CD4+ T cells kill HLA-class-II-antigen-positive melanoma cells presenting peptide in vitro

Purpose: Most melanoma cell lines express HLA class II antigens constitutively or can be induced to do so with interferon γ (IFNγ). We have previously demonstrated that peptide-specific CD4⁺ T cells proliferate in response to HLA-class-II-antigen-mediated peptide presentation by melanoma cells in vitro and produce interleukin-10 (IL-10) and (IFNγ). We asked whether the responding T cells kill the tumor cells and, if so, whether direct cell contact was required. Methods: Two HLA class II⁺ melanoma cell lines derived from metastases were co-cultured with a human CD4⁺ T cell clone specific for influenza hemagglutinin peptide (HA). T cells, melanoma, and HA were co-cultured for 48 h. Melanoma cells with and without HA and/or T cells served as controls. After 36 h, the medium was removed for cytokine analysis by enzyme-linked immunosorbent assay (ELISA). Twelve hours later non-adherent cells were washed away and the adherent melanoma cells were trypsinized and counted. Dual-chamber culture plates were used to determine whether cell contact and/or exposure to cytokine were required for tumor cell death. Results: Melanoma cell counts were over 80% lower in wells containing T cells than in wells with melanoma and peptide alone (P < 0.05). ELISA of supernatants revealed production of IFNγ and IL-10 by the responding T cells. Direct T cell contact with tumor cells was not required for tumor cell death, as melanoma cells were killed when they shared medium but had no contact with T cells responding to peptide presentation by HLA-class-II-antigen-positive melanoma cells in a separate chamber. Blocking antibody to IFNγ but not IL-10 prevented melanoma cell death at levels of cytokine similar to that present in co-culture assays. Conclusions: Peptide-specific CD4⁺ T cells kill melanoma cells in vitro when they recognize peptide presented by the tumor cell in the context of HLA class II antigen. Direct cell contact is not required, suggesting that it is a cytokine-mediated event. Immunotherapy, using primed CD4⁺ T cells and peptide, may be beneficial in patients whose tumors express HLA class II antigens or can be induced to do so with IFNγ. Received: 1 July 1999 / Accepted: 17 September 1999     

Genetic mapping of ossification of the posterior longitudinal ligament of the spine.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is recognized as a common disorder among Japanese and throughout Asia. Estimates of its prevalence are in the range of 1. 9%-4.3%. Although its etiology is thought to involve a multiplicity of factors, epidemiological and family studies strongly implicate genetic susceptibility in the pathogenesis of OPLL. In this study we report an identification of a predisposing locus for OPLL, on chromosome 6p, close to the HLA complex. The evidence for this localization is provided by a genetic-linkage study of 91 affected sib pairs from 53 Japanese families. In this sib-pair study, D6S276, a marker lying close to the HLA complex, gives evidence for strongly significant linkage (P = .000006) to the OPLL locus. A candidate gene in the region, that for collagen 11A2, was analyzed for the presence of molecular variants in affected probands. Of 19 distinct variants identified, 4 showed strong statistical associations with OPLL (highest P = .0004). These observations of linkage and association, taken together, show that a genetic locus for OPLL lies close to the HLA region, on chromosome 6p.     

Body odor evoked potentials: a new method to study the chemosensory perception of self and non‐self in humans

A new method will be presented which allows the perception of body odors in humans to be studied objectively. The analysis of body odor‐evoked potentials was used to investigate if and how the human brain is able to differentiate self from non‐self body odor for the first time. Six subjects (three females) participated in two experimental sessions. In each session, two body odors (axillary hair) were presented within an olfactory oddball paradigm. One of the odors was collected from the subject and the other from an odor donor of the same sex. In the first session the subjects' attention was distracted to a secondary task (passive paradigm), in the second session the subjects were asked to actively differentiate the odors (active paradigm). For the EEG recordings the odors were presented within a constantly flowing airstream. The results show that the subjects could hardly differentiate the body odors subjectively. However, it could be demonstrated that the central nervous processing of one's own odor was faster than the processing of the chemosensory non‐self signal. Moreover, in the active paradigm, the potentials appeared to be larger when the subjects perceived their own body odor. The conclusion is reached that the measurement of chemosensory event‐related potentials (CSERP) is the method of choice for the investigation of HLA‐associated body odors. This revised version was published online in July 2006 with corrections to the Cover Date.     

