To understand global effector mechanisms of CTL therapy, we performed microarray gene expression analysis in a murine model using pmel-1 TCR transgenic T cells as effectors and B16 melanoma cells as targets. In addition to up-regulation of genes related to antigen presentation and the MHC class I pathway, and cytotoxic effector molecules, cell cycle-promoting genes were down-regulated in the tumor on days 3 and 5 after CTL transfer. To investigate the impact of CTL therapy on the cell cycle of tumor cells in situ, we generated B16 cells expressing fluorescent ubiquitination-based cell cycle indicator (B16-fucci). We then performed CTL therapy in B16-fucci tumor-bearing mice. We observed that diffuse infiltration of CTLs into the tumor was associated with large numbers of tumor cells arrested at G1 of the cell cycle, in addition to the presence of spotty apoptotic or necrotic areas, 3 days after CTL transfer. Thus, tumor growth suppression was largely dependent on G1 cell cycle arrest rather than killing by CTLs. Tumor growth inhibition and G1 arrest were both prevented by IFN-γ neutralizing antibody. The mechanism of G1 arrest involved downregulation of S-phase kinase-associated protein 2 (Skp2) and accumulation of its target Cyclin-Dependent Kinase Inhibitor p27 in the B16-fucci tumor cells. Because tumor-infiltrating CTLs are far fewer in number than the tumor cells, we propose that CTLs predominantly regulate tumor growth via IFN-γ-mediated profound cytostatic effects rather than cytotoxicity. This dominance of G1 arrest over other mechanisms may be widespread but not universal because IFN-γ sensitivity varied among tumors.
- Received May 16, 2014.
- Revision received July 22, 2014.
- Accepted August 5, 2014.
- Copyright © 2014, American Association for Cancer Research.