Localization of class i histocompatibility molecule assembly by subfractionation of the early secretory pathway

Class I molecules of the major histocompatibility complex bind peptides derived from cytosolic proteins and display them on the cell surface. This function alerts cytotoxic T cells to the presence of intracellular pathogens. Class I molecule assembly requires the association of the heavy chain with β₂-microglobulin, accompanied by peptide loading via specific transporters. This study localizes where these assembly steps take place, using monoclonal antibodies recognizing class I molecules in different assembly states to analyze subcellular fractions of the early secretory pathway. The distribution of peptide-loaded class I molecules was more localized than the distribution of the total pool of class I molecules in the early secretory pathway. Loaded molecules colocalized with the peptide transporter, free heavy chains, and the chaperone calnexin in high density rough endoplasmic reticulum (RER) membranes. These data suggest that subunit assembly and peptide acquisition occur at the same intracellular site. Class I molecules also localized to less dense subfractions of the early secretory pathway, which contained comparatively less peptide-loaded molecules than the high density RER fractions, at steady state. Following a 15 °C temperature block, class I molecules accumulated in these less dense membrane fractions, indicating that these fractions represent the intermediate compartment where empty class I molecules are trapped in mutant cells. In the presence of cycloheximide, a pool of class I molecules recycling to the RER was detected, suggesting empty molecules recycle to acquire peptide.     

The rational design of TAP inhibitors using peptide substrate modifications and peptidomimetics

The major histocompatibility complex (MHC)-encoded transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum. This step precedes the binding of peptides to MHC class I molecules and is essential for cell surface expression of the MHC class I/peptide complex. TAP has a broad sequence specificity and a preference for peptides of around 9 amino acids. To synthesize inhibitors for TAP, we studied various alterations of the peptide substrate. The results indicate that TAP is stereospecific and that peptide bonds engineered into isosteric structures can improve translocation of the peptide. Furthermore, TAP is able to translocate peptides with large side chains that correspond to a peptide of ～ 21 amino acids in extended conformation. Peptides with longer side chains compete for the peptide binding site of TAP but fail to be translocated. Therefore, they represent the first rationally designed inhibitors of TAP.     

New TAP2 polymorphisms in Africans

Abstract: Polymorphisms in genes encoding transporters associated with antigen processing (TAP) have been associated with heterogeneity of disease progression in HTV-l-infected homosexual men. In our recent AIDS-related studies of cohorts from Rwanda and Zambia, four new polymorphic sites in the TAP2 coding region were detected by single-strand conformation polymorphism (SSCP) and confirmed by bi-directional nucleotide sequencing and restriction enzyme digestion. The first, a substitution of Thr (GCC) for Ala (ACC) at codon position 374 in exon 5, was found in about 13% of Rwandans and Zambians ( n =213). The remaining 3 new polymorphisms were seen in the 7th exon with changes of 458Thr-ACG to ACA, 466Gly-GGG to GGA, and 467Val-GTT to Ile-ATT, respectively. These 3 variants occurred exclusively on the same chromosome and appeared to have arisen together from the 374Thr-bearing allele. Analyses of the relationship between the 374Thr-457Ile segment and the nearby markers in DQB1 and DRB1 suggested the existence of a unique extended haplotype related to these newly identified variants.     

上海人群中TAP与IgA肾炎相关性的研究

目的探讨上海人群中抗原处理相关转运蛋白（ＴＡＰ）与ＩｇＡ肾炎的相关性。方法用ＰＣＲ－ＳＳＯ方法对上海地区８８名正常人及４０名ＩｇＡ肾炎患者ＴＡＰ进行分型。结果在病人组及对照组中共发现３种ＴＡＰ１（ＴＡＰ１Ａ、１Ｂ、１Ｃ）及４种ＴＡＰ２（ＴＡＰ２＊０１０１、＊０１０２、＊０２０１、＊０２０２）等位基因。未发现病人组与对照组之间ＴＡＰ等位基因分布的差异。结论本文结果未能证明ＴＡＰ基因与ＩｇＡ肾炎相关，但不排除未检测的多态位点与ＩｇＡ肾炎关联的可能性。     

An integrative approach to CTL epitope prediction: a combined algorithm integrating MHC class I binding, TAP transport efficiency, and proteasomal cleavage predictions

Reverse immunogenetic approaches attempt to optimize the selection of candidate epitopes, and thus minimize the experimental effort needed to identify new epitopes. When predicting cytotoxic T cell epitopes, the main focus has been on the highly specific MHC class I binding event. Methods have also been developed for predicting the antigen-processing steps preceding MHC class I binding, including proteasomal cleavage and transporter associated with antigen processing (TAP) transport efficiency. Here, we use a dataset obtained from the SYFPEITHI database to show that a method integrating predictions of MHC class I binding affinity, TAP transport efficiency, and C-terminal proteasomal cleavage outperforms any of the individual methods. Using an independent evaluation dataset of HIV epitopes from the Los Alamos database, the validity of the integrated method is confirmed. The performance of the integrated method is found to be significantly higher than that of the two publicly available prediction methods BIMAS and SYFPEITHI. To identify 85% of the epitopes in the HIV dataset, 9% and 10% of all possible nonamers in the HIV proteins must be tested when using the BIMAS and SYFPEITHI methods, respectively, for the selection of candidate epitopes. This number is reduced to 7% when using the integrated method. In practical terms, this means that the experimental effort needed to identify an epitope in a hypothetical protein with 85% probability is reduced by 20-30% when using the integrated method.The method is available at http://www.cbs.dtu.dk/services/NetCTL. Supplementary material is available at http://www.cbs.dtu.dk/suppl/immunology/CTL.php.     

