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Background: Invariant natural killer T (iNKT) cells have been shown as an ideal immunotherapeutic target of which the activation via α-Galactosylceramide (α-Gal) conjugated with the MHC class 1-like CD1d molecule induces both innate and adaptive immune system. With transfection of antigen information, α-Gal loaded CD1d expressing cells can act as a vector to enhance the antigen specific immune responses. Though transfection of antigen information by using antigen coding mRNA or tumor peptide is common, the process of extracting mRNA or identifying tumor peptide is sometimes too complicated. Electroporation (EP) has been reported as a way to induce substances easily into cells. We applied EP as a simple method to deliver the antigen into the vector for iNKT cell activation.In this study, we aimed to show a feasibility of EP as a method to deliver the antigen into α-Gal loaded dendritic cells (DCs), a naturally CD1d expressing vector, in terms of inducing antigen specific antitumor responses and innate immunity. Methods: DCs were generated from bone marrow of C57BL/6 mice. Ovalbumin (OVA) was electroporated to DCs as a tumor model antigen. To load α-Gal, DCs were cultured with α-Gal for 24 hours before cultured with LPS. We compared the antitumor responses of α-Gal loaded DCs with or without electroporated OVA in C57BL/6 mice. In subcutaneous tumor model, mice were prophylactically administered each type of DCs intravenously followed by subcutaneous injection of EG7, which is OVA induced EL4, thymoma (n = 5). We evaluated the tumor size and the survival rate. We also evaluated the innate immunity which induced by α-Gal loaded DCs with or without electroporated OVA. Results: With electroporated OVA, tumors didn't grow in mice administered α-Gal loaded DCs whereas tumors in mice administered α-Gal loaded DCs without electroporated OVA aggressively progressed(P < 0.05). In mice administered α-Gal loaded DCs with electroporated OVA, no death was observed during the whole observation period. Survival rate was significantly better for mice administered α-Gal loaded DCs with electroporated OVA than those administered α-Gal loaded DCs without electroporated OVA(P < 0.05). On the other hand, α-Gal loaded DCs with or without electroporated OVA showed no difference in inducing NK cells. But, they either induced more NK cells than α-Gal unloaded DCs(P < 0.05). Conclusion: α-Gal loaded DCs with electroporated OVA showed significantly higher rate of tumor rejection and longer survival in the mice model inoculated OVA-induced tumor comparing to α-Gal loaded DCs without electroporated OVA. Also, EP didn't impair the ability of inducing innate immunity. EP was feasible as a way to deliver the antigen into the vector which induced antigen specific antitumor responses. We are trying to show the activity of antigen-specific cytotoxic T lymphocytes following the administration of α-Gal loaded DCs with electroporated antigen. Citation Format: Akihiro Watanabe, Kimihiro Yamashita, Akira Arimoto, Kouta Yamada, Kyosuke Agawa, Takashi Kamigaki, Rishu Takimioto, Yoshihiro Kakeji. Antigen electroporated DC loaded with NKT-cell ligand induces the antigen specific antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 698.

Background: The enhancement of antitumor immune response with radiotherapy was reported recently, but there are few reports on the mechanism of the effect in rectal cancer. In neoadjuvant chemoradiotherapy (NACRT) for rectal cancer, we reported that CD8+T cells in tumor tissues were highly induced and the density of CD8+T cells were a favorable prognostic factor. The aim of the present study was to elucidate the dynamics of CD8+T cells in the tumor after irradiation. Methods: For this purpose, we established a therapeutic model of irradiation for BALB/c mice inoculated with CT26 colon cancer cell line. They are irradiated with a single dose of 10Gy after 14 days. We analyzed CD8+ T cells infiltration of both irradiated (mice) and no treated (control) mice by flow cytometry. Intratumor CD8+ T cells were fractionated into three populations according to the expression of PD-1 and Tim-3. The numbers and ratios were measured and their dynamics were analyzed. Next, purified CD8+T cells from the tumor with or without irradiation and intracellular cytokines such as IFNγ, TNF-α, and IL-2 were measured. Finally, we analyzed mice treated with FTY720 to test newly infiltrating T cell infiltration after irradiation delivered locally into tumors. Results: In our model, elevated numbers of tumor-infiltrating CD8+ T cells between day11 and day18 after irradiation were observed in tumors of