onto class I molecules. In this study, we show that ERp57 does indeed participate in oxidative folding of the heavy chain. Depletion of ERp57 by RNA interference delayed heavy chain disulfide bond formation, slowed folding of the heavy chain alpha(3) domain, and caused slight delays in the transport of class I molecules from the endoplasmic reticulum to the Golgi apparatus. In contrast, heavy chain-beta(2)-microglobulin association kinetics were normal, suggesting that the interaction between heavy chain and beta(2) -microglobulin does not depend on an oxidized alpha(3) domain. Likewise, the peptide loading complex assembled properly, and peptide loading appeared normal upon depletion of ERp57. These studies demonstrate that ERp57 is involved in disulfide formation in vivo but do not support a role for ERp57 in peptide loading of class I molecules. Interestingly, depletion of another thiol oxidoreductase, ERp72, had no detectable effect on class I biogenesis, consistent with a specialized role for ERp57 in this process.     

Probing the stability of class I major histocompatibility complex (MHC) molecules on the surface of human cells

 We used binding of a fluorescent adduct of β₂-microglobulin, fluorescein β₂m, to probe the stability of class I HLA molecules on the surface of human cells. The weight of the literature suggests that this ligand binds to heavy chains that have lost β₂m and possibly peptide as well. Hence Fl-β₂m reports on the stability of the class I HLA trimer. A small fraction of HLA molecules, ∼5%, binds Fl-β₂m on both resting and activated T cells. A larger fraction of all HLA molecules binds Fl-β₂m in FO-1 cells, β₂m-deficient cells, transfected with various B2m genes. HLA molecules of FO-1 cells are more stable when expressed with human β₂m, than when expressed with mouse β₂m. The non-covalent association of HLA heavy chains, β₂m and peptide implies that eventually every molecule of HLA trimer ought to dissociate and bind Fl-β₂m. In fact, the extent of exchange is limited by the lifetime of a given molecule at the cell surface. β₂m exchange decreases as cell concentration increases, suggesting that some density-dependent process acts to enhance degradation or denaturation of β₂m-free HLA heavy chains.     

Domain structures of cell surface glycopeptides encoded by class I and class II beta genes of the major histocompatibility complex

Published sequence data of MHC genes, cDNAs and MHC products were analyzed for their sequence homologies. Alignment statistics revealed that class I gene products consist of four mutually homologous domains, and that class II beta gene products is composed of three mutually homologous domains. Not only extracellular domains but also newly discovered C-terminal shorter domains of class I and class II beta gene products were found to have evolved from a one-domain-long beta 2-microglobulin-like protein by repeated exon duplications and splittings.     

Peptide-receptive major histocompatibility complex class I molecules cycle between endoplasmic reticulum and cis-Golgi in wild-type lymphocytes

Prior to binding to a high affinity peptide and transporting it to the cell surface, major histocompatibility complex class I molecules are retained inside the cell by retention in the endoplasmic reticulum (ER), recycling through the ER-Golgi intermediate compartment and possibly the cis-Golgi, or both. Using fluorescence microscopy and a novel in vitro COPII (ER-to-ER-Golgi intermediate compartment) vesicle formation assay, we find that in both lymphocytes and fibroblasts that lack the functional transporter associated with antigen presentation, class I molecules exit the ER and reach the cis-Golgi. Intriguingly, in wild-type T1 lymphoma cells, peptide-occupied and peptide-receptive class I molecules are simultaneously exported from ER membranes with similar efficiencies. Our results suggest that binding of high affinity peptide and exit from the ER are not coupled, that the major histocompatibility complex class I quality control compartment extends into the Golgi apparatus under standard conditions, and that peptide loading onto class I molecules may occur in post-ER compartments.     

Influence of major histocompatibility complex H-2LD class I molecules on spleen colony-forming units

To test whether the major histocompatibility complex class I genes are involved in the regulation of hemopoiesis, the stem cell activities of BALB/c-H-2dm2 (Dm2) mice, which are defective in the expression of H-2L antigens, have been compared with those of the wild-type, BALB/c-Kh, in in vivo and in vitro stem cell assays. In spleen colony-forming unit assays, Dm2 as hosts consistently supported a smaller number of colonies than did BALB/c-Kh. However, both Dm2 and BALB/c-Kh supported a comparable number of colonies in in vitro granulocyte-macrophage colony-forming unit and erythroid colony-forming unit assays. These observations together suggest that the mutation in Dm2 has not affected the hemopoietic potential of the stem cells but may probably affect the hemopoietic microenvironment for the development of the stem cells.     

