title: Role of parvovirus MVMp in generation of antitumor immune responses
creator: Grekova, Svitlana
subject: ddc-570
subject: 570 Life sciences
description: Rhodent autonomous parvoviruses display a pronounced tropism for tumors and their infection may ultimately result in lysis of established tumors. This work evaluated the efficacy of Minute Virus of Mice prototype (MVMp) as a stimulus of specific antitumor immune response against mouse melanoma and glioblastoma tumors.  MVMp was shown to effectively infect both melanoma B78/H1 and glioma GL261cell lines and induce cell death. MVMp was more effective inducing cell death in glioma GL261 cultures where 10% live cells were detected 96h after virus infection at multiplicity of infection (MOI) 10. In comparison only 40% cell death was observed in the melanoma B78/H1 cells after virus infection at high MOI 100. MVMp infected glioma GL261 cells displayed characteristics of apoptosis, such as caspase-3 activation and DNA fragmentation, which were not detected in melanoma.  Activation of antigen presenting cells (APC) provides the initial cue for an innate and adaptive immune response. Therefore, we generated tumor cell lysates after MVMp-infection and investigated their capacity to induce activation of dendritic cells (DCs) and microglia, specific subtypes of APC. Whereas MVMp infected B78/H1 melanoma cells were unable to activate DCs, virus infected glioma cells efficiently induced DC activation. Two different DC subpopulations (myeloid and lymphoid) were activated after co-culture with MVMp-infected glioma GL261 cells, and activation was monitored by upregulation of specific activation markers such as CD80, CD86, and MHC class II. In addition, the release of proinflammatory cytokines such as TNF-α and IL-6 was measured. Similarly, microglia were activated, when co-cultured with MVMp-infected glioma GL261 cells, measured by upregulation of activation markers and cytokine production. Taken together these results demonstrate that glioma GL261 cell lysates generated after MVMp infection can substantially activate DCs as well as microglia. Toll-like receptors (TLRs) expressed in DCs and microglia mediate crucial signaling pathways initiating effective innate and adaptive immune responses. To assess the contribution of TLR signaling in the context of PV-based virotherapy, stable cell lines expressing single murine TLRs were established. These cells lines were used as a model to correlate specific TLR activation with the exposure to virus induced tumor cell lysates. A direct downstream target of the TLR signaling cascade is the transcription factor NF-κB and thus a reporter approach was utilized to measure NF-κB activation. Upon culture of single TLR expressing stable cell lines with MVMp-mediated tumor cell lysates, the cell line stably expressing TLR3 had an eight fold higher activation of NF-κB reporter gene expression compared to control. This finding called for a role of TLR3 mediated signaling in the activation NF-κB and downstream target genes such as pro- and inflammatory cytokines.  The in vitro findings were extended to in vivo studies and antitumoral effects mediated by MVMp were assessed in immunedeficient RAG2-/- and immunocompetent C57BL/6 mice. Immunocompetent mice completely (100%) rejected MVMp-infected glioma GL261 at MOI 3 and MOI 30 with greater efficiency than control tumor cells (17%). Eradication of tumor cells mediated by the oncolytic effect of MVMp at MOI 30 amounted to only 20% in immunodeficient RAG2-/- animals. In addition, the release of IFN-γ from splenocytes of mice, which were injected with MVM-infected glioma cells, in response to uninfected glioma cells was assessed ex vivo and was significantly higher (2.3 times more IFN-γ producing cells) compared to control splenocytes. These findings provide evidence of a specific immune response towards glioma GL261 cells and highlight the importance of a T cell mediated immune response to obtain a strong antitumor effect. Furthermore, it was shown in re-challenge experiments that mice were protected from tumor growth. This highlights the induction of a tumor specific memory response. In conclusion, MVMp induced GL261 tumor cell lysates stimulate activation of APC, initiate cytokine production via NF-κB signaling and in vivo initiate primary and secondary tumor specific immune responses. These findings could place PV as promising candidates to break tumor tolerance and treat glioma. 
date: 2008
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/8712/1/Thesis_Final_Version.pdf
identifier: DOI:10.11588/heidok.00008712
identifier: urn:nbn:de:bsz:16-opus-87126
identifier:   Grekova, Svitlana  (2008) Role of parvovirus MVMp in generation of antitumor immune responses.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/8712/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng