Fig. 5

Inhibition of lymphocyte proliferation by cells from tumor-bearing mouse spleen expressing the CD11b/Mac-1 surface antigen requires cell contact

(A) Mixing experiments were performed to evaluate inhibition by cells or soluble factors. Responder spleen cells from naive mice were incubated with either CD90/Thy-1.2, CD45R/B220 spleen cells (80% CD11b/Mac1 positive by FACS analyses) from tumor-bearing mice or supernatant after incubation for 24 hr at a 9:1 ratio in 96-well plates. These treated responder cells were then incubated with an equivalent number of irradiated allogeneic stimulator cells. [³H]thymidine was added 4 days later and incorporation was measured after an additional 16 hr. Results are reported as mean CPM ± SEM from triplicate wells. Experiments are representative of results from at least three independent experiments and at a 5:1 ratio. (B) Membrane separation was performed to evaluate inhibition by soluble factors. Responder spleen cells from naive mice were incubated with spleen cells from tumor-bearing mice at a 3:1 ratio in the same well or separated by a semipermeable membrane. The mixtures were then incubated with an equivalent number of irradiated allogeneic stimulator cells. [³H]thymidine was added 4 days later and the cells were placed into wells in a 96-well dish and incorporation was measured after an additional 16 hr. Results are reported as mean CPM ± SEM from triplicate wells.