Combined blockade of immune checkpoint proteins increased PD-1 expression on CD8 T cells and increased frequency of tumor specific CTL. The experimental design in Figure 3 was used. Mice were treated with 3 doses of control IgG, anti-PD-L1 only or the combination of anti-PD-L1 with anti-TIM-3, or anti-LAG-3 or anti-CTLA4. (A) CD8+ T cells were isolated from spleens and bone marrow 21 days after tumor cell injection and analyzed for PD-1 expression by flow cytometry. (B) IFN-γ produced by CD8 T cells was determined by IFN-γ ELISPOT assays. T cells were incubated with tumor cells and 10 μg/ml IgG1 or tumor cells and 10 μg/ml anti-PD-L1. The graphs represent 2 independent experiments in which the CD8 T cells for each group were pooled from 5 to 7 individual mice. P values were determined by the Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.