Cowpox virus evades CTL recognition and inhibits the intracellular transport of MHC class I molecules

Orthopoxviruses evade host immune responses by using a number of strategies, including decoy chemokine receptors, regulation of apoptosis, and evasion of complement-mediated lysis. Different from other poxviral subfamilies, however, orthopoxviruses are not known to evade recognition by CTL. In fact, vaccinia virus (VV) is used as a vaccine against smallpox and a vector for eliciting strong T cell responses to foreign Ags. and both human and mouse T cells are readily stimulated by VV-infected APC in vitro. Surprisingly, however, CD8(+) T cells of mice infected with cowpox virus (CPV) or VV recognized APC infected with VV but not APC infected with CPV. Likewise, CD8(+) T cells from vaccinated human subjects could not be activated by CPV-infected targets and CPV prevented the recognition of VV-infected APC upon coinfection. Because CD8(+) T cells recognize viral peptides presented by MHC class I (MHC I), we examined surface expression, total levels, and intracellular maturation of MHC I in CPV- and VV-infected human and mouse cells. Although total levels of MHC I were unchanged, CPV reduced surface levels and inhibited the intracellular transport of MHC I early during infection. CPV did not prevent peptide loading of MHC I but completely inhibited MHC I exit from the endoplasmic reticulum. Because this inhibition was independent of viral replication, we conclude that an early gene product of CPV abrogates MHC I trafficking, thus rendering CPV-infected cells "invisible" to T cells. The absence of this immune evasion mechanism in VV likely limits virulence without compromising immunogenicity.     

Two different primate species express an identical functional MHC class I allele

 The products of the highly polymorphic and variable major histocompatibility complex (MHC) class I loci play a crucial role in host defenses against infectious disease. While similar alleles have been found in closely related species, sharing of a functional MHC class I allele between two species has never been reported. Here we show that an identical functional MHC class I molecule is present in two different primate species with an approximate divergence time of 0.7 million years. Lymphocytes from the red-crested tamarin (Saguinus geoffroyi) expressed an MHC class I allele (Sage-G ^* 01) that was identical in coding sequence to an MHC class I allele (Saoe-G ^* 08) found in the cotton-top tamarin (Saguinus oedipus). Furthermore, influenza virus-specific cytotoxic T lymphocytes (CTLs) generated in the cotton-top tamarin killed lymphocytes expressing the influenza virus nucleoprotein (NP) from the red-crested tamarin. Since the influenza virus NP epitope is bound by Saoe-G^*08 in the cotton-top tamarin, it is likely that this molecule is functional in both species. These data provide the first evidence that functional MHC class I molecules can be maintained entirely intact in two separate species. Received: 6 June 1997 / Revised: 21 July 1997     

Competition between MHC class I alleles for cell surface expression alters CTL responses to influenza A virus

Mammalian cells express up to six different MHC class I alleles, many of which differ in terms of their interaction with components of the Ag presentation pathway and level of cell surface expression. However, it is often assumed in Ag presentation studies that class I alleles function independently of each other. We have compared cell surface expression levels and function of MHC class I molecules in F(1) hybrid mice with those in the homozygous parental strains. The level of cell surface expression of certain alleles in F(1) mice differed significantly from 50% of that found on the same cell type in the corresponding parental strain, suggesting allele-specific competition for cell surface expression, and not expression solely according to gene dosage. The strongest effect was observed in H-2(b) x H-2(k) F(1) mice, in which the H-2(b) class I molecules dominated over the H-2(k) class I molecules. The magnitude of H-2(k)-restricted CTL responses to influenza A virus infection was similar in the F(1) hybrid and parental H-2(k) mice. However, in H-2(k) mice expressing a K(b) transgene, cell surface levels of the endogenous class I molecules were down-regulated to a greater degree than in F(1) hybrid mice, and H-2(k)-restricted CTL responses against influenza A virus were greatly reduced, although the CTL repertoire was apparently present. Therefore, certain MHC class I molecules compete with each other for cell surface expression, and the resulting low cell surface expression of specific alleles can lead to a severe reduction in the ability to generate a CTL response.     

