Fig 2: T cell-specific deletion of Senp3 perturbs T cell homeostasis. a Flow cytometric analysis of the frequency of naive (CD44loCD62Lhi) and memory-like (CD44hiCD62Llo for CD4+ and CD44hi for CD8+ T cells) CD4+ and CD8+ T cells in total splenocytes from 8-week-old Senp3+/+Cd4-Cre and Senp3fl/flCd4-Cre mice. b Flow cytometric analysis of the percentage of IFN-γ-producing and IL-17-producing CD4+ and CD8+ T cells in the spleen of 8-week-old Senp3+/+Cd4-Cre and Senp3fl/flCd4-Cre mice. c, d Flow cytometric analysis of the frequency of naive and memory-like (c) or IFN-γ-producing and IL-17-producing (d) T cells in total splenocytes from 8-month-old Senp3+/+Cd4-Cre and Senp3fl/flCd4-Cre mice. e Hematoxylin-eosin staining of the indicated tissue sections from 8-month-old Senp3+/+Cd4-Cre and Senp3fl/flCd4-Cre mice, showing immune cell infiltrations into the Senp3fl/flCd4-Cre tissues (arrows). Bars, 100 μm. f, g Quantification of CD4+ and CD8+ T cells (f) and percentage of CD4+ and CD8+ T cells expressing CD69 (g) in the lungs and livers of 8-month-old Senp3+/+Cd4-Cre and Senp3fl/flCd4-Cre mice. h Survival curves of Senp3+/+Cd4-Cre and Senp3fl/flCd4-Cre mice (n = 10). Data are representative of three or more independent experiments. Error bars are the mean ± SEM values. n = 5 or 10. Two-tailed unpaired Student’s t tests were performed. *P < 0.05; **P < 0.01

Fig 3: BACH2 deSUMOylation regulates its nuclear export in Treg cells. a IB analysis of the indicated proteins in whole (WL), nuclear (NE), and cytoplasmic (CE) extracts of Senp3+/+Foxp3-Cre and Senp3fl/flFoxp3-Cre Treg cells stimulated with anti-CD3 and anti-CD28 antibodies for 0, 4 or 8 h. bIB analysis of the indicated proteins in anti-CD3 and anti-CD28 antibody-stimulated naive CD4+ T cells from Bach2fl/flCd4-Cre mice transduced with the indicated lentiviral vector encoding Flag-WT BACH2 or Flag-mutant BACH2 under Treg cell conditions. c IB analysis of the indicated proteins in different lentivirus-transduced BACH2-deficient CD4+ T cells under Treg cell conditions treated with 10 ng/ml of leptomycin B for 4 h. d IB analysis of Senp3+/+Foxp3-Cre and Senp3fl/flFoxp3-Cre splenic Treg cells stimulated with anti-CD3 and anti-CD28 antibodies in the presence of leptomycin B for 4 h. e Splenic Treg cells (CD4+CD25+GFP+) from Rag1−/− mice reconstituted (for 8 weeks) with Bach2+/+Cd4-Cre or Bach2fl/flCd4-Cre bone marrow (BM) cells transduced with empty vector (EV) or vector expressing WT or 2KR BACH2 were stimulated with anti-CD3 and anti-CD28 antibodies for 24 h and subjected to flow cytometric analysis of Foxp3 expression. f Splenic Treg cells from Rag1−/− mice reconstituted with Senp3+/+Cd4-Cre or Senp3fl/flCd4-Cre BM cells transduced with EV, WT or 2KR BACH2 were stimulated with anti-CD3 and anti-CD28 antibodies for 24 h and subjected to flow cytometric analysis of Foxp3 expression. Numbers above graphs indicate MFI of Foxp3 (e, f). Data shown are representative of three independent experiments. Error bars are the mean ± SEM values (e, f). Two-tailed unpaired Student’s t tests were performed (e, f). *P < 0.05

Fig 4: Immunofluorescence staining of paraffin sections of nasal mucosa intraoperatively collected from patients with rhinitis. On comparing the nasal mucosal inflammation without NP group (A, C, E, and G) with the nasal mucosal inflammation with NP group (B, D, F, and H), same as the flow cytometry，the number of CD68+CD206+ cells was found to be increased in the nasal mucosal inflammation with NP group (C, D, I) (p = .0026). At the same time, the number of CD68+SENP3− cells without the expression of CD206 was less than those with the expression of CD206 (p = .0176) and the number of CD68+SENP3+ cells without the expression of CD206 was more than those with the expression of CD206 (p = .0009) (G, H, J). The orange arrow indicates CD68+SENP3+CD206−cells, and the yellow arrow indicates CD68+SENP3−CD206+ cells. The cells were counted by three different technicians under random fields. Positive cells were counted, and the data were expressed as mean ± standard error of mean for each group. The statistical analysis was performed using the t test *p < .05, **p < .01, ***p < .001. The scale in the figure is 20 μm.

Fig 5: SENP3 is required for the suppressive function of Treg cells. a CFSE-labeled wild-type CD4+CD25–YFP– T cells were cultured with the indicated number of Senp3+/+Foxp3-Cre or Senp3fl/flFoxp3-Cre Treg cells and stimulated with anti-CD3 and anti-CD28 antibodies for three days. Summary graphs of the percentage of proliferating CFSE-labeled T cells. b–d Disease phenotype of 6-week-old RAG-1-deficient mice given adoptive transfer of wild-type naive CD45RBhi CD4+ T cells together with Senp3+/+Foxp3-Cre or Senp3fl/flFoxp3-Cre Treg cells. Body weight was presented relative to initial weight (b). Frequency of cytokine-producing CD4+ T cells in the mesenteric lymph nodes (c) or hematoxylin-eosin staining of colon sections (d) from recipient mice at 12 weeks after adoptive transfer. Bars, 500 μm. e Tumor growth in Senp3+/+Foxp3-Cre or Senp3fl/flFoxp3-Cre mice subcutaneously (s.c.) injected with B16-F10 melanoma cells (n = 8). f, g Flow cytometric analysis of the frequency of IFN-γ-producing CD4+ and CD8+ T cells or Foxp3+CD4+ T cells in tumors of Senp3+/+Foxp3-Cre and Senp3fl/flFoxp3-Cre mice (day 14 after B16-F10 tumor injection). h Tumor growth in Senp3+/+Foxp3-Cre or Senp3fl/flFoxp3-Cre mice subcutaneously (s.c.) injected with MC38 colon cancer cells (n = 8). i, j Flow cytometric analysis of the frequency of IFN-γ-producing CD4+ and CD8+ T cells or Foxp3+CD4+ T cells in tumors of Senp3+/+Foxp3-Cre and Senp3fl/flFoxp3-Cre mice s.c. injected with MC38 colon cancer cells (day 14). Data are representative of at least three independent experiments. Error bars are the mean ± SEM values. n = 5 or 8. Two-tailed unpaired Student’s t tests were performed. *P < 0.05; **P < 0.01