Figure 4. C5a promotes cytotoxicity of SKOV-3 tumor cells while Gr-1⁺CD11b⁺ cells from SKOV-3 C5a tumors are significantly less immunosuppressive.

(A) SKOV-3 C5a and CV tumor cells were cultured overnight, and the following day non-adherent leukocytes from naïve SCID mice as effector cells were added at a ratio of 20:1 (E:T). After 16 hours of co-culture, percent of cytotoxicity was calculated (n=6). Data indicate that effector cells kill significantly more SKOV-3 C5a cells than SKOV-3 CV cells. **p<0.01. (B) Similarly, purified NK cells from naïve SCID mice were added to SKOV-3 C5a or CV tumor cells in vitro, and percent of cytotoxicity was determined following 24 hr co-culture. *p<0.05. (C) SKOV-3 tumor cells were co-cultured with non-adherent leukocytes as effector cells (20:1) in the presence or absence of Gr-1⁺CD11b⁺ cells sorted from CV or C5a-transfected tumor (1:1), or without effectors but with sorted Gr-1⁺CD11b⁺ cells from tumor. The innate leukocytes demonstrated effective cytotoxicity of SKOV-3 tumor cells and cytotoxicity was significantly decreased in the presence of Gr-1⁺CD11b⁺ cells sorted from tumors. However, Gr-1⁺CD11b⁺ cells from SKOV-3 C5a tumors were significantly less suppressive. **p<0.01. (D) Cytospin and stain of the Gr-1⁺CD11b⁺ cells sorted from the SKOV-3 tumors. Images were acquired at 20X and 40X magnification.