重组腺病毒 Ad–sh–SOCS1 增强宫颈癌树突状 细胞疫苗免疫反应的作用研究

〔摘 要〕 目的：研究沉默树突状细胞（DCs）中细胞因子信号抑制因子 1（SOCS1）能否强化宫颈癌癌蛋白 E7 的表位 肽 E7.49–57 脉冲 DCs 疫苗的免疫反应。方法：制备的重组腺病毒 Ad–sh–SOCS1 和 Ad–sh–mSOCS1 感染体外培养的小鼠 骨髓来源的树突状细胞（BMDCs）,用实时荧光定量聚合酶链式反应（RT–PCR）检测 SOCS1 的表达,用酶联免疫吸附试 验（ELISA）检测培养上清液肿瘤坏死因子 α（TNF–α）、白细胞介素（IL）–12、IL–6 的水平;并用表位肽 E7.49–57 脉冲 被感染的 DCs,体内、外研究对小鼠抗组成式表达 HPV16E6E7 的小鼠肺上皮肿瘤细胞（TC–1）的抗肿瘤效果。结果：重 组腺病毒 Ad–sh–SOCS1 能显著抑制 DCs 中 SOCS1 的表达。与 DCs 组和 Ad–sh–mSOCS1 感染 DCs 组比较,感染重组病毒 Ad–sh–SOCS1 的 DCs 分泌的细胞因子 IL–6、IL–12 和 TNF–α 显著增多,差异具有统计学意义（P ＜ 0.05）。E7.49–57 多 肽脉冲的 Ad–sh–SOCS1 感染的 DCs 在体内及体外均能显著抑制 TC–1 肿瘤细胞的生长,差异具有统计学意义（P ＜ 0.05）。 结论：重组腺病毒 Ad–sh–SOCS1 能增强宫颈癌 DC 疫苗的抗瘤免疫反应。     

Murine CD8(+)T cell cytotoxicity against schistosomula induced by inoculation of schistosomal 22.6/26GST coupled Sepharose 4B beads

Schistosomasis is a world-wide parasitic disease. Although chemotherapy is the main treatment method for schistosomasis currently, it cannot prevent schistosome reinfection. Up to now no effective vaccine is available to prevent schistosomiasis. Dendritic cells (DCs) are one of the key players in the cellular immune response and play an important role in antigen presentation as antigen-presenting cells. Here we reported a novel large particulate antigen, in which Sepharose 4B beads were coated with Sj22.6/26GST. Our results showed that this particulate antigen could be cross-presented by DCs to CD8(+)T cells. Furthermore, CD8(+)T cells stimulated by particulate antigen directly exerted cytotoxicity against Schistosoma japonicum schistosomula. We also demonstrated that S. japonicum schistosomula acquired the MHC class I molecules from host blood serum and presented the molecules at the larval surface. While it may help them escape from the host immune surveillance, these MHC I-antigen complexes presented on the surface render schistosomula the potential targets of the CD8(+)T cell cytotoxicity induced by particulate antigen-based vaccine. Finally we evaluated the protective immunity of this particulate vaccine in a mouse infection challenge model. Our data clearly showed that the particulate vaccine induced a partial reduction in both worm burdens and egg loads. Taken together, these results suggest that this large particulate vaccine could be a potential vaccine for the prevention of schistosome infection.     

Induction of antiviral cytotoxic T cells by plasmacytoid dendritic cells for adoptive immunotherapy of posttransplant diseases

Virus-associated hematologic malignancies (EBV lymphoproliferative disease) and opportunistic infections (CMV) represent a major cause of hematopoietic stem cell and solid organ transplantation failure. Adoptive transfer of antigen-specific T lymphocytes appears to be a major and successful immunotherapeutic strategy, but improvements are needed to reliably produce high numbers of virus-specific T cells with appropriate requirements for adoptive immunotherapy that would allow extensive clinical use. Since plasmacytoid dendritic cells (pDCs) are crucial in launching antiviral responses, we investigated their capacity to elicit functional antiviral T-cell responses for adoptive cellular immunotherapy using a unique pDC line and antigens derived from Influenza, CMV and EBV viruses. Stimulation of peripheral blood mononuclear cells from HLA-A*0201(+) donors by HLA-A0201 matched pDCs pulsed with viral-derived peptides triggered high levels of multi-specific and functional cytotoxic T-cell responses (up to 99% tetramer(+) CD8 T cells) in vitro. Furthermore, the central/effector memory cytotoxic T cells elicited by the pDCs strongly display antiviral activity upon adoptive transfer into a humanized mouse model that mimics a virus-induced malignancy. We provide a simple and potent method to generate virus-specific CTL with the required properties for adoptive cellular immunotherapy of post-transplant diseases.     

