Fig 2: SENP7-mediated PTEN deSUMOylation facilitates PTEN degradation.(A) HEK293T cells cotransfected with Flag-tagged PTEN, Ubc9, and RH-SUMO3 in the presence of WT or catalytically inactive (C979S) mutant (M) SENP7 were immunoprecipitated with anti-Flag antibody and assessed by immunoblotting (IB) with anti-SUMO2/3. HA, hemagglutinin; RH, arginine(R)-glycine(G)-serine(S)-histidine. (B) PTEN SUMOylation assays were performed by immunoprecipitating PTEN under denaturing conditions followed by detection of SUMOylated PTEN using anti-SUMO2/3 antibodies in CD8+ T cells stimulated with anti-CD3 and anti-CD28 antibodies. (C) Quantifications of PTEN-SUMO2/3:PTEN levels in CD8+ T cells stimulated with anti-CD3 and anti-CD28 antibodies (n = 3). (D) Immunoblot analysis of the indicated proteins in CD8+ T cells stimulated with anti-CD3 and anti-CD28 antibodies for 1 hour following incubation with CHX (50 μg/mL) for the indicated durations. (E) PTEN ubiquitination assays in CD8+ T cells stimulated with anti-CD3 and anti-CD28 antibodies in the presence of MG132. (F) Immunoblot analysis of the indicated proteins in CD8+ T cells from WT mice stimulated with anti-CD3 and anti-CD28 antibodies plus LMB for 2 hours. (G) PTEN SUMOylation assays using nuclear and cytoplasmic fractions of WT and KO CD8+ T cells stimulated with anti-CD3 and anti-CD28 antibodies for 2 hours. (H) Quantification of PTEN-SUMO2/3:PTEN levels in the cytoplasmic fractions of WT and KO CD8+ T cells stimulated with anti-CD3 and anti-CD28 antibodies for 2 hours (n = 4). (I) Immunoblot analysis of the indicated proteins in CD8+ T cells from WT mice stimulated with anti-CD3 and anti-CD28 antibodies plus LMB for 0, 2, and 4 hours. (J and K) PTEN SUMOylation (J) and ubiquitination (K) assays using nuclear and cytoplasmic fractions of CD8+ T cells from WT mice stimulated with anti-CD3 and anti-CD28 antibodies plus LMB for 2 hours. Data are representative of 3 independent experiments and presented as the mean ± SEM. *P < 0.05 and **P < 0.01, by Student’s t test.

Fig 3: SENP7 ablation dampens CD8+ T cell antitumor responses in vivo.(A) Tumor growth in Senp7fl/fl (WT) and Senp7fl/fl Cd4-Cre (KO) mice injected s.c. with MC38 murine colon cancer cells (n = 10 mice per group). (B) Flow cytometric analysis of the frequency of IFN-γ–producing CD8+ or CD4+ T cells in the draining lymph nodes of WT and KO mice injected s.c. with MC38 murine colon cancer cells (day 14, n = 6). (C) CD8+ T cell numbers in tumors (TILs) of WT and KO mice injected s.c. with MC38 murine colon cancer cells (day 14, n = 4) were normalized to 100 mg tumor tissue. (D and E) Flow cytometric analysis of IFN-γ–producing, TNF-α–producing, or granzyme B–producing CD8+ T cells in the tumors of WT and KO mice injected s.c. with MC38 murine colon cancer cells (day 14, n = 5). The data are presented as summary graphs in D and as representative plots in E. (F) Tumor growth in WT and KO mice injected s.c. with B16-F10 melanoma cells (n = 10 mice per group). (G) Frequency of IFN-γ–producing CD4+ T cells or CD8+ T cells in the tumors of WT and KO mice injected s.c. with B16-F10 melanoma cells (day 14, n = 5). (H and I) Tumor growth and survival curves for B6.SJL mice injected s.c. with MC38-OVA cancer cells adoptively transferred with WT OT-I or Senp7fl/fl Cd4-Cre (KO) OT-I CD8+ T cells on day 7 after tumor cell inoculation (n = 10 mice per group). Representative data are shown from 3 independent experiments. Data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01, by 2-tailed Student’s t test (A–D and F–H) and log-rank (Mantel-Cox) test (I).

