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The genes of major histocompatibility complex (MHC) are important to vertebrate immune system. In this study, two new MHC class I genes, designated as Egeu-UAA and Egeu-UBA, were discovered in the vulnerable Chinese egret (Egretta eulophotes). Using a full length DNA and cDNA produced by PCR and RACE methods, these two MHC class I loci were characterized in the genome of the Chinese egret and were also found to be expressed in liver and blood. Both new genes showed the expected eight exons and were similar to two copies of the minimal essential MHC complex of chicken. In genetic diversity, 14 alleles (8 for UAA and 6 for UBA) in the MHC class I gene exon 3 were found in 60 individuals using locus-specific primers and showed little polymorphism. Only three potential amino acid residues were detected under positive selection in potential peptide-binding regions (PBRs) by Bayesian analysis. These new results provide the fundamental basis for further studies to elucidate the molecular mechanisms and significance of MHC molecular adaptation in vulnerable Chinese egret and other ardeids, finding that have not been previously reported.

Major Histocompatibility Complex (MHC) class I molecules enable cytotoxic T lymphocytes to destroy virus-infected or cancerous cells, thereby preventing disease progression. MHC class I molecules provide a snapshot of the contents of a cell by binding to protein fragments arising from intracellular protein turnover and presenting these fragments at the cell surface. Competing fragments (peptides) are selected for cell-surface presentation on the basis of their ability to form a stable complex with MHC class I, by a process known as peptide optimization. A better understanding of the optimization process is important for our understanding of immunodominance, the predominance of some T lymphocyte specificities over others, which can determine the efficacy of an immune response, the danger of immune evasion, and the success of vaccination strategies. In this paper we present a dynamical systems model of peptide optimization by MHC class I. We incorporate the chaperone molecule tapasin, which has been shown to enhance peptide optimization to different extents for different MHC class I alleles. Using a combination of published and novel experimental data to parameterize the model, we arrive at a relation of peptide filtering, which quantifies peptide optimization as a function of peptide supply and peptide unbinding rates. From this relation, we find that tapasin enhances peptide unbinding to improve peptide optimization without significantly delaying the transit of MHC to the cell surface, and differences in peptide optimization across MHC class I alleles can be explained by allele-specific differences in peptide binding. Importantly, our filtering relation may be used to dynamically predict the cell surface abundance of any number of competing peptides by MHC class I alleles, providing a quantitative basis to investigate viral infection or disease at the cellular level. We exemplify this by simulating optimization of the distribution of peptides derived from Human Immunodeficiency Virus Gag-Pol polyprotein., Author Summary Major Histocompatibility Complex (MHC) class I molecules bind to protein fragments (peptides) within the cell and present these fragments at the cell surface, thus providing a snapshot of the cell contents that can subsequently be used to trigger an immune response. Only a fraction of the potentially billions of peptides inside a cell are selected for presentation, and the process is optimized to select for peptides that form a stable complex with MHC class I. The mechanisms of the optimization process are important for predicting the efficacy of an immune response and for designing effective vaccines, yet are still not well-understood. In this article we present a dynamical systems model of peptide optimization by MHC class I. We show that peptide optimization can be quantified and mechanistically explained by a peptide filtering relation, which relates cell surface abundance to peptide supply, peptide unbinding and interactions with the chaperone molecule tapasin. The filtering relation also accounts for differences in optimization across MHC class I alleles. Finally, we show how the filtering relation can be used to quantify the cell-surface presentation of virus-derived peptides for immune system surveillance.

