Abstract 3963: Chemo-resistantregulatory T cells suppress the development of antitumor immunity aftercytotoxic regimens.
Authors: Saida Yu , Watanabe Satoshi , Tanaka Tomohiro , Baba Junko , Satoh Koh , Shoji Satoshi , Igarashi Natsue , Nozaki Koichirou , Okajima Masaaki , Miura Satoru , Tanaka Junta , Kagamu Hiroshi , Yoshizawa Hirohisa , Narita Ichiei 
Published: August 2014

Previous studies demonstrated that naïve T cells transferred into lymphopenic hosts develop into memory like T cells and acquire some effector functions. We and others have shown that sublethal whole body irradiation and transfer of naïve T cells augment antitumor immunity and inhibited tumor progression. Further, we found a significant increase of CD4+CD25+Foxp3+ regulatory T cells (Treg) in irradiated mice. Depletion of those radio-resistant Treg after irradiation and transfer of naïve T cells enhanced generation of antitumor effector T cells and suppressed tumor progression (Baba J, et al. 2012, Blood). The combination of lymphodepletion and transfer of naïve T cells seems to be a promising strategy. However, whole body irradiation has not been routinely used in clinical settings. Previously, we demonstrated that the sublethal doses of cyclophosphamide (CPA) efficiently depleted lymphocytes in mice, and enhanced antitumor effects of transferred naïve T cells. In this study, we examined the effect of lymphodepleting doses of CPA on immune suppressor cells. Similar to the irradiated lymphopenic hosts, a significant increase of Treg was observed in mice treated with CPA. The combination of CPA, transfer of naïve T cells and Treg depletion with anti-CD25 monoclonal antibodies succeeded to cure advanced skin tumors.To evaluate the proliferation and apoptosis of Treg increasing in CPA-treated mice, we performed BrdU incorporation, Ki-67 expression and Annexin V apoptosis assay. The results indicated that the increased percentage of Treg during recovery from lymphopenia is due to the rapid proliferation of Treg that survive CPA treatment.

Next, we investigated which subsets of donor T cells are necessary in this combination therapy. CD4+ or CD8+ T cells were depleted from donor T cells before transfer into lymphopenic skin tumor bearing mice. Depletion of CD4+ T cells, but not CD8+ T cells eliminated the antitumor effects. Intracellular cytokine FACS revealed that transfer of CD4+ T cells following CPA treatment induced tumor specific effector CD4+ T cells in lymphopenic hosts. Interestingly, transfer of CD4+ T cells also induced CD8+ effector T cells from CPA treated recipient cells. Depletion of CD8+ recipient cells with anti-CD8 monoclonal antibodies abrogated the antitumor effects of lymphodepletion and CD4+ T cells transfer. Furthermore, Transfer of CD4+ T cells into Rag-2 knockout mice showed no antitumor effects. These results suggested that donor CD4+ T cells induce effector CD8+ T cells originate from recipients after CPA treatment and suppress tumor progression. Our results showed that the combination of CPA treatment, naïve CD4+ T cell transfer, and Treg depletion had potent antitumor efficacy. Both of donor CD4+ T cells and recipient CD8+ T cells were necessary for this augmentation of antitumor immunity.

Citation Format: Yu Saida, Satoshi Watanabe, Tomohiro Tanaka, Junko Baba, Koh Satoh, Satoshi Shoji, Natsue Igarashi, Koichirou Nozaki, Masaaki Okajima, Satoru Miura, Junta Tanaka, Hiroshi Kagamu, Hirohisa Yoshizawa, Ichiei Narita. Chemo-resistantregulatory T cells suppress the development of antitumor immunity aftercytotoxic regimens. [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 3963. doi:10.1158/1538-7445.AM2013-3963