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 Fc?Rs at increasing Fc?R concentrations with immobilized IgG variants; inset graphs show interaction of immobilized IgG variants with aggregated low-affinity Fc?Rs 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.