β-榄香烯联合DC/DRibble疫苗促进脾细胞增殖及活化研究

目的:通过观察β-榄香烯联合DC/DRibble疫苗对小鼠脾细胞增殖和IFN-γ分泌水平的影响,探讨β-榄香烯抗肿瘤的免疫机制。方法:制备Balb/c小鼠脾脏来源的树突状细胞(dentritic cell,DC)并鉴定其表型,以Balb/c小鼠来源的肝癌细胞株BNL1MEA.7R.1(BNL)为靶细胞,募集富含肿瘤"信息"的抗原载体—自噬小体(Drips in blebs,DRibble),制备DC/DRibble疫苗。用CCK-8法检测β-榄香烯联合DC/DRibble疫苗对脾细胞的增殖作用,用ELISA法检测培养上清液中IFN-γ的水平。结果:β-榄香烯在合适浓度下联合DC/DRibble疫苗能促进脾细胞增殖并刺激效应性淋巴细胞产生高水平的IFN-γ。结论:β-榄香烯可能通过增强DC抗原递呈功能以发挥抗肿瘤作用。     

Human Langerhans cells capture measles virus through Langerin and present viral antigens to CD4⁺ T cells but are incapable of cross-presentation

Langerhans cells (LCs) are a subset of DCs that reside in the upper respiratory tract and are ideally suited to sense respiratory virus infections. Measles virus (MV) is a highly infectious lymphotropic and myelotropic virus that enters the host via the respiratory tract. Here, we show that human primary LCs are capable of capturing MV through the C-type lectin Langerin. Both immature and mature LCs presented MV-derived antigens in the context of HLA class II to MV-specific CD4(+) T cells. Immature LCs were not susceptible to productive infection by MV and did not present endogenous viral antigens in the context of HLA class I. In contrast, mature LCs could be infected by MV and presented de novo synthesized viral antigens to MV-specific CD8(+) T cells. Notably, neither immature nor mature LCs were able to cross-present exogenous UV-inactivated MV or MV-infected apoptotic cells. The lack of direct infection of immature LCs, and the inability of both immature and mature LCs to cross-present MV antigens, suggest that human LCs may not be directly involved in priming MV-specific CD8(+) T cells. Immune activation of LCs seems a prerequisite for MV infection of LCs and subsequent CD8(+) T-cell priming via the endogenous antigen presentation pathway.     

The subcellular location of antigen expressed by adenoviral vectors modifies adaptive immunity but not dependency on cross-presenting dendritic cells

Adenoviral (Ad) vaccine vectors can generate protective immunity to various pathogens in animal studies. However, recent failures in clinical vaccine trials have underscored the need for a better understanding of how mucosal immune responses to Ad-encoded vaccine Ags are generated in vivo. In this study, we addressed whether directing Ad-encoded ovalbumin (OVA) to different subcellular compartments influences the generation of OVA-specific acquired immunity and the APCs required following i.n. immunization of mice. We show that both secreted and membrane-anchored OVA activate CD4(+) T cells, induce cytotoxic CD8(+) T lymphocytes (CTLs) and generate serum IgG. Additionally, vaginal IgG is induced when OVA is expressed at these subcellular locations, but only the secreted form generates a significant IgA response in the lungs. On the contrary, intracellular expression of OVA efficiently expands CD8(+) T cells but fails to activate CD4(+) T cells, results in poor CTL activity, and does not generate Abs. Finally, we show that regardless of the subcellular localization of OVA, conventional DCs (cDCs) are required for the activation of T cells. However, the direct transduction of conventional DCs is not essential. These findings have important implications for the improvement of Ad vector design and vaccine-induced mucosal immunity.     

