Fig 2: CTLA-4, GITR, ICOS and OX40 Are Highly Expressed by Tumor-Infiltrating Treg Cells(A–C) Mice (n = 5) were injected subcutaneously (s.c.) with B16, MCA205, MC38 (C57BL/6 mice) or CT26 (Balb/c mice) cells. Ten days later, cell suspensions of PBMC, draining LNs and tumor-infiltrating lymphocytes (TILs) were stained and analyzed by flow cytometry. (A) Representative histograms of CTLA-4 expression detected by intracellular staining of individual T cell subsets in mice with MCA205 tumors. Dotted lines represent the gates, numbers indicate the percentage of CTLA-4+ cells. (B and C) Percentage (B) and MFI (C) of CTLA-4-expressing cells in murine PBMCs, LNs, and TILs in different tumor models.(D) Representative histograms of CTLA-4 expression detected by intracellular staining of T cell subsets in PBMCs and TILs in a patient with advanced melanoma.(E and F) Percentage (E) and MFI (F) of CTLA-4 expression in T cells in PBMCs and TILs of patients with advanced melanoma (n = 8), early-stage NSCLC (n = 8) and RCC (n = 8).(G) Heatmap demonstrating the percentage of cells expressing co-inhibitory and co-stimulatory molecules within the indicated T cell subsets quantified by flow cytometry. Each row represents an individual murine or human tumor (n = 5).(H) MFI of the indicated co-inhibitory and co-stimulatory molecules in PBMCs and TILs in patients with melanoma. Horizontal bars represent the mean; error bars show ± standard error of the mean (SEM).∗p < 0.05; ∗∗∗∗p < 0.0001. See also Table S1 and Figure S1.

Fig 3: Anti-CTLA-4 Antibodies of IgG1 and IgG2 Isotype Mediate Depletion of CTLA-4-Expressing Target Cells In Vitro(A) SPR analysis of anti-murine CTLA-4 with human IgG variants. Large graphs demonstrate interaction of free monomeric FcγRs at increasing FcγR concentrations with immobilized IgG variants; inset graphs show interaction of immobilized IgG variants with aggregated low-affinity FcγRs at increasing concentrations. RU, response units.(B) Schematic representation of the mechanism of action of chimeric anti-mCTLA-4 antibodies and predicted ADCC activity for each human IgG variant.(C) In vitro ADCC assay with human monocyte-derived macrophages and mCTLA-4+ target cells in the presence of anti-mCTLA-4 mAbs with different human IgG variants.(D) ADCC assay in the presence of CD32a or CD32b-blocking F(ab’)2 antibody fragments and with a deglycosylated IgG2 mAb (IgG2EndoS).Results are representative of three independent experiments. Error bars show ±SEM of experimental triplicates. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. See also Figure S3.

Fig 4: Transferrin suppresses T cell activation. (A) Immunoblot analysis of total and phosphorylated CD3ξ, zeta-chain-associated protein kinase 70 (ZAP70), and lymphocyte cytosolic protein 2 (LCP2) in human T cells. Corresponding quantifications of (A) are shown at right. GAPDH was used as loading control. Data represent the mean ± SD of 3 independent experiments. ns, no significance; **P < 0.01 by one-way ANOVA with Fisher’s protected t test. (B and C) ELISA of the expression of IL-2 (B) and IFN-γ (C). Isolated T cells were stimulated with anti-CD3/CD28 (10 μg/ml) for 24 h. Similar results were acquired in multiple donors (4). Data represent the mean ± SD of 4 independent experiments. ns, no significance; *P < 0.05 and **P < 0.01 by one-way ANOVA with Fisher’s protected t test. (D) Schematic for generation of TEX cells in vitro. The model is based on the repeated stimulation of isolated T cells with anti-CD3/CD28 beads over a period of 8 d. (E and F) Flow cytometry analysis of the expression of CTLA-4, LAG-3, PD-1, TIM-3, and TIGIT in human T cells (E). As control, freshly isolated (unstimulated) or single-stimulated T cells were employed. Frequency and MeanFI are summarized in (F). Example plots are gated on viable CD3+ T cells. Similar results were acquired in multiple donors (3). Data represent the mean ± SD of 3 independent experiments. ns, no significance; *P < 0.05 and **P < 0.01 by 2-way ANOVA. (G and H) Isolated T cells were repeatedly stimulated with anti-CD3/CD28 beads (4 times in total). After each stimulation, the cells were counted, supernatants were collected, and the expression of IL-2 (G) and IFN-γ (H) was assessed by ELISA. Data represent the mean ± SD of 3 independent experiments. Similar data were acquired in multiple donors (3). p-CD3ξ; phosphorylated CD3ξ; p-ZAP70, phosphorylated ZAP70; p-LCP2, phosphorylated LCP2; PBMCs, peripheral blood mononuclear cells; PE, phycoerythrin; Percp, peridinin–chlorophyll–protein complex; FITC, fluorescein isothiocyanate; Tf, transferrin.

Fig 5: Transferrin overexpression and knockdown aggravate or attenuate liver metastasis, respectively. (A) Representative images of livers in liver metastasis mice of B16F10 tumor (left) and tumor weights (right) are shown. Scale bar, 1 cm. Images of 1 representative experiment of 5 are shown. Data represent the mean ± SD of 5 independent experiments; **P < 0.01 by one-way analysis of variance (ANOVA) with Fisher’s protected t test. (B) Hematoxylin-and-eosin (H&E) staining of liver sections (left) from liver metastasis mice of EL4 tumor. The tumor area is shown (right). T, tumor region; L, adjacent liver tissue. Scale bars: 500 or 50 μm. Images of 1 representative experiment of 3 are shown. (C) Representative images of tumors (top) and quantifications of tumor weights (bottom) in BALB/c mice with PLP-Tf or RNR-Tf intravenous on day 30 after subcutaneous inoculation of 1 × 106 EL4 cells (n = 3). Data represent the mean ± SD of 3 independent experiments; *P < 0.05 and **P < 0.01 by one-way ANOVA with Fisher’s protected t test. (D) Immunohistochemical analysis of Granzyme B (Grz B) in liver tissues of EL4 tumor. Scale bars, 50 μm. Images of 1 representative experiment of 5 are shown. Data represent the mean ± SD of 5 independent experiments; *P < 0.05 and **P < 0.01 by one-way ANOVA with Fisher’s protected t test. (E) Flow cytometry analysis of the expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene 3 protein (LAG-3), programmed cell death protein 1 (PD-1), T cell immunoglobulin domain and mucin domain protein 3 (TIM-3), and T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) in liver metastasis mice of B16F10 tumor. Frequency and mean fluorescence intensity (MeanFI) are summarized in (E). (F) Flow cytometry analysis of the expression of interleukin-2 (IL-2) and interferon-γ (IFN-γ) in liver metastasis mice of B16F10 tumor. Frequency and MeanFI are summarized in (F). (G) Flow cytometry analysis of the expression of CTLA-4, LAG-3, PD-1, TIM-3, and TIGIT in liver metastasis mice of EL4 tumor. Frequency and MeanFI are summarized in (G). (H) Flow cytometry analysis of the expression of IL-2 and IFN-γ in liver metastasis mice of EL4 tumor. Frequency and MeanFI are summarized in (H). Data represent the mean ± SD of 3 independent experiments. ns, no significance; *P < 0.05 and **P < 0.01 by 2-way ANOVA. Tf, transferrin; PLP-Tf, Tf overexpression and its blank PLP; RNR-Tf, Tf knockdown and its blank RNR.