Immunosuppresive properties of peptidyl-fluoromethyl ketones and the role of oxidative stress in T cell activation and proliferation

It is well established that caspases are involved in T cell activation and proliferation. Many of the early studies relied on peptidyl-fluoromethylketone (FMK) caspase inhibitors, such as z-VAD-FMK. Recently, z-VAD-FMK and z-IETD-FMK were shown to block T cell proliferation without inhibiting caspase activation. These results suggest that the inhibition of T cell proliferation by these peptidyl-FMK caspase inhibitors is independent of their caspase inhibition properties. In contrast, z-FA-FMK, commonly used as a negative control peptide for the FMK-containing caspase inhibitors, blocked T cell proliferation as well as inhibiting caspase processing. In the present study, several analogues of z-FA-FMK were examined to determine whether peptidyl-FMKs block mitogen-induced T cell activation and proliferation through the inhibition of specific targets or due to non-specific effects of the FMK group. The results showed that only z-FA-FMK blocks T cell proliferation whereas z-FA-diazomethylketone (DMK) has little effect while z-FA-chloromethylketone (CMK) was toxic and killed all the cells. This suggests that the FMK moiety may play an important role in blocking T cell activation and proliferation. Interestingly, replacing the alanine in z-FA-FMK with phenylalanine, z-FF-FMK, blocked T cell proliferation to a higher degree than z-FA-FMK. Similar to z-FA-FMK, z-FF-FMK inhibited T cell blast formation, CD25 and CD69 expression and cell cycle entry while inhibiting caspase processing in activated T cells. When peptidyl-methyl ketones were tested on Jurkat T cells, it was found that only z-FA-FMK and z-FF-MK blocked T cell proliferation. Next the mechanism of z-FA-FMK-, z-FF-FMK- and z-VAD-FMK-induced block of proliferation in primary and Jurkat T cells was examined. Several studies have shown that the cellular redox state plays an important role in T cell activation and proliferation. Thus, the effect of these three compounds on intracellular glutathione (GSH) levels and reactive oxygen species (ROS) generation was explored. The data showed that intracellular GSH was depleted while ROS generation was increased following treatment with the peptidyl-FMK inhibitors in primary and Jurkat T cells. The addition of the low molecular weight thiols GSH, N-acetylcysteine (NAC) and L-cysteine, readily reversed the inhibition of T cell proliferation induced by these peptidyl-FMK inhibitors, whereas D-cysteine, which cannot be metabolised to GSH had no effect. Furthermore, GSH, NAC and L-cysteine fully restored the caspase-8 and caspase-3 processing in anti-CD3-activated primary T cells treated with z-FA-FMK and z-FF-FMK. In conclusion, the results suggest that the immunosuppressive effect of z-FA-FMK, z-FF-FMK and z-VAD-FMK is likely due to the induction of oxidative stress and GSH depletion in T cells.