There is spontaneous release of Th1 and Th2 cytokines from splenic CD4 T cells harvested from mice treated with anti-PD-L1 and anti-CTLA4. The experimental design shown in Figure 3 was used. Mice were treated with anti-PD-L1, or the combination of anti-PD-L1 with anti-TIM-3, or anti-LAG-3 or anti-CTLA4. T cells were isolated from spleens 21 days after tumor cell injection (14 days after irradiation). CD4 T cells were purified by immunomagnetic cell sorting, then stimulated with MHC class II− 5T33 wild-type cells, 5T33-CIITA MHC class II+ cells or T cells only for 48 hours. Supernatants were collected and cytokine levels from were determined using a multiplex cytokine assay. The graphs are representative of two independent experiments in which the CD4+ T cells for each group were pooled from 5 individual mice. p < 0.05, **p < 0.01.