红景天苷通过TLR4调控DC提高T细胞对肺癌3LL细胞的杀伤力

目的：探讨红景天苷（salidroside，SAL）对树突状细胞（dendritic cell，DC）表型及细胞毒性T细胞（cytotoxic T lympho‐cyte，CTL）抗肿瘤能力的影响。方法：选用Lewis肺癌细胞株3LL、野生型C57BL/6和TLR4-/-C57BL/6小鼠，获取小鼠骨髓来源的DC前体细胞，经过培养分化成未成熟DC，收获第6天的DC，经磁珠分选后获得纯度较高的CD11c+DC。将细胞分成PBS组、SAL组和脂多糖（lipopolysaccharide，LPS）组，培养48 h后用流式细胞术检测SAL体外对DC表面分子、吞噬功能、TLR4通路和对T细胞杀伤能力的影响。结果：与 PBS 组比较，SAL组DC的表面分子CD80、CD86、MHC Ⅱ表达水平显著升高（均 P<0.05）、吞噬功能显著下降（P<0.05）、TLR4 表达水平显著升高（P<0.01）；与野生型组比较，TLR4-/-组DC经SAL或LPS处理后，其表面分子CD80、CD86、MHC Ⅱ的表达水平显著降低（均P<0.05）；与PBS组比较，SAL组刺激活化的CTL对肺癌3LL细胞的杀伤效应显著升高（P<0.05）。结论：SAL可以通过调控TLR4诱导DC成熟，从而提高T细胞的杀伤能力     

Inactivated influenza vaccine formulated with single-stranded RNA-based adjuvant confers mucosal immunity and cross-protection against influenza virus infection

Influenza vaccination is considered the most valuable means to prevent and control seasonal influenza infections, which causes various clinical symptoms, ranging from mild cough and fever to even death. Among various influenza vaccine types, the inactivated subunit type is known to provide improved safety with reduced reactogenicity. However, there are some drawbacks associated with inactivated subunit type vaccines, with the main ones being its low immunogenicity and the induction of Th2-biased immune responses. In this study, we investigated the role of a single-stranded RNA (ssRNA) derived from the intergenic region in the internal ribosome entry site of the Cricket paralysis virus as an adjuvant rather than the universal vaccine for a seasonal inactivated subunit influenza vaccine. The ssRNA adjuvant stimulated not only well-balanced cellular (indicated by IgG2a, IFN-γ, IL-2, and TNF-α) and humoral (indicated by IgG1 and IL-4) immune responses but also a mucosal immune response (indicated by IgA), a key protector against respiratory virus infections. It also increases the HI titer, the surrogate marker of influenza vaccine efficacy. Furthermore, ssRNA adjuvant confers cross-protective immune responses against heterologous influenza virus infection while promoting enhanced viral clearance. Moreover, ssRNA adjuvant increases the number of memory CD4⁺ and CD8⁺ T cells, which can be expected to induce long-term immune responses. Therefore, this ssRNA-adjuvanted seasonal inactivated subunit influenza vaccine might be the best influenza vaccine generating robust humoral and cellular immune responses and conferring cross-protective and long-term immunity.     

Reduced oligomeric and vascular amyloid-β following immunization of TgCRND8 mice with an Alzheimer's DNA vaccine

Immunization with amyloid-β (Aβ) peptide reduces amyloid load in animal studies and in humans; however clinical trials resulted in the development of a pro-inflammatory cellular response to Aβ. Apoptosis has been employed to stimulate humoral and Th2-biased cellular immune responses. Thus, we sought to investigate whether immunization using a DNA vaccine encoding Aβ in conjunction with an attenuated caspase generates therapeutically effective antibodies. Plasmids encoding Aβ and an attenuated caspase were less effective in reducing amyloid pathology than those encoding Aβ alone. Moreover, use of Aβ with an Arctic mutation (E22G) as an immunogen was less effective than wild-type Aβ in terms of improvements in pathology. Low levels of IgG and IgM were generated in response to immunization with a plasmid encoding wild-type Aβ. These antibodies decreased plaque load by as much as 36 ± 8% and insoluble Aβ42 levels by 56 ± 3%. Clearance of Aβ was most effective when antibodies were directed against N-terminal epitopes of Aβ. Moreover, immunization reduced CAA by as much as 69 ± 12% in TgCRND8 mice. Finally, high-molecular-weight oligomers and Aβ trimers were significantly reduced with immunization. Thus, immunization with a plasmid encoding Aβ alone drives an attenuated immune response that is sufficient to clear amyloid pathology in a mouse model of Alzheimer's disease.     

