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Direct Addition of Aminophylline to Effector-Target CeU Cultures in Vitro The direct addition of concentrations of aminophyl-line that exceeded 5 X 10 “M inhibited NK cell activity in a dose-dependent manner. Representative data obtained from three independent experiments on one individual are shown in Table 1. The degree of inhibition was similar in two other individuals, and the inhibitory effect occurred at various effector-target ratios (data not shown). Fifty percent inhibition was achieved at between 1 and 5x 10_4M. The inhibition was not due to direct cytotoxicity of this compound because the viability of PBLs after incubation with aminophylline (1 x 10~M) for 4 h was greater than 95 percent of untreated control PBLs. Although aminophylline contains ethylenediamine, this compound could not inhibit NK cell activity even at 1 x 10-3M (data not shown). Effect of Preincubation on the Effector Cells The effector cells were preincubated with aminophylline at 10~M for 30, 60, 120, and 180 minutes and then washed extensively and assayed for NK cell activity. As shown in Figure 1, the NK cell activity was significantly inhibited (30 to 54 percent inhibition) by preincubation with aminophylline. As short as 30 minutes of preincubation was effective for inhibition. Table 1—Dose-Dependent Inhibition ofNK Cell Activity against K562 by Direct Addition of Aminophylline

Molar  Concentration	Percentage of Cytotoxicity	Percentage of Inhibition
None	57.6 ±7.9
1 x 10~	6.1 ± 1.8	92.0±4.0
5xl0<	14.7±2.8	79.1 ±3.9
1 x 10	33.3±6.5	42.0±4.9
5x10	47.7 ±5.6	16.7 ±5.6
1 x 10	49.2 ±2.0	8.7±4.1
5X10	51.3±1.9	2.1±1.9
1 x 10	51.7±4.3	2.0±4.3

Figure 1. Preincubation of effector cells with theophylline induces inhibition of NK cell activity. Peripheral blood lymphocytes (1 x 10*/ ml) were incubated with 1 x 10-3M aminophylline. After extensive washing, NK cell activity was assessed using those cells. Inhibition occurred in time-dependent manner.