Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States.
Division of Infectious Diseases, Center for Tuberculosis Research, Department of Medicine, The Johns Hopkins Hospital, Baltimore, MD, United States.
Front Immunol. 2023 Jan 19;13:1074644. doi: 10.3389/fimmu.2022.1074644. eCollection 2022.
DNA vaccines containing a fusion of the gene encoding chemokine MIP-3α (CCL20), the ligand for CCR6 on immature dendritic cells (DCs), to melanoma-associated antigen genes have enhanced anti-tumor immunity and efficacy compared to those lacking the chemokine gene. Previous work has shown that type-I interferon (IFNα or IFN) and 5-Aza-2'-deoxycytidine (5Aza) significantly enhance the therapeutic benefit of DNA vaccines as measured by reduced tumor burden and improved mouse survival.
Here, we explored mouse intratumoral immune correlates underlying the therapeutic benefit of this combination regimen (vaccine, IFN, and 5Aza) as compared to vaccine alone and IFN and 5Aza without vaccine, focusing on chemokine mRNA expression by qRT-PCR and inflammatory cellular infiltration into the tumor microenvironment (TME) by flow cytometry and immunohistochemistry (IHC).
The combination group significantly upregulated intratumoral mRNA expression of key immune infiltration chemokines XCL1 and CXCL10. Flow cytometric analyses of tumor suspensions exhibited greater tumor infiltration of CD8+ DCs, CCR7+ DCs, and NK cells in the combination group, as well as reduced levels of myeloid-derived suppressor cells (MDSCs) in vaccinated groups. The mice receiving combination therapy also had greater proportions of effector/memory T-cells (Tem), in addition to showing an enhanced infiltration of Tem and central memory CD8+ T-cells, (Tcm). Tem and Tcm populations both correlated with smaller tumor size. Immunohistochemical analysis of tumors confirmed that CD8+ cells were more abundant overall and especially in the tumor parenchyma with combination therapy.
Efficient targeting of antigen to immature DCs with a chemokine-fusion vaccine offers a potential alternative approach to classic and dendritic cell-based vaccines. Combining this approach with IFNα and 5Aza treatments significantly improved vaccine efficacy. This treatment creates an environment of increased inflammatory chemokines that facilitates the trafficking of CD8+ DCs, NK cells, and CD8+ T-cells, especially memory cells, while reducing the number of MDSCs. Importantly, in the combination group, CD8+ cells were more able to penetrate the tumor mass in addition to being more numerous. Further analysis of the pathways engaged by our combination therapy is expected to provide additional insights into melanoma pathogenesis and facilitate the development of novel treatment strategies.
含有趋化因子 MIP-3α（CCL20）基因融合的 DNA 疫苗，该基因编码的趋化因子与未成熟树突状细胞（DCs）上的 CCR6 配体结合，与缺乏趋化因子基因的疫苗相比，增强了抗肿瘤免疫和疗效。以前的工作表明，I 型干扰素（IFNα 或 IFN）和 5-Aza-2'-脱氧胞苷（5Aza）显著增强了 DNA 疫苗的治疗益处，表现为肿瘤负担减少和小鼠存活率提高。
在这里，我们探索了这种联合方案（疫苗、IFN 和 5Aza）治疗益处的小鼠肿瘤内免疫相关性，与单独疫苗和没有疫苗的 IFN 和 5Aza 相比，重点关注 qRT-PCR 检测的趋化因子 mRNA 表达和流式细胞术和免疫组织化学（IHC）检测的肿瘤微环境（TME）中的炎症细胞浸润。
联合组显著上调了肿瘤内关键免疫浸润趋化因子 XCL1 和 CXCL10 的 mRNA 表达。肿瘤混悬液的流式细胞分析显示，联合组中 CD8+DC、CCR7+DC 和 NK 细胞的肿瘤浸润程度更高，而接种组的髓系来源抑制细胞（MDSCs）水平降低。接受联合治疗的小鼠还具有更高比例的效应/记忆 T 细胞（Tem），此外还显示 Tem 和中央记忆 CD8+T 细胞（Tcm）的浸润增强。Tem 和 Tcm 群体都与肿瘤体积减小相关。肿瘤的免疫组织化学分析证实，与单独使用联合疗法相比，CD8+细胞总体上更丰富，尤其是在肿瘤实质中。
用趋化因子融合疫苗将抗原有效靶向未成熟 DC，为经典和树突状细胞疫苗提供了一种潜在的替代方法。将这种方法与 IFNα 和 5Aza 治疗相结合，显著提高了疫苗的疗效。这种治疗创造了一个炎症趋化因子增加的环境，促进了 CD8+DC、NK 细胞和 CD8+T 细胞的迁移，尤其是记忆细胞，同时减少了 MDSCs 的数量。重要的是，在联合组中，CD8+细胞不仅更多，而且更能穿透肿瘤块。对我们联合治疗所涉及的途径的进一步分析预计将为黑色素瘤发病机制提供更多的见解，并促进新的治疗策略的发展。
