Fig 1.
In vitro effects of cladribine on cell survival of PBMCs and proliferation of PBMCs, CD4+ cells and CD8+ cells Human immune cells were stimulated with anti-CD3/anti-CD28 antibodies (a, n = 4; c, n = 6) or PHA (b, n = 4; d, n = 3) in the absence or presence of cladribine (1×10−8 M to 1×10−5 M) for 72 h.
Determination of cell survival in PBMCs (a, b) was performed by standard trypan blue exclusion method. Proliferation was determined separately in PBMC, CD4+ cells and CD8+ cells (c, d) by the incorporation of tritiated thymidine. Stimulation indices were calculated as the ratios of the counts per minute of stimulated samples to unstimulated and untreated samples. Data are depicted as mean + standard deviation (SD).
Fig 2.
Apoptosis of stimulated PBMCs, CD4+ cells and CD8+ cells caused by cladribine in vitro PBMCs (a, n = 3), CD4+ cells (b, n = 3) and CD8+ cells (c, n = 3) were stimulated with anti-CD3/anti-CD28 antibodies and were incubated in the absence or presence of cladribine (1×10−8 M to 5×10−7 M) for 12, 24, 48 and 72 h.
Apoptotic cells were defined as Annexin V positive cells. Data are depicted as mean + standard deviation (SD).
Table 1.
Apoptosis of stimulated PBMCs caused by cladribine in vitro.
Table 2.
Apoptosis of stimulated CD4+ cells caused by cladribine in vitro.
Table 3.
Apoptosis of stimulated CD8+ cells caused by cladribine in vitro.
Fig 3.
In vitro effects of initial cladribine treatment on the proliferation of PBMCs stimulated at Days 9, 16, 23, 30, 44 and 58 after transfer into cladribine-free medium PBMCs were initially incubated in the absence or presence of cladribine (1×10−8 M to 5×10−7 M) for 72 h.
Cells were washed three times and then transferred into cladribine-free medium. Cells were restimulated with anti-CD3/anti-CD28 antibodies (a, n = 5) or PHA (b, n = 4) for 48h at Days 9, 16, 23, 30, 44 and 58; proliferation was determined by the incorporation of tritiated thymidine. Stimulation indices were defined as the ratios of the counts per minute of stimulated samples to unstimulated and untreated samples. Data are depicted as mean + standard deviation (SD).
Fig 4.
In vitro effects of initial cladribine treatment on cytokine secretion of PBMCs restimulated at Days 9, 16, 23, 30, 44 and 58 after transfer into cladribine-free medium.
PBMCs from healthy blood donors (n = 5) were incubated in the absence or presence of cladribine. (1×10−8 M to 5×10−7 M) for 72 h. Then cells were washed three times and transferred into cladribine-free medium. Cells were restimulated with anti-CD3/anti-CD28 antibodies for 48 h at days 9, 16, 23, 30, 44 and 58; supernatants were collected and cytokine concentrations were determined. Data are depicted as box plot diagrams. Whiskers represent maximum and minimum values. The IL-4/IFN-γ ratio was defined as the ratio of IL-4 (pg/ml) to IFN-γ (pg/ml). *: p<0.05; **: p<0.01.
Fig 5.
In vitro effects of initial cladribine treatment on cytokine secretion of PBMCs restimulated at Days 9, 16, 23 and 30 after transfer into cladribine-free medium.
PBMCs from healthy blood donors (n = 4) were incubated in the absence or presence of cladribine. (1×10−8 M to 5×10−7 M) for 72 h. Then cells were washed three times and transferred into cladribine-free medium. Cells were restimulated with PHA for 48 h at days 9, 16, 23 and 30; supernatants were collected and cytokine were determined. Data are depicted as box plot diagrams. Whiskers represent maximum and minimum values. The IL-4/IFN-γ ratio was defined as the ratio of IL-4 (pg/ml) to IFN-γ (pg/ml). *: p<0.05; **: p<0.01.