The MHC class I (MHC-I) region in New World monkeys (Platyrrhini) has remained relatively understudied. To evaluate the diversification patterns and transcription behavior of MHC-I in Platyrrhini, we first analyzed public genomic sequences from the MHC-G-like subregion in Saimiri boliviensis, Ateles geoffroyi and Callicebus moloch, and from the MHC-B subregion in Saimiri boliviensis. While S. boliviensis showed multiple copies of both MHC-G-like (10) and –B (15) loci, A. geoffroyi and C. moloch had only three and four MHC-G-like genes, respectively, indicating that not all Platyrrhini species have expanded their MHC-I loci. We then sequenced MHC-G-like and -B cDNAs from nine Platyrrhini species, recovering two to five unique cDNAs per individual for both loci classes. In two Saguinus species, however, no MHC-B cDNAs were found. In phylogenetic trees, MHC-G-like cDNAs formed genus-specific clusters whereas the MHC-B cDNAs grouped by Platyrrhini families, suggesting a more rapid diversification of the former. Furthermore, cDNA sequencing in 12 capuchin monkeys showed that they transcribe at least four MHC-G-like and five MHC-B polymorphic genes, showing haplotypic diversity for gene copy number and signatures of positive natural selection at the peptide binding region. Finally, a quantitative index for MHC:KIR affinity was proposed and tested to predict putative interacting pairs. Altogether, our data indicate that i) MHC-I genes has expanded differentially among Platyrrhini species, ii) Callitrichinae (tamarins and marmosets) MHC-B loci have limited or tissue-specific expression, iii) MHC-G-like genes have diversified more rapidly than MHC-B genes, and iv) the MHC-I diversity is generated mainly by genetic polymorphism and gene copy number variation, likely promoted by natural selection for ligand binding.

In our body, cells are continuously exposed to physical forces that can regulate different cell functions such as cell proliferation, differentiation and death. In this work, we employed two different strategies to mechanically stress cancer cells. The cancer and healthy cell populations were treated either with mechanical stress delivered by a micropump (fabricated by deep X-ray nanolithography) or by ultrasound wave stimuli. A specific down-regulation of Major Histocompatibility Complex (MHC) class I molecules expression on cancer cell membrane compared to different kinds of healthy cells (fibroblasts, macrophages, dendritic and lymphocyte cells) was observed, stimulating the cells with forces in the range of nano-newton, and pressures between 1 and 10 bar (1 bar5100.000 Pascal), depending on the devices used. Moreover, Raman spectroscopy analysis, after mechanical treatment, in the range between 700–1800 cm-1, indicated a relative concentration variation of MHC class I. PCA analysis was also performed to distinguish control and stressed cells within different cell lines. These mechanical induced phenotypic changes increase the tumor immunogenicity, as revealed by the related increased susceptibility to Natural Killer (NK) cells cytotoxic recognition.

The human herpesvirus-7 (HHV-7) U21 gene product binds to class I major histocompatibility complex (MHC) molecules and reroutes them to a lysosomal compartment. Trafficking of integral membrane proteins to lysosomes is mediated through cytoplasmic sorting signals that recruit heterotetrameric clathrin adaptor protein (AP) complexes, which in turn mediate protein sorting in post-Golgi vesicular transport. Since U21 can mediate rerouting of class I molecules to lysosomes even when lacking its cytoplasmic tail, we hypothesize the existence of a cellular protein that contains the lysosomal sorting information required to escort class I molecules to the lysosomal compartment. If such a protein exists, we expect that it might recruit clathrin adaptor protein complexes as a means of lysosomal sorting. Here we describe experiments demonstrating that the μ adaptins from AP-1 and AP-3 are involved in U21-mediated trafficking of class I molecules to lysosomes. These experiments support the idea that a cellular protein(s) is necessary for U21-mediated lysosomal sorting of class I molecules. We also examine the impact of transient versus chronic knockdown of these adaptor protein complexes, and show that the few remaining μ subunits in the cells are eventually able to reroute class I molecules to lysosomes.

