Serum exosomes and cytokines promote a T-helper cell type 2 environment in the peripheral blood of glioblastoma patients

Abstract

Background: Glioblastoma (GBM) is an aggressive infiltrative brain tumor with a particularly poor prognosis that is characterized by microvascular proliferation, necrotic tissue, and significant infiltration of M2-like monocytes. Compromised barrier function in tumor vasculature might be expected to permit communication between the tumor microenvironment and peripheral blood.

Methods: To ascertain whether tumor-derived vesicles and/or factors might reach the bloodstream and what effects these molecules have on the peripheral compartment, we analyzed blood samples collected from primary GBM patients.

Results: Notably, a significant number of patient sera samples contained tumor exosome-reactive immunoglobulin (Ig)G2 and IgG4 antibody isotypes, which are consistent with Th2 immunity. M2-like monocytes expressing CD14+ and CD163+, another indicator of Th2 bias, are elevated in GBM patient blood and associated with high serum concentrations of colony-stimulating factor 2 and 3, as well as interleukin-2, -4, and -13, the latter 2 cytokines being hallmarks of Th2 immunity. GBM patient sera samples induce high levels of CD163 expression when added to normal monocytes, providing mechanistic evidence of a basis for Th2 bias. Fractionation of GBM patient sera into samples enriched for exosomes or soluble factors proved that both fractions are capable of inducing CD163 expression in normal monocytes.

Conclusions: The results of the current study indicate a Th2 bias in the periphery of GBM patients, likely as a result of products elaborated by the tumor. Consequentially, through immune modulation these brain tumors exert systemic effects beyond the confines of the CNS.

Keywords: M2 monocytes; Th2 immunity; exosomes; glioblastoma immunity; peripheral blood mononuclear cells.

Fig. 1.

Tumor exosome-reactive antibodies from glioblastoma patient sera are biased toward Th2. Exosomes isolated from 3 primary GBM cell lines were coated onto 96-well plates. Binding of antibodies in GBM patient sera collected before surgery was detected in duplicate wells with biotinylated, isotype-specific antibodies followed by fluorescently conjugated streptavidin. Isotype MFI is plotted with standard deviation. Statistical significance between patient and control samples was determined by ANOVA and Dunnett's posttest analysis. Numbers represent P-values (*P < .001; #P < .01; †P < .5) for antibody responses in GBM patients when compared with control (ctrl) human antibody serum samples.

Fig. 2.

An increased frequency of blood monocytes in glioblastoma patients correlates with high serum levels of G-CSF and GM-CSF. (A) A live cell gate is established on forward scatter (FSC) and side scatter (SSC) flow cytometry contour plots. Monocytes are defined as CD11b+ and CD14+ cells. Numbers indicate the frequency of gated cells, and axes are presented as log scale. (B) A summary chart containing the frequency of monocytes in PBMC of patients with GBM (n = 25) or normal blood donors (n = 23) as measured by flow cytometry. Statistical significance was assessed by Student's t-test, and numbers indicate P-values. (C) Luminex analysis was used to quantify sera cytokine concentrations (pg/mL) in GBM patients segregated based on monocyte frequency (high and low) as determined by CBC performed by Thomas Jefferson University Hospital. The horizontal black line indicates published sera cytokine levels in healthy individuals. Patient samples are plotted with group medians (diamond center line) and 95% CIs (diamond points). Statistical significance was assessed by the Mann-Whitney test, and numbers indicate P-values.

Fig. 3.

Classical monocytes (CD14+, CD16−) are enriched in GBM patient blood and express high levels of the M2 marker CD163. (A) CD14 and CD16 contour plots define 3 monocyte subset gates. Numbers indicate the frequency of gated cells, and axes are presented as log scale. (B) Summary data of CD14+, CD16−; CD14+, CD16+; and CD14−, CD16+ monocyte subset frequency in normal and GBM patient PBMC. Statistical significance was assessed by Student's t–test, and numbers indicate P-values. (C) CD163 MFI of individual monocyte subsets is overlayed in flow cytometry histogram plots. Filled histograms provide intensity levels and proportion of CD163 expression in ungated PBMC. Axes are presented as log scale. (D) Summary data of CD163 MFI for gated monocyte subsets in PBMC samples. Statistical significance was assessed by Student's t–test, and numbers indicate P-values.

Fig. 4.

Th2 cytokine concentrations are elevated in the sera of GBM patients with high proportions of monocytes. Luminex analysis was used to quantify sera cytokine concentrations (pg/mL) in GBM patients segregated based on monocyte frequency (high and low) as determined by CBC performed by Thomas Jefferson University Hospital. The horizontal black line indicates published sera cytokine levels in healthy individuals. Patient samples are plotted with group medians (diamond center line) and 95% CIs (diamond points). Statistical significance was assessed by the Mann–Whitney test, and numbers indicate P-values.

Fig. 5.

Sera from GBM patient with high levels of tumor antigen (Ag)-specific sera IgG induce M2 monocyte differentiation of normal blood monocytes. GBM patients were divided into groups based on levels of tumor Ag-specific sera IgG. (A) Primary GBM cell line exosomes were coated onto 96-well plates. Binding of antibodies in GBM patient sera collected before surgery was detected in duplicate wells with anti–human IgG–fluorescein isothiocyanate antibodies. IgG MFI is plotted. Boxes separate GBM sera samples with low and high exosome-reactive IgG. (B and C) Normal donor PBMC were cultured in the presence of normal human antibody sera (Hab) or GBM patient sera containing various levels of exosome-reactive IgG. Flow cytometry histograms (B) and summary chart (C) document CD163 MFI in gated monocytes (CD11b+ and CD14+ PBMC). Statistical significance was assessed by Student's t–test, and numbers indicate P-values. (D) Sera cytokine concentrations (pg/mL) in GBM patient sera, as measured by Luminex, were separated based on high and low IgG and plotted with box and whisker plots. Statistical significance was assessed by the Mann-Whitney test.

Fig. 6.

Exosomes and soluble sera fractions from GBM patients induce M2 monocyte differentiation of normal human monocytes that correlates with levels of tumor exosome-specific sera IgG and quantities of circulating exosomes. GBM patients were divided into groups based on levels of tumor exosome-reactive sera IgG. (A) IgG MFI and (B) serum vesicles were labeled with DiD and CD63 and analyzed by flow cytometry. DiD MFI of gated exosomes (CD63+) is plotted with sample median (diamond center line) and 95% CIs (diamond points). Statistical significance was assessed using ANOVA and Tukey's posttest analysis, and numbers represent P-value. (C and D) Normal donor PBMC were cultured in the presence of normal human antibody sera (ctrl) or fractionated GBM serum enriched for exosomes (exo) or soluble factors (SF). CD163 MFI in gated monocytes (CD11b+ and CD14+ PBMC) is plotted with sample median (diamond center line) and 95% CIs (diamond points). Statistical significance was assessed by ANOVA and Tukey's posttest analysis (letter groupings, P < .0001). (D) Frequency of gated monocytes with high or low CD163 MFI in fractionated sera cocultures from patients with different amounts of tumor exosome-reactive serum IgG (low, intermediate, or high). A cutoff of 100 was calculated as the upper 95th percentile of CD163 MFI in normal donor PBMC cultured in the presence of normal human antibody sera.