Recognition of MHC class I allodeterminants regulates the generation of MHC class II-specific CTL

We have analyzed the signals influencing the generation of major histocompatibility complex (MHC) class II allospecific cytolytic T lymphocytes (CTL) and have found that the development of these CTL is actively regulated in primary in vitro cultures by Lyt-2+ T cells triggered in response to MHC class I alloantigens. Class II allospecific CTL can be readily stimulated in primary cultures, but the presence of a simultaneous class I MHC stimulus in these cultures causes a marked reduction of class II-specific CTL activation. This reduction can be prevented by adding to culture a dose of monoclonal anti-Lyt-2 antibody (in the absence of complement) that does not block the generation of class I-specific CTL. The role of MHC class I alloantigens in the regulation of class II allospecific responses illustrates that T cells recognizing class I and class II MHC antigens in mixed leukocyte cultures interact in a complex and nonreciprocal manner to influence the final effector T cell repertoire elicited by this complex immunogenic challenge.     

Evolution of a new nonclassical MHC class I locus in two Old World primate species

 HLA-G is a nonclassical major histocompatibility complex (MHC) class I molecule that is expressed only in the human placenta, suggesting that it plays an important role at the fetal-maternal interface. In rhesus monkeys, which have similar placentation to humans, the HLA-G orthologue is a pseudogene. However, rhesus monkeys express a novel placental MHC class I molecule, Mamu-AG, which has HLA-G-like characteristics. Phylogenetic analysis of AG alleles in two Old World primate species, the baboon and the rhesus macaque, revealed limited diversity characteristic of a nonclassical MHC class I locus. Gene trees constructed using classical and nonclassical primate MHC class I alleles demonstrated that the AG locus was most closely related to the classical A locus. Interestingly, gene tree analyses suggested that the AG alleles were most closely related to a subset of A alleles which are the products of an ancestral interlocus recombination event between the A and B loci. Calculation of the rates of synonymous and nonsynonymous substitution at the AG locus revealed that positive selection was not acting on the codons encoding the peptide binding region. In exon 4, however, the rate of nonsynonymous substitution was significantly lower than the rate of synonymous substitution, suggesting that negative selection was acting on these codons. Received: 22 April 1998 / Revised: 15 July 1998     

MHC class I A region diversity and polymorphism in macaque species

The HLA-A locus represents a single copy gene that displays abundant allelic polymorphism in the human population, whereas, in contrast, a nonhuman primate species such as the rhesus macaque (Macaca mulatta) possesses multiple HLA-A-like (Mamu-A) genes, which parade varying degrees of polymorphism. The number and combination of transcribed Mamu-A genes present per chromosome display diversity in a population of Indian animals. At present, it is not clearly understood whether these different A region configurations are evolutionarily stable entities. To shed light on this issue, rhesus macaques from a Chinese population and a panel of cynomolgus monkeys (Macaca fascicularis) were screened for various A region-linked variations. Comparisons demonstrated that most A region configurations are old entities predating macaque speciation, whereas most allelic variation (>95%) is of more recent origin. The latter situation contrasts the observations of the major histocompatibility complex class II genes in rhesus and cynomolgus macaques, which share a high number of identical alleles (>30%) as defined by exon 2 sequencing.     

Characterization of different B-F (MHC class I) molecules in the chicken

B-F alloantisera recognized distinct 45-Kd molecules on peripheral red blood cells (RBC) from embryonic chickens and heterogeneous molecules of approximately 40 to 44 Kd on peripheral RBC from adult chickens, provisionally referred to as type 1 and type 2, respectively. Type 2 molecules migrated to the basic end of isoelectric focusing gels, exhibited multiple isomorphic variants, and were associated with a smaller polypeptide of approximately 11 to 12 Kd assumed to be beta-2-microglobulin. Type 1 molecules migrated to the acidic end of isoelectric focusing gels, exhibited limited heterogeneity, and were not associated with a smaller polypeptide. Type 1 and type 2 molecules were also shown to be distinct by peptide mapping and serological analyses. In addition, two distinct molecular-weight forms of the type 2 molecules were distinguished, provisionally referred to as 2A (45 Kd) and 2B (42 Kd). In vivo-derived avian erythroblastosis virus (AEV)-transformed erythroleukemia cells expressed type 2A molecules. In vitro-derived AEV-transformed erythroleukemia cells expressed very low levels of B-F molecules; however, they expressed type 2B molecules when induced to differentiate. Normal bursa-derived lymphoid cells expressed type 2A molecules, whereas normal thymus-derived lymphoid cells expressed type 2B molecules. Cloned reticuloendotheliosis virus (REV)-transformed immature lymphoid cells expressed either type 2A or type 2B molecules.     

