Supplemental Figure 1. Bioluminescence plots of individual animals following treatment with immune checkpoint blocking mAbs against (A) CTLA-4 and PD-1, (B) CTLA-4 and PD-L1, (C) PD-1 and PD-L1, and (D) PD-L1 and PD-L2. Each experiment includes 8 mice per cohort and also includes IgG controls.
Supplemental Figure 2. Immune cell infiltrates within intracranial GL261-luc2 tumor following treatment with isotype control IgG or combination therapy with CTLA-4 plus PD-1 mAb therapy. Mice were treated and cells prepared as in Figure 5 with leukocyte populations collected from brain tissue on Day 24. Gating strategies are indicated and numbers in the plot represent the percentage of positive cells within that panel. Representative contour plots are shown for the following live cell populations: activated NK cells (CD45+NK1.1+CD86+); MDSCs (CD45+CD11b+Gr-1+); exhausted T cells (CD45+CD3+PD-1+Tim3+); Tregs (CD45+CD3+CD4+FoxP3+); and CD8 effector (CD45+CD3+CD8+granzyme B+).
Supplemental Figure 3: Treatment antibody and detection antibody for PD-1 do not share same the epitope. 300.19 cells stably transfected to express PD-1 were incubated for 30 min. with increasing concentrations of unlabeled PD-1 treatment antibody (clone: 332.8H3), a different PD-1 antibody (clone: 332.5E12), or the IgG1 control antibody. PE-conjugated PD-1 detection antibody (clone: RMP1-30) or PE-conjugated isotype control was added, and cells were incubated for an additional 30 min., washed, and analyzed. Graph depicts mean fluorescent intensities (MFI) of PE-conjugated detection antibody.
Supplemental Figure 4. Representative immunohistochemistry results of intratumoral CD3+, CD4+ and CD8+ T cell infiltrates following immune checkpoint therapy with mAbs against CTLA-4, PD-1 and both CTLA-4 and PD-1.
Supplemental Figure 5. Immune cell infiltration within the draining cervical lymph node (cLN) of GL261-luc2 tumor-bearing mice following treatment with isotype control IgG or combination therapy with mAbs against CTLA-4 plus PD-1. Mice were treated and cells prepared as in Figure 5 with leukocyte populations collected from cLN tissue on Day 24. Gating strategies are indicated and numbers in the plot represent the percentage of positive cells within that panel. Representative contour plots are shown for the following live cell populations: activated NK cells (CD45+NK1.1+CD86+); MDSCs (CD45+CD11b+Gr-1+); exhausted T cells (CD45+CD3+PD-1+Tim3+); Tregs (CD45+CD3+CD4+FoxP3+); and CD8 effector (CD45+CD3+CD8+granzyme B+).
Supplemental Figure 6. Comparisons of chemokine expression levels in sera of mice treated with checkpoint blockade. Tumor was established with advanced disease and treated as described in Figure 4 with isotype control IgG or blocking immune checkpoint mAbs against CTLA-4, PD-1 or the combination of CTLA-4 plus PD-1. Serum was prepared from whole blood on Day 24. The Mouse Chemokine Antibody Array (R&D Systems) was use to detect chemokine levels. Representative densitometry images for one mouse per treatment group and spots indicate chemokine protein levels and reference controls in duplicate (A). (see Supplemental Table 1 for list/location of chemokines) The mean pixel density of chemokines relative to respective reference spots for individually analyzed mice are presented as a box plot for each treatment group(B) (n=9). One-way ANOVA was used to determine statistical significance (*P<0.05; **P P<0.01; ***P<0.001)
Supplemental Figure 7: Expression of checkpoint ligands and receptors on GL261-Luc cells in vitro. GL261-Luc cells were stained with fluorochrome-conjugated antibodies to PD-L1, PD-L2, PD-1, TIM-3, and CTLA-4 (red) or the corresponding IgG isotype control antibody (blue). Representative histograms show the mean fluorescent intensity of antibody staining.