BAC/YAC contigs from the H2-M region of mouse Chr 17 define gene order as Znf173-Tctex5-Mog-D17Tu42-M3-M2

 A yeast artificial chromosome (YAC) contig from the C57BL/6 (H2 ^b ) mouse was created from the major histocompatibility complex (Mhc, H2 in mouse) class Ib subregion, H2-M. It spans approximately 1.2 megabase (Mb) pairs and unites the previous >1.5-Mb YAC contigs (Jones et al. 1995) into a single contig, which includes 21 Mhc class I genes distal to H2-T1. A bacterial artificial chromosome (BAC) contig from the 129 (H2 ^bc ) mouse, spanning approximately 600 kilobases, was also built from Znf173 (Afp, a gene for acid finger protein), through Tctex5 (t-complex testis expressed-5) and Mog (myelin oligodendrocyte glycoprotein), to H2-M2. Twenty-four sequence-tagged site (STS) markers were newly developed, and 35 markers were mapped in the YAC/BAC contigs, which define the marker order as Cen –Znf173–Tctex5 – Mog–D17Tu42–D17Mit232–H2-M3–D17Leh525–H2-M2– Tel. The gene order of Znf173 – Tctex5 – Mog – D17Tu42 is conserved between mouse and human, showing that the middle H2-M region corresponds to the subregion of the human Mhc surrounding HLA-A. Received: 25 July 1997 / Revised: 10 September 1997     

PCR-SSP技术对广东汉族人HLA-DR基因分型

探索具有高分辨率、高特异性和简捷快速的方法对ＨＬＡ－ＤＲ基因分型,为临床器官移植配型和疾病相关性分析提供实用的方法和基础资料．利用ＤＲ１～ＤＲｗ１８序列特异性的１９组引物及１对内参照引物进行ＰＣＲ扩增即ＰＣＲ－ＳＳＰ对ＨＬＡ－ＤＲ进行基因分型,扩增产物经琼脂糖凝胶电泳,溴乙锭染色,在紫外光下观察分型结果．每个被检个体的ＤＲ型别可由特异引物扩增出现的电泳谱带直接判断．双盲检测２２例的结果１００％正确．在１０２例中国广东地区汉族人中,ＤＲ９和ＤＲ２的基因频率最高,分别为０．２２０５和０．１９１２,ＤＲ１０为最低（０．００９８）．与用ＰＣＲ－ＳＳＯ方法分型获得的结果比较,基因型别分布基本一致,但一些等位基因的频率有差异,表明ＨＬＡ－ＤＲ基因频率的分布在不同地区、不同种族的人群间存在着差异．ＰＣＲ－ＳＳＰ法分辨率和特异性虽不及ＰＣＲ－ＳＳＯ法但比血清学方法精细,分型的全过程只需２～４ｈ能满足临床器官移植配型的要求．基因频率调查结果为器官移植配型和疾病相关性分析提供了基础资料．     

Significance of tumour cell HLA‐G5/‐G6 isoform expression in discrimination for adenocarcinoma from squamous cell carcinoma in lung cancer patients

Human leucocyte antigen (HLA)‐G has seven isoforms, of which HLA‐G1‐G4 are membrane‐bound and HLA‐G5‐G7 are soluble. Previous studies reinforced HLA‐G expression was strongly related to poor prognosis in different types of cancers. Among these studies, the monoclonal antibody (mAb) 4H84 was used which detects all HLA‐G isoform heavy chain; unfortunately, leaves the specific types of isoforms expressed in lesions undistinguished and its clinical significance needs to be clarified. To explore clinical significance of lesion soluble HLA‐G (sHLA‐G) in non‐small‐cell lung cancer (NSCLC), mAb 5A6G7 recognizing HLA‐G5/‐G6 molecules was used. Tumour cell sHLA‐G expression in 131 primary NSCLC lesions (66 squamous cell carcinoma, 55 adenocarcinoma and 10 adenosquamous carcinoma) were analysed with immunohistochemistry. Data showed that sHLA‐G expression was observed in 34.0% (45/131) of the NSCLC lesions, which was unrelated to patient age, sex, lymph nodal status, tumour–node–metastasis stage and patient survival. However, tumour cell sHLA‐G expression in lesions was predominately observed in adenocarcinoma lesions (73.0%, 40/55) which was significantly higher than that in squamous cell carcinoma (6.0%, 4/66) and adenosquamous carcinoma lesions (10.0%, 1/10, P < 0.001). The area under the receiver operating characteristic curve for lesion sHLA‐G was 0.833 (95% CI: 0.754–0.912, P < 0.001) for adenocarcinoma versus squamous cell carcinoma. Our findings for the first time showed that tumour cell sHLA‐G was predominately expressed in lung adenocarcinoma, which could be a useful biomarker to discriminate adenocarcinoma from squamous cell carcinoma in NSCLC patients.     