Genetic diversity at class II DRB loci of the primate MHC

The evolution of polymorphism at loci encoding the beta-chains of the MHC class II DR Ag was studied in primates by DNA amplification (polymerase chain reaction). Phylogenetic analysis of 63 DRB sequences from the polymorphic second exon (first domain) of nonhuman primates and 53 human sequences indicates the presence of five DRB loci in primates, derived from a DRB1-like ancestral locus over 20 million yr ago. Many of the allelic types at the DRB1 locus predate the divergence of hominoids (5 million yr ago) and some (DR4, DR3, 5, 6) predate the divergence of Old world monkeys and hominoids (20 million yr ago). The DRB3 locus appears to have arisen before the divergence of hominoids on an ancestral DRB1 lineage. The DRB2 and DRB5 loci were generated more than 20 million yr ago and the DRB4 locus more than 5 million yr ago. The DRB2 locus, a pseudogene in humans, is polymorphic in the nonhuman primates.     

MHC class I antigen processing of an adenovirus CTL epitope is linked to the levels of immunoproteasomes in infected cells

Proteasomes are the major source for the generation of peptides bound by MHC class I molecules. To study the functional relevance of the IFN-gamma-inducible proteasome subunits low molecular mass protein 2 (LMP2), LMP7, and mouse embryonal cell (MEC) ligand 1 in Ag processing and concomitantly that of immunoproteasomes, we established the tetracycline-regulated mouse cell line MEC217, allowing the titrable formation of immunoproteasomes. Infection of MEC217 cells with Adenovirus type 5 (Ad5) and analysis of Ag presentation with Ad5-specific CTL showed that cells containing immunoproteasomes processed the viral early 1B protein (E1B)-derived epitope E1B192-200 with increased efficiency, thus allowing a faster detection of viral entry in induced cells. Importantly, optimal CTL activation was already achieved at submaximal immunosubunit expression. In contrast, digestion of E1B-polypeptide with purified proteasomes in vitro yielded E1B192-200 at quantities that were proportional to the relative contents of immunosubunits. Our data provide evidence that the IFN-gamma-inducible proteasome subunits, when present at relatively low levels as at initial stages of infection, already increase the efficiency of antigenic peptide generation and thereby enhance MHC class I Ag processing in infected cells.     

Assembly of MHC class I molecules analyzed in vitro

Recent evidence suggests that peptide ligands take part in the assembly of class I molecules in living cells. We now describe a simple system for studying class I assembly in vitro. Detergent extracts of the mutant cells RMA-S and .174, in which class I assembly does not occur spontaneously, will support assembly in vitro when specific peptides are added. Peptides stabilize a conformational change in the class I heavy chain and association with beta 2-microglobulin, at concentrations approximately 100-fold lower than required in "peptide feeding" experiments with whole cells. We show that peptides bind class I molecules during assembly and demonstrate that the conformational change induced in the heavy chain is influenced by the concentrations of both peptide and beta 2-microglobulin.     

Mutations in a dominant Nef epitope of simian immunodeficiency virus diminish TCR:epitope peptide affinity but not epitope peptide:MHC class I binding