mice compared to control. We observed that the number of PD-1+Tim-3+ CD8+ T cells, that comprised the major population among intratumoral CD8+ T cells, were markedly increasing in irradiated mice. Functionally, intratumoral CD8+ T cells in irradiated mice produced more INF-γ compared to control. Especially, PD-1+Tim-3+ CD8+ T cells enhanced INF-γ production. Among the three populations, PD-1+Tim-3+ CD8+ T cells contained the largest fraction of effector memory (CD44hiCD62Llow) cells, but the lowest fraction of central memory (CD44hiCD62Lhi) cells. Finally, after administration of FTY-720, the decrease rate of intratumoral CD8+ T cells in irradiated mice was much higher than in control. This results suggest that newly infiltrating CD8+ T cell number in irradiated mice was higher than in control. Conclusions: In mouse model of irradiation, there were newly infiltrating CD8+ T cells which was markedly increased compared to the control. This population may be involved in the increase of intratumor CD8+ T cells and may contribute to antitumor effects. This change is expected to trigger further therapeutic intervention. Citation Format: Kimihiro Yamashita, EIJI Fukuoka, Yutaka Sugita, Akira Arimoto, Akihiro Watanabe, Gousuke Takiguchi, Naoki Urakawa, Hiroshi Hasegawa, Masashi Yamamoto, Shingo Kanaji, Yoshiko Matsuda, Takeru Matsuda, Takeru Matsuda, Taro Oshikiri, Tetsu Nakamura, Satoshi Suzuki, Yoshihiro Kakeji. Radiotherapy enhances newly infiltrating CD8+T cells into tumor tissue to increase intratumor CD8+T cells and exert an anti-tumor effects [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2260.

Background: Invariant natural killer T (iNKT) cells have been shown as an ideal immunotherapeutic target of which the activation via α-galactosylceramide (α-Gal) conjugated with the MHC class I- like CD1d molecule induces both innate and adaptive immune system. With transfection of antigen information, α-Gal loaded CD1d expressing cells can act as a vector to enhance the antigen specific immune responses. Though transfection of antigen information coded as mRNA is popular, the process of extracting mRNA or identifying tumor peptide is sometimes too complicated. Electroporation (EP) has been reported as a way to induce substances easily into cells. We applied EP to deliver the antigen into the vector for iNKT cell activation just as a simple procedure. In this study, we aimed to show a feasibility of EP as a method to deliver the antigen into α-Gal loaded dendritic cells (DCs), a naturally CD1d expressing vector, in terms of inducing antigen specific antitumor responses. Methods: DCs were generated from bone marrow of C57BL/6 mice. Ovalbumin (OVA) was electroporated to DCs as a tumor model antigen. To load α-Gal, DCs were cultured with α-Gal for 48 hours. We compared the antitumor responses of α-Gal loaded DCs with or without electroporated OVA in C57BL/6 mice. In subcutaneous tumor model, mice were prophylactically administered each type of DCs intravenously followed by subcutaneous injection of EG7, which is OVA induced EL4, thymoma (n = 5, respectively). We evaluated the tumor size and the survival rate. We also evaluated the antitumor responses of which EP density of OVA can induce efficient antitumor response. Results: With electroporated OVA, tumors didn't grow in mice administered α-Gal loaded DCs whereas tumors in mice administered α-Gal loaded DCs without electroporated OVA aggressively progressed (P < 0.05). In mice administered α-Gal loaded DCs with electroporated OVA, no death was observed during the whole observation period. Survival rate was significantly better for mice administered α-Gal loaded DCs with electroporated OVA than those administered α-Gal loaded DCs without electroporated OVA (P < 0.05). The α-Gal loaded DCs also induce more memory progenitor cells than control group (P < 0.01). Conclusion: α-Gal loaded DCs with electroporated OVA showed significantly higher rate of tumor rejection and longer survival in the mice model inoculated OVA-induced tumor comparing to α-Gal loaded DCs without electroporated OVA. EP was feasible as a way to deliver the antigen into the vector which induced antigen specific antitumor responses. We are trying to show the activity of antigen-specific cytotoxic T lymphocytes following the administration of α-Gal loaded DCs with electroporated antigen. Citation Format: Akihiro Watanabe, Kimihiro Yamashita, Akira Arimoto, Yutaka Sugita, Eiji Fukuoka, Takashi Kamigaki, Rishu Takimoto, Yoshihiro Kakeji. Antigen specific antitumor effect induced by antigen-electroporated, natural killer T cell ligand-loaded dendritic cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6534.

