Small molecule inhibitors selectively targeting the immunoproteasome subunit β5i are currently being developed for the treatment of autoimmune disorders. However, patients carrying loss‐of‐function mutations in the gene encoding β5i (Psmb8) suffer from the proteasome‐associated autoinflammatory syndromes (
PRAAS
) emphasizing the need to study pharmacological inhibition of immunoproteasome function in human cells. Here, we characterized the immunomodulatory potential of the selective β5i inhibitor
ONX
0914 and Bortezomib, a pan‐proteasome inhibitor, in human peripheral blood mononuclear cells (
PBMC
s). Both compounds efficiently blocked pro‐inflammatory cytokine secretion in human whole blood and
PBMC
cultures stimulated with toll‐like receptor (
TLR
) agonists. Furthermore, the compounds inhibited T cell cytokine production induced by recall antigen
CMV
pp65 or by polyclonal stimulation. The viability of
PBMC
s, however, was rapidly decreased in the presence of
ONX
0914 and Bortezomib demonstrated by decreased residual cytosolic
ATP
and increased Annexin V surface binding. Interestingly,
HLA
‐
DR
+
monocytes were rapidly depleted from the cultures in the presence of
ONX
0914 as a β5i‐selective inhibitor and Bortezomib. In conclusion, the anti‐inflammatory potential of β5i‐selective inhibitors is correlating with a cytotoxicity increase in human
PBMC
subsets ex vivo. Our results provide important insights into the anti‐inflammatory mechanism of action of β5i‐inhibitors which currently hold the promise as a novel therapy for autoinflammatory diseases.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.