Table 2b.

Inhibition of PHA-induced cell surface expression of IL2 receptor alpha (CD25) by BTP2: Four consecutive experiments^*

PBMC	PBMC + BTP2	PBMC + PHA	PBMC + PHA + BTP2	P Value†		P Value‡ PBMC Vs.	P Value‡ PBMC + BTP2 Vs.	P Value‡ PBMC + PHAVs.	P Value‡ PBMC + PHA + BTP2 Vs.
(n = 4)	(n = 4)	(n = 4)	(n = 4)
Percentage of CD25 Positive PBMC Median (25th and 75th percentile)
7.33	7.56	68.65	29.65	< 0.0001	PBMC	-	-	-
					PBMC + BTP2	0.89	-	-	0.03
(6.81 – 8.8)	(7.48 – 8.28)	(55.9 – 78.2)	(21.6 – 36.2)		PBMC + PHA	0.03	0.03	-	0.03
					PBMC + PHA + BTP2	0.03	0.03	0.03	-
MCF of CD25 Positive PBMC Median (25th and 75th percentile)
1636	1599	7513	2064	0.003	PBMC	-	-	-	0.2
					PBMC + BTP2	0.34	-	-	0.2
(1501 – 1652)	(1402 – 1636)	(4316 – 7834)	(1588 – 2865)		PBMC + PHA	0.03	0.03	-	0.03
					PBMC + PHA + BTP2	0.2	0.2	0.03	-

^*Peripheral blood mononuclear cells (PBMC, 1x10⁶ cells/ml of RPMI 1640 + 5% heat inactivated fetal bovine serum) were incubated without BTP2 or PHA (PBMC), with 1000 nM BTP2 (PBMC + BTP2), with 2 μg/ml PHA (PBMC + PHA), or with 1000 nM BTP2 + 2 μg/ml PHA (PBMC + PHA + BTP2) for 16 hours at 37°C in 95% air and 5% CO₂ atmosphere. At the end of incubation, the cells were washed and labeled with FITC-IgG2a isotype control mAb and PE IgG1 isotype control mAb or FITC IgG2a mouse anti-human CD3 mAb and PE IgG1 mouse anti-human CD25 mAb. Flow cytometry data were acquired on a FACSCanto II using FACSDiva software (8.0.1) and the FCS files were analyzed using FlowJo 10.8.1 software. The percentage and the mean channel fluorescence (MCF) of CD25 positive cells from each experiment (Table 2A) and the median (25th and 75th percentile) values from 4 consecutive experiments (Table 2B) are shown.

^†P values calculated using Kruskal-Wallis test of no differences in the percentage or MCF of CD25 positive cells (dependent variable) among the PBMC incubated without BTP2 or PHA (PBMC), PBMC incubated with 1000 nM BTP2, PBMC incubated with 2 μg/ml PHA or PBMC incubated with 1000 nM BTP2 + 2 μg/ml PHA groups.

^‡P values calculated using Mann-Whitney test of no difference in the percentage or MCF of CD25 positive cells (dependent variable) between two groups.