Figure 5

Signal 1 determines resistance to ABT-737 in activated T cells. (a) Selective inhibitors of signal 1, 2 and 3 were added to an MLR during the stimulation phase with BM3.3 splenocytes reacting against CD8 T-cell-depleted B6 splenocytes to investigate the role of the different T-cell activation pathways for resistance to ABT-737(1 μM, during additional 12 h of culture). The calcineurin inhibitor and signal 1 blocker CsA prevented resistance to ABT-737 in a concentration-dependent manner, whereas inhibition of signal 2 by CTLA4Ig and signal 3 by rapamycin did not influence this process. (b) The results obtained with CsA were confirmed by other inhibitors of this same pathway, namely the alternative calcineurin inhibitor tacrolimus and the NFAT inhibitor VIVIT-R. Cell viability of BM3.3 CD8 T cells was assessed by PI exclusion in FACS in at least three independent experiments. Percentage of cells treated with vehicle is given. Statistical comparison of ABT-737 versus vehicle: *P<0.05, **P<0.01. (c and d) Exposure to CsA during the stimulation phase inhibited the upregulation of A1 as assessed by qRT-PCR (c) and western blot (d)