Analysis of HLA DR2&;DQ6 (DRB1*1501, DQA1*0102, DQB1*0602) Haplotypes in Iranian Patients with Multiple Sclerosis

Multiple sclerosis (MS) is prototype of inflammatory demyelinating disease of the central nervous system .The etiology of MS remains unclear, but according to current data the disease develops in genetically susceptible individuals and may require additional environmental triggers. The human leukocyte antigen (HLA) class II alleles (DRB1*1501, DQA1*0102, DQB1*0602) may have the strongest genetic effect in MS. In this study, the role of these alleles were investigated in 183 Iranian patients with multiple sclerosis and compared with 100 healthy individuals. HLA typing for DRB1*1501, DQA1*0102, DQB1*0602 was performed by polymerase chain reaction (PCR) amplification with sequence-specific primers (PCR-SSP) method. The results show that, HLA DR B1*1501 was significantly more frequent among MS patients (46% vs. 20%, PV = 0.0006) but DQA1*0102 haplotype was negatively associated with MS (30% vs. 50%, PV = 0.0049) and no significant association was found with DQB1*0602 and MS patients in comparison with control group (24% and 30%, PV = 0.43). No significant correlation was observed among these alleles with sex, type of disease; initial symptoms, expanded disability status scale (EDSS), as well as age at onset and familial MS. This study therefore indicates that there is no association of above HLA haplotypes with clinical presentation, disease duration, and disability in Iranian patients with MS which is in line with other previous studies in different ethnic groups.     

敲减XAGE-1b基因表达降低ACCM和A673肿瘤细胞的增殖能力

XAGE是通过生物信息学方法发现的新一类肿瘤 睾丸抗原(cancer/testis antigen,CT抗原)基因,其主要表达亚型为XAGE-1b.已有的研究表明,该基因可能与肿瘤细胞的生长相关．本研究构建了针对XAGE-1b进行RNA干扰的重组腺病毒载体Adv-Sh1和Adv-Sh2．半定量和定量RT-PCR显示,Adv-Sh1比Adv-Sh2对XAGE-1b的干扰效率更高. 细胞荧光干扰实验显示,Adv-Sh1对XAGE-1b有显著的干扰作用．在细胞增殖和集落形成实验中,感染Adv-Sh1后的细胞增殖能力显著降低(Ｐ<0.01),形成的单克隆集落数也显著减少．研究结果表明,下调XAGE-1b基因表达使ACCM和A673肿瘤细胞的增殖能力均有降低,提示XAGE-1b在肿瘤生长过程中可能发挥重要作用．本研究为进一步深入研究XAGE-1b的功能和致癌机制打下了良好基础．     

超抗原葡萄球菌肠毒素A、B和C1的T细胞抗原HLA表位预测

为预测超抗原葡萄球菌肠毒素（SE）家族中的SEA、SEB和SEC1的HLAⅠ和HLAⅡ抗原结合表位,并对其活化T细胞作用的机理进行探讨,根据已发表的SEA、SEB和SEC1基因全序列,用T细胞抗原表位预测软件Guotif2.0对其进行T细胞抗原表位预测,统计与HLAⅠ和HLAⅡ各抗原位点结合的SEA、SEB和SEC1肽段的出现次数。结果显示,SEA、SEBT SEC1具有共同的特点,即都是主要与HLAⅠ类分子的A3位点和HLAⅡ类分子的DR1位点具有较强的结合。说明SEA、SEB和SEC1与HLAⅠ类分子和HLAⅡ类分子都有很强的结合性。三者在HLAⅠ和HLAⅡ结合位点上具有较强的同源性。本研究为SE活化T细胞作用机制的功能实验提供了依据。     

Study of HLA class I restriction and the directed antigens of cytotoxic T lymphocytes at the tumor sites of ovarian cancer

The molecular basis of T-cell-mediated recognition of ovarian cancer cells remains to be fully addressed. In this study we investigated HLA class I restriction and directed antigens of cytotoxic T lymphocytes (CTL) at the sites of ovarian cancer. Three HLA-class-I-restricted CTL lines were established from the tumor sites of ovarian cancer by culturing tumor-infiltrating lymphocytes or tumor-associated ascitic lymphocytes with interleukin-2: (1) HLA-A2402-restricted and ovarian-adenocarcinoma-specific CTL, (2) HLA-A2-restricted CTL recognizing histologically different cancers, and (3) HLA-B52-restricted and ovarian-cancer-specific CTL. HLA-A0201, HLA-A0206 and HLA-A0207 tumor cells were lysed by the HLA-A2-restricted CTL. HLA-B52 restriction of the third CTL line was confirmed by the transfection of HLA-B5201 cDNA into the tumor cells. The HLA-A2-restricted CTL recognized the SART-1, but not the MAGE-1 or MAGE-3 antigen. These results may facilitate a better understanding of the molecular basis of tumor-specific immunity at the tumor site of ovarian cancer. Received: 30 December 1998 / Accepted: 2 March 1999     

