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Comparative Study
. 2006 Jul;8(3):261-79.
doi: 10.1215/15228517-2006-008. Epub 2006 Jun 14.

The role of human glioma-infiltrating microglia/macrophages in mediating antitumor immune responses

S Farzana Hussain  1 , David Yang, Dima Suki, Kenneth Aldape, Elizabeth Grimm, Amy B Heimberger
Affiliations
Comparative Study

The role of human glioma-infiltrating microglia/macrophages in mediating antitumor immune responses

S Farzana Hussain et al. Neuro Oncol. 2006 Jul.

Abstract

Little is known about the immune performance and interactions of CNS microglia/macrophages in glioma patients. We found that microglia/macrophages were the predominant immune cell infiltrating gliomas ( approximately 1% of total cells); others identified were myeloid dendritic cells (DCs), plasmacytoid DCs, and T cells. We isolated and analyzed the immune functions of CD11b/c+CD45+ glioma-infiltrating microglia/macrophages (GIMs) from postoperative tissue specimens of glioma patients. Although GIMs expressed substantial levels of Toll-like receptors (TLRs), they did not appear stimulated to produce pro-inflammatory cytokines (tumor necrosis factor alpha, interleukin 1, or interleukin 6), and in vitro, lipopolysaccharides could bind TLR-4 but could not induce GIM-mediated T-cell proliferation. Despite surface major histocompatibility complex class II expression, they lacked expression of the costimulatory molecules CD86, CD80, and CD40 critical for T-cell activation. Ex vivo, we demonstrate a corresponding lack of effector/activated T cells, as glioma-infiltrating CD8+ T cells were phenotypically CD8+CD25-. By contrast, there was a prominent population of regulatory CD4 T cells (CD4+CD25+FOXP3+) infiltrating the tumor. We conclude that while GIMs may have a few intact innate immune functions, their capacity to be stimulated via TLRs, secrete cytokines, upregulate costimulatory molecules, and in turn activate antitumor effector T cells is not sufficient to initiate immune responses. Furthermore, the presence of regulatory T cells may also contribute to the lack of effective immune activation against malignant human gliomas.

