Fig 2: The regulation of TRAPPC4 on PD-L1 affects T-cell-mediated antitumor immunity in vitro and in vivo.a Flow cytometry detection of PD-1 bound by cell surface PD-L1 on RKO cells treated with siRNAs targeting TRAPPC4 or control siRNA. The y axis indicates the MFI of fluorescence conjugated PD-1. This figure represents the result of three independent experimental replicates. Values indicate means ±SD. b Schematic illustration of the experimental procedures for the T-cell-mediated cytotoxicity assay. CRC cells transfected with siRNAs targeting TRAPPC4 or control siRNA were incubated with activated PBMCs. An apoptosis assay was further conducted to determine the T-cell-mediated cytotoxicity. PBMC, peripheral blood mononuclear cell. c T-cell cytotoxicity assay against RKO cells treated with a siRNA for TRAPPC4 and control siRNA. Flow cytometry detection of apoptotic cells was conducted after co-incubation of PBMCs with CRC cells. Total apoptotic cells were defined as the proportion of cells in quadrant Q2 and Q3. This experiment was performed three times independently and values indicate means ±SD. Statistical analysis was conducted using a two-sided Student’s t test. d–f Stable MC38 cells transduced with the indicated lentiviruses were injected into C57BL/6 mice. Statistical analysis of tumor volumes (d), representative images of tumors (e), and tumor weight (f) of mice in different groups (n = 9 per group) were shown. Values indicate means ±standard error of the mean (SEM) in (d) and means ±SD in (f), compared by the two-sided Student’s t test. (P = 1.3e-5 in d). g–j The activation and function of the tumor-infiltrating T cells in each group were analyzed using flow cytometry. The percentages of CD3+ CD8+ T cells (g) (n = 9 per group), CD3+ CD8+ CD69+ T cells (h, the left panels were gated in CD45+ subsets) (n = 7 per group), CD3+ CD8+ CD107a+ T cells of CD45+ cells (i) (n = 9 per group) and mean fluorescence intensity (MFI) of IFN-γ of CD8+ T cells (j) (n = 9 per group) were plotted as means ±SD compared by the two-sided Student’s t test. Source data are provided as a Source Data file.

Fig 3: GSK2857916 enhances intratumor immune cell infiltration and activation. Quantification by flow cytometry of immune cells infiltrating C57BL/6 mice bearing EL4-hBCMA tumors following treatment with GSK2857916 or isotype control (at 15 mg/kg), or saline vehicle. A, CD8+, CD4+, and regulatory (CD4+CD25+FoxP3+) T cells, and DC (CD11c+). B, T-cell activation markers CD25, CD69, and ICOS and the exhaustion marker PD-1. C, Quantification of the DC costimulatory molecule CD86, T-cell proliferation (Ki67) and cytotoxicity (granzyme B, GrzB) in TILs and NK cells (CD49b+). A one-way ANOVA statistical analysis was performed. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P ≤ 0.0001. D, IHC for CD8 reactive cells in mice bearing EL4-hBCMA tumors treated with GSK2857914 (top) or GSK2857916 (bottom) at 15 mg/kg (as in Fig. 4). Representative images, 20× magnification. E, Quantification of calreticulin cell surface expression (MFI) on tumor cells in mice. F, Heatmap depicting a type I IFN-related gene signature following treatment with saline, isotype control or GSK2857916 (at 15 mg/kg) from the same experiment as in (A–C, E). Each column represents an individual mouse and each row an individual gene.

Fig 4: Overexpression of Trappc4 affects tumor microenvironment composition and immunotherapy efficacy.a Schematic representation of the xenograft mouse model and dosage regimen. Mice bearing control or Trappc4-OE MC38 tumors were treated with anti-PD-L1 mAb. b–d Statistical analysis of tumor volumes (b) (vector+α-PD-L1, n = 13; all other groups n = 14), representative images of tumors (c) and tumor weights (d) (vector + IgG, n = 12, Trappc4-OE + IgG, n = 12, Vector+α-PD-L1, n = 10, Trappc4-OE + α-PD-L1, n = 10) of the mice with subcutaneous tumors in different groups. Values indicate means ±SEM in (b) and means ±SD in (d), compared by nonparametric Mann–Whitney test, two-sided. (*P = 7.2e-5) e–h The percentages of CD8+ T cells (e), CD69+ CD8+ T cells (f), CD107a+ CD8+ T cells of CD45+ cells (g) and IFN-γ+ cells of CD8+ T cells (h) in each group (n = 6 per group) were analyzed by flow cytometry. The statistics were plotted as means ±SD, compared by two-sided Student’s t test in e, g, h, and nonparametric Mann–Whitney test in f. OE, overexpression. (**P = 3.6e-5). Source data are provided as a Source Data file.

Fig 5: EGFR Expression by T Cells Contributes to Host Resistance against Gastro-intestinal Helminth Infections(A–C) WT, Egfrfl/fl, and Egfrfl/flxCd4-cre (EGFRΔCD4) mice were infected with H. polygyrus or N. brasiliensis or left untreated. Percentage and absolute number of EGFR expressing CD4+ T helper cells in (A) duodena, (B) mLN, and (C) spleen, and EGFR expression on total CD4+ and CD69+ and CD69– mLN-derived T cells from naive or H. polygyrus-infected mice was determined by flow cytometry analysis on day 14 post infection.(D) Comparison of GATA3 expression on CD4+ T cells from mLN and spleen of H. polygyrus-infected Egfrfl/fl and EGFRΔCD4 mice.(E) Time-dependent egg load in feces and worm burden 4 weeks post infection with H. polygyrus (n = 4 mice).(F) Time-dependent egg load in feces and worm burden 9 days post infection with N. brasiliensis (n = 3 mice).All data are representative of at least two independent experiments (mean ± SEM); results for individual mice are shown as dots. See also Figures S1–S3.