Amyloid precursor-like protein 2 association with HLA class I molecules

Amyloid precursor-like protein 2 (APLP2) is a ubiquitously expressed protein. The previously demonstrated functions for APLP2 include binding to the mouse major histocompatibility complex (MHC) class I molecule H-2K^d and down regulating its cell surface expression. In this study, we have investigated the interaction of APLP2 with the human leukocyte antigen (HLA) class I molecule in human tumor cell lines. APLP2 was readily detected in pancreatic, breast, and prostate tumor lines, although it was found only in very low amounts in lymphoma cell lines. In a pancreatic tumor cell line, HLA class I was extensively co-localized with APLP2 in vesicular compartments following endocytosis of HLA class I molecules. In pancreatic, breast, and prostate tumor lines, APLP2 was bound to the HLA class I molecule. APLP2 was found to bind to HLA-A24, and more strongly to HLA-A2. Increased expression of APLP2 resulted in reduced surface expression of HLA-A2 and HLA-A24. Overall, these studies demonstrate that APLP2 binds to the HLA class I molecule, co-localizes with it in intracellular vesicles, and reduces the level of HLA class I molecule cell surface expression.     

Detailed characterization of the peptide binding specificity of five common Patr class I MHC molecules

The chimpanzee (Pan troglodytes) is an important model for studying the immune response to several human pathogens, but the study of correlates of immunity has been hindered by the fact that little is known about the epitope-binding specificity of chimpanzee (Patr) class I MHC. In the present study we have characterized the peptide binding specificity of several common Patr class I molecules. Using single amino acid substitution analogs and large peptide libraries, quantitative peptide binding motifs have been derived for Patr A*0101, A*0701, A*0901, B*0101, and B*2401. Each molecule was found to bind peptides using position 2 and the C terminus as main anchor contacts. On the other hand, each Patr molecule is associated with a unique binding specificity, and the range of specificities is similar to that seen amongst HLA alleles. A high degree of cross-reactivity was noted between Patr A*0701 and Patr A*0901, suggesting the existence of a Patr-specific supertype. Consistent with previous studies suggesting that some cross-reactivity may exist between HLA and Patr alleles, Patr A*0901 was found to have an appreciable degree of cross-reactivity with molecules of the HLA A24-supertype. Finally, utilizing motif scans and peptide binding and intracellular cytokine staining assays, 77 hepatitis B virus (HBV)-derived epitopes were identified in five chimpanzees that were recently convalescent from acute HBV infection. Because the Patr alleles studied herein were found to be very common in two different chimpanzee populations, the present data should facilitate the use of chimpanzees for immunological studies.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Influence of the tapasin C terminus on the assembly of MHC class I allotypes

Several endoplasmic reticulum proteins, including tapasin, play an important role in major histocompatibility complex (MHC) class I assembly. In this study, we assessed the influence of the tapasin cytoplasmic tail on three mouse MHC class I allotypes (H2-K^b, -K^d, and -L^d) and demonstrated that the expression of truncated mouse tapasin in mouse cells resulted in very low K^b, K^d, and L^d surface expression. The surface expression of K^d also could not be rescued by human soluble tapasin, suggesting that the surface expression phenotype of the mouse MHC class I molecules in the presence of soluble tapasin was not due to mouse/human differences in tapasin. Notably, soluble mouse tapasin was able to partially rescue HLA-B8 surface expression on human 721.220 cells. Thus, the cytoplasmic tail of tapasin (either mouse or human) has a stronger impact on the surface expression of murine MHC class I molecules on mouse cells than on the expression of HLA-B8 on human cells. A K408W mutation in the mouse tapasin transmembrane/cytoplasmic domain disrupted K^d folding and release from tapasin, but not interaction with transporter associated with antigen processing (TAP), indicating that the mechanism whereby the tapasin transmembrane/cytoplasmic domain facilitates MHC class I assembly is not limited to TAP stabilization. Our findings indicate that the C terminus of mouse tapasin plays a vital role in enabling murine MHC class I molecules to be expressed at the surface of mouse cells.     

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.     

