Processing of MHC class I presented antigens

The immune defences of our organism against pathogens and malignant transformation rely to a large extent on surveillance by cytotoxic T lymphocytes. This surveillance in turn depends on the antigen processing system, which provides peptide samples of the cellular protein composition to MHC (major histocompatibility complex) class I molecules displayed on the cell surface. To continuously and almost in real time provide a representative sample of the array of proteins synthesized by the cell, this system exploits some fundamental pathways of the cellular metabolism, with the help of several dedicated players acting exclusively in antigen processing. Thus, a key element in the turnover of cellular proteins, protein degradation by cytosolic proteasome complexes, is exploited as source of peptides, by recruiting a minor fraction of the produced peptides as ligands for MHC class I molecules. These peptides can be further processed and adapted to the precise binding requirements of allelic MHC class I molecules by enzymes in the cytosol and endoplasmic reticulum. The latter compartment is equipped with several dedicated players helping peptide assembly with class I molecules. These include the TAP (transporter associated with antigen processing) membrane transporter pumping peptides into the ER, and tapasin, a chaperone with a structure similar to MHC molecules that tethers class I molecules awaiting peptide loading to the TAP transporter, and mediates optimization of MHC class I ligand by a still somewhat mysterious mechanism. Additional "house-keeping" chaperones that are known to act in concert in ER quality control, assist and control correct folding, oxidation and assembly of MHC class I molecules. While this processing system handles exclusively endogenous cellular proteins in most cells, dendritic cells employ one or several special pathways to shuttle exogenous, internalized proteins into the system, in a process referred to as cross-presentation. Deciphering the cell biological mechanism creating the link between the endosomal and secretory pathways that enables cross-presentation is one of the challenges faced by contemporary research in the field of MHC class I antigen processing.     

MHC class I antigens as surface markers of adult erythrocytes during the metamorphosis of Xenopus

An alloantiserum produced against Xenopus MHC class I antigens has been used to distinguish different erythrocyte populations at metamorphosis. By analysis using a fluorescence-activated cell sorter (FACS) analyzer, tadpole (stage 55) and adult erythrocytes have distinct volume differences and tadpole cells have no MHC antigens on the cell surface. Both tadpole and adult erythrocytes express a "mature erythrocyte" antigen marker, recognized by its monoclonal antibody (F1F6). During metamorphosis, immature erythrocytes, at various stages of differentiation, which express adult levels of cell-surface MHC antigens by 12 days after tail resorption, are found in the bloodstream. These immature cells are biosynthetically active, produce adult hemoglobin, and mature by 60 days after the completion of metamorphosis. Percoll gradient-density fractionation has shown that all of the cells in the new erythrocyte series express adult levels of MHC antigens but there is only a gradual increase in the amount of "mature erythrocyte" antigen. Tadpole erythrocytes, which are biosynthetically active during larval stages, produce small amounts of surface MHC antigens before the metamorphic climax and then become metabolically inactive. They are completely cleared from the circulation by 60 days after metamorphosis. Erythrocytes from tadpoles arrested in their development for long periods of time express intermediate levels of MHC antigens, suggesting a "leaky" expression of these molecules in the tadpole cells. The most abundant erythrocyte cell-surface proteins from tadpoles and adults, as judged by two-dimensional gel electrophoresis, are very different.     

Carbohydrate moieties of rat MHC class I antigens

The rat major histocompatibility complex class I antigens RT1.A^u and RT1.E^u from the u haplotype and RT1.Aⁿ from the n haplotype were labeled with ¹⁴C-asparagine or with ³H-fucose, mannose, galactose, and N-acetylglucosamine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed complete removal of radioactivity from the sugar-labeled antigen heavy chains by digestion with glycopeptidase F, an enzyme that removes N-linked glycans completely. High performance liquid chromatography analysis of the tryptic digests of the mixed sugar-labeled and asparagine-labeled antigens demonstrated that all the sugar-labeled peptides were coincident with asparagine-labeled peptides. The Aⁿ antigen showed three glycopeptides, each of which had different amounts of sugar radioactivity. The antigens A^u and E^u showed two glycopeptides with different amounts of radioactivity but at identical positions in the two antigens. Antigen E^u had an additional glycopeptide with a lower amount of radioactivity. The positions of the glycopeptides from the A^u and E^u antigens were different from those of the Aⁿ antigen. The peptide profiles of the ¹⁴C-asparagine-labeled A^u and E^u antigens demonstrated distinct differences between the molecules. The results of this study show that: (a) all the glycans on rat class I antigens are N-linked, as they are on H-2 and HLA class I antigens; (b) there are compositional differences among the glycans in each of the three antigens; (c) the glycosylation pattern of the rat class I antigens is similar to that of the mouse class I antigens, which contain two or three glycans, in contrast to that of the human class I antigens, which contain only one glycan; and (d) the antigens A^u and E^u from the same haplotype are more closely related to each other than they are to the Aⁿ antigen.     

