Lymphocyte infiltrate in tumors from IL-10 deficient mice (A, C and D) or from anti-IL10/IL-10R neutralizing antibody treated mice (B, E and F). A. Single cell suspensions were stained with the indicated antibodies plus anti-CD45 and anti-F4/80. Lymphocytes were analyzed in the CD45+F4/80- population. B. After 6 hours incubation in tissue culture flasks, non adherent cells from total tumor suspension were harvested and stained with the indicated antibodies, tetramer and anti-CD45. C and E. Immunofluorescence of cryo-sections from tumors harvested from IL-10 KO and wild type mice or neutralizing anti-IL10/IL-1R and irrelevant IgG antibodies, as indicated. Sections were stained with anti-CD8 (in FITC, green) and PE (red) conjugated HPV16 E749-57 tetramer - E749-57 Tet, and irrelevant tetramer - Irrelevant Tet. Cells were counterstained with DAPI (blue). Arrowheads indicate double positive cells in yellow, arrows indicate cells positive only for CD8. D and F. Granzyme B expression in tumor cryo-sections from IL-10 KO and wild type mice or mice treated with anti-IL10/IL-10R neutralizing antibodies and irrelevant IgG, as indicated. Sections were stained with anti-CD8 (green), followed by fixation, permeabilization and staining with anti-Granzyme B (red). Tissue was counterstained with DAPI. Solid arrows indicate double positive cells, arrowhead indicate CD8 only positive cell. Insets show details of cells positive only for CD8 (green) or double positive CD8/tetramer (C and E), CD8/Granzyme B (D and F) in yellow or orange due to the sobreposition of emission from FITC and PE (tetramer) or alexa 594 (Granzyme B).
Bolpetti et al. BMC Immunology 2010 11:27 doi:10.1186/1471-2172-11-27