Figure 3.

Proliferation of tumor-infiltrating T cells in various conditions. Six patients were included and each bar represents one subject. (A) Effect of pre-incubating with various concentrations of IL-2 on the proliferation of tumor T cells. Tumor leukocytes were pre-incubated with 0, 2, 5 or 20 U/ml IL-2 for 3 days in culture medium, and then the cells were washed, stained with CFSE, washed again and stimulated with 2 µg/ml anti-CD3 antibody. After 6 days, the cells were harvested and the percentages of CFSE^lo cells in CD3⁺ cells were examined by flow cytometry. Data were normalized against the results with 0 U/ml IL-2 in each patient (repeated measures one-way analysis of variance, followed by Dunnett's test; **P<0.01). (B) Effect of the lengths of pre-incubation with IL-2 on the proliferation of tumor T cells. Tumor leukocytes were pre-incubated with 20 U/ml IL-2 for 0, 3, 6, or 9 days in culture medium, and then the cells were stained with CFSE and stimulated with 2 µg/ml anti-CD3 antibody. The percentages of CFSE^lo cells were examined after 6 days. Data were normalized against the results without IL-2 pre-incubation (0 day) in each patient (repeated measures one-way analysis of variance, followed by Dunnett's test; **P<0.01 and *P<0.05). (C) Proliferation of tumor-infiltrating T cells without or with IL-10 depletion. Tumor leukocytes were stained with CFSE and stimulated with 2 µg/ml anti-CD3 antibody for 6 days. Concurrent with the addition of anti-CD3 antibodies, 2 µg/ml anti-human IL-10 monoclonal antibody or isotype control antibody were added (paired t-test). NS, not significant; IL, interleukin; CD, cluster of differentiation; CFSE, carboxyfluorescein succinimidyl ester.