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.     

Role of TAP-1 and/or TAP-2 antigen presentation defects in tumorigenicity of mouse melanoma

Mutations in transporters associated with antigen processing (TAP-1 and -2) required for the transport of cytosolic endogenous peptides to the endoplasmic reticulum correlate with increased metastatic potential and reduced host survival in several malignancies. To address the possible function of TAP as a "tumor suppressor" gene, we show that correction of TAP-1 and/or TAP-2 defects in B16 mouse melanoma enhanced the cell surface expression of MHC class I molecules and significantly reduced the rate of subcutaneous tumor growth and pulmonary metastatic burden. Cytotoxic assays confirmed increased sensitivity of TAP-1 and/or TAP-2 transfected clones of B16 melanoma to cytotoxic T lymphocytes. These results indicate that the expression of TAP limits the malignant potential of tumors with implications for CD8(+) T cell-based immunotherapy in controlling growth of certain TAP-deficient malignancies.     

Classification of A1- and A24-supertype molecules by analysis of their MHC-peptide binding repertoires

At the functional level, the majority of human leukocyte antigen (HLA) class I MHC variants can be classified into about ten different major groups, or supertypes, characterized by overlapping peptide binding motifs and repertoires. Previous studies have detailed the peptide binding specificity of the HLA A2, A3, B7, and B44 supertypes, and predicted, on the basis of MHC pocket structures, known motifs, or the sequence of T cell epitopes, the existence of the HLA A1 and A24 supertypes. Direct experimental validation of the A1 and A24 supertypes, however, has been lacking. In the current study, the peptide-binding repertoires and main anchor specificities of several common HLA A molecules (A*0101, A*2301, A*2402, A*2601, A*2902, and A*3002) predicted to be members of the A1 or A24 supertypes were analyzed and defined using single amino acid substituted peptides and a large peptide library. Based on the present findings, the A1 supertype includes A*0101, A*2601, A*2902, and A*3002, whereas the A24 supertype includes A*2301 and A*2402. Interestingly, A*2902 is associated with a motif and peptide binding repertoire that overlaps significantly with those of all of the A1- and A24-supertype molecules studied, representing—to our knowledge—the first report of significant cross-reactivity among molecules belonging to different supertypes.     

Assembly and cell surface expression of TAP-independent,chloroquine-sensitive and interferon-gamma-inducible class I MHC complexes in transformed fibroblast cell lines are regulated by tapasin

Antigen processing and presentation by class I MHC molecules generally require assembly with peptide epitopes generated by the proteasome and transported into the ER by the transporters associated with antigen presentation (TAP). Recently, TAP-independent pathways supporting class I MHC-mediated presentation of exogenous antigens, as well as of endogenously synthesized viral antigens, were described. We now characterize a TAP-independent pathway that is operative in both TAP1- and TAP2-deficient Adenovirus (Ad)-transformed fibroblast cell lines. To the best of our knowledge, this is the first time that the existence of such a pathway has been described in non-infected cells that do not belong to the hematopoietic lineage. We show that this pathway is proteasome-independent and chloroquine-sensitive. Cell surface expression of these TAP-independent class I complexes is modulated by tapasin levels and is enhanced by IFN-gamma. The data imply that IFN-gamma increases the relative level of TAP-independent high affinity class I complexes that exit the ER on their way to the cell surface and to vacuolar compartments where peptide cleavage/exchange might take place before recycling to the cell surface. Since both TAP and tapasin expression are altered in numerous tumors and in virus-infected cells, TAP-independent class I complexes may be a valuable target source for immune responses.     

Sequence, linkage to H2-K, and function of mouse tapasin in MHC class I assembly

 Assembly of major histocompatibility complex (MHC) class I molecules in human cells is dependent on the accessory protein tapasin, which mediates their interaction with the transporters associated with antigen processing (TAP) and thereby ensures efficient peptide binding. Analysis of a mouse tapasin complementary DNA defined a conserved polypeptide sharing sequences diagnostic of a transmembrane protein related to the immunoglobulin superfamily, and an endoplasmic reticulum retention motif. The mouse tapasin gene was mapped about 70 kilobases from H2-K at the centromeric end of the mouse MHC. Expression of mouse tapasin in a tapasin-deficient human mutant cell line restored the normal assembly and expression of class I alleles. Thus, tapasin is a structurally and functionally conserved component of the MHC class I antigen processing pathway. Its genetic linkage to the class I and TAP subunit genes in the MHC may be of significance in the coordinate expression and functional coadaptation of the diverse gene products. Received: 1 February 1998 / Revised: 23 March 1998     

Regulation of antigen presentation machinery in human dendritic cells by recombinant adenovirus

