Fig 2: USP5 interacts with PD-1 and deubiquitinates PD-1.a–c IB analysis of WCL and anti-PD-1 IPs derived from Jurkat cells (a), mouse primary CD3+ T cells (b), or glutathione S-transferase (GST) pull-down precipitates from HEK293T cell lysates with ectopic expression of PD-1-cHA incubated with recombinant GST or GST-USP5 protein (c). Jurkat cells were stimulated with PHA (150 ng/mL) for 3 days. Mouse primary CD3+ T cells were stimulated with anti-CD3/CD28 (2 µg/mL) for 48 h. d, g A schematic illustration of USP5 (d) or PD-1 (g) protein sequence to show its different domains and deletion truncations we generated. e, f, h IB analysis of WCL and anti-HA IPs (e) or GST pull-down precipitates (f, h) from HEK293T cell lysates co-transfected with indicated constructs. i–k IB analysis of WCL and Ni-NTA pull-down products derived from lysates of HEK293T cells transfected with the indicated constructs. Cells were treated with 10 µM MG132 for 12 h before harvesting. l For in vitro deubiquitination assay, HEK293T cells transfected with His-ubiquitin and PD-1-cFlag were treated with 10 µM MG132 for 12 h. Ubiquitinated PD-1 was purified with IP using anti-Flag beads and was incubated without or with purified recombinant USP5. IB analysis with indicated antibodies. m IB analysis of WCL and anti-PD-1 denatured-IPs derived from lysates of shGFP- or shUSP5-treated Jurkat cells using indicated antibodies. Cells were treated with PHA (150 ng/mL) for 3 days and 5 µM MG132 for 6 h. n IB analysis of WCL and anti-PD-1 denatured-IPs derived from naive CD3+ T cells isolated from spleens of Usp5fl/fl (WT) or Usp5fl/fl Cd4-Cre (cKO) mice. CD3+ T cells were stimulated with anti-CD3/CD28 (2 µg/mL) for 48 h and with 5 µM MG132 for 4 h before harvesting. All data are representative of two independent experiments. Source data are provided as a Source data file.

Fig 3: Identifying the deubiquitinase USP5 as a positive regulator for PD-1.a Immunoprecipitation (IP) coupled with mass spectrometry (MS) analysis to identify potential PD-1-interacting protein including indicated deubiquitinases. Potential PD-1-interacting proteins purified from HEK293T cells transfected with pcDNA3.1-PD-1-cHA were analyzed by MS. All proteins were identified in Supplementary Data 1. b–d Immunoblotting (IB) analysis of whole-cell lysates (WCL) from HEK293T cells co-transfected with indicated constructs (b) or MOLT-4 cells stably expressing shUSP5, shUSP10, shUSP15 or shGFP (d). The mRNA level of PD-1 was measured using quantitative reverse transcription PCR (qRT-PCR) (c). e–h IB analysis of WCL from HEK293T cells transfected with indicated constructs (e) or Jurkat cells stably expressing shUSP5 as well as shGFP (g). Cells were treated with 200 µg/mL cycloheximide (CHX) for indicated time points. PD-1 band intensity was quantified by Image J, which was normalized to Vinculin/GAPDH and then compared to the t = 0 time point (f, h). i, j IB analysis of WCL from MOLT-4 cells (i) or mouse primary CD3+ T cells (j) treated with indicated deubiquitinase inhibitors (1.5 µM) for 8 h. Mouse primary CD3+ T cells were pre-stimulated with anti-CD3/CD28 (2 µg/mL) for 48 h. k–n IB analysis of WCL from Jukat or MOLT-4 cells treated with EOAI3402143 (1 or 1.5 µM) for 8 h (k, m). The mRNA level of PD-1 was measured using qRT-PCR (l, n). o, p Cell surface PD-1(o) and relative mean fluorescence intensity (MFI) of PD-1 (p) on MOLT-4 cells was analyzed by flow cytometry. q Representative multiplex immunohistochemistry (mIHC) images of USP5 (red), PD-1 (green), CD3 (Orange), CK (cyan), and DAPI nuclear staining (blue) in human colon tumor sections. White arrows indicate positive cells for PD-1 and USP5 colocalization. Yellow color is considered overlapping for PD-1 and USP5 staining. n = 5. Scale bars, 50 µm. For g, k, Jurkat cells were stimulated with PHA (150 ng/mL) for 3 days. For c, f, h, l, n, and p, data were presented as mean ± S.D. n = 3 biologically independent samples. Two-tailed unpaired t-test. All IB data are representative of three independent experiments. Source data are provided as a Source data file.