Yu Saida

DEAD/H (Asp–Glu–Ala–Asp/His) box polypeptide 3, X-linked is an immunogenic target of cancer stem cells

Abstract Accumulating evidence suggests that most solid malignancies consist of heterogeneous tumor cells and that a relatively small subpopulation, which shares biological features with stem cells, survives through potentially lethal stresses such as chemotherapy and radiation treatment. Since the survival of this subpopulation of cancer stem cells (CSC) plays a critical role in recurrence, it must be eradicated in order to cure cancer. We previously reported that vaccination with CD133+ murine melanoma cells exhibiting biological CSC features induced CSC-specific effector T cells. These were capable of eradicating CD133+ tumor cells in vivo, thereby curing the parental tumor. In the current study, we indicated that DEAD/H (Asp–Glu–Ala–Asp/His) box polypeptide 3, X-linked (DDX3X) is an immunogenic protein preferentially expressed in CD133+ tumor cells. Vaccination with DDX3X primed specific T cells, resulting in protective and therapeutic antitumor immunity. The DDX3X-primed CD4+ T cells produced CD133+ tumor-specific IFNγ and IL-17 and mediated potent antitumor therapeutic efficacy. DDX3X is expressed in various human cancer cells, including lung, colon, and breast cancer cells. These results suggest that anti-DDX3X immunotherapy is a promising treatment option in efforts to eradicate CSC in the clinical setting.

Critical Roles of Chemoresistant Effector and Regulatory T Cells in Antitumor Immunity after Lymphodepleting Chemotherapy.

Antitumor immunity is augmented by cytotoxic lymphodepletion therapies. Adoptively transferred naive and effector T cells proliferate extensively and show enhanced antitumor effects in lymphopenic recipients. Although the impact of lymphodepletion on transferred donor T cells has been well evaluated, its influence on recipient T cells is largely unknown. The current study demonstrates that both regulatory T cells (Tregs) and effector CD8+ T cells from lymphopenic recipients play critical roles in the development of antitumor immunity after lymphodepletion. Cyclophosphamide (CPA) treatment depleted lymphocytes more efficiently than other cytotoxic agents; however, the percentage of CD4+CD25+ Foxp3+ Tregs was significantly increased in CPA-treated lymphopenic mice. Depletion of these chemoresistant Tregs following CPA treatment and transfer of naive CD4+ T cells augmented the antitumor immunity and significantly suppressed tumor progression. Further analyses revealed that recipient CD8+ T cells were responsible for this augmentation. Using Rag2−/− mice or depletion of recipient CD8+ T cells after CPA treatment abrogated the augmentation of antitumor effects in CPA-treated reconstituted mice. The transfer of donor CD4+ T cells enhanced the proliferation of CD8+ T cells and the priming of tumor-specific CD8+ T cells originating from the lymphopenic recipients. These results highlight the importance of the recipient cells surviving cytotoxic regimens in cancer immunotherapies.

Recurrent Interstitial Lung Disease Induced By Various Therapies forNon-Small Cell Lung Cancer

Erlotinib is a human epidermal growth factor receptor type 1 tyrosine kinase inhibitor which is used for non-small cell lung cancer treatment. Interstitial lung disease has been reported as an adverse event of erlotinib. We report the case of a 39-year-old man with erlotinib-induced interstitial lung disease in a non-small cell lung cancer patient. Although interstitial lung disease had improved by steroid therapy, palliative radiotherapy recalled the pneumonitis beyond the radiation fields. After the pneumonitis was well controlled, the patient was started on irinotecan, but the interstitial lung disease recurred shortly thereafter. We may have to abandon further cytotoxic therapies to avoid the recurrence of interstitial lung disease in patients who develop erlotinib-induced interstitial lung disease once.