Expression of MHC class I, MHC class II, and cancer germline antigens in neuroblastoma

Background: Neuroblastoma is the most common solid extracranial tumor in childhood, still with poor survival rates for metastatic disease. Neuroblastoma cells are of neuroectodermal origin and express a number of cancer germline (CG) antigens. These CG antigens may represent a potential target for immunotherapy such as peptide-based vaccination strategies. Objective: The purpose of this study was to analyze the presence of MAGE-A1, MAGE-A3/A6, and NY-ESO-1 on an mRNA and protein level and to determine the expression of MHC class I and MHC class II antigens within the same tumor specimens. Methods: A total of 68 tumors were available for RT-PCR, and 19/68 tumors were available for immunohistochemical (IHC) analysis of MAGE-A1, MAGE-A3/A6, and NY-ESO-1. In parallel, the same tumors were stained with a panel of antibodies for MHC class I and MHC class II molecules. Results: Screening of 68 tumor specimens by RT-PCR revealed expression of MAGE-A1 in 44%, MAGE-A3/A6 in 21%, and NY-ESO-1 in 28% of cases. Immunohistochemistry for CG antigens of selected tumors showed good agreement between protein and gene expression. However, staining revealed a heterogeneous expression of CG antigens. None of the selected tumors showed MHC class I or MHC class II expression. Conclusions: mRNA expression of MAGE-A1, MAGE-A3/A6, and NY-ESO-1 is congruent with the protein expression as determined by immunohistochemistry. The heterogeneous CG-antigen expression and the lack of MHC class I and II molecules may have implications for T-cell–mediated immunotherapy in neuroblastoma.     

High-resolution one-dimensional isoelectric focusing of mouse MHC class I antigens

A method is described for a biochemical comparison of mouse class I antigens utilizing antisera with a monomorphic pattern of reaction and high-resolution one-dimensional isoelectric focusing (1D-IEF). The most commonly occurring and studied H-2K and D alleles were identified in a comparison of over 40 mouse strains. By comparing H-2 mutant mouse strains, cell lines transfected with defined class I genes, or mice transgenic for a mouse class I gene and H-2 recombinant mouse strains, unambiguous identification of class I alleles was possible. The complex pattern presented by H-2-heterozygous mice was readily resolved into the contribution by the individual parental alleles. The H-2 ^b bm series of mutants was analyzed, and for those mutants where a charge difference was predicted based on their known sequence, a change in isoelectric point (IEP) was indeed observed. Based on analysis by IEF, the bm8 mutant may contain (an) amino acid substitution(s) in addition to those reported. The present method further appears useful in elucidating defects in class I antigen synthesis and post-translational modifications, as these cannot be easily characterized when using surface staining with monoclonal antibodies (mAbs).     

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     

Allogeneic T cell activation triggering by MHC class I antigens

The role of MHC-encoded class I molecules in allogeneic activation and proliferation of human T lymphocytes was investigated. The study was performed by using primary mixed culture of lymphocytes from MHC recombinant siblings identical for MHC class II Ag (DR, DP, DQ) and displaying MHC class I disparity. The results indicate that such allogeneic combination is sufficient to trigger early activation steps within responder T cells without promoting a significant proliferation. After MHC class I allosensitization, a significant proportion of cells entered the cell cycle (G0----G1). The stimulatory potential of MHC class I Ag was further stressed by the specific induction on responder cells of IL-2R (22% T cell activation Ag positive). Under the same experimental conditions, transferrin receptor expression and IL-2 activity were not detectable. This is consistent with the low T cell proliferation. Exogenous rIL-1 did not improve IL-2 production and the subsequent T cell proliferation indicating that these two events were not associated with a defective accessory cell function involving IL-1 release. MHC class I disparity can also prime precursor CTL to differentiate into IL-2-dependent functional MHC restricted cytotoxic T cells. Conversely IFN-gamma had no effect. Addition to the culture of W6/32, a mAb specifically directed against a monomorphic determinant on human class I HLA-A, -B, and -C Ag was able to block all these activation events. These data clearly indicate a role of HLA class I Ag involvement in the early events triggering allogeneic T cell activation.     

