Fig 2: 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.

Fig 3: Usp5 conditional knockout (cKO) mice have effective tumor control.a Conditional knockout strategy of Usp5fl/flCd4-Cre (cKO) mice. b Protein levels of Usp5 in WT or cKO CD8+ T cells of spleens. c–e The production of IFN-?, TNF, or GzmB in WT and cKO CD8+ T cells with anti-CD3/CD28 (0 or 2 µg/mL) stimulation for 96 h and Brefeldin A (BFA, 5 µg/mL) treatment for 5 h. n = 3 biologically independent samples. f–i The growth of subcutaneous MC38 tumors and survival of WT or cKO mice. Tumor growth was measured every 2 days and plotted (f). Endpoint tumor weight (g) and images (h) of MC38 tumors. Kaplan–Meier survival curves for WT and cKO mice bearing MC38 tumors (i). Data were presented as mean ± S.D. n = 5 mice per group (f, g). Two-way ANOVA for (f). For i, WT = 15 mice, cKO = 10 mice, log-rank test. j–l Quantification of CD44+/CD8+ T cells (j) or cellular surface PD-1 on CD8+ T cells (k, l) in subcutaneous MC38 tumors derived from WT or cKO mice. n = 5 mice per group. m–p Quantification of CD8+/CD3+ T cells (m), CD8+/Foxp3+CD4+ T cells (n), CD4+/CD3+ T cells (o), or Foxp3+/CD4+ T cells (p) in subcutaneous MC38 tumors derived from WT or cKO mice. n = 5 mice per group. q–s The growth of subcutaneous LLC tumors in WT or cKO mice. Tumor growth was measured every 2 days and plotted (q). Endpoint tumor weight (r) and images (s) of LLC tumors. n = 6 mice per group. Data were presented as mean ± S.D. Two-way ANOVA for (q). t–v Flow cytometry analysis of cellular surface PD-1 on CD8+ T cells in subcutaneous LLC tumors from WT or cKO mice (t–v). Quantification of CD4+/CD3+ T cells (w), Foxp3+/CD4+ T cells (x), or CD8+/CD3+ T cells (y) in subcutaneous LLC tumors derived from WT or cKO mice. n = 4 mice per group. For c–e, g, j, l–p, r, u–y, data were presented as mean ± S.D. Two-tailed unpaired t-test. For b, data are representative of two independent experiments. Source data are provided as a Source data file.