Glioma-associated cytomegalovirus mediates subversion of the monocyte lineage to a tumor propagating phenotype

Abstract

Purpose: Cytomegalovirus (CMV) has been ubiquitously detected within high-grade gliomas, but its role in gliomagenesis has not been fully elicited.

Experimental design: Glioblastoma multiforme (GBM) tumors were analyzed by flow cytometry to determine CMV antigen expression within various glioma-associated immune populations. The glioma cancer stem cell (gCSC) CMV interleukin (IL)-10 production was determined by ELISA. Human monocytes were stimulated with recombinant CMV IL-10 and levels of expression of p-STAT3, VEGF (vascular endothelial growth factor), TGF-β, viral IE1, and pp65 were determined by flow cytometry. The influence of CMV IL-10-treated monocytes on gCSC biology was ascertained by functional assays.

Results: CMV showed a tropism for macrophages (MΦ)/microglia and CD133+ gCSCs within GBMs. The gCSCs produce CMV IL-10, which induces human monocytes (the precursor to the central nervous system MΦs/microglia) to assume an M2 immunosuppressive phenotype (as manifested by downmodulation of the major histocompatibility complex and costimulatory molecules) while upregulating immunoinhibitory B7-H1. CMV IL-10 also induces expression of viral IE1, a modulator of viral replication and transcription in the monocytes. Finally, the CMV IL-10-treated monocytes produced angiogenic VEGF, immunosuppressive TGF-β, and enhanced migration of gCSCs.

Conclusions: CMV triggers a feedforward mechanism of gliomagenesis by inducing tumor-supportive monocytes.

Fig. 1. CMV antigen expression has a tropism for subsets of immune cells within GBMs

(A) Flow cytometry staining of GBM in single cell suspension shows only a subset of cells are positive for the CMV antigen pp65. The percentage of cells positive is denoted in the upper right quadrant of the histogram. (B) Flow cytometry gating strategy used to distinguish cells of monocyte lineage (such as microglia and MΦs [TAMs]) and lymphocytes based on CD11b and CD45 expression profile and intracellular staining of pp65. Each phenotype was separately analyzed for pp65 expression, as indicated by the histograms. The gray line represents the isotype control, and the black line shows the cells with positive staining for pp65. Percentages of cells are denoted in each quadrant and by their respective gates. The percent positive compared to the isotype control is shown in the right upper quadrant in the pp65 sub analysis panel. This data shown here is a representative of the five newly diagnosed glioblastoma samples analyzed.

Fig. 2. CMV antigen expression is enhanced in CD133 gCSCs

(A) Representative flow cytometry analysis of a gCSC sample stained for intracellular expression of CMV antigen expression of pp65, IE1, and gB as well as surface expression of US28. The percentage in the upper quadrant is positive cells relative to isotype. Similar data were obtained in three other gCSCs. (B) Flow cytometry gating strategy for CD133-positive gCSCs demonstrating the rectangular gate-capturing cells staining positively for CD133 relative to the isotype control. The round gates capture the CD133^low and CD133^high phenotypes. The shift in histograms (solid black line) reflects increased CMV protein expression in the CD133^high population relative to the CD133^low (grey line).

Fig. 3. CMV IL-10 induces CMV transcriptional activity in monocytes

(A) Expression levels of pp65 in the PBMCs from GBM patients were found to be higher than in PBMCs from healthy donor controls although this was not statistically significant. (B) Purified CD14+ monocytes demonstrated expression of pp65 but relatively low levels of IE1. The dashed line represents isotype control and the gray line untreated monocyte staining. Upon exposure of the monocytes to CMV IL-10 as shown by the black line, there was up regulation of CMV IE1 but no further increase in pp65 expression.

Fig. 4. CMV IL-10 induces an immune suppressive phenotype in monocytes

(A) In monocytes exposed to CMV IL-10 for 24 hours, there was down modulation in percentage of monocytes expressing MHC II and CD86 with up regulation of the costimulatory inhibitory molecule B7-H1. (B) The exposure of the monocytes to CMV IL-10 caused an up regulation of intracellular p-STAT3, TGF-β1, and VEGF. The dashed line represents the isotype control, the gray line shows monocytes cultured without CMV IL-10, and the black line shows monocytes cultured in medium containing CMV IL-10 at a dose of 10ng/mL. Analysis was performed after 48 hours in culture. This experiment was done with triplicate donor monocytes with similar results. The number in the right upper quadrant denotes percentage of positive cells upon exposure to CMV IL-10 (black) and the percentage of positive cells without exposure to CMV IL-10 (grey )

Fig. 5. CMV IL-10 exposed monocytes enhance gCSC migration

The gCSCs were exposed to medium that was conditioned for 24 hours by monocytes treated with CMV IL-10 and appropriate controls. The CMV IL-10-treated monocyte supernatant increased gCSC migration in a chemotaxis assay compared with those grown in control medium. This experiment was done in duplicate. *P=0.013

Fig. 6. Schema demonstrating the interrelationship between the gCSC and the monocyte

CMV infects the neural progenitor cell, the precursor to the gCSC, by surface attachment to the PDGF-α receptor. The CMV may possibly integrate into the host genome or interact with the STAT3 pathway to induce gliomagenesis. Viral activity, including the production of CMV IL-10, together with monocyte chemokines elaborated by the gCSC result in the preferential migration and conversion of the monocyte to an immunosuppressive, tumor-supportive M2 MΦ/microglia (M2; tumor-associated MΦ).