Membrane-bound and water-soluble nonclassical class I MHC antigens on rat placenta

We have used mouse monoclonal antibodies to different determinants on rat class I major histocompatibility complex (MHC) antgiens in order to identify water-soluble and membrane-bound nonclassical (i.e., non-RT1.A) class I MHC antigens on the spongiotrophoblast and labyrinthine trophoblast of rat placenta. Initial immunohistological studies with monoclonal antibodies reacting with determinant restricted to classical (RT1.A) rat class I antigens confirmed the presence of these antigens on spongiothrophoblast, but not on labyrinthine trphoblast. Staining with another monoclonal antibody, which reacts with both classical and at least some nonclassical rat class I antigens, gave strong staining of both the labyrinthine and spongiotrophoblast. To distinguish membrane-bound from water-soluble class I molecules, quantitative adsorption analyses were carried out using both placental cell membranes and ultracentrifuged aqueous extracts of placenta. The aqueous placental extract had no absorptive capacity for the RT1.A-specific antibodies, but it had very strong absorptive capacity for the more broadly reactive antibody. This strongly suggests the presence of large quantities of a soluble nonclassical class I MHC antigen in rat placenta. The placental cell membranes had four to fivefold greater absorptive capacity for the broadly reactive antibody when compared to the antibodies to classical class I antigens, a result that was consistent with the presence of membrane-bound non-classical class I antigens on rat placenta. The membrane-bound nonclassical class I antigen was purified from detergent extracts of DA rat placental membranes using monoclonal antibody affinity and lentil lectin affinity chromatography. The putative nonclassical class I antigen had a heavy chain of M _r 43 000, which is 2000 smaller than the amino acid sequence analysis demonstrated that the nonclassical placental antigen differed at three amino acid residues from the classical RT1.A class I molecule and also from the Q10-like class I molecule of the DA strain. It differed also from the pAR 1.5 cDNA sequence, the only full-length rat class I DNA sequence available so far. Address correspondence and offprint requests to: J. Fabre.     

Tat-mediated protein delivery can facilitate MHC class I presentation of antigens

We have previously shown that the tat protein of HIV-1 can be used as a carrier to promote the intracellular delivery of heterologous proteins. Here we have tested if the tat-delivery technology can be used to direct MHC class I presentation of native protein, using ovalbumin (OVA) as a model system. We show that a tat-ovalbumin conjugate (tatOVA) can be delivered into cells and that subsequent processing and presentation occurs, resulting in effective and specific killing of these target cells by an OVA specific cytotoxic T-lymphocyte (CTL) line. Comparison with the E.G7 line that expresses the OVA gene indicates that tat-mediated delivery is as efficient as endogenous expression in this system. Tat-mediated antigenic protein delivery may be useful both as a research technique and, potentially, as a therapeutic or prophylactic vaccine.     

Expression of CD1 and class I MHC antigens by human thymocytes

The acquisition of surface class I MHC molecules is associated with the maturation of thymocytes. Here, surface expression of class I MHC and CD1, which represents a family of MHC-related molecules, was analyzed on various human immature and mature thymocyte subpopulations. Class I expression was inversely related to the expression of CD1. The majority of CD4+ CD8+ cortical type thymocytes expressed low levels of class I MHC Ag, the previously described CD4+ CD8+ thymocyte subpopulation with low CD8 expression exhibited intermediate levels of class I MHC, whereas most of the single positive CD4 and CD8 thymocytes displayed high levels of class I MHC. Biochemical comparison of CD1 and class I showed that thymic class I molecules were post-translationally modified by phosphorylation, whereas CD1 was not phosphorylated. Furthermore, our studies suggested that in addition to CD1/CD8 complexes, thymocytes bear CD8/class I complexes. Chemical cross-linking and peptide mapping studies clearly identified the CD8-associated protein on thymic clones as the class I MHC molecule.     

Allele-specific down-regulation of MHC class I antigens in Burkitt lymphoma lines

We have reported that Burkitt lymphomas (BL) that arise in HLA-A11 positive individuals are resistant to lysis by HLA-A11-specific and HLA-A11-restricted CTLs(10,11). Here we show that this phenomenon can be explained by a selective loss of the HLA-A11 polypeptide. The HLA-A11 negative phenotype is due to a regulatory phenomenon, rather than a structural defect, as proven by the ability to rescue expression of HLA-A11 in in vitro Epstein-Barr virus (EBV)-converted sublines of EBV negative BLs.     

