Fig. 5
PP2A inhibition and PD-1 blockade synergistically decrease tumor growth of B16 melanoma. a C57BL/6 mice were randomized into four treatment groups. 2.5 × 105 B16F10 cells were inoculated 2 days after initiation of treatment subcutaneously in the right thoracic flank. Mice were treated every two days until survival end point. b Tumor growth curves: control (blue), LB-100 (red), aPD-1 (green), and combination (purple). Individual and mean tumor volume over time. c Quantitation of B16 tumor volume 15 days after inoculation. (*P < 0.05, one-way ANOVA with Tukey’s multiple comparison test). d Cumulative survival over time. *P < 0.05, (log-rank test). e At survival end point, organs of the mice were harvested for fixation and staining. Representative images of hematoxylin and eosin staining of the skin and salivary gland of each treatment group (n = 2 per group). Scale bars, 100 μm. f LB-100 enhanced Ag-specific CTL cytotoxicity. OT-1 CD8 cells were isolated from spleens of OT-1 TCR transgenic mice and activated with CD3/CD28 for 3 days. CTLs were treated with LB-100 for 4 h before being mixed with DiO-labeled B16-Ova target cells (TCs). After 3 h of incubation in the presence of PI, cells were analyzed by flow cytometry. Dead TCs were DiO+ PI+. % of effector cell induced cell death was calculated. Ag-specific cell-mediated cytotoxicity was significantly increased when the CTLs were pre-treated with 2 μM LB-100 compared to control. (**P < 0.01, one-way ANOVA with Tukey’s multiple comparison test). g LB-100 did not change level of conjugation formation. OT-1 CD8 cells were similarly isolated and activated as above. Cells were then pre-treated with LB-100 for 5 h and labeled with green florescent CMFDA dye. B16-Ova TCs were labeled with orange florescent CMRA dye. The CTLs and TC were then mixed and incubated for 1 h before analyzed by FACS. Conjugate formation was determined as % of CMFDA+ CMRA+ double positive cells. LB-100 at concentration up to 2 μM did not affect the degree of conjugate formation using both ET of 1 and 5. Data are from one experiment representative of two independent experiments with similar results