Fig 4: ROS trigger cytosolic translocation of SENP7 in tumor-infiltrating CD8+ T cells.(A and B) Immunoblot analysis of the indicated proteins (A) and quantification of SENP7 expression (B, n = 6) in CD4+ and CD8+ T cells from CRC tissues. (C) Immunoblot analysis of the indicated proteins in CD4+ and CD8+ T cells from WT mice stimulated with anti-CD3 and anti-CD28 antibodies (α-CD3/28). (D–F) Immunoblot analysis using whole-cell lysates (WL) and nuclear (NF) and cytoplasmic (CF) fractions of CD8+ T cells from WT mice stimulated with anti-CD3 and anti-CD28 antibodies (D), CD8+ T cells from WT mice treated with 0.2 mM H2O2 for 1 hour (E), and CD8+ T cells from WT mice stimulated with anti-CD3 and anti-CD28 antibodies plus 10 mM NAC for 1 hour (F). (G and H) Histogram shows the MFI of ROS (G) and quantification of the MFI of ROS (H, n = 7) in CD8+ T cells from the spleens (Spl) and tumors (TIL) of tumor-bearing mice (day 7 after injection of tumors with MC38 cells). (I and J) Immunoblot analysis using CD8+ T cells from the spleens and tumors of tumor-bearing mice (I) and tumor-infiltrating CD8+ T cells treated with 10 mM NAC for 1 hour (J). (K and L) Histogram shows the MFI of ROS (K) and quantification of the MFI of ROS (L, n = 4) in CD8+ T cells from patient-derived PBMCs and CRC tissues. (M and N) Immunoblot analysis of the indicated proteins in CD8+ T cells from patient-derived PBMCs and CRC tissues (M) and CD8+ T cells from CRC tissues treated with 10 mM NAC for 1 hour (N). Representative data are shown from 2 (A, M, and N) and 3 (C–F, I, and J) independent experiments. *P < 0.05 and **P < 0.01, by Student’s t test (B, H and L).

Fig 5: SENP7-dependent reduction of PTEN controls CD8+ T cell metabolism and function.(A) Immunoblot analysis of the indicated proteins in WT and KO CD8+ T cells after stimulation with anti-CD3 and anti-CD28 antibodies. (B) PI3K activity in WT and KO CD8+ T cells stimulated with anti-CD3 and anti-CD28 antibodies was analyzed using an Echelon kit (n = 3). PIP3, phosphatidylinositol (3,4,5)-trisphosphate. (C) Immunoblot analysis of the indicated proteins in WT and KO CD8+ T cells after stimulation with anti-CD3 and anti-CD28 antibodies. (D and E) Flow cytometric analysis of p-S6 (D) and Ki-67 (E) expression in WT, Senp7fl/fl Cd4-Cre (KO), Ptenfl/fl Cd4-Cre (Pten-KO), and Ptenfl/fl Senp7fl/fl Cd4-Cre (DKO) CD8+ T cells stimulated with anti-CD3 and anti-CD28 antibodies for 2 hours (D, n = 3) or 2 days (E, n = 4). (F) Flow cytometric analysis of the division of WT, KO, Pten-KO, and DKO CD8+ T cells labeled with CFSE upon stimulation with anti-CD3 and anti-CD28 antibodies for 72 hours (n = 3). (G and H) ECAR (G) and OCR (H) of WT, KO, Pten-KO, and DKO CD8+ T cells stimulated for 6 hours with anti-CD3 and anti-CD28 antibodies. (I) Tumor growth of MC38 tumor–bearing Rag1-KO mice injected with 2 × 106 WT, KO, Pten-KO, or DKO CD8+ T cells along with 2 × 106 WT CD4+ T cells on day 7 after tumor cell inoculation (n = 10). (J) Flow cytometric analysis of IFN-γ–producing CD8+ T cells in the tumors of Rag1-KO mice from I (day 12, n = 4). Data shown are representative of 3 independent experiments and are presented as the mean ± SEM. *P < 0.05 and **P < 0.01, by 1-way ANOVA with Tukey’s multiple-comparison test (D–H and J); 2-way ANOVA with Geisser-Greenhouse correction (I); and 2-tailed Student’s t test (B).