Hybrid Ia antigens: Genetic,serologic, and biochemical analyses

Ia specificities 22 and 23 were found to be determinants on hybrid Ia molecules, formed by the noncovalent binding of a 26,000–28,000 dalton beta polypeptide chain (A_e) coded by the I-A subregion and a 32,000–35,000 dalton alpha chain (E_α) coded by the I-E subregion. For expression of Ia. 23 the A_e chain, coded by the I-A subregion, must be derived from the H-2^d haplotype, while A^b, A^s, or A^k can provide the complementing beta chain for the expression of Ia. 22. For expression of Ia. 22 and Ia. 23, most Ia. 7 positive strains can provide the complementing alpha chain (E_α), with the one exception of B 10. PL (E^u), which is Ia. 7 positive but will not complement with A^d to express Ia. 23. Antisera were also produced against hybrid Ia antigens by immunizing with F₁ cells expressing Ia. 22 or Ia. 23 generated by transcomplementation. These antisera detect the same specificities as conventional anti-Ia. 22 and anti-Ia. 23 sera produced against cis-complementing Ia antigens. It is postulated that hybrid Ia determinants are involved in recognition and generation of immune response to antigens under dual Ir gene control. It is also suggested that there are 2 types of Ia specificities: (1) allotypic Ia specificities expressed on the alpha or beta chains (for example, Ia. 7 on the E_α chain) and (2) hybrid Ia specificities, which are unique interaction determinants formed by the association of alpha and beta chains (for example, Ia. 22 and Ia. 23). These interaction gene products may be involved in antigen recognition and presentation.     

Pseudomonas exotoxin-mediated delivery of exogenous antigens to MHC class I and class II processing pathways

Peptides associated with class II MHC molecules are normally derived from exogenous proteins, whereas class I MHC molecules normally associate with peptides from endogenous proteins. We have studied the ability of Pseudomonas exotoxin A (PE) fusion proteins to deliver exogenously added antigen for presentation by both MHC class I and class II molecules. A MHC class II-restricted antigen was fused to PE; this molecule was processed in a manner typical for class II-associated antigens. However, a MHC class I-restricted peptide fused to PE was processed by a mechanism independent of proteasomes. Furthermore, we also found that the PE fusion protein was much more stable in normal human plasma than the corresponding synthetic peptide. We believe that effective delivery of an antigen to both the MHC class I and class II pathways, in addition to the increased resistance to proteolysis in plasma, will be important for immunization.     

MHC class I antigens,immune surveillance,and tumor immune escape

Oncogenic transformation in human and experimental animals is not necessarily followed by the appearance of a tumor mass. The immune system of the host can recognize tumor antigens by the presentation of small antigenic peptides to the receptor of cytotoxic T-lymphocytes (CTLs) and reject the nascent tumor. However, cancer cells can sometimes escape these specific T-cell immune responses in the course of somatic (genetic and phenotypic) clonal evolution. Among the tumor immune escape mechanisms described to date, the alterations in the expression of major histocompatibility complex (MHC) molecules play a crucial step in tumor development due to the role of MHC antigens in antigen presentation to T-lymphocytes and the regulation of natural killer cell (NK) cell function. In this work, we have (1) updated information on the mechanisms that allow CTLs to recognize tumor antigens after antigen processing by transformed cells, (2) described the altered MHC class I phenotypes that are commonly found in human tumors, (3) summarized the molecular mechanisms responsible for MHC class I alteration in human tumors, (4) provided evidence that these altered human leukocyte antigens (HLA) class I phenotypes are detectable as result of a T-cell immunoselection of HLA class I-deficient variants by an immunecompetent host, and (5) presented data indicating the MHC class I phenotype and the immunogenicity of experimental metastatic tumors change drastically when tumors develop in immunodeficient mice.     

