Fig 1.
PHA stimulation leads to long-term CD25 expression in human blood T lymphocytes.
(A) The representative histograms of nine experiments on PBL from different donors are shown. PBL were cultivated with 10 μg/ml PHA in the absence or presence of 80 μM WHI-P131 (WHI) or 1.0 μM PP2 and after 5, 24 or 48 h were analyzed by flow cytometry. The dot plots have been obtained with Win MDI Version 2.8. In order to identify the axis labels, the additional vertical axis is shown in the upper row of histograms. Y-axis is 4 decades logarithmic scale. 1, 2 –The gate was chosen considering the cell size increase during blasttransformation: 1- CTR—control, non-stimulated PBL, 2 –after 48 h of culture with 10 μg/ml PHA. X-axis–forward scatter (FSC), Y-axis–side scatter (SSC). (B) Time-course of CD25+ expression in PHA-induced PBL. 1 –Total number of CD25+ cells, 2—large CD25+ cells, 3—small CD25+ cells. (C) WHI-P131- and PP2-inibitable portions of CD25+ cells in PHA-stimulated PBL. Summary of independent experiments is presented as mean ±SEM (n = 9, p˂0.05). In each experiment, when analyzing the PHA-induced CD25 expression changes, the background CD25 expression in resting cells (CTR) was subtracted. Summary data are presented as mean ± SEM (n = 10, p˂0.05).
Table 1.
The growth, proliferation and viability of human blood T lymphocytes, stimulated with PHA or IL-2 in the absence or presence of WHI-P131 and PP2 for 48 h.
Fig 2.
The inhibitory effect of WHI-P131 and PP2 on CD25 expression in PHA-stimulated human blood T lymphocytes.
(A) The PP2-inhibitable expression of CD25 is timely associated with the initial stages of PBL activation. Cells were cultivated with 10 μg/ml PHA without or with 80 μM WHI-P131 (WHI) or 1.0 μM PP2 for 19 h (middle row) or cells were cultivated with 10 μg/ml PHA for 19 h and thereafter WHI-P131 or PP2 were added for the next 21 h (bottom row). Additional Y-axis is 4 decades logarithmic scale. Representative data on PBL from one donor are shown. (B) Summary data of four independent experiments are shown as mean + SEM (n = 4, p˂0.05). (C) The inhibitory effects of WHI-P131 (WHI) and PP2 on CD25 expression are additive. Cells were cultivated with 10 μg/ml PHA without or with WHI-P131 (WHI, 20 and 50 μM), or PP2 (0.4 and 0.8 μM), or WHI-P131 (20 μM) and PP2 (0.4 μM) were given simultaneously for 24 h. Summary of independent experiments are presented as mean + SEM (n = 4). ⃰, p<0.05. CTR—control, non-stimulated PBL.
Fig 3.
Stimulation with non-mitogenic PHA and IL-2 leads to long-term CD25 expression and proliferation in human blood T lymphocytes.
(A) The representative histograms of one experiment on PBL from one donor are shown. PBL were not stimulated (CTR) or stimulated with 0.7 μg/ml (0.7PHA), or 10 μg/ml (10PHA) PHA, or 200 U/ml IL-2 (IL-2) for 48 h. In the same experiment, PBL were preincubated in culture medium with 0.7 μg/ml PHA and 80 μM WHI-P131 (WHI) or 1.0 μM PP2 for 24 h prior to IL-2 stimulation, or drugs were added simultaneously with 0.7 μg/ml PHA for 24 h and thereafter IL-2 was added for the next 24 h. Additional Y-axis is 4 decades logarithmic scale. (B) CD25 expression in resting PBL in response to 0.7 μg/ml PHA (2) and 10 μg/ml PHA (8), or in competent PBL in response to IL-2 in the absence (3) or presence of WHI-P131 (4) or PP2 (5), or in response to IL-2 in PBL preactivated with 0.7 μM PHA in the presence of WHI-P131 (6) or PP2 (7). Summary data of independent experiments on PBL from different donors are shown as mean + SEM (n = 4, p˂0.05). (C) Proliferation of resting PBL in response to 0.7 μg/ml PHA (2) and 10 μg/ml PHA (6, 7), or competent PBL in response to IL-2 in the absence (3) or presence of PP2 (4) or WHI-P131 (5). Bars represent the number of cells in (S+G2+M) phases of cell cycle at 48h after PBL stimulation. Summary data of independent experiments on PBL from different donors is shown as mean + SEM (n = 4, p˂0.05).
Fig 4.
Expression of mRNA encoding for IL-2Rα in human blood T lymphocytes stimulated by PHA or IL-2.
(A) The representative PCR amplification of IL-2Rα in PBL, resting (Ctrl) or stimulated with 10 μg/ml PHA (10PHA) for 4 and 24 h, or in competent lymphocytes treated with 0.7 μg/ml PHA (0.7PHA) for 24 h and then stimulated by IL-2 (0.7PHA+IL-2) for 4 and 24 h. β-glucuronidase (GUS) was used as an internal control. (B) Quantification of the results presented in (A). The mRNA expression is shown relative to the optical signals obtained with the RNAs in resting or competent PBL. The summary data of independent experiments on PBL of four donors, mean ± SEM (n = 4).
Fig 5.
PHA induces delayed and sustained STAT5 activation in human blood T lymphocytes.
(A) Representative Western blots of total lysates of PBL stimulated with 100 units/ml IL-2 (IL-2) or 10 μg/ml PHA (PHA) for 2, 24 or 48 h in the absence or presence of 80 μM WHI-P131 (W). (B) and (C) Densitometric analysis of STAT5 (B) and STAT3 (C) tyrosine phosphorylation in PBL stimulated with PHA (line 1) or IL-2 (line2) in the absence or presence of 80 μM WHI-P131 (W, lines 3 and 4). At each time point P-STAT5 and P-STAT3 levels relative to total STAT5 and total STAT3 levels were calculated and these values were normalized to those from samples at 24 h on the same protein gel blot to determine fold change. Summary of independent experiments on PBL from six donors are presented as mean ± SEM (n = 6, p˂0.05). Significant changes from P-STAT/STAT ratio at 24 h are indicated with an asterisk, p˂0.05. (D) Sustained STAT5 activity is provided by JAK3 kinase in mitogen-induced PBL. PBL were stimulated with 10 μg/ml PHA (10PHA) or with 100U/ml IL-2 (IL-2) or IL-2 after pretreatment of resting PBL with 0.7 μg/ml PHA (0.7PHA) in the absence or presence of 80 μM WHI-P131 (WHI). Data are representative of three experiments on PBL from different healthy donors. Ctrl—control, unstimulated PBL.
Fig 6.
Sustained STAT5 phosphorylation is necessary to induce long-term CD25 expression in human T lymphocytes.
(A) CD25+ cell number in PBL population stimulated with 100 units/ml IL-2 (IL-2) or 10 μg/ml PHA (10PHA) for 24 or 48 h or 0,7 μg/ml PHA(0.7PHA) for 48 h in the absence or presence of IL-2, %. Summary of independent experiments on PBL from four donors are presented as mean ± SEM (n = 4, p˂0.05). (B) Relative optical density in bands corresponding to normalized STAT5 tyrosine phosphorylation (P-STAT5). Summary of 11 independent experiments on PBL from different healthy donors are presented as mean + SEM (n = 11, p˂0.05). Ctrl—control, unstimulated PBL.