Natural Killer (NK) cells are crucial in early resistance to murine cytomegalovirus (MCMV) infection. In B6 mice, the activating Ly49H receptor recognizes the viral m157 glycoprotein on infected cells. We previously identified a mutant strain (MCMVG1F) whose variant m157 also binds the inhibitory Ly49C receptor. Here we show that simultaneous binding of m157 to the two receptors hampers Ly49H-dependent NK cell activation as Ly49C-mediated inhibition destabilizes NK cell conjugation with their targets and prevents the cytoskeleton reorganization that precedes killing. In B6 mice, as most Ly49H+ NK cells do not co-express Ly49C, the overall NK cell response remains able to control MCMVm157G1F infection. However, in B6 Ly49C transgenic mice where all NK cells express the inhibitory receptor, MCMV infection results in altered NK cell activation associated with increased viral replication. Ly49C-mediated inhibition also regulates Ly49H-independent NK cell activation. Most interestingly, MHC class I regulates Ly49C function through cis-interactions that mask the receptor and restricts m157 binding. B6 Ly49C Tg, β2m ko mice, whose Ly49C receptors are unmasked due to MHC class I deficient expression, are highly susceptible to MCMVm157G1F and are unable to control a low-dose infection. Our study provides novel insights into the mechanisms that regulate NK cell activation during viral infection., Author Summary We previously identified a viral murine cytomegalovirus (MCMV) strain whose variant m157 immunoevasin can bind the inhibitory Ly49C NK cell receptor in addition to activating Ly49H receptor in B6 mice. Here we show that simultaneous engagement of the two receptors by m157 hampers NK cell activation. Most Ly49H+ NK cells lack Ly49C in B6 mice, as a result, NK cell population efficiently controls MCMV infection; however, the anti-viral response is reduced in transgenic mice where all NK cells express Ly49C. MHC I masks Ly49C through cis-interactions, which restricts its inhibitory function. Indeed, B6 Ly49C Tg, β2m ko mice, that are deficient for MHC I, are highly susceptible to low dose of MCMV. Our findings indicate that both the NK cell repertoire and MHC I molecules control susceptibility vs resistance to viral infections.

CD8(+) T cells are responsible for killing cells of the body that have become infected or oncogenically transformed. In order to do so, effector CD8(+) T cells must recognize their cognate antigenic peptide bound to a MHC class I molecule that has been directly presented by the target cell. Due to the rapid nature of antigen presentation, it is believed that antigenic peptides are derived from a subset of newly synthesized proteins which are degraded almost immediately following synthesis and termed Defective Ribosomal Products or DRiPs. We have recently reported on a bioassay which can distinguish antigen presentation of DRiP substrates from other forms of rapidly degraded proteins and found that poly-ubiquitin chain disassembly may be necessary for efficient DRiP presentation. The AAA ATPase p97 protein is necessary for efficient cross-presentation of antigens on MHC class I molecules and plays an important role in extracting mis-folded proteins from the endoplasmic reticulum. Here, we find that genetic ablation or chemical inhibition of p97 does not diminish DRiP antigen presentation to any great extent nor does it alter the levels of MHC class I molecules on the cell surface, despite our observations that p97 inhibition increased the levels of poly-ubiquitinated proteins in the cell. These data demonstrate that inhibiting poly-ubiquitin chain disassembly alone is insufficient to abolish DRiP presentation.

The transporter associated with antigen processing (TAP) translocates the cytosol-derived proteolytic peptides to the endoplasmic reticulum lumen where they complex with nascent human leukocyte antigen (HLA) class I molecules. Non-functional TAP complexes and viral or tumoral blocking of these transporters leads to reduced HLA class I surface expression and a drastic change in the available peptide repertoire. Using mass spectrometry to analyze complex human leukocyte antigen HLA-bound peptide pools isolated from large numbers of TAP-deficient cells, we identified 334 TAP-independent ligands naturally presented by four different HLA-A, -B, and -C class I molecules with very different TAP dependency from the same cell line. The repertoire of TAP-independent peptides examined favored increased peptide lengths and a lack of strict binding motifs for all four HLA class I molecules studied. The TAP-independent peptidome arose from 182 parental proteins, the majority of which yielded one HLA ligand. In contrast, TAP-independent antigen processing of very few cellular proteins generated multiple HLA ligands. Comparison between TAP-independent peptidome and proteome of several subcellular locations suggests that the secretory vesicle-like organelles could be a relevant source of parental proteins for TAP-independent HLA ligands. Finally, a predominant endoproteolytic peptidase specificity for Arg/Lys or Leu/Phe residues in the P1 position of the scissile bond was found for the TAP-independent ligands. These data draw a new and intricate picture of TAP-independent pathways. © 2013 Lorente et al., Ministerio de Ciencia e Innovacion; FIPSE Foundation; Israel Science Foundation 916/05.

