BackgroundThe mechanisms by which vaccinia virus (VACV) interacts with the innate immune components are complex and involve different mechanisms. iNOS-mediated NO production by myeloid cells is one of the central antiviral mechanisms and this study aims to investigate specifically whether iNOS-mediated NO production by myeloid cells, is involved in tumor eradication following the virus treatment.MethodsHuman colon adenocarcinoma (HCT-116) xenograft tumors were infected by VACV. Infiltration of iNOS+ myeloid cell population into the tumor, and virus titer was monitored following the treatment. Single-cell suspensions were stained for qualitative and quantitative flow analysis. The effect of different myeloid cell subsets on tumor growth and colonization were investigated by depletion studies. Finally, in vitro culture experiments were carried out to study NO production and tumor cell killing. Student’s t test was used for comparison between groups in all of the experiments.ResultsInfection of human colon adenocarcinoma (HCT-116) xenograft tumors by VACV has led to recruitment of many CD11b+ ly6G+ myeloid-derived suppressor cells (MDSCs), with enhanced iNOS expression in the tumors, and to an increased intratumoral virus titer between days 7 and 10 post-VACV therapy. In parallel, both single and multiple rounds of iNOS-producing cell depletions caused very rapid tumor growth within the same period after virus injection, indicating that VACV-induced iNOS+ MDSCs could be an important antitumor effector component. A continuous blockade of iNOS by its specific inhibitor, L-NIL, showed similar tumor growth enhancement 7–10 days post-infection. Finally, spleen-derived iNOS+ MDSCs isolated from virus-injected tumor bearing mice produced higher amounts of NO and effectively killed HCT-116 cells in in vitro transwell experiments.ConclusionsWe initially hypothesized that NO could be one of the factors that limits active spreading of the virus in the cancerous tissue. In contrast to our initial hypothesis, we observed that PMN-MDSCs were the main producer of NO through iNOS and NO provided a beneficial antitumor effect, The results strongly support an important novel role for VACV infection in the tumor microenvironment. VACV convert tumor-promoting MDSCs into tumor-killing cells by inducing higher NO production.
Oncolytic virotherapy is safe and clinically active in solid tumors, however its efficacy in hematologic malignancies as well as in combination with checkpoint inhibitors and radiation is unexplored.To simulate advanced lymphoma, A20 lymphoma cells were injected subcutaneously on bilateral flanks of BALB/c mice and treatment initiated on day 17 to only the right flank tumor with local irradiation (Irr), intratumoral (i.t.) vaccinia virus (VACV) and i.t. anti-CTLA-4 mAb (Irr-VACV-CTLA4, Figure 1). The Irr-VACV-CTLA4 regimen was the most effective in eradicating or shrinking both treated and untreated tumors and extending survival, followed by the Irr-VACV regimen. Treatment with Irr-VACV-CTLA4, led to initially a mature, activated NK cell (KLRG1+CD27+) infiltrate (day 6 post-treatment) followed by a CD8+T cells infiltrate (day+13) in treated tumors. In contrast, treatment with VACV-CTLA4 led to activated NK-cell accumulation (day +6) followed by a CD8+T-cell infiltrate (day+13) in non-treated tumors. Importantly, CD8+CD44hi T cells isolated from the blood and spleens of the treated mice showed functional specificity to A20 lymphoma cells, but not to MHC-matched tumor cells (CT26) in intra-cellular stains for IFN-g. Splenocyte-derived A20-specific CD8+CD44hi T cells were induced most efficiently in the Irr-VACV-CTLA4 regimen-treated mice, while blood-derived A20-specific CD8+CD44hi T cells were induced most efficiently in the Irr-VACV regimen-treated mice. Viral plaque assays (VPA) showed lack of live viral particles in both treated and untreated tumors upon sacrificing mice 4 to 10 weeks after treatment initiation. Surprisingly, VPA assays identified live virus in the livers of Irr-VACV-CTLA4 regimen-treated mice, which paralleled a reduced metastatic load. Our findings are the first to demonstrate the potential of combination immunotherapy with oncolytic viruses and checkpoint inhibitors in hematologic malignancies. The antitumor activity is attributed to the induction of an effective and specific immune response. This finding is corroborated by the significant infiltration with mature activated NK cells, followed by CD8+T cells, in both treated and untreated tumors. Importantly, the tumor-specific CD8+T cells showing a memory phenotype (CD44hi) suggest the effective induction of a potent immune memory response. Effective targeting of distant metastases after intratumoral administration is also an important finding with significant clinical implications. This novel combination immunotherapy with oncolytic viruses and checkpoint inhibitors following local tumor irradiation is now being translated to a Phase 1 proof-of-concept clinical trial in non-Hodgkin's lymphoma at our institution. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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