Evolution of major histocompatibility complex class I genes in Cetartiodactyls

Previous studies of cattle MHC have suggested the presence of at least four classical class I loci. Analysis of haplotypes showed that any combination of one, two or three genes may be expressed, although no gene is expressed consistently. The aim of this study was to examine the evolutionary relationships among these genes and to study their phylogenetic history in Cetartiodactyl species, including cattle and their close relatives. A secondary aim was to determine whether recombination had occurred between any of the genes. MHC class I data sets were generated from published sequences or by polymerase chain reaction from cDNA. Phylogenetic analysis revealed that MHC class I sequences from Cetartiodactyl species closely related to cattle were distributed among the main cattle gene "groups", while those from more distantly related species were either scattered (sheep, deer) or clustered in a species-specific manner (sitatunga, giraffe). A comparison between gene and species trees showed a poor match, indicating that divergence of the MHC sequences had occurred independently from that of the hosts from which they were obtained. We also found two clear instances of interlocus recombination among the cattle MHC sequences. Finally, positive natural selection was documented at positions throughout the alpha 1 and 2 domains, primarily on those amino acids directly involved in peptide binding, although two positions in the alpha 3 domain, a region generally conserved in other species, were also shown to be undergoing adaptive evolution.     

Metformin rescues cell surface major histocompatibility complex class I (MHC-I) deficiency caused by oncogenic transformation

Active avoidance by tumor cells from attack and elimination by immune cells is an emerging cancer hallmark that is achieved primarily through decreasing the levels of major histocompatibility complex class I (MHC-I) at the cancer cells’ surface. Deficiencies in MHC-I antigen-restricted immunosurveillance may be intertwined with an altered, Warburg-like cancer cell-intrinsic metabolism, another emerging hallmark of cancer that involves a switch from mitochondrial respiration to glycolysis to efficiently support large-scale biosynthetic programs that are required for active cell proliferation. We recently envisioned that intervention strategies aimed at reversing the bioenergetic signature of cancer cells (e.g., the antidiabetic biguanide metformin) should correct oncogene (e.g., HER2)-driven MHC-I defects, thus preventing immune escape of oncogene transformants. First, we explored how metformin treatment impacted mitochondrial biogenesis in cultured breast cancer cells overexpressing the membrane tyrosine kinase receptor HER2, the best-characterized downregulator of MHC-I. Metformin exposure was found to dose-dependently increase the expression levels of cytochrome c oxidase I and mitochondrial succinate dehydrogenase, which are encoded by mitochondrial and nuclear DNA, respectively. Second, we explored whether metformin-enhanced mitochondrial biogenesis might significantly alter the MHC-I status in breast carcinoma cells. MHC-I expression, as assessed by flow cytometry using an anti-HLA-ABC monoclonal antibody, was fully restored (up to ~25-fold upregulation) in MHC-I-negative HER2 gene-amplified carcinoma cells. These findings may help delineate a previously unrecognized mechanism through which metformin (and metformin-like drugs) may enable a cancer patient’s own immune system to mount an efficient anti-metastasis response that can prevent or delay disease recurrence. Restored antigenicity and immunogenicity of tumor cells may represent a previously unrecognized primary mode of action underlying the cancer-preventive effects of metformin.     

Human major histocompatibility complex class II invariant chain is expressed on the cell surface.

Class II major histocompatibility complex antigens are intracellularly associated with a nonpolymorphic polypeptide referred to as the invariant chain. Before the class II heterodimer appears on the cell surface, the invariant chain dissociates but it has so far been unclear as to whether or not a proportion of the invariant chain also appears on the plasma membrane. We describe a study with three monoclonal antibodies which recognize an extracytoplasmic determinant present on all forms of the invariant chain and use them to demonstrate its presence on the surface of the intact cells. The determinants recognized by two of the antibodies were found to be located within the 60 amino acids at the extreme C-terminal (extracytoplasmic) end of the invariant chain. The invariant chain-specific monoclonal antibody, VIC-Y1, was found to bind a determinant located between amino acids 1 and 73, which correspond to mainly cytoplasmic residues. Using the C-terminal specific antibodies, the number of antibody binding sites on the surface of two B lymphoma lines was estimated to be 10(5) per cell. The results of this study appear to resolve the highly disputed question of whether or not the invariant chain can appear as a plasma membrane protein. The results are discussed in the context of a possible role for the invariant chain in antigen processing and presentation.