Flow cytometry and in vitro analysis of human glioma–associated macrophages

Abstract

To date, glioma immunotherapy has been focused mostly on stimulating antitumor peripheral lymphocyte responses; however, some data suggest that microglia and/or macrophages (not lymphocytes) are the predominant inflammatory cells infiltrating gliomas. To study this hypothesis further, the authors analyzed inflammatory cell infiltrates in fresh human malignant glioma specimens and primary cultures. Single-cell suspensions from fresh operative malignant glioma specimens, obtained by stereotactic localization, were analyzed for CD11b and CD45 by using flow cytometry. A comparison was made with peripheral blood mononuclear cells. In a subset of patients, a more detailed flow cytometry analysis of Class I and II major histocompatibility complex, B7-1, B7-2, CD11c, and CD14 expression was performed. Macrophage-like cells in primary glioma cultures were similarly assessed. Operative samples were obtained from 9 newly diagnosed malignant gliomas. The mean percent of CD45(+)/CD11b(-) cells (lymphocytes) was 2.48% (range 0.65-5.50%); CD45(dim)/CD11b(+) cells (microglia), 1.65% (range 0.37-3.92%); and CD45(bright)/CD11b+ (monocytes/macrophages), 6.25% (range 1.56-15.3%). More detailed fluorescence-activated cell sorting suggested that macrophage-like cells expressed Class I and II major histocompatibility complex, B7-2, and CD11c but not CD14 or B7-1. Primary human glioma cultures contained significant numbers of macrophage-like (CD45(bright)/CD11b(+)) cells, but these cells were lost with successive passages. These cells maintained the immunomarker profiles of macrophage-like cells from fresh specimens only if they were cultured in serum-free media. The CD45(+)/CD11b(+) cells are the predominant inflammatory cell infiltrating human gliomas. Of this type, the CD45(bright)/CD11b(+) cells, a phenotype compatible with circulating macrophages in rodent models, and not microglia, are the most common. Their immunomarker profile is compatible with an immature antigen-presenting cell. They are present in primary glioma cultures but are lost in successive passages. Their role is enigmatic, and they may prove an important target for future glioma immunotherapy studies.