Summary The proteasome constitutes the central proteolytic component of the highly conserved ubiquitin–proteasome system, which is required for the maintenance and regulation of basic cellular processes, including differentiation, proliferation, cell cycling, gene transcription and apoptosis. Here we show that inhibition of proteasomal proteolytic activity by the proteasome inhibitors bortezomib and lactacystin suppresses essential immune functions of human CD4+ T cells activated by allogeneic dendritic cells (DCs). In activated CD4+ T cells, proteasome inhibition induces apoptosis accompanied by rapid accumulation and stabilization of the tumour suppressor protein p53. Activated CD4+ T cells surviving proteasome inhibition undergo inhibition of proliferation by induction of G1 phase cell‐cycle arrest. Induction of G1 arrest is accompanied by the accumulation of cyclin‐dependent kinase inhibitors p21WAF1/CIP1 and p27KIP1 and the disappearance of cyclin A, cyclin D2 and proliferating cell nuclear antigen, proteins known to regulate G1 to S phase cell‐cycle transitions. Expression of the activation‐associated cell surface receptors CD25, CD28, CD120b and CD134 as well as production of interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α), interleukin‐4 (IL‐4) and IL‐5 is suppressed in response to proteasome inhibition in CD4+ T cells activated by DCs. Expression of CD25, IFN‐γ, TNF‐α, IL‐4 and IL‐5 is known to be mediated by the transcriptional activity of nuclear factor of activated T cells (NFAT), and we show here that proteasome inhibition suppresses activation and nuclear translocation of NFATc2 in activated CD4+ T cells. Thus, the proteasome is required for essential immune functions of activated CD4+ T cells and can be defined as a molecular target for the suppression of deregulated and unwanted T‐cell‐mediated immune responses.

We have previously shown that inhibition of the proteolytic activity of the proteasome induces apoptosis and suppresses essential functions of activated human CD4(+) T cells, and we report now the detailed mechanisms of apoptosis following proteasome inhibition in these cells. Here we show that proteasome inhibition by bortezomib activates the mitochondrial pathway of apoptosis in activated CD4(+) T cells by disrupting the equilibrium of pro-apoptotic and anti-apoptotic proteins at the outer mitochondrial membrane (OMM) and by inducing the generation of reactive oxygen species (ROS). Proteasome inhibition leads to accumulation of pro-apoptotic proteins PUMA, Noxa, Bim and p53 at the OMM. This event provokes mitochondrial translocation of activated Bax and Bak homodimers, which induce loss of mitochondrial membrane potential (DeltaPsim). Breakdown of DeltaPsim is followed by rapid release of pro-apoptotic Smac/DIABLO and HtrA2 from mitochondria, whereas release of cytochrome c and AIF is delayed. Cytoplasmic Smac/DIABLO and HtrA2 antagonize IAP-mediated inhibition of partially activated caspases, leading to premature activation of caspase-3 followed by activation of caspase-9. Our data show that proteasome inhibition triggers the mitochondrial pathway of apoptosis by activating mutually independent apoptotic pathways. These results provide novel insights into the mechanisms of apoptosis induced by proteasome inhibition in activated T cells and underscore the future use of proteasome inhibitors for immunosuppression.