Human T cell recognition of the Mycobacterium leprae LSR antigen: epitopes and HLA restriction

We have in this work mapped epitopes and HLA molecules used in human T cell recognition of the Mycobacterium leprae LSR protein antigen. HLA typed healthy subjects immunized with heat killed M. leprae were used as donors to establish antigen reactive CD4+ T cell lines which were screened for proliferative responses against overlapping synthetic peptides covering the C-terminal part of the antigen sequence. By using this approach we were able to identify two epitope regions represented by peptide 2 (aa 29-40) and peptide 6 (aa 49-60), of which the former was mapped in detail by defining the N- and C-terminal amino acid positions necessary for T cell recognition of the core epitope. MHC restriction analysis showed that peptide 2 was presented to T cells by allogeneic cells coexpressing HLA-DR4 and DRw53 or DR7 and DRw53. In contrast, peptide 6 was presented to T cells only in the context of HLA-DR5 molecules. In conclusion, the M. leprae LSR protein antigen can be recognized by human T cells in the context of multiple HLA-DR molecules, of which none are reported to be associated with the susceptibility to develop leprosy. The results obtained are in support of using the LSR antigen in subunit vaccine design.     

T cell recognition of melanoma antigens in association with HLA-A1 on allogeneic melanoma cells

Previous studies have shown that recognition of melanoma by cytotoxic T lymphocytes may be restricted by HLA-A1, A2 and other HLA antigens. The present study examined the cytotoxic specificity and major histocompatibility complex restriction of cloned cytotoxic T lymphocytes (CTL) isolated from a patient with the HLA phenotype A3,31 who had been immunized with a vaccine prepared from HLA-A1,3 melanoma cells. Cytotoxic assays against HLA-typed allogeneic melanoma cells indicated that cloned CTL from the patient were able to kill allogeneic melanoma cells expressing HLA-A1 but not other HLA-A1-positive cells. Studies on a representative clone indicated that proliferation and cytokine (tumour necrosis factor ) production in response to melanoma cells was also associated with HLA-A1 on melanoma cells. Response to the melanoma cells was associated with interleukin-4 (IL-4) rather than IL-2 production. The antigen recognized in the context of HLA-A1 on allogeneic melanoma cells was detected in cytotoxic assays on cells from 9 of 12 HLA-A1⁺ melanoma cell lines and did not appear to be the product of the MAGE-1 or-3 genes. These findings suggest that T cells can recognize melanoma antigens in the context of alloantigens and that allogeneic vaccines containing immunodominant alloantigens may generate CTL that are ineffective against autologous melanoma. The study does not, however, exclude the possibility that CTL with specificity to the latter may be activated by allogeneic vaccines, and further studies are needed to answer this question.     

Tumor antigen-specific T helper cells in cancer immunity and immunotherapy

Historically, cancer-directed immune-based therapies have focused on eliciting a cytotoxic T cell (CTL) response, primarily due to the fact that CTL can directly kill tumors. In addition, many putative tumor antigens are intracellular proteins, and CTL respond to peptides presented in the context of MHC class I which are most often derived from intracellular proteins. Recently, increasing importance is being given to the stimulation of a CD4+ T helper cell (Th) response in cancer immunotherapy. Th cells are central to the development of an immune response by activating antigen-specific effector cells and recruiting cells of the innate immune system such as macrophages and mast cells. Two predominant Th cell subtypes exist, Th1 and Th2. Th1 cells, characterized by secretion of IFN- and TNF-, are primarily responsible for activating and regulating the development and persistence of CTL. In addition, Th1 cells activate antigen-presenting cells (APC) and induce limited production of the type of antibodies that can enhance the uptake of infected cells or tumor cells into APC. Th2 cells favor a predominantly humoral response. Particularly important during Th differentiation is the cytokine environment at the site of antigen deposition or in the local lymph node. Th1 commitment relies on the local production of IL-12, and Th2 development is promoted by IL-4 in the absence of IL-12. Specifically modulating the Th1 cell response against a tumor antigen may lead to effective immune-based therapies. Th1 cells are already widely implicated in the tissue-specific destruction that occurs during the pathogenesis of autoimmune diseases, such as diabetes mellitus and multiple sclerosis. Th1 cells directly kill tumor cells via release of cytokines that activate death receptors on the tumor cell surface. We now know that cross-priming of the tumor-specific response by potent APC is a major mechanism of the developing endogenous immune response; therefore, even intracellular proteins can be presented in the context of MHC class II. Indeed, recent studies demonstrate the importance of cross-priming in eliciting CTL. Many vaccine strategies aim to stimulate the Th response specific for a tumor antigen. Early clinical trials have shown that focus on the Th effector arm of the immune system can result in significant levels of both antigen-specific Th cells and CTL, the generation of long lasting immunity, and a Th1 phenotype resulting in the development of epitope spreading.     

