Extended Data Fig. 4: Targeting the p97-Npl4 interaction skewed Treg-Th17 cell balance.

a, Gating strategy for TI-Treg and Th17 cells gated from tumor-infiltrated CD4⁺ T cells, with a fluorescence minus one (FMO) sample used as negative control. b, Absolute numbers of intratumoral Treg (TI-Treg, Foxp3⁺) and Th17 cells (IL17a⁺) per milligram tumor from tumor-bearing control (Ctrl) and TB-intraperitoneally (i.p.)-injected mice. n = 4 biologically independent samples examined over two independent experiments. c, Absolute numbers of TI-Treg and Th17 cells per milligram tumor from tumor-bearing Ctrl and TB-intravenously (i.v.)-injected mice. n = 4 biologically independent samples examined over two independent experiments. d, Representative flow cytometry plots and percentages of TI-Treg cells from tumor-bearing Ctrl or TB-intratumorally (i.t.)-injected mice. n = 4 biologically independent samples examined over two independent experiments. e, Absolute number of TI-Treg cells per milligram tumor in each of the indicated mice. n = 4 biologically independent samples examined over two independent experiments. f, Representative plots and percentages of tumor-infiltrated Th17 cells from tumor-bearing Ctrl or TB-intratumorally (i.t.)-injected mice. n = 4 biologically independent samples examined over two independent experiments. g, Absolute number of tumor-infiltrated Th17 cells per milligram tumor in each of the indicated mice. n = 4 biologically independent samples examined over two independent experiments. h, Schematic illustration of the iTreg-cell-transfer assay procedure. CD45.1 tumor-bearing mice were intravenously injected with Vcp^3A/3A iTreg (WT) or Vcp^3A/3A Foxp3^Cre (3A) iTreg cells. i, Flow cytometry analysis of the purity of iTreg cells derived from WT and 3A mice for adoptive transfer assay. j, Absolute numbers of TI-Treg cells per milligram tumor from tumor-bearing Vcp^3A/3A, Foxp3^Cre and Vcp^3A/3A Foxp3^Cre mice. n = 10 biologically independent samples examined over four independent experiments. k, Absolute numbers of intratumoral Th17 cells per milligram tumor from tumor-bearing Vcp^3A/3A, Foxp3^Cre and Vcp^3A/3AFoxp3^Cre mice. n = 10 biologically independent samples examined over four independent experiments. l, Absolute numbers of tumor-infiltrated IFNγ⁺CD8⁺ T cells per milligram tumor from tumor-bearing Vcp^3A/3A, Foxp3^Cre and Vcp^3A/3AFoxp3^Cre mice. n = 10 biologically independent samples examined over four independent experiments. Data shown as means ± s.e.m. (b-g,j-l). Data were analyzed for significant differences by performing one-way ANOVA with Dunnett’s post hoc analysis (b–g) or two-tailed, unpaired Student’s t-test (j–l).

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