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PBMCs from six healthy controls were examined for the relative frequencies of IL-10-producing cells after stimulation with PMA and ionomycin (Table 1). The frequencies of IL-10-producing CD3+ cells after 4 h, 8 h and 24 h of stimulation were all significantly higher than that of the control samples (P<0.05). A typical dot plot is shown in Figure 1. Similarly, the frequencies of IL-10-producing CD4+ cells after 4 h, 8 h and 24 h of stimulation were significantly higher than that of the control samples (P<0.05). The other method for enumerating CD4+ cells, gated as CD3+CD8- cells, also showed similar kinetics (data not shown). The frequencies of IL-10-producing CD8+ cells after 4 h and 24 h were significantly higher than that of the control samples (P<0.05). These results suggest that the frequencies of IL-10-producing cells reached a maximum after 24 h of stimulation. Therefore, we examined the frequencies of IL-10-producing cells at 4 h without stimulation (prestimulation) and 24 h with stimulation (poststimulation). Proportions of CD3+, CD4+ and CD8+ cells among subject groups Cell viability at the beginning of incubation ranged from 97% to 100%, and no significant difference was observed among subject groups. There was no difference in the proportions of CD3+, CD4+ or CD8+ cells among subject groups (Table 2). Table 1. Time course of interleukin-10 production (median count) in six healthy controls after stimulation with phorbol 12-myristate 13-acetate and ionomycin

	Control	4 h after stimulation	8 h after stimulation	24 h after stimulation
CD3+ (%) CD4+ (%) CD8+ (%)	0.00 (0.90) 0.02 (0.54) 0.03 (0.67)	3.81 (4.82)* 3.13 (7.34)* 1.10 (1.55)*	3.51 (2.54)* 2.74 (4.13)* 1.05 (2.41)	3.41 (4.11)* 5.20 (7.83)* 2.29 (4.07)*

Values are presented as medians with interquartile ranges in parenthesis. *P<0.05 compared with control Table 2. Frequencies (median count) of interleukin (IL)-10-producing cells among subject groups

	Healthy  controls (n=12)	Atopic  nonasthmatics (n=10)	Atopic  asthmatics (n=13)
Mononuclear cells (%)
CD3+	64.7 (11.6)	66.6 (15.4)	62.8 (11.9)
CD4+	37.3 (10.4)	36.6 (16.6)	38.2 (13.8)
CD8+	19.6 (8.0)	21.2 (8.8)	20.1 (15.9)
Baseline IL-10 (+) cells (%)
CD3+	0.00 (0.90)	0.40 (1.54)	1.61 (2.37)*
CD4+	0.02 (0.54)	1.39 (3.07)*	1.48 (2.45)*
CD8+	0.03 (0.67)	0.12 (1.80)	1.04 (2.45)*
Stimulated IL-10 (+) cells (%)
CD3+	3.41 (4.11)t	2.76 (2.64)t	2.10 (3.54)
CD4+	5.20 (7.42)t	1.88 (3.22)	2.01 (2.26)
CD8+	2.29 (4.07)t	2.01 (2.77)	1.62 (2.99)

Values are presented as medians with interquartile ranges in parenthesis. *P<0.05 compared with normal controls; tP<0.05 compared with baseline IL-10 (+) cells Figure 1) Typical dot plots of interleukin (IL)-10-producing CD3+cells in a healthy control subject. Panel A and B show unstimulated samples stained with isotype control immunoglobulin (Ig)G2a and anti-IL-10 antibody, respectively. Panel C shows a sample of the same subject stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 24 h, and then stained with anti-IL-10 antibody. PE-IL-10 Phycoerythrin interleukin-10; SSC Side scatter