Transfer of in vitro-expanded naïve T cells after lymphodepletion enhances antitumor immunity through the induction of polyclonal antitumor effector T cells

The adoptive transfer of effector T cells combined with lymphodepletion has demonstrated promising antitumor effects in mice and humans, although the availability of tumor-specific T cells is limited. We and others have also demonstrated that the transfer of polyclonal naïve T cells induces tumor-specific effector T cells and enhances antitumor immunity after lymphodepletion. Because tumors have been demonstrated to induce immunosuppressive networks and regulate the function of T cells, obtaining a sufficient number of fully functional naïve T cells that are able to differentiate into tumor-specific effector T cells remains difficult. To establish culture methods to obtain a large number of polyclonal T cells that are capable of differentiating into tumor-specific effector T cells, naïve T cells were activated with anti-CD3 mAbs in vitro. These cells were stimulated with IL-2 and IL-7 for the CD8 subset or with IL-7 and IL-23 for the CD4 subset. Transfer of these hyperexpanded T cells after lymphodepletion showed significant antitumor efficacy, and tumor-specific effector T cells were primed from these expanded T cells in tumor-bearing hosts. Moreover, these ex vivo—expanded T cells maintained T cell receptor diversity and showed long-term persistence of memory against specific tumors. Further analyses revealed that combination therapy consisting of vaccination with dendritic cells that were co-cultured with irradiated whole tumor cells and the transfer of ex vivo—expanded T cells significantly enhanced antitumor immunity. These results indicate that the transfer of ex vivo—expanded polyclonal T cells can be combined with other immunotherapies and augment antitumor effects.

Effect of lymphodepletion on donor T cells and the role of recipient cells persisting after cytotoxic treatments in cancer immunotherapies

The effectiveness of lymphodepletion in antitumor immunity has been well established. Although recent studies have elucidated some of the broad mechanisms underlying the augmentation of antitumor immunity by lymphodepletion, such as increased availability of cytokines due to the elimination of cellular elements and improvement in tumor antigen presentation, the precise mechanisms remain unclear. Previous studies have focused on the enhancement of the functions of transferred antitumor CD8+ T cells after lymphodepletion. In this review, we discuss the important role of other immune cells in the effectiveness of lymphodepletion. Recent studies have demonstrated that lymphodepletion enhances not only transferred tumor-specific CD8+ T cells but also tumor-specific CD4+ T cells and polyclonal naïve T cells. Moreover, recipient immune cells, including CD8+ T cells, regulatory T cells, dendritic cells, and macrophages, are involved in the augmentation of antitumor effects by lymphodepletion. These host cells can survive lymphodepletive therapies and play a role in the development of antitumor immunity after lymphodepletion. Improvements in the understanding of lymphodepletion allow us to design effective cancer immunotherapy.

Abstract 3206: Programmed death receptor-1/programmed death receptor ligand-1 blockade improves priming of antitumor effector T cells after cytotoxic therapies

Cytotoxic lymphodepletion therapies, such as chemotherapy and radiotherapy, have been established to augment antitumor immunity. Naive T cells elicit effector-like phenotypes and functions during recovery from lymphopenia. We and others have repeatedly demonstrated that transfer of naive T cells into lymphopenic-tumor bearing mice delayed tumor growth. This enhancement of naive T cells is required an activation through T-cell receptor (TCR). Programmed death receptor-1 (PD-1)/programmed death receptor ligand-1 (PD-L1) blockade therapy has been demonstrated to augment antitumor immunity. Previous studies have shown that PD-1/PD-L1 blockade therapy stimulates or restores the function of antitumor T cells in the effector phase. Because engagement of programmed death receptor-1 (PD-1) by ligand suppresses TCR signaling and inhibits T cell activation and function, there is a possibility that PD-1 regulates activation of T cells during recovery from lymphopenia after cytotoxic therapies. In the current study, we transferred naive T cells into tumor-bearing mice following whole-body irradiation for lymphodepletion. These mice were further treated with anti-PD-1 antibodies. PD-1/PD-L1 blockade therapy after lymphodepletion significantly suppressed tumor progression. Next, we tested several kinds of cytotoxic agents to induce lymphopenia in mice. We found that the kind of cytotoxic agents affected the augmentation of antitumor efficacies of PD-1/PD-L1 blockade. Analyses of tumor-draining lymph-nodes (TDLNs) revealed that the number of tumor-specific effector T cells was significantly increased in mice treated with anti-PD-1 antibodies. By contrast, the number of effector T cells in spleens was not increased by PD-1/PD-L1 blockade therapy. These results indicate that PD-1 regulates a priming of antitumor effector T cells in TDLNs after cytotoxic lymphodepletion therapies. PD-1/PD-L1 blockade therapy is able to enhance antitumor T cell responses not only in the effector phase but also in the priming phase. Citation Format: Miho Takahashi, Satoshi Watanabe, Ko Sato, Tomohiro Tanaka, Yu Saida, Junko Baba, Aya Ohtsubo, Miyuki Sato, Rie Kondo, Masaaki Okajima, Junta Tanaka, Hiroshi Kagamu, Hirohisa Yoshizawa, Toshiaki Kikuchi. Programmed death receptor-1/programmed death receptor ligand-1 blockade improves priming of antitumor effector T cells after cytotoxic therapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3206.

