The frequencies of tumor-reactive spleen and bone marrow-derived CD8 and CD4 T cells were increased in mice treated with a combination of checkpoint blockade. The experimental design shown in Figure 3 was used. Myeloma bearing 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) T cells were isolated from spleens (top row) and bone marrow (bottom row) 21 days after tumor cell injection (i.e., 14 days after irradiation). CD4+ and CD8+ T cells were purified by immunomagnetic cell sorting and were tested for IFN-γ production in ELISPOT assays using 5T33-CIITA MHC class II+ or MHC class II− 5T33 wild-type cells as stimulators, respectively. Purified CD8+ or CD4+ T cells for each group were pooled from 5-7 individual mice. The graphs are representative of three independent experiments. (B) Purified splenic CD8 T cells were stimulated with 5T33 tumor cells for 48 hours. Supernatants were collected and cytokine levels were determined using a multiplex cytokine assay. The graphs are representative of two independent experiments in which the CD8+ T cells for each group were pooled from 5 individual mice. For A and B: *p < 0.05, **p < 0.01 as compared with T cells from mice treated with anti-PD-L1 alone.