Recombinant adenoviral vectors (AdV) are potent vehicles for antigen engineering of dendritic cells (DC). DC engineered with AdV to express full length tumor antigens are capable stimulators of antigen-specific polyclonal CD8+ and CD4+ T cells. To determine the impact of AdV on the HLA class I antigen presentation pathway, we investigated the effects of AdV transduction on antigen processing machinery (APM) components in human DC. Interactions among AdV transduction, maturation, APM regulation and T cell activation were investigated. The phenotype and cytokine profile of DC transduced with AdV was intermediate, between immature (iDC) and matured DC (mDC). Statistically significant increases in expression were observed for peptide transporters TAP-1 and TAP-2, and HLA class I peptide-loading chaperone ERp57, as well as co-stimulatory surface molecule CD86 due to AdV transduction. AdV transduction enhanced the expression of APM components and surface markers on mDC, and these changes were further modulated by the timing of DC maturation. Engineering of matured DC to express a tumor-associated antigen stimulated a broader repertoire of CD8+ T cells, capable of recognizing immunodominant and subdominant epitopes. These data identify molecular changes in AdV-transduced DC (AdV/DC) that could influence T cell priming and should be considered in design of cancer vaccines.     

Targeting tumour cells with defects in the MHC Class I antigen processing pathway with CD8+ T cells specific for hydrophobic TAP- and Tapasin-independent peptides: the requirement for directed access into the ER

It is becoming increasingly apparent that the majority of tumours display defects in the MHC class I antigen processing pathway, particularly low levels of the transporters-associated with antigen processing (TAP) and tapasin. Thus, immunotherapy approaches targeting such tumours with CD8+ cytotoxic T lymphocytes (CTL) requires strategies to overcome these defects. Previously we had identified an antigen processing pathway by which cytosolically derived hydrophobic peptides could be presented in the absence of TAP. Here we show in the tapasin-negative cell line 721.220 that a number of these hydrophobic TAP-independent peptides can also be presented in a tapasin-independent manner. Yet when these experiments were extended to tumour cell lines derived from small cell lung cancer (SCLC), which we show to be tapasin deficient in addition to TAP-negative, the TAP-, tapasin-independent peptides were not presented. This lack of presentation could be rectified by pre-treatment of SCLC cells with IFNgamma. Alternatively, by directing the TAP-, tapasin-independent peptides into the endoplasmic reticulum (ER) via an ER signal sequence, these peptides were presented efficiently by SCLC cells. We infer from this data that the TAP-independent pathway for presentation of hydrophobic peptides generates a low concentration of peptide in the ER and, for tumour cells which also lack tapasin, this concentration of antigenic peptide is insufficient to load onto MHC class I molecules. Thus, for immunotherapeutic approaches to target SCLC and other tumours with defects in the MHC class I antigen processing pathway it will be important to consider strategies that address tapasin-defects.     

Organization of the histone genes in the rainbow trout (Salmo gairdnerii)

Summary Twelve clones containing histone genes were isolated from a genomic trout library constructed in the vector Charon 4A. Each of the clones was found to contain a conserved 10.2-kb Eco RI fragment that contained one copy of each of the histones in the order H4-H2B-H1-H2A-H3, all of which are transcribed from the same strand. Genomic Southern blots indicate that these clusters are representative of the vast majority of the histone genes in the trout. Tandemly linked clusters were not found. Approximately 145 copies of this cluster are present in a trout sperm cell. Sequence analysis has shown the genes to be without introns and to show strong selection for codons ending in C or G. Consensus signals similar to those found in other histone genes are present in the flanking regions.     

The spectrum of HLA-DQ and HLA-DR alleles, 2006: a listing correlating sequence and structure with function

The list of alleles in the HLA-DRB, HLA-DQA, and HLA-DQB gene loci has grown enormously since the last listing in this journal 8 years ago. Crystal structure determination of several human and mouse HLA class II alleles, representative of two gene loci in each species, enables a direct comparison of ortholog and paralog loci. A new numbering system is suggested, extending earlier suggestions by [Fremont et al. in Immunity 8:305-317, (1998)], which will bring in line all the structural features of various gene loci, regardless of animal species. This system allows for structural equivalence of residues from different gene loci. The listing also highlights all amino acid residues participating in the various functions of these molecules, from antigenic peptide binding to homodimer formation, CD4 binding, membrane anchoring, and cytoplasmic signal transduction, indicative of the variety of functions of these molecules. It is remarkable that despite the enormous number of unique alleles listed thus far (DQA = 22, DQB = 54, DRA = 2, and DRB = 409), there is invariance at many specific positions in man, but slightly less so in mouse or rat, despite their much lower number of alleles at each gene locus in the latter two species. Certain key polymorphisms (from substitutions to an eight-residue insertion in the cytoplasmic tail of certain DQB alleles) that have thus far gone unnoticed are highly suggestive of differences or diversities in function and thus call for further investigation into the properties of these specific alleles. This listing is amenable to supplementation by future additions of new alleles and the highlighting of new functions to be discovered, providing thus a unifying platform of reference in all animal species for the MHC class II allelic counterparts, aiding research in the field and furthering our understanding of the functions of these molecules.     