Background; The relationship between tumor immunological microenvironment and irradiation has been studied so far. Based on these findings, we examined a tumor microenvironment of rectal cancer performed by neoadjuvant chemoradiotherapy (NACRT) with immunohistochemical analysis of surgical specimens. But, for introduction to clinical settings, the analysis is not yet sufficient. So, in more detail, we investigate tumor infiltrating CD8+ T cells by irradiation in mouse CT26 colon tumor model. Methods; Locally advanced rectal cancer were enrolled and 68 were eligible for this study. Immunohistochemical staining of programmed cell death- lignad1(PD-L1), CD8, and CD163 was performed on specimens of all cases. In a mouse model, CT26 tumor cells were inoculated in mouse, tumors are irradiated with a single dose of 10Gy after 14 days. We analyzed CD8+ T cells infiltration of both irradiated mouse and no treated mouse by flow cytometry. Results; There were 44 cases in which NACRT was performed (NA group) and 24 cases in which surgery alone was performed (SA group). In the SA group, there was no significant change in the infiltration of PD-L1+immune cells (PD-L1+IC) nor CD8 +cells before and after treatment, but in the NA group, the infiltrations of them were significantly increased. In multivariate analysis, low infiltration of CD8 +cells were identified as poor prognostic factors in disease-free survival. Moreover, in our mouse model, elevated numbers of tumor-infiltrating CD8+ T cells between day11 and day18 after irradiation were observed in tumors of irradiated mice compared to no treated mouse. We observed that among CD8+ T cells, cells that coexpress PD-1 and Tim-3 comprise the major population in irradiated mouse. PD-1+Tim-3+ CD8+ T cells produced more INF-γ compared to that by PD-1+Tim-3-CD8+ T cells and PD-1-Tim-3- CD8+ T cells in irradiated mouse and no treated mouse. Among the three populations PD-1+Tim-3+ population contained the largest fraction of central memory (CD44hiCD62Llow) cells, but the lowest fraction of central memory (CD44hiCD62Lhi) cells. Conclusion; The infiltration of PD-L1+IC and CD8 +cells was induced by NACRT. In the mouse model, we confirmed the kinetical and phenotypical changes of tumor-infiltrating CD8+ T cells. Citation Format: Eiji Fukuoka, Kimihiro Yamashita, Yutaka Sugita, Akira Arimoto, Tetsu Nakamura, Yoshihiro Kakeji. The induction of CD8+ T lymphocytes and PD-L1+ immune cells by neoadjuvant chemoradiotherapy for rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2809.

Background: Myeroid-derived suppressor cells (MDSCs) are heterogeneous cell populations derived from bone marrow, and two subsets of M-MDSC and PMN-MDSC are known in mice. They accumulate in the tumor-bearing host, and suppresse cell-mediated immunity mainly on T cells, and inhibit anti-tumor immune responses. We have previously reported that (1) the increase of MDSCs in lung, peripheral blood, and spleen reflects the cancer progression, (2) MDSCs in peripheral blood would be useful as a marker of recurrence as it increases in recurrent cases after surgical resection. On the other hand, no detailed report on the dynamics of intraperitoneal MDSCs has been made. In this study, we made peritoneal dissemination mouse models and monitored intraperitoneal MDSCs, and investigated whether MDSCs are involved in the progression of peritoneal dissemination. Methods: MC38 colon cancer cell line was intraperitoneally injected to C57BL / 6J mice and the change in the number and ratio of immune cells (MDSC, Macrophage, T-cell, B-cell) in the spleen, peripheral blood and peritoneal cavity was monitored over time. We examined the correlation between transition of immune cells and cancer progression. Results: MDSCs increased with cancer progression in all spleen, peripheral blood, and intraperitoneal cavity. The proportion of PMN-MDSCs in MDSCs increased in peripheral blood. In contrast, the proportion of M-MDSCs increased initially, then the PMN-MDSCs increased in the abdominal cavity. Conclusion: Intraperitoneal MDSCs were thought to reflect the progression of peritoneal dissemination as well as spleen and peripheral blood. The proportion of M-MDSCs reflects the disease state in the microperitoneal dissemination with serous ascitic fluid, and ascites became bloody and the proportion of PMN-MDSCs increased with the progression of seeding. We are examining treatment for peritoneal dissemination with this feature in mind. Citation Format: Yutaka Sugita, Kimihiro Yamashita, Rishu Takimoto, Tomoko Tanaka, Akira Arimoto, Eiji Fukuoka, Tetsu Nakamura, Satoshi Suzuki, Yoshihiro Kakeji. Intraperitoneal myeloid-derived suppressor cells in peritoneal dissemination mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1527.