Abstract 3153: Critical roles of chemo-resistant effector and regulatory T cells in cancer immunotherapy during hemostatic proliferation

Antitumor immunity has been well established to be augmented by cytotoxic lymphodepletion therapies. Adoptively transferred naive and effector T cells proliferate extensively and show enhanced antitumor effects during homeostatic proliferation when they were adoptively transferred into tumor-bearing hosts that were lymphodepleted with cytotoxic agents or by whole body irradiation. Although the impact of lymphodepletion on transferred donor T cells has been well evaluated, its influence on recipient T cells is largely unknown. The current study demonstrates that both regulatory T cells (Tregs) and effector CD8 + T cells from lymphopenic recipients play critical roles in the development of antitumor immunity after lymphodepletion. Cyclophosphamide (CPA) treatment depleted lymphocytes more efficiently than other cytotoxic agents, such as fludarabine, cisplatin, etoposide, paclitaxel or gemcitabine; however, the percentage of CD4 + CD25 + Foxp3 + Tregs was significantly increased in CPA-treated lymphopenic mice. Depletion of these chemo-resistant Tregs following CPA treatment and transfer of naive CD4 + T cells augmented the antitumor immunity and significantly suppressed tumor progression. Further analyses revealed that recipient CD8 + T cells were responsible for this augmentation. Using Rag2 −/− mice or depletion of recipient CD8 + T cells after CPA treatment abrogated the augmentation of antitumor effects in CPA-treated reconstituted mice. The transfer of donor CD4 + T cells enhanced the proliferation of CD8 + T cells and the priming of tumor-specific CD8 + T cells originating from the lymphopenic recipients. These results highlight the importance of the recipient cells surviving cytotoxic regimens in cancer immunotherapies. Citation Format: Ko Sato, Satoshi Watanabe, Yu Saida, Tomohiro Tanaka, Junko Baba, Aya Ohtsubo, Satoshi Shoji, Daisuke Ishikawa, Rie Kondo, Masaaki Okajima, Satoru Miura, Junta Tanaka, Hiroshi Kagamu, Hirohisa Yoshizawa, Ichiei Narita. Critical roles of chemo-resistant effector and regulatory T cells in cancer immunotherapy during hemostatic proliferation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3153. doi:10.1158/1538-7445.AM2015-3153

Abstract 2818: Dendritic cell vaccination and regulatory T-cell depletion augment antitumor immunity after cytotoxic therapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The ability of lymphodepleting cytotoxic regimens to augment antitumor immune responses has been well established. We and others have previously reported that naive T cells, as well as effector T cells, transferred into lymphopenic tumor-bearing mice acquire memory-like phenotypes and show antitumor effects. The transfer of naive T cells into irradiated lymphopenic mice inhibited tumor progression. Further analyses revealed that the transfer of CD4+ naive T cells was essential for this augmentation of antitumor immunity after lymphodepletion. To enhance the antitumor efficacy of lymphodepletion and transfer of naive T cells, we have tested anti-CTLA-4 mAbs and anti-CD25 mAbs for depletion of regulatory T cells. Mice were irradiated with 500 cGy to deplete lymphocytes and were transferred i.v. with T cells (2 x 107). On the same day, mice were inoculated with MCA205 tumor cells (1 x 105) followed by the injection of anti-CTLA-4 mAbs (UC10) or the injection of anti-CD25 mAbs (PC61). Although the injection of anti-CTLA-4 mAbs showed minimal augmentation of the antitumor effects of the combination of lymphodepletion and naive T cell transfer, anti-CD25 mAbs significantly enhanced antitumor immune responses. Next, we evaluated whether dendritic cell vaccination augments this combination therapy. Different from previous experiments, 3-day tumor-bearing mice were irradiated and reconstituted with T cells. Dendritic cells were generated from bone marrow of naive mice and were co-cultured with irradiated MCA205 tumor cells followed by stimulation with agonistic anti-CD40 mAbs. We found that dendritic cell vaccination significantly inhibited tumor growth in irradiated reconstituted mice. Previously, we reported that a significant increase of CD4+CD25+Foxp3+ regulatory T cells in irradiated mice. We demonstrated that these radio-resistant regulatory T cells from irradiated recipient mice suppressed the development of antitumor immunity. To clarify the roles of other recipient cells in antitumor immunity during recovery from lymphopenia, Rag-2 knockout mice were irradiated and reconstituted with T cells. These mice were then inoculated MCA205 tumor cells. In Rag-2 knockout mice, antitumor effects were not observed after lymphodepletion and reconstitution. These findings indicate that recipient T cells or B cells were required to augment antitumor immunity in lymphopenic mice in our model. Citation Format: Tomohiro Tanaka, Satoshi Watanabe, Ko Sato, Yu Saida, Junko Baba, Koichiro Nozaki, Daisuke Ishikawa, Natsue Igarashi, Satoshi Shoji, Masaaki Okajima, Satoru Miura, Junta Tanaka, Hiroshi Tanaka, Hiroshi Kagamu, Hirohisa Yoshizawa, Ichiei Narita. Dendritic cell vaccination and regulatory T-cell depletion augment antitumor immunity after cytotoxic therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2818. doi:10.1158/1538-7445.AM2014-2818

