干扰素α增强HCA587长肽疫苗的抗肿瘤效应及机制研究

Synthetic long peptide (SLP) vaccines are economical and safe for tumor patients. However the clinical effects often fail, which is possibly duo to scarce CD8+ T cell response induced by SLP vaccines. Cross-presentation is the key to the activation of CD8+ T cell response elicited by synthetic long peptide (SLP) vaccines. IFN-α can promote DC cross-prime CD8+ T cells, beneficial to overcome the bottleneck of inefficient anti-tumor effects of SLP vaccines. This study will immunize wide-type (WT) mice with the combination of IFN-α and HCA587 SLP vaccine, and evaluate CD8+ T cell responses through enzyme-linked immune spot assay (ELISpot), intracellular cytokine staining and cytotoxicity assay; treat melanoma-bearing WT mice and IFNAR-/- mice with the combination of IFN-α and HCA587 SLP vaccine, and with or without deletion of CD8+ T cells, analyzed the phenotype, percentage and function of CD8+ T cells in tumor sites, and their correlation with tumor burden and the survival of tumor-bearing mice, to make sure the anti-tumor effects of HCA587 SLP vaccine-induced CD8+ T cells enhanced by IFN-α; isolate DC, find out the target molecules with differential expression by cDNA microarray assay screening the molecules involved in signal transduction and cross presentation, thus through further knock-down of the target molecules, analyze the activation of CD8+ T cells by DC cross-presenting HCA587 SLP, to explore the mechanisms of IFN-α enhancing the activation of CD8+ T cells elicited by HCA587 SLP vaccine. Our study will provide the new thread and experimental foundation for enhancing the clinical effects of therapeutic tumor SLP vaccines.

长肽疫苗是一种经济安全的肿瘤疫苗，但是临床治疗效果不佳，这与其诱导CD8+ T细胞应答低下有关。交叉提呈是决定长肽疫苗活化CD8+ T细胞效能的关键环节。IFN-α促进DC交叉活化CD8+ T细胞，有助于克服长肽疫苗抗肿瘤效能不佳的瓶颈。本课题拟采用IFN-α联合HCA587长肽（HCA587 SLP）疫苗的免疫模型（WT小鼠），通过ELISpot，ICCS和杀伤实验检测CD8+ T细胞的活化情况；及肿瘤治疗模型（WT和IFNAR-/-），分析肿瘤部位CD8+ T细胞的表型，比例和功能，及与肿瘤大小，生存期的关系；分选DC，芯片检测DC信号通路和交叉提呈途径相关分子的表达差异，找出差异表达分子，进行沉默干扰，观察DC交叉提呈HCA587 SLP活化CD8+ T细胞的变化，初步探讨IFN-α影响DC交叉提呈长肽活化CD8+ T细胞的机制，为提高长肽疫苗的肿瘤治疗效果提供新的思路及实验基础。

长肽（SLP）疫苗是一种经济安全的肿瘤疫苗，但是临床治疗效果不佳，这与其诱导CD8+ T细胞应答低下有关。HCA587 SLP与TLR9配体CpG ODN1826和弗氏佐剂联合应用可产生较强的Th1型CD4+ T细胞免疫应答，而CD8+ T细胞未能有效活化，可能是导致抗肿瘤效果欠佳的原因。促使DC交叉提呈活化CD8+ T细胞是解决该问题的关键，IFN-α具备活化DC交叉激活CD8+ T细胞应答的特征。CpG ODN 2395是IFN-α的有效诱导剂，本研究使用ODN 2395， HCA587 SLP 和AddaVax乳化后免疫小鼠，通过ELISpot，ICCS检测，发现该疫苗组合可产生较强的特异性细胞免疫应答，且可激发CD8+T细胞分泌IFN-γ和Granzyme B；进一步通过B16-HCA587黑色素瘤的肿瘤治疗模型观察HCA587长肽+AddaVax+ODN2395疫苗的抗肿瘤效果。与AddaVax+ODN2395佐剂对照组和不治疗组相比，HCA587长肽+AddaVax+ODN2395疫苗治疗组的小鼠肿瘤体积显著减小，生长显著减慢；疫苗治疗组中荷瘤小鼠的生存期也较佐剂组和不治疗组的显著延长。在接种B16-HCA587肿瘤细胞后58天，HCA587长肽+AddaVax+ODN2395疫苗治疗组仍有1只荷瘤小鼠存活，检测该小鼠脾细胞的免疫应答情况，发现CD8+T细胞分泌Granzyme B，在其他HCA587 SLP治疗组的荷瘤小鼠中观察到脾细胞和肿瘤浸润淋巴细胞均有IFN-γ的分泌（CD4+ T细胞）和颗粒酶B的分泌（CD8+T细胞），且颗粒酶B的分泌与肿瘤生长大小存在一定的相关性。.我们的研究发现HCA587长肽与AddaVax+ODN2395的联合疫苗可交叉激活CD8+T细胞分泌颗粒酶B和IFN-γ，并产生显著的抗肿瘤效应。