Identification of new immunogenic peptides in conserved regions of hepatitis C virus (HCV) 1b with the potentiality to generate cytotoxic T lymphocytes in HCV1b+ HLA-A24+ patients

Aim: Hepatitis C virus (HCV) 1b is resistant to standard interferon therapy and has a high risk of developing into hepatocellular carcinoma at the late stage of infection. Therefore, new therapeutic modalities for HCV1b infection must be developed. One approach would be active specific immunotherapy with highly immunogenic HCV1b peptides. Methods: HCV1b-derived 44 synthetic peptides were selected based on their binding scores to HLA-A24. Peptide-specific IgG were measured by ELISA. Peptide-specific cytotoxic T-lymphocytes (CTLs) were induced in vitro by repeated peptide-stimulation. Results: We identified three novel candidate peptides of HCV1b proteins containing HLA-A24 binding motifs. Each of them had the ability to induce HLA-A24-restricted and peptide-specific CTL activity, and IgGs specific to each of them were detected in the plasma of HCV1b patients. Among these three peptides, a peptide NS5A 2132-2142 was recognized by both cellular and humoral immunities in the majority of blood samples of patients tested. More importantly, the peptide-stimulated peripheral blood mononuclear cells (PBMCs) showed cytotoxicity against cells cotransfected with NS5A and HLA-A2402 genes in an HLA-restricted manner. This is an additional report to our previous study. Conclusion: These findings may provide a new insight into the development of a peptide-based specific immunotherapy for HCV1b-infected patients.     

利用目的基因随机表位库筛选葡激酶抗原决定簇

目的：用目的基因随机表位库方法寻找葡激酶(straphylokinase SAK)的显性表位。方法：①SAK免疫BALB／C小鼠，免疫亲和层析纯化抗血清后，抗SAK抗体用生物素标记；②构建SAK随机表位肽库，随机挑取12个独立克隆测序，分析库DNA片段的分布和碱基含量情况；③以多抗为靶蛋白用克隆原位杂交法筛选肽库；④构建SAK的缺失突变体mSAK，Western blot分析mSAK的免疫反应性。结果：①筛选肽库得到一个由19个氨基酸组成的免疫显性表位区，命名为A1区；②mSAK不能与抗SAK多抗反应。结论：用简便、有效的方法筛选得到了SAK的一个表位A1区，初步确定A1区是SAK引起免疫反应的重要区域。     

Antigen-specific cytolysis of infected cells in murine candidiasis

Immune L3T4+ and Lyt-2+ lymphocytes play an important role in the acquired resistance of mice to challenge with virulent Candida albicans, and release macrophage-activating cytokines in response to yeast cells in vitro. To determine whether antigen (Ag)-specific cytotoxic T lymphocytes are generated during fungal infection, purified L3T4+ and Lyt-2+ lymphocytes from immunized mice were cultured in the presence of syngeneic accessory cells, Candida Ag, and IL-2. Yeast-infected bone marrow macrophages and peritoneal exudate neutrophils were used as target cells in a standard ⁵¹Cr release assay. Ag-specific, MHC-unrestricted lysis of infected macrophages was evident with immune Lyt-2+ cells after 5–10 days in culture. Under the same experimental conditions, the cytotoxic activity of L3T4+ cells was negligible, but its expression could be induced by the addition of anti-CD3 antibody.Culturing immune Lyt-2+ cells for shorter periods of time (1–2 days) resulted in preferential lysis of infected neutrophils. In addition, at limiting effector cell numbers, Ag-specific MHC-restricted lymphocytes with cytotoxic activity to infected macrophages could be identified. We suggest that C. albicans infection stimulates multiple cytotoxic T-cell precursors with varying recognition stringency, wich may have an important role in antifungal resistance in vivo.     

Epitope Context and Reshaping of Activated T Helper Cell Repertoire

In recent years, a growing interest in the study of peptide antigenicity in relation to the role of flanking sequences and protein topology in processing, presentation, and recognition has been observed. However, the information available on the antigenicity of recombinant fusion proteins and their effect on the selection of antigen receptor repertoires is limited. To analyze the role of molecular topology of T epitopes in a system relevant to human pathology, we have used the bacterially expressed Schistosoma japonicum glutathione S transferase (GST) to construct recombinant antigens containing HIV-1 derived T cell determinants, and human T cell clones specific for these determinants. We found that antigenicity of a given GST—peptide combination was not the same when T cells and antigen presenting cells from different individuals were tested. Our results show that differences in processing and presentation of chimeric proteins are not dictated by the use of diverse restriction elements. We also found that the context in which an antigenic peptide is delivered affects the recruited repertoire as defined according to T cell receptor Vβ usage and fine specificities of selected T cells.     

