Fig. 5

Flow cytometry analysis of cell infiltrate in R-848 + poly(I:C) + L. monocytogenes-SMCC + mannan-SMCC melanoma treatment. The transplantation of melanoma B16-F10 was performed as described in Fig. 1. Twelve days after tumor transplantation, mice were randomized in two groups of 24. Therapy based on intratumoral application of corresponding therapeutic mixtures (50 microliters/mouse) started immediately. Four therapeutic pulses were applied on days 0, 1, 2…8, 9, 10…16, 17, 18…24, 25, 26. The composition of therapeutic mixture was: 1 billion L. monocytogenes-SMCC + 0.5 mg R-848, HCl form + 0.5 mg poly(I:C)/ml 0.2 mM mannan-SMCC in PBS, PBS was used as a control. Three mice from each group were euthanized on days 3, 7, 11, 15, 19 after the start of the therapy. Three mice were killed without any application at time 0 (negative control). The analysis of cell infiltrate of excised tumors was performed using flow cytometry and expressed as cells/mm³ of tumor mass. The following labeled antibodies were used: a anti-mouse Ly-6G (Gr-1) Alexa Fluor 700 (granulocyte detection), b anti-mouse CD4 APC; clone GK1.5 (CD4+ Th lymphocytes), c anti-mouse CD8a; clone 53–6.7 (CD8+ Tc lymphocytes), d anti-Mouse CD11c PE; clone N418, anti-Mouse MHCII (I-A/I-E) Alexa Fluor 700; clone M5/114.15.2 (dendritic cells). * P ≤ 0.05 compared to control (PBS)