The connection of innate and adaptive anti-cancer immunity

使用重组鸡痘病毒靶向传递小鼠干扰素-γ：自然杀伤(NK)细胞在区域淋巴结中的募集和肿瘤特异性宿主免疫的启动。干扰素是一种促炎细胞因子，也是一种有效的免疫调节剂。在这项研究中，一种复制缺陷的重组禽类(鸡痘)病毒被设计成表达小鼠干扰素-γ基因(RF-Mu干扰素-γ)，其原理是将细胞因子的集中水平输送到局部组织微环境中。皮下(S.C.)给药后，干扰素-γ的产生仅限于注射部位的组织微环境，(2)具有生物活性，主要组织相容性复合体(MHC)在S.C.的表达水平显著增加。射频-干扰素-伽玛刀治疗后肿瘤生长。用RF-MuIFN-γ感染高致瘤性小鼠细胞系MC38，可以作为一种有效的肿瘤细胞疫苗，保护小鼠免受原发肿瘤的形成和后续的肿瘤攻击。如果用MC38细胞接种小鼠，无论是经过rIFN-γ预处理的MC38细胞还是感染野生型鸡痘病毒(FP-WT)的小鼠，基于细胞的疫苗都是完全无效的。对注射RF-MuIFN-γ的肿瘤细胞疫苗注射部位的区域淋巴结的分析显示，存在肿瘤特异性细胞裂解(CTL)以及大量针对NK敏感的YAC-1细胞的裂解。流式细胞仪分析结合功能分析证实，在注射RF-MuIFN-Gamma后，NK1.1(+)细胞在这些引流淋巴结内持续存在长达5天。在注射感染了RF-MuIFN-Gamma的MC38肿瘤细胞之前，用NK细胞耗尽抗体治疗的小鼠并没有受到原发肿瘤生长的保护；对NK耗竭小鼠注射部位的淋巴引流的分析显示，伴随着肿瘤特异性CTL活性的丧失。这些发现提供的证据表明，NK细胞也提供了发展适应性免疫反应所需的免疫调节信号，这反过来又保护了接种疫苗的小鼠免受肿瘤生长。NK细胞以其对宿主天然免疫的贡献而闻名。干扰素-α是一种多效性细胞因子，具有免疫调节特性，可提高治疗性癌症疫苗的疗效。本项目的目的是评估重组干扰素与针对人类癌胚抗原(CEA)的痘病毒疫苗在以CEA为自身抗原的结直肠癌和胰腺癌小鼠模型中的有效性和兼容性。在无肿瘤小鼠的引流腹股沟淋巴结中，评估了干扰素的表型和功能效应。我们研究了干扰素注射部位(局部和远端)对痘病毒疫苗抗原特异性免疫反应的影响。机制研究是评估干扰素-a和CEA导向的痘病毒疫苗在荷瘤CEA转基因小鼠中的效果。我们确定了干扰素的剂量和时间表，该剂量和时间表可以诱导引流腹股沟淋巴结的局部扩张，并改善细胞毒性(自然杀伤和CD8+)和抗原呈递。通过在接种部位远端注射干扰素，可避免抑制痘苗病毒。干扰素和疫苗的组合抑制了肿瘤的生长，提高了存活率，并在CEA+腺癌的小鼠中引发了CEA特异性的CTL反应。在患有胰腺肿瘤的小鼠中，干扰素可减缓肿瘤生长，诱导CTL活性，并增加CD8+肿瘤浸润性淋巴细胞。这些数据表明，干扰素可用作抗原导向的痘病毒疫苗的生物反应调节剂，以产生显著的治疗性抗肿瘤免疫反应。这项研究提供了理论基础和机制见解，支持在CEA表达的癌症患者中进行这种免疫治疗策略的临床试验。

Targeted delivery of murine interferon (IFN)-gamma using a recombinant fowlpox virus: natural killer (NK) cell recruitment to regional lymph nodes and priming of tumor-specific host immunity. IFN-gamma is a proinflammatory cytokine that also acts as a potent immunomodulatory agent. In this study, a replication-deficient recombinant avian (fowlpox) virus was engineered to express the murine IFN-gamma gene (rF-MuIFN-gamma) with the rationale of delivering concentrated levels of the cytokine to a local tissue microenvironment. Subcutaneous (s.c.) rF-MuIFN-gamma administration resulted in IFN-gamma production that was restricted to the tissue microenvironment of the injection site and (2) was biologically active, as evidenced by a significant increase of class I major histocompatibility complex (MHC) expression levels in s.c. growing tumors following rF-MuIFN-gamma administration. Infection of a highly tumorigenic murine cell line, MC38, with rF-MuIFN-gamma functioned as an effective tumor cell-based vaccine by protecting mice from the formation of primary tumors and from subsequent tumor challenge. The cell-based vaccine was completely ineffective if mice were vaccinated with MC38 cells either pretreated with rIFN-gamma or infected with the wild-type fowlpox virus (FP-WT). Analysis of the regional lymph nodes draining the site of injection of the rF-MuIFN-gamma-based tumor cell vaccine revealed the presence of tumor-specific cell lysis (CTL) as well as a significant amount of lysis directed at NK-sensitive YAC-1 cells. Flow cytometric analyses coupled with functional assays confirmed the sustained presence of NK1.1(+) cells within those draining lymph nodes for up to 5 days after rF-MuIFN-gamma injection. Mice treated with NK cell-depleting antibodies prior to the injection of the rF-MuIFN-gamma-infected MC38 tumor cells were not protected from primary tumor growth; analysis of the lymph nodes draining the injection site in NK-depleted mice revealed an accompanying loss of the tumor-specific CTL activity. The findings provide evidence that NK cells, known for their contributions to host innate immunity, also provide immunoregulatory signals required for the development of an adaptive immune response, which, in turn, protected vaccinated mice against tumor growth. Interferon-alpha (IFN-α) is a pleiotropic cytokine possessing immunomodulatory properties that may improve the efficacy of therapeutic cancer vaccines. The aim of this project was to evaluate the effectiveness and compatibility of combining recombinant IFN-α with poxvirus vaccines targeting the human carcinoembryonic antigen (CEA) in murine models of colorectal and pancreatic adenocarcinomas, where CEA is a self-antigen. The phenotypic and functional effects of IFN-α were evaluated in the draining inguinal lymph nodes of tumor-free mice. We studied the effect of the site of IFN-α administration (local versus distal) on antigen-specific immune responses to poxvirus vaccination. Mechanistic studies were conducted to assess the efficacy of IFN-a and CEA-directed poxvirus vaccines in tumor-bearing CEA transgenic mice. We identified a dose and schedule of IFN-α that induced a locoregional expansion of the draining inguinal lymph nodes and improved cellular cytotoxicity (natural killer and CD8+) and antigen presentation. Suppression of the vaccinia virus was avoided by administering IFN-α distal to the site of vaccination. The combination of IFN-α and vaccine inhibited tumor growth, improved survival, and elicited CEA-specific CTL responses in mice with CEA+ adenocarcinomas. In mice with pancreatic tumors, IFN- α slowed tumor growth, induced CTL activity, and increased CD8+ tumor-infiltrating lymphocytes. These data suggest that IFN- α can be used as a biological response modifier with antigen-directed poxvirus vaccines to yield significant therapeutic antitumor immune responses. This study provides the rationale and mechanistic insights to support a clinical trial of this immunotherapeutic strategy in patients with CEA-expressing carcinomas.