Proteasome inhibition suppresses essential immune functions of human CD4+ T cells.

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 G(1) phase cell-cycle arrest. Induction of G(1) arrest is accompanied by the accumulation of cyclin-dependent kinase inhibitors p21(WAF1/CIP1) and p27(KIP1) and the disappearance of cyclin A, cyclin D2 and proliferating cell nuclear antigen, proteins known to regulate G(1) 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-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), 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-gamma, TNF-alpha, 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.

Proteasomal chymotrypsin-like peptidase activity is required for essential functions of human monocyte-derived dendritic cells.

The ubiquitin-proteasome pathway is the principal system for extralysosomal protein degradation in eukaryotic cells, and is essential for the regulation and maintenance of basic cellular processes, including differentiation, proliferation, cell cycling, gene transcription and apoptosis. The 26S proteasome, a large multicatalytic protease complex, constitutes the system's proteolytic core machinery that exhibits different proteolytic activities residing in defined proteasomal subunits. We have identified proteasome inhibitors - bortezomib, epoxomicin and lactacystin - which selectively inhibit the proteasomal beta5 subunit-located chymotrypsin-like peptidase activity in human monocyte-derived dendritic cells (DCs). Inhibition of proteasomal chymotrypsin-like peptidase activity in immature and mature DCs impairs the cell-surface expression of CD40, CD86, CD80, human leucocyte antigen (HLA)-DR, CD206 and CD209, induces apoptosis, and impairs maturation of DCs, as demonstrated by decreased cell-surface expression of CD83 and lack of nuclear translocation of RelA and RelB. Inhibition of chymotrypsin-like peptidase activity abrogates macropinocytosis and receptor-mediated endocytosis of macromolecular antigens in immature DCs, and inhibits the synthesis of interleukin (IL)-12p70 and IL-12p40 in mature DCs. As a functional consequence, DCs fail to stimulate allogeneic CD4(+) and CD8(+) T cells and autologous CD4(+) T cells sufficiently in response to inhibition of chymotrypsin-like peptidase activity. Thus, proteasomal chymotrypsin-like peptidase activity is required for essential functions of human DCs, and inhibition of proteasomal chymotrypsin-like peptidase activity by selective inhibitors, or by targeting beta5 subunit expression, may provide a novel therapeutic strategy for suppression of deregulated and unwanted immune responses.