Background: In anti-tumor immunity, T cells play a central role to eliminate cancer cells. Immune responses of T cells are initiated by communication with antigen presenting cells (APCs). It became clear that an immunological synapse (IS) is formed at the T cell-APC interface and is made up of a supramolecular activation cluster (SMAC) containing the T cell receptors (TCR) microclusters (MCs) and CD28 MCs. TCR MCs consist of TCR, kinases, and adaptor molecules, and are the minimal unit of T cell signal transduction. CD28 MCs also function as the signalosomes of CD28 activation. By using total internal reflection fluorescence microscopy to observe the interaction of T cell and planar bilayer, it was elucidated that TCR activation is regulated by the amount of MCs aggregation, which shows the extent of IS formation. Based on this finding, we hypothesized that ex vivo T cells could be examined IS formation and that exhausted T cells of advanced cancer patients should be low in response to a stimulus. Method: To simplify IS formation of T cells, we used human monoclonal T cells by αCD3 and/or αCD28 antibody and replaced to evaluate the aggregation of MCs by detecting the CD3 or CD28 molecules on the surface of T cells. This phenomenon was defined as IS-like formation. In this method, IS-like formation was detected by confirming an image of each cell with imaging cytometry. After these conditions was established, it was verified whether it is possible to measure IS-like formation rate of T cells. Additionally, the correlation between IS-like formation and interferon-γ cytokine production after this activation was examined. In the same manner as above, IS - like formation rates of T cells derived from PBMCs of healthy donors or esophageal and gastric cancer patients were measured. Result: We have succeeded in observing that CD3 or CD28 molecules on a T cell aggregated in one area after stimulation as IS-like formation by imaging cytometry. In dot plot study, positive area of CD3 or CD28 decreased after the stimulation, which showed the aggregation of these molecules. Accordingly, we could measure IS-like formation rate of a population of T cell clones. In addition, the rate of IS-like formation showed a positive correlation with interferon-γ production. Similar results were obtained T cell from PBMC in healthy donors. Moreover, IS-like formation rate of T cell from cancer patients decreased compared with healthy donors. Conclusion: IS-like formation of T cell succeeded to observe by CD3 and/or CD28 antibody stimulation. We established the evaluation method of IS-like formation degree by imaging cytometry. Moreover, IS-like formation rate correlated with cytokine production of IFN-g. We established new method to evaluate the strength of T cell after activation. Citation Format: Tomoko Tanaka, Kimihiro Yamashita, Yutaka Sugita, Eiji Fukuoka, Akira Arimoto, Yoshihiro Kakeji. A new method for evaluating T cell response after stimulation on gastrointestinal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5699.

Background; Immunotherapy, particularly blockade of programmed cell death-1 (PD-1) has shown unprecedented success in treating various types of cancers. In recent clinical trial of PD-1 blockade, clinicians focused on the characteristics of metastatic colorectal cancer patients with MSI-H (microsatellite instability- high) tumor associated with somatic hypermutation and confirmed a benefit of this immunotherapy in the disease. On the other hand, for MSS patients who account for most of colorectal cancer patient, new immunological strategies are needed. From before, radiation therapy is expected as an approach to overcome this relative resistance across colorectal cancers. Some reports on the relationship between tumor immunological microenvirment and irradiation have also been reported. Based on these findings, we examined a tumor immunological microenvironment of rectal cancer performed by chemoradiotherapy with immunohistochemical analysis of biopsy and surgical specimens. Methods; From January 2005 to December 2016, 77 lower rectal cancer cases with cT3-4 or cN (+) were surgically treated in our institution and enrolled and 68 were eligible for this study. Immunohistochemistry of PD-L1, CD8, and CD163 was performed on biopsy specimens and surgically resected specimens of all cases, and their changes of expression before and after treatment were examined. We also examined the relationship between the expression of these tumor immunity-related factors and