Towards understanding of human cytomegalovirus in glioblastoma

Human Cytomegalovirus (HCMV) is a widely prevalent herpesvirus. Following an active primary infection, often asymptomatic or subclinical in immunocompetent individuals, the virus establishes latency. HCMV produces several hundreds of proteins with immunomodulatory and immunosuppressive abilities to interfere with the immune defenses of the host in order to stay undetected and avoid elimination. During recent decades, HCMV proteins and nucleic acids have been detected in a variety of tumors. Through a combination of various immunosuppressive abilities, HCMV has been suggested to be a pathogen potentially contributing to the development of cancer. Glioblastoma Multiforme (GBM) is the most common primary intracranial tumor in adults and is associated with poor prognosis despite treatment. It has been shown that 99% of GBM express HCMV-IE (Immediate early) epitopes and that the level of HCMV-IE expression is a prognostic factor for patient survival. Therefore the main goal of my thesis work was to characterize the immune phenotype of HCMV infected GBM patients and to investigate the possible role of HCMV in gliomagenesis. This work is important for further development of therapies against GBM. As it is well accepted that T cells are central players in the immune response, we investigated whether HCMV subverts the immune system by directly inhibiting proliferation and activation of CD4-positive T cells (Paper 1). Our observations suggest that HCMV silences T cells, which has clinical consequences for both humoral and cell-mediated immunity and may explain the general immunosuppression observed in HCMV-infected patients.

To investigate the role of HCMV in gliomagenesis, we examined the ability of HCMV to induce a more aggressive cancer stem cell (CSC)-like phenotype in primary GBM cell lines (Paper 2). HCMV infection induced a stem cell phenotype in primary GBM cell lines as determined by changes in the cellular gene expression profile and by the conferred ability of cells to grow as neurospheres in vitro. As CSCs are known to be resistant to chemotherapy, our results imply that HCMV may enhance the malignancy grade of the tumor, and possibly contribute to therapy resistance.

To understand whether the immunological phenotype of GBM patients with HCMV infected tumors has an impact on overall survival, we investigated the T cell phenotypes of these patients (Paper 3). We found that patients with lower levels of CD3-positive cells had significantly shorter overall survival. GBM patients with signs of immunosenescence, as indicated by CD57 expression and loss of CD28 expression on CD4-positive T cells, had shorter overall survival. As the CD57+CD28- CD4+T cells only have been described in HCMV seropositive individuals, our findings suggest that the signs of immunosenescence in GBM patients may be linked to HCMV infection and indicate poor patient survival.

Since HCMV is carried by a large part of the population and by most of GBM patients, we investigated the serology status, the levels of HCMV DNA and RNA in blood and T cell activity against HCMV peptides in blood of GBM patients (Paper 4). All GBM patients were positive for HCMV proteins in the tumors and HCMV DNA in blood and T cell reactivity against HCMV peptides, but despite this, 29% of patients were HCMV IgG negative using three commercial ELISA tests. Some of these HCMV seronegative patients were IgG positive, when tested in an ELISA assay using antigens prepared from an HCMV clinical isolate. Thus, our results suggest that commercial serology tests are not reliable to detect an ongoing or previous HCMV infection in GBM patients.

We investigated cytokine patterns in blood of GBM patients with HCMV infected tumors and studied whether neutrophil activation is associated with HCMV infection and GBM progression (Paper 5). We found that GBM patients with higher neutrophil activity had shorter time to tumor progression and shorter survival. In conclusion, considering the immunomodulatory abilities of HCMV and its CSC-phenotype inducing potential in primary GBM cell lines, our results suggest an active role of HCMV in GBM and that this virus may be capable of driving GBM progression

List of scientific papers

I. Fornara O, Odeberg J, Khan Z, Stragliotto G, Peredo I, Butler L, Söderberg-Nauclér C. Human cytomegalovirus particles directly suppress CD4 T-lymphocyte activation and proliferation. Immunobiology. 2013 Aug;218:1034-40.
https://doi.org/10.1016/j.imbio.2013.01.002

II. Fornara O, Bartek J Jr, Rahbar A, Odeberg J, Khan Z, Peredo I, Hamerlik P, Bartek J, Stragliotto G, Landázuri N, Söderberg-Nauclér C. Cytomegalovirus infection induces a stem cell phenotype in human primary glioblastoma cells : prognostic significance and biological impact. Cell Death Differ. 2015 Jul 3.
https://doi.org/10.1038/cdd.2015.91

III. Fornara O, Odeberg J, Wolmer Solberg N, Tammik C, Skarman P, Peredo I, Stragliotto G, Rahbar A, Söderberg-Nauclér C. Poor survival in glioblastoma patients is associated with early signs of immunosenescence in the CD4 T-cell compartment after surgery. Oncoimmunology. 2015 Jun 5;4(9):e1036211.
https://doi.org/10.1080/2162402X.2015.1036211

IV. Rahbar A, Peredo I, Solberg NW, Taher C, Dzabic M, Xu X, Skarman P, Fornara O, Tammik C, Yaiw K, Wilhelmi V, Assinger A, Stragliotto G, Söderberg-Naucler C. Discordant humoral and cellular immune responses to Cytomegalovirus inglioblastoma patients whose tumors are positive for CMV. Oncoimmunology. 2015 Feb 25;4:e982391.
https://doi.org/10.4161/2162402X.2014.982391

V. Afsar Rahbar, Madeleine Cederarv, Nina Wolmer-Solberg, Charlotte Tammik, Giuseppe Stragliotto, Inti Peredo, Fornara O, Xinling Xu, Mensur Dzabic, Chato Taher, Petra Skarman, Cecilia Söderberg-Nauclér. Enhanced neutrophil activity is associated with shorter time to tumor progression in glioblastoma patients. Oncoimmunology. 2016 Aug.
https://doi.org/10.1080/2162402X.2015.1075693