Normal human monocytes exposed to glioma cells acquire myeloid-derived suppressor cell-like properties

Abstract

Glioblastoma patients are immunosuppressed, yet glioblastomas are highly infiltrated by monocytes/macrophages. Myeloid-derived suppressor cells (MDSC; immunosuppressive myeloid cells including monocytes) have been identified in other cancers and correlate with tumor burden. We hypothesized that glioblastoma exposure causes normal monocytes to assume an MDSC-like phenotype and that MDSC are increased in glioblastoma patients. Healthy donor human CD14(+) monocytes were cultured with human glioblastoma cell lines. Controls were cultured alone or with normal human astrocytes. After 48 hours, glioblastoma-conditioned monocytes (GCM) were purified using magnetic beads. GCM cytokine and costimulatory molecular expression, phagocytic ability, and ability to induce apoptosis in activated lymphocytes were assessed. The frequency of MDSC was assessed by flow cytometry in glioma patients' blood and in GCM in vitro. As predicted, GCM have immunosuppressive, MDSC-like features, including reduced CD14 (but not CD11b) expression, increased immunosuppressive interleukin-10, transforming growth factor-beta, and B7-H1 expression, decreased phagocytic ability, and increased ability to induce apoptosis in activated lymphocytes. Direct contact between monocytes and glioblastoma cells is necessary for complete induction of these effects. In keeping with our hypothesis, glioblastoma patients have increased circulating MDSC compared with normal donors and MDSC are increased in glioma-conditioned monocytes in vitro. To our knowledge, this has not been reported previously. Although further study is needed to directly characterize their origin and function in glioblastoma patients, these results suggest that MDSC may be an important contributor to systemic immunosuppression and can be modeled in vitro by GCM.

Fig. 1.

Decreased CD14 expression in GCM and glioblastoma-infiltrating monocytes. (A) Representative flow cytometry dot plots of CD14 and CD11b expression for fresh lymphocytes and monocytes, cultured monocytes, and bulk (unsorted) monocyte and astrocyte (NHA) or glioma (U87, U251) cocultures. Double negatives indicate glioma cells or astrocytes, CD14^+/−/CD11b⁺ indicate monocytes. The number of CD14⁺/CD11b⁺ monocytes as percentage of total cell number is indicated in bold. Note that the autofluorescence for cultured monocytes was slightly greater than for fresh monocytes or lymphocytes (Supplementary Material, Fig. S1A), resulting in slightly different quadrant placement. (B) Bar graph showing the average number of double positive (CD14⁺/CD11b⁺) cells among fresh monocytes, cultured monocytes, and monocyte/glioma cocultures expressed as a percentage of total monocytes (CD11b⁺ cells) in 4 separate experiments. *P < .05 compared with fresh monocytes. ⁺P < .05 compared with cultured monocytes alone. (C) Bar graph showing the average number of CD14⁺ and CD14⁻ cells expressed as a percentage of CD11b⁺ glioblastoma-infiltrating monocytes from 6 different operative specimens. A significant majority of glioblastoma-infiltrating monocytes were CD14⁻ (***P < .001). Graphs are mean ± SEM.

Fig. 2.

In vitro GCM have an immunosuppressive molecular profile. (A) Representative histograms showing B7-H1 expression in GCM, but not naïve or astrocyte-conditioned monocytes. NM, naïve monocytes; NHA-CM, normal human astrocytes-conditioned monocytes; U87-CM, U87-conditioned monocytes; U251-CM, U251-conditioned monocytes. (B) Graph showing percentage of cells expressing B7-H1 among NM and CM. U87-CM and U251-CM expressed significantly more B7-H1 than NM or NHA-CM. (C) Graph showing percentage of cells expressing IL-10 among NM and CM. U87-CM and U251-CM express significantly more IL-10 than NM. (D) Graph showing percentage of cells expressing TGF-β among NM and CM. U251-CM expressed significantly more TGF-β than NM. (E) Graph showing percentage of cells expressing IL-12 among NM and CM. IL-12 expression is minimal under all conditions and does not vary significantly. (F) Graph showing percentage of cells expressing IFN-γ among NM and CM. IFN-γ expression is minimal under all conditions and does not vary significantly. The scale in (C)–(F) have been kept uniform in order to allow easier relative comparisons between immunosuppressive (IL-10, TGF-β) and proinflammatory (IL-12, IFN-γ) cytokines. All graphs show mean ± SEM of three separate experiments using different PBMC donors. *P < .05; **P < .01; ns,not significant.

Fig. 3.