clinicopathological factors and prognosis. Results; There were 44 cases in which NACRT was performed (NA group) and 24 cases in which surgery alone was performed (S group). In the S group, there was no significant change in the infiltration of PD-L1 positive immune cells (PD-L1+IC) nor CD8 positive tumor-infiltrating lymphocytes (CD8+TIL) before and after treatment, but in the NA group, the infiltrations of them were significantly increased (p = 0.038 and p = 0.0027, respectively). Although no PD-L1 positive tumor cells (PD-L1+TC) were observed in the biopsy specimens, there were 5 cases in which PD-L1+TC were observed in the surgically resected specimens, and all of which were cases of NACRT group (not significant). In multivariate analysis, low infiltration of PD-L1+IC (p = 0.033) was revealed as poor prognostic factor in overall survival, and low infiltration of PD-L1+IC (p = 0.041) and CD8+TIL (p = 0.0094) were identified as poor prognostic factors in disease-free survival. Conclusion; The infiltration of PD-L1+IC and CD8+TIL was induced by NACRT. The presence of PD-L1+IC in the tumor tissue was revealed to improve the prognosis of rectal cancer. In NACRT for rectal cancer, PD-1 inhibitor combination therapy should be introduced to tumors with PD-L1 positive IC. Citation Format: Kimihiro Yamashita, Akio Nakagawa, Tomoko Tanaka, Akira Arimoto, Eiji Fukuoka, Yutaka Sugita, Junko Mukohyama, Piero Dalerba, Hiroshi Hasegawa, Takeru Matsuda, Satoshi Suzuki, Yoshihiro Kakeji. The induction of PD-L1 positive immune cells and CD8-positive T lymphocytes by neoadjuvant chemoradiotherapy for rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4572.

Background: Invariant natural killer T (iNKT) cells have been shown as an ideal immunetherapeutic target of which the activation via α-Galactosylceramide (α-GalCer) conjugated with the MHC class I-like CD1d molecule induces both the innate and adaptive immune system. With transfection of antigen-coding mRNA, α-GalCer loaded CD1d-expressing cells can act as a vector to enhance the antigen-specific immune responses. However, the method using the transfection of antigen-coding mRNA is sometimes too complicated. Electroporation (EP) has been reported as a way to induce substances easily into cells. We applied EP as a simple method to deliver the antigen into the vector for iNKT activation. In this study, we aimed to show the feasibility of EP as a method to deliver the antigen into α-GalCer loaded DCs, a naturally CD1d-expressing vector, in terms of inducing antigen-specific antitumor responses. Methods: DCs were generated from bone marrow of C57BL/6 mice. Ovalbumin (OVA) was electroporated to DCs as a tumor model antigen. To load α-GalCer, DCs were cultured with α-GalCer for 48 hours. We compared the antitumor responses of α-GalCer loaded DCs with or without electroporated OVA in C57BL/6 mice. Mice were prophylactically administered each type of DCs intravenously followed by subcutaneous injection of EG7, which is OVA induced EL4, thymoma (n = 5, respectively). We evaluated the tumor size and the survival rate. Results: With electroporated OVA, tumors did not grow in mice administered α-GalCer loaded DCs whereas tumors in mice administered α-GalCer loaded DCs without electroporated OVA aggressively progressed (tumor size on day 28 after tumor inoculation; 3 ± 3 vs 4176 ± 1571 mm3, P < 0.05). In mice administered α-GalCer loaded DCs with electroporated OVA, no death was observed during the whole observation period. Survival rate was significantly better for mice administered α-GalCer loaded DCs with electroporated OVA than those administered α-GalCer loaded DCs without electroporated OVA (P < 0.05). Conclusion: α-GalCer loaded DCs with electroporated OVA showed significantly higher rate of tumor rejection and longer survival in the mice model inoculated OVA-induced tumor compared to α-GalCer loaded DCs without electroporated OVA. EP was feasible as a way to deliver the antigen into the vector, which induced antigen-specific antitumor responses. We are trying to show the activity of antigen-specific cytotoxic T lymphocytes following the administration of α-GalCer loaded DCs with electroporated antigen. Citation Format: Akira Arimoto, Masayasu Nishi, Kimihiro Yamashita, Yutaka Sugita, Eiji Fukuoka, Tomoko Tanaka, Takashi Kamigaki, Rishu Takimoto, Yoshihiro Kakeji. Electroporation as a feasible method for antigen delivering into vectors loaded with NKT cell ligand [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5642.