Technologies for MHC class I immunoproteomics

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

Activated mouse T-cells synthesize MHC class II, process, and present morbillivirus nucleocapsid protein to primed T-cells

A pivotal step in the initiation of T-cell immunity is the presentation of antigenic peptides by major histocompatibility complex (MHC) expressed on antigen presenting cells. The expression of MHC class II molecules by mouse T-cells has not been shown unequivocally. In the present work, we demonstrate that activated mouse T-cells synthesize MHC class II molecules de novo and express them on their surface. Further, we have demonstrated that in vitro activated T-cells take up extra-cellular soluble nucleocapsid protein of a morbillivirus. The internalized antigen goes to antigen processing compartment as shown by co-localization of antigen and LAMP-1 using confocal microscopy. We show that activated T-cells express H2M, a chaperone molecule known to have a role in antigen presentation. Further, we demonstrate that activated T-cells process and present internalized extra-cellular antigen to primed T-cells as shown by IL-2 secretion and in vitro proliferation. The presentation of antigen by T-cells may have implications in immuno-regulation, control of infection by lymphotropic viruses and maintenance of immunological memory.     

Use of selected reaction monitoring mass spectrometry for the detection of specific MHC class I peptide antigens on A3 supertype family members

The development of peptide-based vaccines that are useful in the therapeutic treatment of melanoma and other cancers ultimately requires the identification of a sufficient number of antigenic peptides so that most individuals, regardless of their major histocompatibility complex (MHC)–encoded class I molecule phenotype, can develop a cytotoxic T lymphocyte (CTL) response against one or more peptide components of the vaccine. While it is relatively easy to identify antigenic peptides that are presented by the most prevalent MHC class I molecules in the population, it is problematic to identify antigenic peptides that are presented by MHC class I molecules that have less frequent expression in the population. One manner in which this problem can be overcome is by taking advantage of known MHC class I supertypes, which are groupings of MHC class I molecules that bind peptides sharing a common motif. We have developed a mass spectrometric approach which can be used to determine if an antigenic peptide is naturally processed and presented by any given MHC class I molecule. This approach has been applied to the A3 supertype, and the results demonstrate that some, but not all, A3 supertype family–associated peptides can associate with all A3 supertype family members. The approach also demonstrates the shared nature of several newly identified peptide antigens. The use of this technology negates the need to test peptides for their ability to stimulate CTL responses in those cases where the peptide is not naturally processed and bound to the target MHC class I molecule of interest, thus allowing resources to be focused on the most promising vaccine candidates.     

Unprecedented intraspecific diversity of the MHC class I region of a teleost medaka, Oryzias latipes

The major histocompatibility complex (MHC) is present at a single chromosomal locus of all jawed vertebrate analyzed so far, from sharks to mammals, except for teleosts whose orthologs of the mammalian MHC-encoded genes are dispersed at several chromosomal loci. Even in teleosts, several class IA genes and those genes directly involved in class I antigen presentation preserve their linkage, defining the teleost MHC class I region. We determined the complete nucleotide sequence of the MHC class I region of the inbred HNI strain of medaka, Oryzias latipes (northern Japan population-derived), from four overlapping bacterial artificial chromosome (BAC) clones spanning 540,982 bp, and compared it with the published sequence of the corresponding region of the inbred Hd-rR strain of medaka (425,935 bp, southern Japan population-derived) as the first extensive study of intraspecies polymorphisms of the ectotherm MHC regions. A segment of about 100 kb in the middle of the compared sequences encompassing two class Ia genes and two immunoproteasome subunit genes, PSMB8 and PSMB10, was so divergent between these two inbred strains that a reliable sequence alignment could not be made. The rest of the compared region (about 320 kb) showed a fair correspondence, and an approximately 96% nucleotide identity was observed upon gap-free segmental alignment. These results indicate that the medaka MHC class I region contains an ∼100-kb polymorphic core, which is most probably evolving adaptively by accumulation of point mutations and extensive genetic rearrangements such as insertions, deletions and duplications. The nucleotide sequence data of HNI MHC class I region reported in this paper have been submitted to the DDBJ/EMBL/GenBank and were assigned the accession number AB183488.     