Tim-1 stimulation of dendritic cells regulates the balance between effector and regulatory T cells

We show that the T-cell immunoglobalin mucin, Tim-1, initially reported to be expressed on CD4(+) T cells, is constitutively expressed on dendritic cells (DCs) and that its expression further increases after DC maturation. Tim-1 signaling into DCs upregulates costimulatory molecule expression and proinflammatory cytokine production, thereby promoting effector T-cell responses, while inhibiting Foxp3(+) Treg responses. By contrast, Tim-1 signaling in T cells only regulates Th2 responses. Using a high-avidity/agonistic anti-Tim-1 antibody as a co-adjuvant enhances the immunogenic function of DCs, decreases the suppressive function of Tregs, and substantially increases proinflammatory Th17 responses in vivo. The treatment with high- but not low-avidity anti-Tim-1 not only worsens experimental autoimmune encephalomyelitis (EAE) in susceptible mice but also breaks tolerance and induces EAE in a genetically resistant strain of mice. These findings indicate that Tim-1 has an important role in regulating DC function and thus shifts the balance between effector and regulatory T cells towards an enhanced immune response. By understanding the mechanisms by which Tim-1 regulates DC and T-cell responses, we may clarify the potential utility of Tim-1 as a target of therapy against autoimmunity, cancer, and infectious diseases.     

Regulation of antigen-expressing dendritic cells by double negative regulatory T cells

TCRαβ(+) CD3(+) CD4(-) CD8(-) NK1.1(-) double negative (DN) Tregs comprise 1-3% of peripheral T lymphocytes in mice and humans. It has been demonstrated that DN Tregs can suppress allo-, xeno- and auto-immune responses in an Ag-specific fashion. However, the mechanisms by which DN Tregs regulate immune responses remain elusive. Whether DN Tregs can regulate DCs has not been investigated previously. In this study, we demonstrate that DN Tregs express a high level of CTLA4 and are able to down-regulate costimulatory molecules CD80 and CD86 expressed on Ag-expressing mature DCs (mDCs). DN Tregs from CTLA4 KO mice were not able to downregulate CD80 and CD86 expression, indicating that CTLA4 is critical for DN Treg-mediated downregulation of costimulatory molecule expression on Ag-expressing mature DCs. Furthermore, DN Tregs could kill both immature and mature allogeneic DCs, as well as Ag-loaded syngeneic DCs, in an Ag-specific manner in vitro and in vivo, mainly through the Fas-FasL pathway. These data demonstrate, for the first time, that DN Tregs are potent regulators of DCs and may have the potential to be developed as a novel immune suppression treatment.     

Steady state migratory RelB+ langerin+ dermal dendritic cells mediate peripheral induction of antigen-specific CD4+ CD25+ Foxp3+ regulatory T cells

Tolerance to self-antigens expressed in peripheral organs is maintained by CD4(+) CD25(+) Foxp3(+) Treg cells, which are generated as a result of thymic selection or peripheral induction. Here, we demonstrate that steady-state migratory DCs from the skin mediated Treg conversion in draining lymph nodes of mice. These DCs displayed a partially mature MHC II(int) CD86(int) CD40(hi) CCR7(+) phenotype, used endogenous TGF-β for conversion and showed nuclear RelB translocation. Deficiency of the alternative NF-κB signaling pathway (RelB/p52) reduced steady-state migration of DCs. These DCs transported and directly presented soluble OVA provided by s.c. implanted osmotic minipumps, as well as cell-associated epidermal OVA in transgenic K5-mOVA mice to CD4(+) OVA-specific TCR-transgenic OT-II T cells. The langerin(+) dermal DC subset, but not epidermal Langerhans cells, mediated conversion of naive OT-II×RAG-1(-/-) T cells into proliferating CD4(+) CD25(+) Foxp3(+) Tregs. Thus, our data suggest that steady-state migratory RelB(+) TGF-β(+) langerin(+) dermal DCs mediate peripheral Treg conversion in response to epidermal antigen in skin-draining lymph nodes.