Beneficial therapeutic effects with different particulate structures of murine polyomavirus VP1-coat protein carrying self or non-self CD8 T cell epitopes against murine melanoma

Polyomavirus-like-particles (PLPs) are empty, non-replicative, non-infectious particles that represent a potent antigen-delivery system against malignant disease. Protective anti-tumour immunity can be induced under therapy conditions by subcutaneous (s.c.) treatment with particulate antigenic structures like chimerical polyomavirus-pentamers (PPs). These PPs displaying an immunodominant H-2K^b-restricted ovalbumin (OVA)_257-264 epitope evoked nearly complete tumour remission in MO5 (B16-OVA) melanoma-bearing C57BL/6 mice by two s.c. applications in a weekly interval. The immunotherapeutic intervention started at day 4 after melanoma implant. Furthermore, 40% of melanoma-bearing mice vaccinated with heterologous PPs carrying a H-2K^b-restricted cytotoxic T lymphocyte (CTL) epitope derived from of tyrosinase-related protein 2 (TRP2) survived similar treatment conditions. However, a late immunotherapeutic onset at day 10 post melanoma inoculation revealed no significant differences between the therapeutic values (40–60% survival) of VP1-OVA_252-270 and VP1-TRP2_180-192 PPs, respectively. These experiments underlined the capacity of PPs to break T cell tolerance against a differentially expressed self-antigen. As a correlate for preventive and therapeutic immunity against MO5 melanoma the number of OVA_257-264- or TRP2_180-188-specific CD8 T cells were significantly increased within the splenocyte population of treated mice as measured by H-2K^b-OVA_257-264-PE tetramer staining or appropriate ELISPOT assays, respectively. These results reveal that heterologous PLPs and even chimerical PPs represent highly efficient antigen carriers for inducing CTL responses underlining their potential as immunotherapeutics against cancer.This article is a symposium paper from the second international conference Strategies for Immune Therapy, 29 February--3 March 2004, Würzburg, Germany, summarized by G. Pawelec and C. Gouttefangeas.     

Identification of a WT1 protein‐derived peptide,WT1187, as a HLA‐A*0206‐restricted,WT1‐specific CTL epitope

The Wilms' tumor gene WT1 is overexpressed in various kinds of hematopoietic malignancies as well as solid cancers, and this protein has been demonstrated to be an attractive target antigen for cancer immunotherapy. WT1‐specific CTL epitopes with a restriction of HLA‐A*2402 or HLA‐A*0201 have been already identified. In the present study it has been demonstrated that a 9‐mer WT1‐derived WT1₁₈₇ peptide, which had already been shown to elicit a WT1‐specific CTL response with a restriction of HLA‐A*0201, can also elicit a CTL response with a restriction of HLA‐A*0206. In all three different HLA‐A*0206⁺ healthy donors examined, WT1₁₈₇ peptide‐specific CTL could be generated from peripheral blood mononuclear cells, and the CTL showed cytotoxic activity that depended on dual expression of WT1 and HLA‐A*0206 molecules. The present study describes the first identification of a HLA‐A*0206‐restricted, WT1‐specific CTL epitope. The present results should help to broaden the application of WT1 peptide‐based immunotherapy from only HLA‐A*0201‐positive to HLA‐A*0206‐positive cancer patients as well.     

A cytokine promoter/yellow fluorescent protein reporter transgene serves as an early activation marker of lymphocyte subsets

A mouse containing an IL-4 promoter linked to the yellow fluorescent protein (YFP) reporter transgene was created to follow aspects of lymphocyte development and function. Following stimulation with phorbol 12-myristate 13-acetate and ionomycin, anti-CD3/CD28, antigen-specific peptide, or allogeneic cells, both CD4 and CD8 T cells expressed the transgene within 24h in a manner that was consistent with cellular activation markers. Transgene induction was inhibited by cyclosporine and FK506, suggesting that its activation occurs in an NFAT-dependent manner. B lymphocytes were also able to express the transgene when stimulated with LPS. This induction was inhibited in part by rapamycin. The results suggest that this transgene can function as an indicator of lymphocyte activation. Because YFP is not toxic and requires no preparation of the cells to view the reporter gene, this system provides a unique tool to follow lymphocyte activation in a number of model systems, such as those involving transplantation, allergy, and vaccine development.