The absence of invariant chain in MHC II cancer vaccines enhances the activation of tumor-reactive type 1 CD4<Superscript>+</Superscript> T lymphocytes

Activation of tumor-reactive T lymphocytes is a promising approach for the prevention and treatment of patients with metastatic cancers. Strategies that activate CD8⁺ T cells are particularly promising because of the cytotoxicity and specificity of CD8⁺ T cells for tumor cells. Optimal CD8⁺ T cell activity requires the co-activation of CD4⁺ T cells, which are critical for immune memory and protection against latent metastatic disease. Therefore, we are developing “MHC II” vaccines that activate tumor-reactive CD4⁺ T cells. MHC II vaccines are MHC class I⁺ tumor cells that are transduced with costimulatory molecules and MHC II alleles syngeneic to the prospective recipient. Because the vaccine cells do not express the MHC II-associated invariant chain (Ii), we hypothesized that they will present endogenously synthesized tumor peptides that are not presented by professional Ii⁺ antigen presenting cells (APC) and will therefore overcome tolerance to activate CD4⁺ T cells. We now report that MHC II vaccines prepared from human MCF10 mammary carcinoma cells are more efficient than Ii⁺ APC for priming and boosting Type 1 CD4⁺ T cells. MHC II vaccines consistently induce greater expansion of CD4⁺ T cells which secrete more IFNγ and they activate an overlapping, but distinct repertoire of CD4⁺ T cells as measured by T cell receptor Vβ usage, compared to Ii⁺ APC. Therefore, the absence of Ii facilitates a robust CD4⁺ T cell response that includes the presentation of peptides that are presented by traditional APC, as well as peptides that are uniquely presented by the Ii⁻ vaccine cells.     

Characterization of the peptide-binding specificity of the chimpanzee class I alleles A*0301 and A*0401 using a combinatorial peptide library

Chimpanzees represent important models for studying several human pathogens. In the present study, we utilized a combinatorial peptide library to characterize the binding specificities of the chimpanzee class I molecules Patr A 0301 and A 0401, both of which are present in about 17% of chimpanzees. Patr A 0301 was found to recognize peptides using the canonical position 2/C-terminus spacing, with the small residues S, T, and A being the most preferred in position 2, and the positively charged residues R and K preferred at the C terminus. Patr A 0401 was found to recognize a more complex motif where the C terminus and then the residue in positions 1 and/or 5 are the primary anchors. Like A 0301, the C-terminal preference of A 0401 is for positively charged residues. At positions 1 and 5, positively charged and large residues are the most preferred, respectively. Coefficient values derived from the combinatorial library proved to be an efficient means for predicting A 0301 and A 0401 binders. The present data provide detailed information to facilitate the identification of potential T cell epitopes recognized in the context of two common chimpanzee class I alleles, and further validate the combinatorial library approach as an efficient method to characterize class I binding specificities.     

35-37 kDa形式可溶性MHC I释放机制研究

目的:探讨35-37 k Da形式的可溶性MHC I释放机制,为开展造血系统恶性肿瘤免疫干预治疗研究奠定理论基础。方法:以细胞表面标记、免疫沉淀、免疫印迹和增强化学发光法探讨EGTA和蔗糖对THP1细胞释放43和35-37 k Da可溶性MHC I的影响;以超速离心法纯化外排小体,并用免疫沉淀、免疫印迹和增强化学发光法检测43和35-37 k Da可溶性MHC I;用Quantity One软件对43和35-37 k Da可溶性MHC I进行相对定量分析。结果:EGTA同时显著抑制43和35-37 k Da可溶性MHC I产生;蔗糖同时显著促进43和35-37 k Da可溶性MHC I产生;43 k Da可溶性MHC I存在于外排小体上,而35-37 k Da可溶性MHC I在外排小体上检测不到。结论:35-37 k Da可溶性MHC I与外排小体都来源于细胞内多泡小体同质膜的溶合后释放,但35-37 k Da可溶性MHC I并不包含在外排小体的泡囊中,而是独立于外排小体释放。     

Human cytomegalovirus immediate early glycoprotein US3 retains MHC class I molecules by transient association

