Histological and immunohistochemical analyses

Hematoxylin and eosin (H&E) staining was performed for an estimation of the gross morphology of tumors and spleen sections.

For immunohistochemistry, sections were stained with Ki67 and CD8 antibodies targeting CD8+ lymphocytes (AK CD8α; eBioscience; Thermo Fisher Scientific, Inc., Waltham, MA, USA; cat. no. 14-0084; AK CD68, Zymed; Thermo Fisher Scientific, Inc.; cat. no. 603–2210; AK Ki67; Zymed; Thermo Fisher Scientific, Inc.; cat. no. RMPD 004). Omission of primary antibodies in the control experiments resulted in the expected absence of any cellular labeling. The extent of infiltration of different immune-cell subsets was quantified by cell counting of five representative high-power fields (HPF) in each section, including the tumor margins.

In order to estimate the putative effects of rhGM-CSF stimulation, macrophages and CD8+ lymphocytes in tumor tissues were counted and the corresponding spleen tissue functioned as positive control. Sections were counted at a magnification of ×100 using a microscope. In each case, five contiguous fields of view were counted and the mean was determined. For brain sections, five visual fields were counted starting from the tumor margin. As ED1 stains macrophages and microglia, only cells with distinct phagocyte morphology were considered. In CD8+ cells, only cells with clear lymphocyte morphology were included.