Fine mapping of human cytotoxic T lymphocyte (CTL) responses against hepatitis C virus (HCV) is based on external loading of target cells with synthetic peptides which are either derived from prediction algorithms or from overlapping peptide libraries. These strategies do not address putative host and viral mechanisms which may alter processing as well as presentation of CTL epitopes. Therefore, the aim of this proof-of-concept study was to identify naturally processed HCV-derived major histocompatibility complex (MHC) class I ligands. To this end, continuous human cell lines were engineered to inducibly express HCV proteins and to constitutively express high levels of functional HLA-A2. These cell lines were recognized in an HLA-A2-restricted manner by HCV-specific CTLs. Ligands eluted from HLA-A2 molecules isolated from large-scale cultures of these cell lines were separated by high performance liquid chromatography and further analyzed by electrospray ionization quadrupole time of flight mass spectrometry (MS)/tandem MS. These analyses allowed the identification of two HLA-A2-restricted epitopes derived from HCV nonstructural proteins (NS) 3 and 5B (NS31406–1415 and NS5B2594–2602). In conclusion, we describe a general strategy that may be useful to investigate HCV pathogenesis and may contribute to the development of preventive and therapeutic vaccines in the future.

Background MHC class I transcription is regulated by two distinct types of regulatory pathways: 1) tissue-specific pathways that establish constitutive levels of expression within a given tissue and 2) dynamically modulated pathways that increase or decrease expression within that tissue in response to hormonal or cytokine mediated stimuli. These sets of pathways target distinct upstream regulatory elements, have distinct basal transcription factor requirements, and utilize discrete sets of transcription start sites within an extended core promoter. Methodology/Principal Findings We studied regulatory elements within the MHC class I promoter by cellular transfection and in vitro transcription assays in HeLa, HeLa/CIITA, and tsBN462 of various promoter constructs. We have identified three novel MHC class I regulatory elements (GLE, DPE-L1 and DPE-L2), located downstream of the major transcription start sites, that contribute to the regulation of both constitutive and activated MHC class I expression. These elements located at the 3′ end of the core promoter preferentially regulate the multiple transcription start sites clustered at the 5′ end of the core promoter. Conclusions/Significance Three novel downstream elements (GLE, DPE-L1, DPE-L2), located between +1 and +32 bp, regulate both constitutive and activated MHC class I gene expression by selectively increasing usage of transcription start sites clustered at the 5′ end of the core promoter upstream of +1 bp. Results indicate that the downstream elements preferentially regulate TAF1-dependent, relative to TAF1-independent, transcription.

There are two main classes of natural killer (NK) cell receptors in mammals, the killer cell immunoglobulin-like receptors (KIR) and the structurally unrelated killer cell lectin-like receptors (KLR). While KIR represent the most diverse group of NK receptors in all primates studied to date, including humans, apes, and Old and New World monkeys, KLR represent the functional equivalent in rodents. Here, we report a first digression from this rule in lemurs, where the KLR (CD94/NKG2) rather than KIR constitute the most diverse group of NK cell receptors. We demonstrate that natural selection contributed to such diversification in lemurs and particularly targeted KLR residues interacting with the peptide presented by MHC class I ligands. We further show that lemurs lack a strict ortholog or functional equivalent of MHC-E, the ligands of non-polymorphic KLR in “higher” primates. Our data support the existence of a hitherto unknown system of polymorphic and diverse NK cell receptors in primates and of combinatorial diversity as a novel mechanism to increase NK cell receptor repertoire., Author Summary Most receptors of natural killer (NK) cells interact with highly polymorphic major histocompatibility complex (MHC) class I molecules and thereby regulate the activity of NK cells against infected or malignant target cells. Whereas humans, apes, and Old and New World monkeys use the family of killer cell immunoglobulin-like receptors (KIR) as highly diverse NK cell receptors, this function is performed in rodents by the diverse family of lectin-like receptors Ly49. When did this functional separation occur in evolution? We followed this by investigating lemurs, primates that are distantly related to humans. We show here that lemurs employ the CD94/NKG2 family as their highly diversified NK cell receptors. The CD94/NKG2 receptors also belong to the lectin-like receptor family, but are rather conserved in “higher” primates and rodents. We could further demonstrate that lemurs have a single Ly49 gene like other primates but lack functional KIR genes of the KIR3DL lineage and show major deviations in their MHC class I genomic organisation. Thus, lemurs have evolved a “third way” of polymorphic and diverse NK cell receptors. In addition, the multiplied lemur CD94/NKG2 receptors can be freely combined, thereby forming diverse receptors. This is, therefore, the first description of some combinatorial diversity of NK cell receptors.