Human cytomegalovirus (HCMV) interferes with major histocompatibility complex (MHC) class I antigen presentation by a sequential multistep process to escape T cell surveillance. During the immediate early phase of infection, the glycoprotein US3 prevents intracellular transport of MHC class I molecules. Interestingly, US3 displays a significantly shorter half-life than US3-retained MHC class I molecules. Here we show that US3 associates only transiently with MHC class I molecules, exits the ER, and is inefficiently retrieved from the Golgi. US3 was degraded in a post-Golgi compartment, most likely lysosomes, because: i) Brefeldin A treatment prolonged the half-life of US3; and ii) US3 co-localized with the lysosomal marker protein LAMP in chloroquine-treated cells. In contrast, MHC class I molecules remained stable in the ER. Upon inhibition of protein synthesis MHC class I molecules were released suggesting that a continuous supply of newly synthesized US3 molecules is required for inhibition of transport. Thus, US3 does not seem to retain MHC class I molecules by a retrieval mechanism. Instead, our observations are consistent with US3 preventing MHC class I trafficking by blocking forward transport.     

Down-regulation of MHC class I expression in human neuronal stem cells using viral stealth mechanism

Due to their unique capacity for self-renewal in addition to their ability to differentiate into cells of all neuronal lineages, neuronal stem cells (NSCs) are promising candidates for cell replacement therapy in neuronal injury and neurodegenerative diseases. However, there are few studies on immune rejection, which is one of the main problems facing successful stem cell therapy. In order to determine if human NSC might be rejected after transplantation the MHC expression level was examined in the HB1.F3 cell line, which has previously been shown to exhibit NSC properties. The results showed low expression levels of the MHC class I molecules on the surfaces of these cells. A dramatic increase in the MHC class I expression level was observed when the cells were treated with IFN-gamma, TNF-alpha, and IL-1beta, alone or in combination. The maximum induction of MHC class I protein expression was observed at above 20ng/ml IFN-gamma 48h after the treatment. The apparent additive effects of TNF-alpha and IL-1beta in combination on the maximum induction of MHC class I expression exerted by IFN-gamma treatment were not observed. The MHC class I levels elevated by IFN-gamma were sustained for 72h after withdrawing the IFN-gamma. Therefore, this study introduced human cytomegalovirus (hCMV) US genes, which are known to be able to reduce the MHC class I expression level on the cell surface after infection, into HB1.F3 cells. The cells transfected with the hCMV US2, US3, US6 or US11 genes showed 20-50% reduction in the MHC class I expression level compared with the mock-transfected cells. These results suggest that NSC expresses high levels of the MHC class I proteins, and unless they are modified, might be rejected upon transplantation. In addition, the various viral stealth mechanisms can be exploited for stem cell transplantation.     

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.     

Cathepsin S controls the trafficking and maturation of MHC class II molecules in dendritic cells

Before a class II molecule can be loaded with antigenic material and reach the surface to engage CD4+ T cells, its chaperone, the class II-associated invariant chain (Ii), is degraded in a stepwise fashion by proteases in endocytic compartments. We have dissected the role of cathepsin S (CatS) in the trafficking and maturation of class II molecules by combining the use of dendritic cells (DC) from CatS(-/-) mice with a new active site-directed probe for direct visualization of active CatS. Our data demonstrate that CatS is active along the entire endocytic route, and that cleavage of the lysosomal sorting signal of Ii by CatS can occur there in mature DC. Genetic disruption of CatS dramatically reduces the flow of class II molecules to the cell surface. In CatS(-/-) DC, the bulk of major histocompatibility complex (MHC) class II molecules is retained in late endocytic compartments, although paradoxically, surface expression of class II is largely unaffected. The greatly diminished but continuous flow of class II molecules to the cell surface, in conjunction with their long half-life, can account for the latter observation. We conclude that in DC, CatS is a major determinant in the regulation of intracellular trafficking of MHC class II molecules.     