Endocytic recycling is required for the presentation of an exogenous peptide via MHC class II molecules

Exogenous antigenic peptides captured and presented in the context of major histocompatibility (MHC) class II molecules on APC, have been employed as potent vaccine reagents capable of activating cellular immune responses. Binding and presentation of select peptide via surface class II molecules has been reported. Here, a role for endocytosis and early endosomes in the presentation of exogenous peptides via MHC class II molecules is described. T cell recognition of a 14 amino acid human serum albumin-derived peptide in the context of HLA-DR4 was observed only with metabolically active APC. The delayed kinetics and temperature dependence of functional peptide presentation via APC, were consistent with a requirement for peptide internalization to early endosomal compartments prior to T cell recognition. Ablating endocytosis by exposing cells to inhibitors of ATP production completely blocked the display of functional peptide:class II complexes on the surface of the APC. Presentation of the peptide was also found to be sensitive to primaquine, a drug that perturbs the recycling of transport vesicles containing endocytic receptors and mature class II complexes. Functional presentation of the endocytosed peptide was dependent upon these mature class II complexes, as inhibitor studies ruled out a requirement for newly synthesized class II molecules. N-terminal processing of the endocytosed peptide was observed upon trafficking through endosomal compartments and linked to the formation of functional peptide:class II complexes. These findings establish a novel mechanism for regulating class II-restricted peptide presentation via the endocytic pathway.     

癌症DNA甲基化调控位点的识别

DNA甲基化是一种重要的表观遗传学修饰,在基因的转录调控方面具有重要的作用。异常的DNA甲基化可以导致癌症等复杂疾病发生,癌基因相关的DNA甲基化调控位点的识别对于解析癌症的发生发展机制及识别新的癌症标记具有重要意义。本研究通过整合The Cancer Genome Atlas(TCGA)的泛癌症基因组的高通量甲基化谱和基因表达谱,识别癌基因相关的DNA甲基化调控位点。对于每种癌症分批次计算Cp G位点甲基化与相关基因表达之间的相关性,并筛选调控下游基因的Cp G位点(包括强调控位点、弱调控位点和不调控位点),结果表明仅有一半的Cp G位点对下游基因具有调控作用;对癌症间共享的调控位点的分析发现不同癌症间共享的调控位点不尽相同,表明癌症特异的甲基化调控位点的存在。进一步地,对差异甲基化和差异表达基因的功能富集分析揭示了受甲基化调控的基因确实参与了癌症发生发展相关的功能。本研究的结果是对当前甲基化调控位点集的重要补充,也是识别癌症新型分子标记特征的重要资源。     

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.     

A deterministic model for the processing and presentation of bacteria-derived antigenic peptides

The amount and the dynamics of antigen supply to the cellular antigen processing and presentation machinery differ largely among diverse microbial antigens and various types of antigen presenting cells. The precise influence, however, of antigen supply on the antigen presentation pattern of cells is not known. Here, we provide a basic deterministic mathematical model of antigen processing and presentation of microbial antigens. The model predicts that different types of antigen presenting cells e.g. cells presenting or cross-presenting exogenous antigens, cells infected with replicating microbes, or cells in which microbial antigen synthesis is blocked after a certain period of time have inherently different antigen presentation patterns which are defined by the kinetics of antigen supply. The reevaluation of existing experimental data [Sijts, A.J., Pamer, E.G., 1997. Enhanced intracellular dissociation of major histocompatibility complex class I-associated peptides: a mechanism for optimizing the spectrum of cell surface-presented cytotoxic T lymphocyte epitopes. J. Exp. Med. 185, 1403-1411] describing the processing and presentation of two antigenic peptides derived from the p60 proteins of the facultatively intracellular bacterium Listeria monocytogenes shows that p60 proteins accumulating intracellularly during bacterial infection of cells play no measurable role as substrate for the cytosolic antigen presentation pathway.     

MHC Class I Presentation of an Exogenous Polypeptide Antigen Encoded by the Murine AIDS Defective Virus

Peptides derived from endogenous proteins are presented by MHC class I molecules, whereas those derived from exogenous proteins are presented by MHC class II molecules. This strict segregation has been reconsidered in recent reports in which exogenous antigens are shown to be presented by MHC class I molecules in the phagocytic pathway. In this report, the presentation pathway of an exogenously added highly antigenic polypeptide encoded by the murine AIDS (MAIDS) defective virus gag p12 gene is investigated. A 25-mer polypeptide (P12–25) encoded within the gag p12 region of the MAIDS defective virus was found to be effective in stimulating unprimed B6 (H-2^b) CD8⁺ T cells in vitro. The presentation of P12–25 is sensitive to cytochalasin B and D, brefeldin A and gelonin, a ribosome-inactivating protein synthesis inhibitor, but less sensitive or resistant to lactacystin, a highly specific inhibitor of the proteasome. Interestingly, CA-074, a selective inhibitor of cathepsin B, inhibited presentation of the polypeptide, indicating its involvement in the degradation of the P12–25 polypeptide. In fact, when P12–25 was digested with purified cathepsin B in vitro, a highly antigenic 11-mer peptide containing the class I (H-2D^b)-binding motif was obtained. Our results favor the phagosome/macropinosome-to-cytosol-to-endoplasmic reticulum (ER)-to-cell surface pathway for exogenous antigens presented by MHC class I molecules. These findings may be relevant to exploiting peptide vaccines that specifically elicit CD8⁺ T cell immunity in vivo.