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.
Lengthy tuberculosis (TB) treatment is required to overcome the ability of a subpopulation of persistent Mycobacterium tuberculosis (Mtb) to remain in a non-replicating, antibiotic-tolerant state characterized by metabolic remodeling, including induction of the Rel Mtb -mediated stringent response. We developed a novel therapeutic DNA vaccine containing a fusion of the rel Mtb gene with the gene encoding the immature dendritic cell-targeting chemokine, MIP-3a/CCL20. To augment mucosal immune responses, intranasal delivery was also evaluated. We found that intramuscular delivery of the MIP-3a/rel Mtb (fusion) vaccine or intranasal delivery of the rel Mtb (nonfusion) vaccine potentiate isoniazid activity more than intramuscular delivery of the DNA vaccine expressing rel Mtb alone in a chronic TB mouse model (absolute reduction of Mtb burden: 0.63 log 10 and 0.5 log 10 colony-forming units, respectively; P=0.0002 and P=0.0052), inducing pronounced Mtbprotective immune signatures. The combined approach involving intranasal delivery of the DNA MIP-3a/rel Mtb fusion vaccine demonstrated the greatest mycobactericidal activity together with isoniazid when compared to each approach alone (absolute reduction of Mtb burden: 1.13 log 10 , when compared to the intramuscular vaccine targeting rel Mtb alone; P<0.0001), as well as robust systemic and local Th1 and Th17 responses. This DNA vaccination strategy may be a promising adjunctive approach combined with standard Frontiers in Immunology frontiersin.org 01
Previous studies in the B16F10 mouse melanoma model have demonstrated that combining a DNA vaccine comprised of regions of gp100 and tyrosinase-related protein 2 fused to Macrophage-inflammatory protein 3-alpha (MIP3α) with recombinant Interferon alpha (IFN) and 5-Aza-2’-Deoxycytidine (5Aza) treatments resulted in significantly greater anti-tumor activity and immunogenicity in the tumor microenvironment (TME). This brief report details that the combination of vaccine with treatments IFN and 5Aza results in both the upregulation of genes expressing CD11c-interacting proteins and an increase in the TME of a distinct CD11c+ CD8+ T cell population. This cell population correlates with tumor size, is primarily comprised of effector or effector memory T cells, and has a more robust response to ex vivo stimulation as compared to CD11c- CD8+ T cells as measured by surface activation markers 4-1BB (CD137) and KLRG1 (Killer cell lectin-like receptor G1) and intracellular IFNγ production. In conclusion, this combination therapy results in greater presence of highly active effector CD8+ T-cells expressing CD11c in the TME that correlate with and are likely primary contributors to treatment efficacy.
Background: The chemokine MIP-3α (CCL20) binds to CCR6 on immature dendritic cells (DCs). DNA vaccines fusing MIP-3α to melanoma-associated antigens gp100 and/or tyrosinase-related protein 2 (Trp2) have delayed tumor growth and increased survival time in the B16F10 mouse melanoma model system compared to vaccines lacking the chemokine. To further enhance the therapeutic effects of the vaccine, our laboratory has added type-I interferon (IFNα) and 5-Aza-2'-deoxycitidine (5Aza) to the therapy. Here, we report that the enhancement previously seen by the combination of IFNα, 5Aza, and a MIP-3α-Gp100-Trp2 (MGpTrp2) DNA vaccine correlates with increases of tumor-infiltrating CD8+T-cells (CD8 TILs) and intratumoral expression of CCL19 but not CCL21. Methods: Beginning on day five post-transplantation of B16F10 melanoma, vaccine was administered intramuscularly (i.m.) by electroporation. CpG adjuvant was given two days later. 5Aza was given intraperitoneally at 1mg/kg and IFNα therapy either intratumorally or i.m. as noted. Tumor sizes, tumor growth, and mouse survival were assessed. Tumor lysate gene expression levels and tumor-infiltrating lymphocytes (TILs) were assessed by qRT-PCR and flow cytometry with intracellular cytokine staining, respectively. Results: Previous work has shown that the combination of IFNα, 5Aza, and MGpTrp2 led to significantly reduced tumor burden and overall increases in mouse survival dependent upon all three components. The addition of 5Aza and IFNα to the vaccine affected T-cell tumor infiltration, increasing the proportion of CD3+CD8+ cells in gated TILs by 92% over vaccine alone (p<0.0001), which correlated with tumor size (r2: 0.507; p<0.0001). Interferon stimulated genes such as Mx1, MHC1, CXCL10, and GranzymeB were modestly upregulated in the tumor lysate. However, CCL19, normally expressed constitutively in lymphoid tissues, was upregulated 10-fold compared to vaccine alone (p<0.0001) and was correlated with tumor size (r2: 0.195; p=0.002). The CCL19 receptor, CCR7, was upregulated to a lesser degree (5-fold over vaccine; p<0.001), and interestingly its partner chemokine CCL21 did not have significantly different expression patterns across groups (p=0.14 to vaccine). Conclusions: Efficient targeting of antigen to immature dendritic cells 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 enhancement was correlated with CD8+ TIL recruitment and with the expression of the T-cell trafficking chemokine CCL19. Further potential therapy optimization currently undergoing investigation offers promise for this line of investigation to become a novel melanoma therapy. Citation Format: James T. Gordy, Avinaash K. Sandhu, Samuel K. Ayeh, Aakanksha Kapoor, Emily Kim, Petros C. Karakousis, Richard B. Markham. The anti-tumor enhancement of a dendritic-cell targeting MIP3α-Gp100-Trp2 DNA vaccine by IFNα and 5-Aza-2'-deoxycytidine treatments correlates with intratumoral CCL19 but not CCL21 expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2198.
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