In vitro GCM are functionally immunosuppressive. (A) Bar graph summarizing experiments where CM induced increased activated T-cell apoptosis over background with NM by flow cytometry for CD3⁺/Annexin V⁺/7AAD⁺ cells. This increase is modest for NHA-CM and U87-CM, but marked for U251-CM. (B) Representative histograms showing decreased CFSE uptake in CD11b⁺ U87-CM and U251-CM compared with NHA-CM or matched (same donor) NM after coculture with CFSE-labeled glioma cells. This indicates decreased tumor cell phagocytosis in GCM. (C) Bar graph summarizing experiments comparing uptake of FITC-labeled E. coli particles by NM and CM measured by flow cytometry. Results are expressed as a percentage of maximum uptake (uptake by LPS-stimulated NM; data not shown). GCM (U251-CM, U87-CM) had significantly decreased phagocytosis of FITC-labeled E. coli particles compared with NM. NHA-CM also demonstrated a modest reduction in phagocytosis. (D) Representative confocal photomicrographs of NM and U251-CM after culture with pHrodo-E. coli particles (red) and counter stained with CD11b-FITC (green). Cells with significant red or yellow staining are defined as phagocytically active. Bar graphs show percent uptake (number of phagocytically active cells/total number of cells). U251-CM have decreased phagocytic activity. This can be decreased further by adding the phagocyotsis inhibitor CCD or increased by adding bacterial LPS. Graphs are mean ± SEM of 3 separate experiments using different PBMC donors. *P < .05; **P < .01.

Fig. 4.

Developing an immunosuppressive phenotype in U251-conditioned monocytes in vitro depends on direct monocyte/glioma cell contact. (A) B7-H1 expression by flow cytometry in naïve (unconditioned) monocytes, monocytes cultured directly with U251 (contact), and monocytes cultured with U251, but separated by a membrane with 0.1 µm pores (noncontact). Both contact and noncontact CM had increased B7-H1 expression compared with unconditioned monocytes (*P < .05), but contact CM had significantly more B7-H1 expression than noncontact-CM (+, *, P < .001). (B) Increased T-cell apoptosis by flow cytometry (CD3⁺/AnV⁺/7AAD⁺) over background (not shown) in activated T cells cultured with U251-CM (contact or noncontact). Only U251-contact CM significantly increase activated T-cell apoptosis over background (*P < .05). (C) E. coli cell wall particle phagocytosis in naïve (unconditioned), contact-U251-conditioned, and noncontact-U251-CM. Contact-U251-conditioned phagocytosis is significantly decreased (*P < .05) compared with unconditioned or noncontact-U251 CM. Graphs are mean ± SEM of three separate experiments using different PBMC donors.

Fig. 5.

MDSC are increased in glioma patients' blood and GCM in vitro. (A) Representative dot plots and histograms showing strategy for determining MDSC frequency in peripheral blood. After gating on monocytes/granulocytes by forward and side scatter, lineage negative/HLA-DR⁻/CD33⁺ MDSC were identified in patients and normal donors. (B) Glioma patients had increased MDSC (expressed as a percentage of Lin⁻ cells) compared with normal donors. (C) Representative dot plots demonstrating CD33 and HLA-DR expression patterns in lineage-negative monocytic/granulocytic cells from among NM and U251-CM. The glioma (U251)-CM have consistently increased CD33⁺/HLA-DR⁻/Lin⁻ MDSC (n = 3).

Fig. 6.

MDSC express increased immunosuppressive B7-H1 compared with monocytes. (A) Representative dot plots demonstrating B7-H1 expression on peripheral MDSC from a healthy donor. Lin⁻/HLA-DR⁻ cells were purified from PBMC using negative selection with magnetic beads. After gating on monocytes/granulocytes by forward/side scatter, CD33⁺ cells (MDSC) were identified. These cells were predominantly B7-H1⁺. (B) Bar graph of costimulatory molecule expression on peripheral blood MDSC (isolated as above) and CD14⁺ monocytes isolated from normal donors (n = 3). B7-H1 expression was significantly increased in MDSC compared with circulating monocytes. Graphs are mean ± SEM. **P < .01.

Fig. 7.

True granulocytes do not contribute to GCM or MDSC although both are enriched for small granular cells. (A) Representative dot plots from 1 of 3 separate experiments performing 3 color flow cytometry for NM and U251-CM. CD11b expression is constant while CD14 expression is reduced (particularly for U251-CM). CD15 expression is absent, ruling out a contribution of granulocytes to our GCM. (B) U251-CM are enriched for small, relatively granular cells (low-forward scatter, relatively high-side scatter). Representative dot plots and pooled data from 3 experiments are shown. (C) MDSC from normal donors and (particularly) glioblastoma patients are also enriched for relatively small, granular cells (low-forward scatter, high-side scatter). Representative dot plots (corresponding to the normal donor and patient shown in Fig. 5A) and pooled data from all patients and donors shown in Fig. 5B. (D) Scatter plot showing increased MDSC in glioblastoma patients compared with normal donors using a lineage cocktail (CD3, CD14, CD16, CD19, CD20, CD56) that excludes granulocytes (CD16⁺) in the lineage negative fraction. This rules out a contribution of granulocytes to the MDSC we have identified.