IFNα and 5-Aza-2’-deoxycytidine combined with a dendritic-cell targeting DNA vaccine alter tumor immune cell infiltration in the B16F10 melanoma model

Abstract

IntroductionDNA 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.MethodsHere, 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).ResultsThe 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.DiscussionEfficient 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.

Combination of a MIP3α-antigen fusion therapeutic DNA vaccine with treatments of IFNα and 5-Aza-2’Deoxycytidine enhances activated effector CD8+ T cells expressing CD11c in the B16F10 melanoma model

“…Previous studies have shown the upregulation of chemokines CCL19, CCR7, XCL1, and CXCL10 in the tumor lysate of mice treated with the combination therapy outlined in Fig. 1A [ 4 , 8 ]. A qRT-PCR panel was performed to elucidate the change in expression of additional immune-related genes in the pooled tumor lysates between the groups receiving vaccine alone and combination therapy.…”

“…1B ). Our previous work showed enhanced infiltration of CD11c + DC[ 8 ], and we hypothesized that other cell types such as T cells may also be expressing CD11c.…”

“…6–12-week-old female C57BL/6 (Charles River, Wilmington, MA) mice were challenged with a lethal dose of B16F10 melanoma (5×10 4 cells, > 95% viability) administered intradermally on the mouse flank on day 0 of therapy[ 2 – 4 , 8 ]. Tumor size was recorded by calipers every 1 – 3 days as square mm (L × W).…”

“…Vaccine antigen is the MIP-3α-Gp100-Trp2 (tyrosinase-related protein 2) DNA construct in the pCMVe mammalian expression plasmid published here[ 8 ]. Therapy including vaccine, recombinant mouse interferon alpha-A (IFNα, R&D Systems, Inc. Minneapolis, MN), and InSolution ™ 5 Aza 2’-deoxycytidine (5Aza, CalBiochem ® , MilliporeSigma, Burlington, MA) has been detailed in prior publications[ 8 ] and is outlined in Fig. 1 A .…”

“…When MIP3α-Gp100-Trp2 vaccine adjuvanted with delayed administration of CpG type C (henceforth referred to as “vaccine”) was combined with the drug treatments low-dose 5Aza-2’Deoxycytidine (5Aza) and a series of high and low doses of recombinant IFNα (IFN) ( Fig. 1A ), significantly greater anti-tumor activity was achieved, including increased dendritic cell (DC) and CD8 + T-cell infiltration into the tumor microenvironment (TME)[ 4 , 8 ].…”