Constitutive macropinocytosis allows TAP-dependent major histocompatibility compex class I presentation of exogenous soluble antigen by bone marrow-derived dendritic cells

Dendritic cells expanded from mouse bone marrow (BMDC) with granulocyte/macrophage-colony-stimulating factor have potent T cell-stimulatory properties both in vitro and in vivo. This has been well documented for major histocompatibility complex (MHC) class II-restricted responses, and more recently using peptide-loaded and protein-pulsed DC for CD8 responses following adoptive transfer in mice. An unresolved question concerns the capacity of BMDC to present exogenous antigen on MHC class I molecules, an unconventional mode of MHC class I loading for which there is now considerable evidence, particularly in macrophages. Here, we show that BMDC exhibit high levels of macropinocytosis driven by constitutive membrane ruffling activity. Up to one-third of actively ruffling and macropinocytosing BMDC transferred pinocytosed horseradish peroxidase into the cytosol following a 15-min pulse, suggesting that they might be capable of presenting exogenous soluble antigen on MHC class I molecules. We show that BMDC presented exogenous ovalbumin to a T cell hybridoma more effectively, more rapidly, and at lower exogenous antigen concentrations than BM macrophages on a cell-for-cell basis. Presentation was TAP dependent, brefeldin A sensitive, and blocked by inhibitors of proteasomal processing, demonstrating use of the classical MHC class I pathway. Although effective presentation of exogenous antigen by BMDC occurred in the absence of agents which stimulate macropinocytosis, treatment with phorbol myristate acetate (PMA) enhanced both pinocytosis and MHC class I presentation by BMDC. Finally, PMA-stimulated BMDC exposed to exogenous ovalbumin in vitro were able to prime an antigen-specific cytotoxic T lymphocyte response following adoptive transfer in vivo.     

Protection from insulin-dependent diabetes mellitus is linked to a peptide transporter gene

HLA class II association with insulin-dependent diabetes mellitus (IDDM) is well established but is still difficult to map to a particular locus. Polymorphism of the genes coding for transporter associated with antigen processing (TAP1 and TAP2), and located in the HLA class II region, was studied in 167 IDDM patients (116 adult-onset and 51 childhood-onset patients) and 98 normal controls using oligotyping after genomic amplification. A dominant protective effect was observed for theTAP2*0201 allele [relative risk (RR)=0.3, corrected probability (pc) < 0.001]. Conversely, susceptibility to IDDM was associated with apparent homozygosity for the TAP2*0101 allele (RR=3.4, pc < 0.001). Protection was independent from but additive to the protection conferred by the DRB1*02 DQB1*0602 haplotype (RR=0.06, pc<0.05), and antagonistic to the DRB1*03 DQB1*0201 and DRB1*04 DQB 1*0302 haplotypes predisposing effect (RR=1.1, not significant), arguing in favor of an absence of linkage disequilibrium between TAP2 and HLA class II genes. This was assessed by x² analysis. TAP1 allelic distribution was not different among diabetics and controls. A significant association was observed between the presence of TAP2*0101 and that of islet cell antibodies (p < 0.05). These data suggest that the TAP2 gene, which encodes protein required for delivery of antigen peptides to class I molecules in the endoplasmic reticulum, could modulate the autoimmune response leading to β cell destruction. From a practical point of view, they make the combined screening of HLA class II and TAP2 loci a highly valuable tool in IDDM prediction.     

超声引导改良肋缘下腹横肌阻滞对腹腔镜胆囊切除术术中术后镇痛的影响

目的探讨超声引导改良肋缘下腹横肌阻滞对腹腔镜胆囊切除术患者术中术后镇痛效果的影响。方法全麻下行腹腔镜胆囊切除术60例,随机分为两组。麻醉诱导后一组在超声引导下行改良肋缘下入路的双侧腹横肌平面(TAP)阻滞,注入0.5%罗哌卡因(0.8 m L/kg,A组),另一组全麻后未行TAP阻滞(B组)。两组术后均应用患者静脉自控镇痛(PCIA)进行镇痛。记录两组手术中瑞芬太尼的用量,手术结束时除去喉罩时的平均动脉压(MAP)和心率(HR),术后2、6、24 h视觉模拟疼痛(VAS)评分,PCIA泵术后36 h总用药量和自控镇痛有效按压次数及镇痛期间不良反应的发生情况。结果与B组比较,A组术中瑞芬太尼的用量明显减少(P<0.05),除去喉罩时MAP、HR波动明显减小(P<0.05),术后2、6 h VAS评分A组明显低于B组(P<0.05),PCIA总用药量和有效按压次数A组明显少于B组(P<0.05),A组镇痛期间不良反应发生也明显少于B组。结论超声引导改良肋缘下TAP阻滞能为腹腔镜胆囊切除术患者提供良好的术中及术后镇痛。