Characterization of the Impact of Oncolytic Vesicular Stomatitis Virus on the Trafficking, Phenotype, and Antigen Presentation Potential of Neutrophils and Their Ability to Acquire a Non-Structural Viral Protein

Abstract

Neutrophils are innate leukocytes that mount a rapid response to invading pathogens and sites of inflammation. Although neutrophils were traditionally considered responders to bacterial infections, recent advances have demonstrated that they are interconnected with both viral infections and cancers. One promising treatment strategy for cancers is to administer an oncolytic virus to activate the immune system and directly lyse cancerous cells. A detailed characterization of how the innate immune system responds to a viral-based therapy is paramount in identifying its systemic effects. This study analyzed how administering the rhabdovirus vesicular stomatitis virus (VSV) intravenously at 1 × 109 PFU acutely influenced neutrophil populations. Bone marrow, blood, lungs, and spleen were acquired three- and 24-h after administration of VSV for analysis of neutrophils by flow cytometry. Infection with VSV caused neutrophils to rapidly egress from the bone marrow and accumulate in the lungs. A dramatic increase in immature neutrophils was observed in the lungs, as was an increase in the antigen presentation potential of these cells within the spleen. Furthermore, the potential for neutrophils to acquire viral transgene-encoded proteins was monitored using a variant of VSV that expressed enhanced green fluorescent protein (GFP). If an in vitro population of splenocytes were exposed to αCD3 and αCD28, a substantial proportion of the neutrophils would become GFP-positive. This suggested that the neutrophils could either acquire more virus-encoded antigens from infected splenocytes or were being directly infected. Five different dosing regimens were tested in mice, and it was determined that a single dose of VSV or two doses of VSV administered at a 24-h interval, resulted in a substantial proportion of neutrophils in the bone marrow becoming GFP-positive. This correlated with a decrease in the number of splenic neutrophils. Two doses administered at intervals longer than 24-h did not have these effects, suggesting that neutrophils became resistant to antigen uptake or direct infection with VSV beyond 24-h of activation. These findings implicated neutrophils as major contributors to oncolytic rhabdoviral therapies. They also provide several clear future directions for research and suggest that neutrophils should be carefully monitored during the development of all oncolytic virus-based treatment regimens.

The Role of Neutrophils in Oncolytic Orf Virus-Mediated Cancer Immunotherapy

“…The analysis of neutrophils performed in the experiments demonstrated that oncolytic OrfV triggered a systemic inflammatory response from neutrophils, including their enhanced expression of trafficking and activation markers, and infiltration of the TME and cytotoxic effector functions. It is possible that the rapid infiltration of neutrophils to the site of metastases following treatment with OrfV was representative of an innate antiviral response to the administration of the virus, as has been previously documented [ 21 , 22 , 23 ]. However, the neutrophil response to the administration of OrfV did not appear to limit therapeutic efficacy but instead directly correlated with enhanced viral amplification, reduced tumor burden, and extended survival.…”

Type I Interferon-Mediated Regulation of Antiviral Capabilities of Neutrophils

“…TLR4, which recognizes lipopolysaccharide (LPS), was shown to be required for neutrophil migration to the lungs [ 7 ]. Neutrophils frequently travel to the lungs after a range of viral infections, including those caused by respiratory syncytial virus (RSV), highly pathogenic avian influenza virus, influenza A virus (IAV) [ 1 ], and vesicular stomatitis virus (VSV) [ 8 ]. However, neutrophils are still capable of killing a range of pathogens independent of TLRs [ 9 ].…”

Disruption of Type I Interferon Signaling Causes Sexually Dimorphic Dysregulation of Anti-Viral Cytokines

“…Since an increase in the frequency of granulocytes was demonstrated in the blood of mice that received recombinant rVSVΔm51, we sought to monitor infiltration of granulocytes into the organs. Oncolytic viruses can be detected in the lungs following intravenous administration [53] and accumulation of neutrophils becomes apparent in the lungs of mice within three hours of intravenous delivery of rVSVΔm51 [54] . Therefore, male and female mice were infected intravenously with 1 × 10 9 pfu of rVSVΔm51 to examine trafficking of neutrophils into the lungs.…”

“…A growing body of evidence describes the existence of biochemically and physically distinct neutrophil subsets in healthy and pathological conditions [87] . Using flow cytometry, we have recently demonstrated that a subset of neutrophils produced IL-12 and gained surface receptors associated with antigen-presenting cell functions [54] , while other groups characterized a subset of neutrophils that produced IL-10 and functioned as an immunomodulatory cell subset [88] . The current study suggests that a subset of neutrophils may play a regulatory role in cytokine responses to rVSVΔm51 when IFNAR signaling is impaired.…”