Viruses like HIV and SIV escape from containment by CD8(+) T lymphocytes through generating mutations that interfere with epitope peptide:MHC class I binding. However, mutations in some viral epitopes are selected for that have no impact on this binding. We explored the mechanism underlying the evolution of such epitopes by studying CD8(+) T lymphocyte recognition of a dominant Nef epitope of SIVmac251 in infected Mamu-A*02(+) rhesus monkeys. Clonal analysis of the p199RY-specific CD8(+) T lymphocyte repertoire in these monkeys indicated that identical T cell clones were capable of recognizing wild-type (WT) and mutant epitope sequences. However, we found that the functional avidity of these CD8(+) T lymphocytes for the mutant peptide:Mamu-A*02 complex was diminished. Using surface plasmon resonance to measure the binding affinity of the p199RY-specific TCR repertoire for WT and mutant p199RY peptide:Mamu-A*02 monomeric complexes, we found that the mutant p199RY peptide:Mamu-A*02 complexes had a lower affinity for TCRs purified from CD8(+) T lymphocytes than did the WT p199RY peptide:Mamu-A*02 complexes. These studies demonstrated that differences in TCR affinity for peptide:MHC class I ligands can alter functional p199RY-specific CD8(+) T lymphocyte responses to mutated epitopes, decreasing the capacity of these cells to contain SIVmac251 replication.     

Impact of MHC class I diversity on immune control of immunodeficiency virus replication

The recent failure of the T-cell-based HIV vaccine trial led by Merck & Co., Inc. prompts the urgent need to refocus on the question of which T-cell responses are required to control HIV replication. The well-described association between the expression of particular MHC class I molecules and successful containment of HIV or, in the macaque model, SIV replication provide a valuable starting point from which to evaluate more precisely what might constitute effective CD8(+) T-cell responses. Here, we review recent studies of T-cell-mediated control of HIV and SIV infection, and offer insight for the design of a successful T-cell-based HIV vaccine in the future.     

Presentation by recycling MHC class II molecules of an influenza hemagglutinin-derived epitope that is revealed in the early endosome by acidification

We investigated the roles of nascent and recycling MHC class II molecules (MHC II) in the presentation of two well-defined I-E(d)-restricted epitopes that are within distinct regions of the influenza virus hemagglutinin (HA) protein. The site 3 epitope (S3; residues 302-313) lies in the stalk region that unfolds in response to mild acidification, while the site 1 epitope (S1; residues 107-119) is situated in the stable globular domain. In a murine B lymphoma cell line and an I-E(d)-transfected fibroblast cell line, presentation from inactivated virus of S3 is inhibited by primaquine, a compound that prevents recycling of cell surface proteins, including MHC II, while S1 presentation is unaffected. In contrast, brefeldin A, an agent that inhibits exit of proteins from the endoplasmic reticulum, selectively inhibited S1 presentation without affecting S3 presentation, suggesting that S1 presentation requires nascent MHC II. The use of agents that perturb endosomal function revealed a requirement for acidification of internalized viral particles for presentation of both epitopes. Notably, all compounds tested had similar effects on presentation of the two epitopes derived from endogenously synthesized HA. Thus, recycling I-E(d) molecules appear to be crucial for capturing and presenting an epitope that is revealed in mild acidic conditions following the uptake of virions or the synthesis of Ag, while nascent I-E(d) molecules are required for presentation of a second epitope located in a structurally constrained region of the same polypeptide. Viral glycoproteins, such as HA, may have been a major impetus for the evolutionary establishment of this recycling pathway.     

Antigen loading of MHC class I molecules in the endocytic tract

Major histocompatibility complex (MHC) class I molecules bind antigenic peptides that are translocated from the cytosol into the endoplasmic reticulum by the transporter associated with antigen processing. MHC class I loading independent of this transporter also exists and involves peptides derived from exogenously acquired antigens. Thus far, a detailed characterization of the intracellular compartments involved in this pathway is lacking. In the present study, we have used the model system in which peptides derived from measles virus protein F are presented to cytotoxic T cells by B-lymphoblastoid cells that lack the peptide transporter. Inhibition of T cell activation by the lysosomotropic drug ammoniumchloride indicated that endocytic compartments were involved in the class I presentation of this antigen. Using immunoelectron microscopy, we demonstrate that class I molecules and virus protein F co-localized in multivesicular endosomes and lysosomes. Surprisingly, these compartments expressed high levels of class II molecules, and further characterization identified them as MHC class II compartments. In addition, we show that class I molecules co-localized with class II molecules on purified exosomes, the internal vesicles of multivesicular endosomes that are secreted upon fusion of these endosomes with the plasma membrane. Finally, dendritic cells, crucial for the induction of primary immune responses, also displayed class I in endosomes and on exosomes.     