HLA-DRB1基因多态性与新疆哈萨克族人群结核病易感性的研究

目的:研究HLA-DRB1基因多态性与新疆哈萨克族人群结核病(TB)的相关性。方法:采用病例-对照的研究方法,应用聚合酶链反应-序列特异性引物(PCR-SSP)技术对231例新疆哈萨克族肺结核患者和230例新疆哈萨克族健康对照者的13个HLA-DRB1等位基因进行分型,比较其等位基因频率(GF)并计算其比值比(OR)。结果:与新疆哈萨克族人群对照组相比,新疆哈萨克族人群结核病例组中HLA-DRB1*04显著增高(11.72%比6.75%,p0.05,OR=1.889),HLA-DRB1*10也增高(2.86%比1.09%),但统计学上无显著性差异(Pc0.05)。结论:HLA-DRB1*04可能是新疆哈萨克族人群结核病的易感基因。     

The diagnostic potential of MPT63‐derived HLA‐A*0201‐restricted CD8+T‐cell epitopes for active pulmonary tuberculosis

MPT63 protein is found only in Mycobacterium tuberculosis complex, including M. tuberculosis and M. bovis. Detection of MPT63‐specific IFN‐γ‐secreting T cells could be useful for the diagnosis of tuberculosis (TB) diseases. In the present study, the HLA‐A*0201 restriction of ten predicted MPT63‐derived CD8 ⁺ T‐cell epitopes was assessed on the basis of T2 cell line and HLA‐A*0201 transgenic mice. The diagnostic potential of immunogenic peptides in active pulmonary TB patients was evaluated using an IFN‐γ enzyme‐linked immunospot assay. It was found that five peptides bound to HLA‐A*0201 with high affinity, whereas the remaining peptides exhibited low affinity for HLA‐A*0201. Five immunogenic peptides (MPT63_18–26, MPT63_29–37, MPT63_20–28, MPT63_5–14 and MPT63_10–19) elicited large numbers of cytotoxic IFN‐γ‐secreting T cells in HLA‐A*0201 transgenic mice. Each of the five immunogenic peptides was recognized by peripheral blood mononuclear cells from 45% to 73% of 40 HLA‐A*0201 positive TB patients. The total diagnostic sensitivity of the five immunogenic peptides was higher than that of a T‐SPOT.TB assay (based on ESAT‐6 and CFP‐10) (93% versus 90%). It is noticeable that the diagnostic sensitivity of the combination of five immunogenic peptides and T‐SPOT.TB assay reached 100%. These MPT63‐derived HLA‐A*0201‐restricted CD8 ⁺ T‐cell epitopes would likely contribute to the immunological diagnosis of M. tuberculosis infection and may provide the components for designing an effective TB vaccine.     

Loss of recognition by cross-reactive T cells and its relation to a C-terminus-induced conformational reorientation of an HLA-B*2705-bound peptide

The human major histocompatibility complex class I antigen HLA‐B*2705 binds several sequence‐related peptides (pVIPR, RRKWRRWHL; pLPM2, RRRWRRLTV; pGR, RRRWHRWRL). Cross‐reactivity of cytotoxic T cells (CTL) against these HLA‐B*2705:peptide complexes seemed to depend on a particular peptide conformation that is facilitated by the engagement of a crucial residue within the binding groove (Asp116), associated with a noncanonical bulging‐in of the middle portion of the bound peptide. We were interested whether a conformational reorientation of the ligand might contribute to the lack of cross‐reactivity of these CTL with a peptide derived from voltage‐dependent calcium channel α1 subunit (pCAC, SRRWRRWNR), in which the C‐terminal peptide residue pArg9 could engage Asp116. Analyses of the HLA‐B*2705:pCAC complex by X‐ray crystallography at 1.94 Å resolution demonstrated that the peptide had indeed undergone a drastic reorientation, leading it to adopt a canonical binding mode accompanied by the loss of molecular mimicry between pCAC and sequence‐related peptides such as pVIPR, pLMP2, and pGR. This was clearly a consequence of interactions of pArg9 with Asp116 and other F‐pocket residues. Furthermore, we observed an unprecedented reorientation of several additional residues of the HLA‐B*2705 heavy chain near the N‐terminal region of the peptide, including also the presence of double conformations of two glutamate residues, Glu63 and Glu163, on opposing sides of the peptide binding groove. Together with the Arg‐Ser exchange at peptide position 1, there are thus multiple structural reasons that may explain the observed failure of pVIPR‐directed, HLA‐B*2705‐restricted CTL to cross‐react with HLA‐B*2705:pCAC complexes.