Abstract 3965: Killing and IFN-γ-dependent G1 cell cycle arrest are the mechanisms of regulation of tumor growth by cytotoxic T lymphocytes.
Authors: Kazuhiro Kakimi , Matsushita Hirokazu , Hosoi Akihiro , Maekawa Ryuji , Ohara Osamu 
Published: August 2014

Adoptive transfer of CTLs leads to tumor regression in animal models and human clinical settings. To understand global effector mechanisms of CTL therapy, we performed microarray gene expression analysis for murine CTL therapy model using pmel-1 TCR transgenic T cells and B16 melanoma cells.

Tumor-specific CTLs were prepared from naïve pmel-1 splenocytes stimulated in vitro with hgp100 peptide-pulsed DCs for 3 days. B16 tumor cells (1×10ˆ6) were implanted subcutaneously in C57BL/6 mice. Nine days later (day 0), 1×10ˆ7 CTLs were intravenously injected into tumor bearing mice. Tumors grew progressively in control mice. In contrast, tumor growth was suppressed in mice receiving CTLs by adoptive transfer from day 3 through day 7. In CTL-treated mice, CD8+CD90.1+CTLs were detected in the tumor as early as day 1 after CTL transfer, peaked on day 3-5 and gradually decreased by day 7. Intracellular IFN-γ staining demonstrated that tumor-infiltrating CTLs produced IFN-γ. The diffuse infiltration of CTLs into the tumor was accompanied by the spotty apoptotic or necrotic area of the tumor.

Tumors were harvested and mRNAs of the tumors were subjected to genome-wide gene expression analysis. Besides up-regulation of genes that are related to antigen presentation, MHC class I pathway, cytotoxic effector molecules, etc., positively regulated cell cycle genes were down-regulated at days 3 and 5 after CTL therapy. To analyze cell cycle of tumor cells, fucci (fluorescence ubiquitination-based cell cycle indicator) was transduced into B16 melanoma cells (B16-fucci), which emit red fluorescence in G1-phase and green fluorescence in S-/G2-/M-phases, respectively. In the same setting of CTL therapy using B16-fucci, tumors were harvested on day 3 and cell cycle was analyzed by calculating Green/Red (G/R) ratio in fluorescence microscopic image as well as flow cytometry. G1 cell cycle arrest was observed in CTL transfer group (G/R ratio: 0.14±0.06, n=3) but not in control untreated group (G/R ratio: 0.40±0.01, n=3) (p=0.014). Infiltrated CTLs and IFN-γ mRNA in the tumor peaked at days 3-5 after CTL therapy. G1 arrest and tumor growth inhibition was abrogated by administration of IFN-γ neutralizing antibody. Furthermore, they were also abrogated when B16-fucci expressing IFN-γ receptor that lacks intracellular component (B16-fucciΔIC) was used in this CTL therapy. These results indicated that IFN-γ by tumor specific CTLs were responsible for the G1 cell cycle arrest of B16 melanoma cells.

Importantly, more tumor cells in cell cycle arrest than those undergo apoptosis were accompanied with the infiltration of CTLs into the tumor. Though infiltrated CTLs are much less in number compared to tumor cells, CTLs can regulate tumor growth dominantly by exerting cytostatic effect on tumor cells (G1 cell cycle arrest) rather than cytocidal effect (killing them one by one).

Citation Format: Kakimi Kazuhiro, Hirokazu Matsushita, Akihiro Hosoi, Ryuji Maekawa, Osamu Ohara. Killing and IFN-γ-dependent G1 cell cycle arrest are the mechanisms of regulation of tumor growth by cytotoxic T lymphocytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3965. doi:10.1158/1538-7445.AM2013-3965