Murine natural killer (NK) cells express inhibitory Ly49 receptors for MHC class I molecules, which allows for "missing self" recognition of cells that downregulate MHC class I expression. During murine NK cell development, host MHC class I molecules impose an "educating impact" on the NK cell pool. As a result, mice with different MHC class I expression display different frequency distributions of Ly49 receptor combinations on NK cells. Two models have been put forward to explain this impact. The two-step selection model proposes a stochastic Ly49 receptor expression followed by selection for NK cells expressing appropriate receptor combinations. The sequential model, on the other hand, proposes that each NK cell sequentially expresses Ly49 receptors until an interaction of sufficient magnitude with self-class I MHC is reached for the NK cell to mature. With the aim to clarify which one of these models is most likely to reflect the actual biological process, we simulated the two educational schemes by mathematical modelling, and fitted the results to Ly49 expression patterns, which were analyzed in mice expressing single MHC class I molecules. Our results favour the two-step selection model over the sequential model. Furthermore, the MHC class I environment favoured maturation of NK cells expressing one or a few self receptors, suggesting a possible step of positive selection in NK cell education. Based on the predicted Ly49 binding preferences revealed by the model, we also propose, that Ly49 receptors are more promiscuous than previously thought in their interactions with MHC class I molecules, which was supported by functional studies of NK cell subsets expressing individual Ly49 receptors.

Whilst the Major Histocompatibility Complex (MHC) is well characterized in the anuran Xenopus, this region has not previously been studied in another popular model species, the common frog (Rana temporaria). Nor, to date, have there been any studies of MHC in wild amphibian host-pathogen systems. We characterise an MHC class I locus in the common frog, and present primers to amplify both the whole region, and specifically the antigen binding region. As no more than two expressed haplotypes were found in over 400 clones from 66 individuals, it is likely that there is a single class I locus in this species. This finding is consistent with the single class I locus in Xenopus, but contrasts with the multiple loci identified in axolotls, providing evidence that the diversification of MHC class I into multiple loci likely occurred after the Caudata/Anura divergence (approximately 350 million years ago) but before the Ranidae/Pipidae divergence (approximately 230 mya). We use this locus to compare wild populations of common frogs that have been infected with a viral pathogen (Ranavirus) with those that have no history of infection. We demonstrate that certain MHC supertypes are associated with infection status (even after accounting for shared ancestry), and that the diseased populations have more similar supertype frequencies (lower F(ST)) than the uninfected. These patterns were not seen in a suite of putatively neutral microsatellite loci. We interpret this pattern at the MHC locus to indicate that the disease has imposed selection for particular haplotypes, and hence that common frogs may be adapting to the presence of Ranavirus, which currently kills tens of thousands of amphibians in the UK each year.

