Fig. 4.

Cytokine secretion. A, B. High cytokine production with IgCD28TCR. Designer T cells (20% modified) were cultured with tumor cells overnight and supernatants harvested and assayed for (A) IL2 and (B) gamma interferon (IFNg) by ELISA. All cultures were negative for cytokine production on MIP101 and MIP101-B7 (not shown). By the international unit definition (WHO standard), 30 ng/ml of IL2 corresponds to 450 IU/ml. C. Tandem T cells die without IL2. T cells from A during log-phase expansion in IL2 were washed and placed in medium -IL2 without tumor cells. T cells were replaced with fresh medium -IL2 on day 3. D. Limited survival of Tandem T cells when depending solely upon their own stimulated IL2 secretion. At time zero and each time point, T cells as in C. were fed irradiated MIPCEA tumor cells and placed in fresh medium, (D1) +IL2 (100 IU/ml) or (D2) -IL2. Note 5X scale difference, with lower cell numbers for -IL2. (Data in D1 reproduced in part from Fig.2B.) E. Exhaustion of IL2 secretion with restimulation. Tandem T cells were incubated with MIPCEA cells as in D2 with medium change and re-plating of viable T cells with fresh irradiated MIPCEA tumor every 48 hours. IL2 measured at each 48-hour time point by ELISA. IL2 was unmeasurable after the third stimulation (days 4–6).