Two tyrosinase nonapeptides recognized on HLA-A2 melanomas by autologous cytolytic T lymphocytes

A number of cytolytic T lymphocyte (CTL) clones derived from several melanoma patients have been found to recognize a majority of melanomas from HLA-A2 patients. We have reported previously that two such CTL clones recognize a product of the tyrosinase gene that is presented by HLA-A2. Here we show that one of these CTL clones recognizes a peptide encoded by the first nine amino acids of the putative signal sequence of tyrosinase. The other CTL clone recognizes a different tyrosinase peptide corresponding to amino acids 368–376. Both peptides contain consensus motifs of HLA-A2 binding peptides.     

Epitope mapping and biological function analysis of antibodies produced by immunization of mice with an inactivated Chinese isolate of severe acute respiratory syndrome-associated coronavirus (SARS-CoV)

Inactivated severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been tested as a candidate vaccine against the re-emergence of SARS. In order to understand the efficacy and safety of this approach, it is important to know the antibody specificities generated with inactivated SARS-CoV. In the current study, a panel of twelve monoclonal antibodies (mAbs) was established by immunizing Balb/c mice with the inactivated BJ01 strain of SARS-CoV isolated from the lung tissue of a SARS-infected Chinese patient. These mAbs could recognize SARS-CoV-infected cells by immunofluorescence analysis (IFA). Seven of them were mapped to the specific segments of recombinant spike (S) protein: six on S1 subunit (aa 12-798) and one on S2 subunit (aa 797-1192). High neutralizing titers against SARS-CoV were detected with two mAbs (1A5 and 2C5) targeting at a subdomain of S protein (aa 310-535), consistent with the previous report that this segment of S protein contains the major neutralizing domain. Some of these S-specific mAbs were able to recognize cleaved products of S protein in SARS-CoV-infected Vero E6 cells. None of the remaining five mAbs could recognize either of the recombinant S, N, M, or E antigens by ELISA. This study demonstrated that the inactivated SARS-CoV was able to preserve the immunogenicity of S protein including its major neutralizing domain. The relative ease with which these mAbs were generated against SARS-CoV virions further supports that subunit vaccination with S constructs may also be able to protect animals and perhaps humans. It is somewhat unexpected that no N-specific mAbs were identified albeit anti-N IgG was easily identified in SARS-CoV-infected patients. The availability of this panel of mAbs also provided potentially useful agents with applications in therapy, diagnosis, and basic research of SARS-CoV.     

Preservation and redirection of HPV16E7-specific T cell receptors for immunotherapy of cervical cancer

Human papilloma virus (HPV) type 16 infections of the genital tract are associated with the development of cervical cancer (CxCa) in women. HPV16-derived oncoproteins E6 and E7 are expressed constitutively in these lesions and might therefore be attractive candidates for T-cell-mediated adoptive immunotherapy. However, the low precursor frequency of HPV16E7-specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer. To overcome this problem, we have isolated T cell receptor (TCR) genes from four different HPV16E7-specific healthy donor and patient-derived human cytotoxic T lymphocyte (CTL) clones. We examined whether genetic engineering of peripheral blood-derived CD8+ T cells in order to express HPV16E711-20-specific TCRs is feasible for adoptive transfer purposes. Reporter cells (Jurkat/MA) carrying a transgenic TCR were shown to bind relevant but not irrelevant tetramers. Moreover, these TCR-transgenic Jurkat/MA cells showed reactivity towards relevant target cells, indicating proper functional activity of the TCRs isolated from already available T cell clones. We next introduced an HPV16E711-20-specific TCR into blood-derived, CD8+ recipient T cells. Transgenic CTL clones stained positive for tetramers presenting the relevant HPV16E711-20 epitope and biological activity of the TCR in transduced CTL was confirmed by lytic activity and by interferon (IFN)-gamma secretion upon antigen-specific stimulation. Importantly, we show recognition of the endogenously processed and HLA-A2 presented HPV16E711-20 CTL epitope by A9-TCR-transgenic T cells. Collectively, our data indicate that HPV16E7 TCR gene transfer is feasible as an alternative strategy to generate human HPV16E7-specific T cells for the treatment of patients suffering from cervical cancer and other HPV16-induced malignancies.