Abstract 3963: Chemo-resistantregulatory T cells suppress the development of antitumor immunity aftercytotoxic regimens.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Previous studies demonstrated that naive 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 naive 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 naive 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 naive 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 naive 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 naive 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, naive 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

Abstract 1550: Successful treatment of advanced tumors with chemo-immunotherapy: a combination of cyclophosphamide and inhibition of chemo-resistant immune suppressor cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL It is well known that naive T cells transferred into lymphopenic hosts develop into memory like T cells and acquire some effector functions. We and others have been shown that transfer of naive T cells following sublethal whole body irradiation augmented antitumor immunity and inhibited tumor progression. Further analyses showed that antitumor effector T cells were primed from transferred naive T cells. Although the exact mechanism underlying this enhancement of antitumor immune responses remains unclear, possible explanations have been proposed; depletion of suppressor cells, improvement of tumor-antigen presentation, and elimination of lymphocytes competing activation cytokines. Combination of lymphodepletion by cytotoxic regimens, such as chemotherapy or radiotherapy, and transfer of naive T cells seems to be a promising strategy. Although whole body irradiation induces lymphopenia to tumor-bearing animals and enhances antitumor immunity, it has not been routinely used in clinical settings. To test whether cytotoxic agents deplete lymphocytes and enhance antitumor immune responses, we infused cyclophosphamide (CPA), fludarabine, cisplatin and etoposide at the sublethal doses to mice. CPA treatment depleted 95% of lymphocytes in mice, and enhanced antitumor effects of transferred naive T cells. Other cytotoxic drugs failed to augment antitumor immunity in combination with transfer of T cells. Previously, we have demonstrated that CD4+CD25+Foxp3+ regulatory T cells (Treg) and CD11b+Gr-1+ myeloid-derived suppressor cells (MDSC) inhibited priming of antitumor effector T cells. Depletion of these suppressor cells increased the number of tumor-specific T cells and augmented antitumor immune responses. To examine the effect of sublethal doses of cyclophosphamide on immune suppressor cells, we injected cyclophosphamide into mice and harvested lymph-nodes and spleens for FACS analyses. Unexpectedly, we found a significant increase in the frequency of Treg and MDSC after CPA treatment. Magnetically isolated Treg and MDSC from cyclophosphamide treated mice suppressed tumor-specific responses of effector T cells in vitro. Depletion of Treg with anti-CD25 monoclonal antibodies following CPA administration and transfer of naive T cells increased the number of antitumor effector T cells. Further, the combination of Treg depletion, CPA treatment and transfer of naive T cells succeeded to cure 20-day established skin tumors in mice. We have also been testing whether inhibition of MDSC after CPA treatment augments antitumor immunity. Our results showed that CPA treatment efficiently depletes lymphocytes and enhanced antitumor immune responses despite an increase of immune suppressor cells. Further inhibition of suppressor cells after CPA administration successfully treated advanced tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1550. doi:1538-7445.AM2012-1550