Fig. 4: Enhancement of immune activation by NEDD8 deficient triple-negative breast cancer (TNBC) cells in response to immunotherapy drugs.

a Label-free quantification of peptides derived from HLA-DRA and HLA-DRB in MDA-MB-231 wild-type (WT) and NEDD8 knock-out (KO) cell lines in proteomics, 4 technical replicates. b WT or NEDD8 KO human TNBC cell lines, i.e., MDA-MB-231 and HCC1937, were treated with PBS (5 independent replicates) or 50 ng/ml rhIFNγ (4 independent replicates) for 24 h. Surface expression of HLA-DR was quantified using flow cytometry. WT or NEDD8 KO MDA-MB-231 cells were co-cultured with CTV-pulsed primary human lymphocytes ±10 μg/ml nivolumab (red) or durvalumab (blue). c Release of soluble IFNγ was tested by ELISA (4 independent donors) or (d) proliferation of T cells was quantified by flow cytometry on day 5 (6 independent donors). e Ctrl or NEDD8 KO HCC1937 cells were co-cultured with primary human lymphocytes ±10 μg/ml nivolumab or durvalumab and release of soluble granzyme B was tested by ELISA on day 5 (4 independent donors). f A truncated NEDD8 protein lacking the C-terminus diglycine residues was re-expressed in NEDD8 KO MDA-MB-231 cells (NEDD8-T) and protein neddylation was measured using Western Blotting. Representative image of 2 independent repeats. g Control (dark gray), NEDD8 KO (open) or NEDD8-T (light gray) cells (2500 cells per well) were co-cultured with primary human lymphocytes ±10 μg/ml nivolumab and release of soluble IFNγ was tested using ELISA on day 5 (4 independent donors). All data in this figure were shown as mean ± SD and unpaired two-tailed T-test was used for statistical analysis. Source data are provided as a source data file.