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.     

Potent cytolytic response by a CD8+ CTL clone to multiple peptides from the same protein in association with an allogeneic class I MHC molecule

CTL clone 2C recognizes the allogeneic class I MHC molecule L(d) in association with peptides derived from alpha-ketoglutarate dehydrogenase (oxoglutarate dehydrogenase (OGDH)), a ubiquitous intracellular protein. One of these peptides, QLSPFPFDL (QL9), elicits more vigorous cytolytic responses than two previously identified naturally processed peptides with overlapping sequences, LSPFPFDL (p2Ca) and VAITRIEQLSPFPFDL (p2Cb), from OGDH. In this study, we show that QL9 forms a more stable complex with cell surface L(d) than does p2Ca or p2Cb and is processed from the longer, naturally occurring peptide p2Cb by 20S proteosomes in vitro. The N-terminal cyclized pyroglutaminyl QL9 (pyroQL9), a form of QL9 to which it is converted at the low pH used for peptide isolation from tissue extracts, is even more active than QL9 in cytotoxicity assays with 2C CTL. Overall, the results indicate that along with the abundant natural peptides p2Ca and p2Cb, the QL9 and other OGDH peptides of various lengths, sharing a conserved C-terminal sequence, are also processed and presented with L(d) as allogeneic ligands for T cells expressing 2C TCR. All these peptides, each available in a low amount, could act in concert at the cell surface, resulting in a high density of cognate ligands that accounts for the exceptionally potent cytolytic response by 2C CTL.     

CLIP-region mediated interaction of Invariant chain with MHC class I molecules

An association between the MHC class II chaperone molecule Invariant chain (Ii) and MHC class I molecules is known to occur, but the basis of the interaction is undetermined. Evidence is presented here that the CLIP region of Ii is involved in this interaction. A peptide encoding residues 91-99 of CLIP (MRMATPLLM) stabilised multiple MHC class I alleles, with the methionine residue at position 99 having a crucial role in binding to H2-K(b), whereas methionine at position 91 also appeared important in binding to RT1-A(a). Ii can also be detected in the class I MHC peptide loading complex. These data provide an explanation for the association of Ii and MHC class I molecules.     

HIV envelope protein inhibits MHC class I presentation of a cytomegalovirus protective epitope

CTL recognize peptides that derive from viral protein Ags by proteolytic processing and are presented by MHC class I molecules. In this study we tested whether coexpression of viral Ags in the same cell leads to competition between them. To this end, two L(d)-restricted epitopes derived from HIV-1 envelope gp160 (ENV) and from CMV pp89 phosphoprotein were coexpressed. HIV ENV strain IIIB, but not MN variant, impaired recognition by specific CTL of CMV pp89 epitope 9pp89. Susceptibility to inhibition after ENV coexpression was inversely related to the amount of antigenic 9pp89 peptide processed from different antigenic constructs. In line with it, competition decreased the yield of naturally processed antigenic 9pp89 peptide bound to MHC class I molecules in coinfected cells. Also, point mutants of the presenting MHC class I molecule differed in their competition pattern. Collectively, the data imply that competition operates at the step of MHC-peptide complex assembly or stabilization. We conclude that, although not the rule, in certain combinations there is interference between different Ags expressed in the same cell and presented by the same MHC class I allele. These studies have implications for vaccine development and for understanding immunodominance.     

The role of class I MHC products in polyclonal activation of CTL function

The generation of CTL function by agents such as lectins, oxidative chemicals, or serum components has been regarded as independent of class I MHC products, because the CTL thus generated are able to lyse virtually any allogeneic target. However, we show here that CTL activation by lectin requires interaction of the pre-CTL with a class I MHC product on the lectin-presenting cell. The lectin-presenting cell can be an irradiated syngeneic or allogeneic spleen cell or, alternatively, lectin can be seen on a neighboring pre-CTL. In either case, there is an absolute requirement for simultaneous (although probably unlinked) perception of a class I MHC product. These results suggest that restricted clonal CTL activation by antigen, and polyclonal activation by so-called "nonspecific" mitogenic agents, may proceed through similar if not identical pathways.