Delineation of three subsets of class I human T antigens (HTA) on Molt-4 cells: serologic and regulatory relationship to HLA class I antigens

Three subsets of class I human T antigens (HTA) were serologically identified on the surface of the Molt-4 T lymphoma cell line. The HTA 1 subset is defined by NAI/34, D47, or 10H3.9 cross-reactive m.Ab. and by BL6 m.Ab. The HTA 2 and HTA 3 subsets are defined by M241 and 4A7.6 m.Ab., respectively. We obtained no evidence of any additional HTA subset. The different HTA antigens share only few epitopes with human leukocyte antigens (HLA-A, -B, and -C). Interestingly, these epitopes all belong to the same cluster defined on HLA class I molecules, but differ from one HTA subset to another. These results would therefore suggest that HTA and HLA class I antigens display a limited structural homology, but have a conserved epitopic area whose detailed structure differs for each HTA subset. Furthermore, the cell surface expression of each HTA class I molecule type is differently enhanced by natural interferon (IFN)-alpha or -gamma. This result additionally supports the serologic delineation of HTA subsets, and suggests that the corresponding genes in Molt-4 cells, are subjected to distinct regulations.     

Structure and expression of two porcine genomic clones encoding class I MHC antigens

Two nonallelic porcine class I MHC (SLA) genes have been isolated and characterized. Both genes are expressed in mouse L cells, directing the synthesis of class I SLA molecules that carry common monomorphic determinants but are serologically distinct. The corresponding DNA sequences have been determined. The organization of both of these genes is similar to that of other class I genes: a leader exon, three exons encoding extracellular domains, a transmembrane exon, and three intracytoplasmic exons. The two genes are highly homologous in both exon and intron segments, with average homologies of 88% and 80%, respectively. Nucleotide changes in exon 2 are clustered, whereas those in the other exons are dispersed throughout. Comparison of the swine DNA sequences with class I genes from other species reveals a generally high conservation of exons 2, 3, 4, and 6 with lower homology in the remaining protein-encoding domains. Introns are markedly less well conserved, although moderate homology is found between swine and human class I MHC genes in both introns and 3' flanking regions. Taken together with comparisons of the deduced protein sequences, these data indicate an order of swine greater than human greater than rabbit greater than mouse in the relationship of class I genes.     

Towards a systems understanding of MHC class I and MHC class II antigen presentation

The molecular details of antigen processing and presentation by MHC class I and class II molecules have been studied extensively for almost three decades. Although the basic principles of these processes were laid out approximately 10 years ago, the recent years have revealed many details and provided new insights into their control and specificity. MHC molecules use various biochemical reactions to achieve successful presentation of antigenic fragments to the immune system. Here we present a timely evaluation of the biology of antigen presentation and a survey of issues that are considered unresolved. The continuing flow of new details into our understanding of the biology of MHC class I and class II antigen presentation builds a system involving several cell biological processes, which is discussed in this Review.     

Analysis of class I MHC antigens in the rat by monoclonal antibodies

Monoclonal antibodies (mAb) were made against class I MHC antigens of the i (mAb 42,70,39) and u (mAb 68-D) haplotypes in the rat by using specific strain combinations in order to obtain reagents for identifying the products of the RT1.An, RT1.Au, and RT1.Eu loci. These antibodies were hemagglutinating only; were IgG except for mAb 68-D3, which had a defective heavy chain; reacted identically with MHC-congenic strains and with their inbred donor strains; and precipitated class I MHC antigens. Strain distribution, sequential immunoprecipitation, and peptide mapping studies were used to define the specificities of the mAb, and the assignments were checked by comparing the specificities of the mAb with those of haplotype-specific alloantisera. The specificities were the following: mAb 42, An; mAb 68-D, Au; mAb 70, Eu; and mAb 39, an antigen encoded by a locus different from A and E. This new locus was designated RT1.F, and the allele detected by mAb 39, as Fa. The serologic data place RT1.F between RT1.A and RT1.D. The plasma membranes of DA.1I(BI) lymphocytes contain comparable amounts of An, Eu, and Fa antigens but express them on the cell surface in the order An much greater than Eu greater than Fa.     

Loading of MHC class I and II presentation pathways by exogenous antigens: a quantitative in vivo comparison

The MHC class I pathway is usually fueled by endogenous Ags, while exogenous Ags reach the MHC class II pathway. Although exogenous epitopes may also enter the MHC class I pathway, quantification of the efficiency of the process has remained a difficult task. In an attempt of such a quantification, we directly compared the amount of exogenous virus-like particles required for induction of cytotoxic T cell responses by cross-priming with the amount of virus-like particles required for induction of Th cell responses by the conventional route of MHC class II loading as an internal standard. Surprisingly, we found that cross-presentation of peptides derived from exogenous Ags on MHC class I molecules is of only marginally lower efficiency ( approximately 1- to 10-fold) than the classical MHC class II pathway in vitro and in vivo. Thus, Ag quantities required for cross-presentation and cross-priming are similar to those required for fueling the MHC class II pathway.