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Figures

Fig. 1
Fig. 1
Gating strategy for immune cell analysis from CNS tissue. Cells were stained with IgG1-FITC, IgG1-PE, and IgG1-APC (isotype control) or CD1c-FITC, CD11c-PE, and BDCA-2-APC (stained) to identify myeloid DCs. In this example, GBM cells were analyzed by using a side-scatter versus forward-scatter dot plot on both the isotype (A) and stained cells (B) to select live cells (G1). The G1 gate was then applied to a CD11c versus CD1c dot plot (D) and its corresponding isotype (C). A statistical gate (G2) was drawn to indicate the CD11c+CD1c+ double positive (C and D). In the alternative gating strategy, isotype cells (E) and stained cells (F) were plotted on side scatter versus FITC, and FITC+ cells were gated (gate G3). By using the G3 gate, isotype and stained cells were analyzed on a CD11c versus CD1c dot plot (G and H, respectively). A statistical gate (G4) was drawn to indicate the CD11c+CD1c+ population. Percentages in C and D and in G and H indicate the percentage of cells within G2 or G4, respectively.
Fig. 2
Fig. 2
Immune cells infiltrating GBMs and normal brain tissue. Dissociated GBM or normal tissue was triple stained with fluorescent conjugated antibodies and analyzed by flow cytometry. The percentage indicated in each respective quadrant/gate denotes the number of gated cells that are positive for the expression of the respective surface markers and subtracts the isotype background. The panel is representative of one glioblastoma tissue specimen and one normal brain specimen. A. All CD11c+ cells were gated, and these cells were then observed for simultaneous CD11b and CD45 surface marker expression. Microglia/macrophages are the CD11c+CD11b+CD45+ cells as indicated by the gate in the upper right quadrant for both GBM tissue and normal tissue. B. CD1c+ gated cells that were BDCA-2− and CD11c+ representing myeloid DCs are indicated by the gate in the upper right quadrant for GBM tissue and normal brain tissue. C. BDCA-2+ gated cells that were CD11c− and CD1c− representing plasmacytoid DCs are in the upper left quadrant for GBM tissue and normal brain tissue. D. The upper right quadrant represents the B cells that are MHC II+ and CD19+ in both gliomas and normal brain. (E) Numbers in each quadrant are CD3+ that are either CD4+ (upper left quadrant) or CD8+ (lower right quadrant) T cells within the normal brain parenchyma or GBM tissue. An autofluorescent population was identified in both normal and GBM tissue samples during flow cytometry analysis and is also consistently present in the respective isotype controls. This population was excluded when calculating the percentages of positive fluorescing cells.
Fig. 3
Fig. 3
GIM can be isolated as a pure population. A. Each interphase from the second Percoll gradient was analyzed for expression of the surface markers CD11b and CD45. The gated cells in the upper right quadrant indicate the percentage of CD45+ gated cells that were positive for CD11b and represent GIM. An autofluorescent population was identified in both normal and GBM tissue samples during flow cytometry analysis and is also consistently present in the respective isotype controls. This population was excluded when calculating the percentages of positive fluorescing cells. B. GIMs express the Fc receptors CD16 and CD32. C. The GIM cells express CD14. D. The GIM population does not contain T cells (CD3+) or NK cells (CD56+). In each histogram of panels B–D, the data plot on the left represents the isotype control, and the plot on the right represents cells that express the respective surface marker. The data are from one tumor specimen but are representative of at least 20 tumor tissue samples from patients with GBM.
Fig. 4
Fig. 4
Expression of TLR on GIM and microglia/macrophages isolated from normal brain. Purified microglia/macrophages from GBM tissue and normal brain tissue were double-stained for CD45 and TLR expression. An autofluorescent population was identified in both normal and GBM tissue samples during flow cytometry analysis and is also consistently present in the respective isotype controls. This population was excluded when calculating the percentages of positive fluorescing cells.
Fig. 5
Fig. 5
Cytokine production by GIM and microglia/macrophages isolated from normal brain. Purified microglia/macrophages were freshly isolated from GBM tissue and stained for the intracellular cytokines IL-6, IL-10, IL-12, TNF-α, and IFN-γ without in vitro stimulation. Cytokines were detected by using fluorescence-labeled antibodies and analyzed by flow cytometry. Positive intracellular cytokine expression was confirmed for all cytokines in either a reporter cell line (A375) or stimulated lymphocytes (data not shown). Microglia/macrophages isolated from normal brain were also analyzed for the same cytokine expression profile.
Fig. 6
Fig. 6
MHC class II and costimulatory marker expression on GIM and microglia/macrophages from normal brain. In each histogram, the data plot on the left represents the isotype control, and the second plot represents CD45+ or CD11b+ gated cells that express the respective surface marker. A. GIMs were gated on CD11b+ expression and were analyzed for the expression of MHC class II antigens (HLA DR, DP, or DQ). GIMs were gated on CD45+ expression and analyzed for the expression of the costimulatory molecules CD80 (B), CD86 (C), and CD40 (D). Microglia/macrophages isolated from normal brain were also assessed for surface expression of the above markers except CD40 (A–C).
Fig. 7
Fig. 7
Functional activity of GIMs: Allo-MLR. Stimulator cells were either GIMs isolated from patients’ tumors or APCs isolated from the peripheral blood of the respective glioma patients as described in Materials and Methods. Responder T cells were from an allogeneic normal donor, CFSE labeled, and incubated with the different stimulators. Following incubation, cells were labeled with PE-conjugated anti-CD3 antibody to further gate out the CFSE-labeled responder T cells for analysis by flow cytometry. The percentage of allo-T cells that underwent proliferation, as determined by dilution of the fluorescence intensity of the CFSE dye on gated CD3+ T cells, is indicated in each data plot. A. Controls included T cells incubated in media alone and T cells stimulated with anti-CD3 antibody. B. CFSE-labeled responder allo-T cells were incubated with the patient’s peripheral blood APCs either with or without LPS. C. CFSE-labeled allo-T cells were incubated with GIMs either with or without LPS.
Fig. 8
Fig. 8
LPS can bind the surface of GIMs via TLR-4. Purified microglia/macrophages from GBMs tissue (n = 2) were incubated with LPS-FITC for 30 min and stained with TLR-4. Controls included GIMs that were stained with TLR-4 but not stimulated with LPS-FITC and GIMs that were incubated with LPS-FITC and stained with isotype to TLR-4 (data not shown). A. Histogram overlay demonstrating LPS binding to the surface of GIM stimulated with LPS (filled graph) compared to GIM without LPS stimulation (clear graph). B. GIMs were gated on LPS+ expression and examined for an LPS+TLR-4+ double-positive population (upper right quadrant).
Fig. 9
Fig. 9
T-cell phenotype in GBM. Patients’ glioma cells and PBMCs were stained with fluorescent conjugated antibodies and analyzed by flow cytometry. An autofluorescent population was identified in GBM tissue samples during flow cytometry analysis and is consistently present in the respective isotype controls. This population was excluded when calculating the percentages of positive fluorescing cells. The panel is representative of one glioblastoma tissue specimen and one PBMC specimen. A. GBM cells were gated on CD3+ cells and examined for CD4+ (upper left quadrant) and CD8+ (lower right quadrant) expression. B. GBM cells were gated on CD8+ expression (by using the R1 gate on Fig. 8A) and examined for CD8+CD25+ expression (upper right quadrant). C. GBM CD4+ T cells were gated (by using the R2 gate on Fig. 8A) and examined for CD4+CD25+ expression in the upper right quadrant. D. GBM CD4+ T cells were gated (by using the R2 gate on Fig. 8A) and examined for FOXP3 expression in the upper right quadrant. E. Patients’ PBMCs were gated on lymphocytes by size and density and examined for CD4+ (upper left quadrant) and CD8+ (lower right quadrant) expression. F. PBMCs were gated on CD8+ expression (by using the R3 gate on Fig. 8A) and examined for CD8+CD25+ expression (upper right quadrant). G. PBMC CD4+ T cells were gated (by using the R4 gate on Fig. 8A) and examined for CD4+CD25+ expression in the upper right quadrant. H. PBMC CD4+ T cells were gated (by using the R4 gate on Fig. 8A) and examined for FOXP3 expression in the upper right quadrant.
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