Figure 5. CY+CpG treatment induces unique tumoricidal properties in myeloid Gr1dim cells.
A.-C. Evaluation of induced tumoricidal properties by CY+CpG treatment. Unfractionated splenocytes “UF”, total T-cells, and myeloid Gr1+ (Gr1hi and Gr1dim) cells from spleens of CY+CpG-treated 4T1 TB mice were co-cultured with non-irradiated 4T1-f cells in the presence or absence of CpG alone (1 μM) or with IFNγ (1000 U/ml), and tumoricidal activity (acellular black areas) was evaluated at 48h, 72h and 7d in culture. The percentage of tumor growth inhibition, denoted in white numbers (top left), was calculated as the average of percentages from 6 non-overlapping fields ± SD. Images without percentages represent confluent monolayers (killing below detection levels). Images were numerically labeled on the bottom right for the purpose of identification in the Results text. The in vitro assays were performed with 2×104 non-irradiated 4T1-f cells and 1×105 leukocytes added to the assays at time 0, and representative photos are shown. Photos were taken using a Zeiss Axio Observer A1 microscope. D. Representative photos of isolated Gr1hi and Gr1dim cells from spleens of CY+CpG-treated mice. Scale bar, 10 μm. For all the assays, 10-12 CY+CpG-treated mice were pooled per experiment at c2d3. Data are representative of three independent experiments run in duplicates or triplicates with similar results. Statistical analysis presented in results was performed using Student's t-Test.