Potential roles of protein oxidation and the immunoproteasome in MHC class I antigen presentation: the 'PrOxI' hypothesis

The major histocompatibility complex (MHC) class I (MHC-I) antigen presentation system is responsible for the cell-surface presentation of self-proteins and intracellular viral proteins. This pathway is important in screening between self, and non-self or infected cells. In this pathway, proteins are partially degraded to peptides in the cytosol and targeted to the cell surface bound to an MHC-I receptor protein. At the cell surface, T cells bypass cells displaying self-peptides but destroy others displaying foreign antigens. Cells contain several isoforms of the proteasome, but it is thought that the immunoproteasome is the major form involved in generating peptides for the MHC-I pathway. How all intracellular proteins are targeted for MHC-I processing is unclear. Oxidative stress is experienced by all cells, and all proteins are exposed to oxidation. We propose that oxidative modification makes proteins susceptible to degradation by the immunoproteasome. This could be called the protein oxidation and immunoproteasome or 'PrOxI' hypothesis of MHC-I antigen processing. Protein oxidation may, thus, be a universal mechanism for peptide generation and presentation in the MHC-I pathway.     

Calreticulin‐dependent recycling in the early secretory pathway mediates optimal peptide loading of MHC class I molecules

Calreticulin is a lectin chaperone of the endoplasmic reticulum (ER). In calreticulin‐deficient cells, major histocompatibility complex (MHC) class I molecules travel to the cell surface in association with a sub‐optimal peptide load. Here, we show that calreticulin exits the ER to accumulate in the ER–Golgi intermediate compartment (ERGIC) and the cis‐Golgi, together with sub‐optimally loaded class I molecules. Calreticulin that lacks its C‐terminal KDEL retrieval sequence assembles with the peptide‐loading complex but neither retrieves sub‐optimally loaded class I molecules from the cis‐Golgi to the ER, nor supports optimal peptide loading. Our study, to the best of our knowledge, demonstrates for the first time a functional role of intracellular transport in the optimal loading of MHC class I molecules with antigenic peptide.     

HER-2/neu mediated down-regulation of MHC class I antigen processing prevents CTL-mediated tumor recognition upon DNA vaccination in HLA-A2 transgenic mice

To study DNA vaccination directed against human HER-2 in the HHD mouse Tg strain, we created a novel HER-2-expressing syngeneic tumor transplantation model. We found that a DNA vaccine encoding the full length HER-2 DNA protected HHD mice from HER-2⁺ tumor challenge by a CTL independent mechanism. A more efficient approach to induce HLA-A2 restricted CTLs, through immunization with a multi-epitope DNA vaccine expressing the HLA-A2 restricted HER-2 369–377, 435–443 and 689–697 epitopes, resulted in high numbers of peptide specific T cells but failed to induce tumor protection. Subsequently we discovered that HER-2 transfected tumor cells down-regulated MHC class I antigen expression and exhibited a series of defects in the antigen processing pathway which impaired the capacity to produce and display MHC class I peptide-ligands to specific CTLs. Our data demonstrate that HER-2 transfection is associated with defects in the MHC class I presentation pathway, which may be the underlying mechanism behind the inability of CTLs to recognize tumors in this HLA-A2 transgenic model. As defective MHC class I presentation may be a common characteristic of HER-2 expressing tumors, vaccines targeting HER-2 should aim at inducing an integrated immune response where also CD4⁺ T cells and antibodies are important components. S. Vertuani and C. Triulzi contributed equally to this work.     

Generation of MHC class I peptide antigens by protein processing in the secretory route by furin

Cytosolic degradation of endogenously synthesized proteins by the proteasome and translocation of processed peptides to the endoplasmic reticulum by the transporters associated with antigen presentation constitutes the classical route for antigen presentation by MHC class I proteins. We have previously defined an alternative pathway in the secretory route involving proteolytic maturation of precursor proproteins for chimeric hepatitis B virus secretory core protein HBe containing a class I epitope at its carboxy-terminus. We extend those results by demonstrating that intracellular delivery of the trans -Golgi network protease furin increases both proteolytic maturation and antigen presentation of the chimeric HBe proteins. An additional class I epitope from the HIV envelope gp160 protein was inserted into this COOH-terminal region of two different chimeric HBe proteins. This epitope was also presented to CTL in a transporter-independent manner involving furin, and protein maturation and antigen presentation were also enhanced by furin over-expression. Presentation of this second epitope was restricted by a different class I allele, thus suggesting that antigen presentation by this new pathway may apply to any antigenic epitope and class I molecule. These results define the furin proteolytic maturation pathway of HBe in the secretory route as a general antigen processing route for MHC class I presentation.