Cd11c 3

For biomarker analysis, 15 mL of blood was collected at baseline and at the time of each disease evaluation. Peripheral blood mononuclear cells (PBMCs) were obtained by standard density gradient centrifugation. To evaluate the prognostic implications of immune cells, multicolour flow cytometry analyses using the CytoFLEX flow cytometry platform (Beckman Coulter, Brea, CA) were performed to determine the proportions of different immune cell populations in the PBMCs. Panels for multicolour flow cytometry included CD3 (UCHT1; BioLegend, San Diego, CA, USA), CD4 (OKT4; BioLegend), CD8 (SK1; BioLegend), CD14 (63D3; BioLegend), CD11c (3.9; BioLegend), CD56 (5.1H11; BioLegend), γδ TCR (B1; BioLegend), HLA-DR (L243; BioLegend), CD69 (FN50; BioLegend), FoxP3 (236A-E7; eBioscience, San Diego), PD-1 (EH12.2H7; BioLegend), LAG-3 (11C3C65; BioLegend), CTLA-4 (L3D10; BioLegend), and TIGIT (A15153G; BioLegend).

The human 20S immunoproteasome core sample was purchased from Boston Biochem. The human i-20s at a concentration of 1.5 mg per ml was incubated with PKS21004 dissolved in DMF with a molar ratio of 1:250 ratio for 1 h at 37 °C, then the mixture was diluted in 50 mM HEPES, pH 7.6, 100 mM NaCl and 1 mM dithiotreitol to a final concentration of 0.1 mg per mL for cryo-EM study. Antibodies used for flow cytometry are described as below (clone, company): CD3 (UCHT1, Biolegend), CD4 (OKT4, Biolegend), CD8 (SK1, Biolegend), CD11c (3.9, Biolegend), CD14 (RMO52, Beckman Coulter), CD16 (3G8, Biolegend), CD19 (HIB19, BD), CD38 (LS198–4–3, Beckman Coulter), CD27 (O323, eBioscience), IgD (IA6–2, eBioscience), HLA-DR (L243, Biolegend), Ki67 (Ki-67, Biolegend).