Epstein-Barr virus (EBV) is a human herpesvirus that persists as a largely subclinical infection in the vast majority of adults worldwide. Recent evidence indicates that an important component of the persistence strategy involves active interference with the MHC class I antigen processing pathway during the lytic replication cycle. We have now identified a novel role for the lytic cycle gene, BILF1, which encodes a glycoprotein with the properties of a constitutive signaling G-protein-coupled receptor (GPCR). BILF1 reduced the levels of MHC class I at the cell surface and inhibited CD8+ T cell recognition of endogenous target antigens. The underlying mechanism involves physical association of BILF1 with MHC class I molecules, an increased turnover from the cell surface, and enhanced degradation via lysosomal proteases. The BILF1 protein of the closely related CeHV15 γ1-herpesvirus of the Rhesus Old World primate (80% amino acid sequence identity) downregulated surface MHC class I similarly to EBV BILF1. Amongst the human herpesviruses, the GPCR encoded by the ORF74 of the KSHV γ2-herpesvirus is most closely related to EBV BILF1 (15% amino acid sequence identity) but did not affect levels of surface MHC class I. An engineered mutant of BILF1 that was unable to activate G protein signaling pathways retained the ability to downregulate MHC class I, indicating that the immune-modulating and GPCR-signaling properties are two distinct functions of BILF1. These findings extend our understanding of the normal biology of an important human pathogen. The discovery of a third EBV lytic cycle gene that cooperates to interfere with MHC class I antigen processing underscores the importance of the need for EBV to be able to evade CD8+ T cell responses during the lytic replication cycle, at a time when such a large number of potential viral targets are expressed., Author Summary Epstein-Barr virus (EBV) is a herpesvirus and an important human pathogen that can cause diseases ranging from non-malignant proliferative disease to fully malignant cancers of lymphocytes and epithelial cells. Nevertheless, the vast majority of people in all populations worldwide are infected with EBV. After primary infection the virus persists for the life of the infected individual, and usually without clinical symptoms. To understand how EBV causes disease, we also need to know how this virus is able to persist in healthy individuals with potent immune-responses. A picture is emerging which suggests that EBV, similar to non-tumorigenic herpesvirus, has multiple co-operating mechanisms to modulate immune responses by interfering with antigen processing pathway in cells undergoing lytic virus replication. We have now identified an EBV-encoded protein, BILF1, which targets MHC class I molecules for lysosomal degradation, leading to impaired recognition by immune T cells. BILF1 is a constitutively active G-protein-coupled receptor, but its effect on MHC class I degradation is independent of its signaling functions, and the molecular mechanisms are distinct from those identified in other viruses that induce degradation of MHC class I. This work aids our understanding of EBV biology and emphasizes the complexity of mechanisms evolved by viruses to enable persistent infection.

Herpes simplex virus (HSV) has a number of genes devoted to immune evasion. One such gene, ICP47, binds to the transporter associated with antigen presentation (TAP) 1/2 thereby preventing transport of viral peptides into the endoplasmic reticulum, loading of peptides onto nascent major histocompatibility complex (MHC) class I molecules, and presentation of peptides to CD8 T cells. However, ICP47 binds poorly to murine TAP1/2 and so inhibits antigen presentation by MHC class I in mice much less efficiently than in humans, limiting the utility of murine models to address the importance of MHC class I inhibition in HSV immunopathogenesis. To address this limitation, we generated recombinant HSVs that efficiently inhibit antigen presentation by murine MHC class I. These recombinant viruses prevented cytotoxic T lymphocyte killing of infected cells in vitro, replicated to higher titers in the central nervous system, and induced paralysis more frequently than control HSV. This increase in virulence was due to inhibition of antigen presentation to CD8 T cells, since these differences were not evident in MHC class I-deficient mice or in mice in which CD8 T cells were depleted. Inhibition of MHC class I by the recombinant viruses did not impair the induction of the HSV-specific CD8 T-cell response, indicating that cross-presentation is the principal mechanism by which HSV-specific CD8 T cells are induced. This inhibition in turn facilitates greater viral entry, replication, and/or survival in the central nervous system, leading to an increased incidence of paralysis., Synopsis While animal models are often instructive in understanding human diseases, many factors that influence disease differ between mouse and man. Although mice can be experimentally infected with HSV-1, this virus has evolved as a human pathogen. One facet of this evolution is HSV's mechanisms to evade the immune response, allowing the virus to persist for the lifetime of the human host. This evasion includes preventing CD8 T cells from recognizing and killing infected cells by inhibiting the expression of the molecule that presents viral peptides to CD8 T cells: major histocompatibility complex (MHC) class I. HSV is unable to inhibit mouse MHC class I, thus rendering this immune-evasion strategy inoperative in the mouse. To better understand the biology of HSV infection and the immune response to this virus in humans, the authors corrected this deficiency by inserting a gene which inhibits murine MHC class I. This recombinant virus demonstrates that MHC class I inhibition is an important determinant of disease progression. The authors found that the recombinant HSV still effectively elicits a CD8 T-cell response, but this response is ineffective in controlling the infection. This finding reveals the important distinction between the size of the immune response and the effectiveness of the response, which may be important to HSV vaccine studies.