Basic medicine

Local radiotherapy induces the systemic anti-tumor immune response by augmented effector function of antigen-specific CD8+ T cells

Author(s)：: Kengo Kuriyama¹, Toshiki Yajima², Arisa Yamaguchi¹, Ryohei Yoshikawa¹, Ryo Muranushi¹, Kunihiko Suga¹, Kouki Hoshino¹, Makoto Sakai¹, Akihiko Sano¹, Hiroomi Ogawa¹, Makoto Sohda¹, Ken Shirabe¹, Hiroshi Saeki¹
Affiliation(s)：: ¹General Surgical Science, Graduate School, Gunma University, ²Innovative Cancer Immunotherapy, Graduate School, Gunma University

Background: Irradiation is known to activate the anti-tumor immune response system. Although irradiation therapy combined with immune checkpoint blockade have received a lot of attention in recent years, some patients do not respond well to treatment. It is necessary to analyze the dynamics of antigen-specific CD8+ T cell during irradiation therapy, however, the number of antigen-specific CD8+ T cells was too few to analyze. We developed the useful mice model to examine the dynamics of antigen-specific CD8+ T cells during radiotherapy using OT-I mice.
Methods: OT-I chimeras were generated by adoptive transfer system of OT-I cells (Ly5.1+) expressing OVA_257-264/K^b-specific T cell receptor into C57BL/6 mice (Ly5.2+) followed by challenge with EL-4 expressing OVA (E.G7). Mice received 10Gy × 1 of radiation therapy on day 14, and their draining lymph nodes, spleens, lungs and livers were harvested on day 21.
Results: The tumor disappeared after radiation of 10Gy. The number of Antigen-specific CD8+ T cells in not only lymphoid organs but also non-lymphoid tissues was higher in irradiated mice than in control mice. OT-I cells increased by radiation had higher IFN-γ production and higher cytotoxicity than those of control mice. Moreover, long-term survival mice after tumor inoculation and radiation had about 10% of OT-I cells expressing memory T cell markers (CD44+, CD127+). Irradiated mice completely rejected re-inoculated E.G7 cells administered 60 days after the first inoculation, and OT-I cells increased on day 8 after re-inoculation, indicating secondary immune response.
Conclusion: Local radiotherapy induced the systemic anti-tumor immune response by augmented effector function of antigen-specific CD8+ T cells. In addition, antigen-specific CD8+ T cells was maintained as memory T cells by irradiation. Further studies are warranted to induce antigen-specific CD8+ T cells by irradiation more efficiently and achieve higher anti-tumor effects.

Kinetic analysis using antigen-specific CD8+ T cell-induced mouse tumor model

Author(s)：: Kouki Hoshino¹, Toshiki Yajima², Norifumi Harimoto¹, Ryo Muranushi¹, Kei Hagiwara¹, Takahiro Yamanaka¹, Norihiro Ishii¹,
Mariko Tsukagoshi², Akira Watanabe¹, Kenichiro Araki¹, Ken Shirabe¹
Affiliation(s)：: ¹Department of General Surgical Science, Graduate School of Medicine, Gunma University, ²Department of innovative cancer immunotherapy, Graduate School of Medicine, Gunma University

Introduction: Recently, the effectiveness of cancer immunotherapy using the PD-1/PD-L1 antibody is widely applied in clinical practice. But the effect is restrictive, it is necessary to analyze the inducted exhaustion mechanism of activated CD8+ T cells more. However, the detailed analysis is difficult because of the little number of derived antigen(Ag)-specific CD8+ T cells. To solve this problem, we constructed the mouse model to increase Ag-specific CD8+ T cells 10-50 times in vivo.
Methods and Results: Using an adoptive transfer system of OT-I cells (Ly5.1+) expressing OVA257-264/Kb-specific TCR into C57BL/6 mice (Ly5.2+) followed by challenge with EL-4 expressing OVA (EG.7), we found that the frequency of Ag-specific CD8+ T cells was increased 10-50 times compared with non-transferred mice on day14 after tumor inoculation. In OT-I cells, PD-1 began to be expressed as early as day7. In addition, functional analysis showed that IFN-γ production and granzyme expression peaked on day14. Furthermore, analysis using CFSE-labelled OT-I cells showed that PD-1 expression and IFN-γ production gradually increased with cell division. Next, we investigated the optimal timing for the administration of anti-PD1 mAb in this mouse model. We administered the anti-PD1 mAb intraperitoneally once on day7 and 14, respectively, and analyzed the results 3 days later. In the LN of the day7 group, Ag-specific CD8+ T cells and IFN-γ production were significantly increased. On the other hand, in the day14 group, no significant changes were observed in any organ. These results suggest that earlier administration of anti-PD-1 mAb may be effective in avoiding exhaustion of Ag-specific CD8+ T cells.
Conclusion: We considered that this model was useful in detail analyzing the molecular mechanism of exhaustion in Ag-specific CD8+ T cells.

Whole exome sequencing of p53 gene revealed the association of the mutation spectrum and the biological characteristics of oral squamous cell carcinoma

Author(s)：: Toshiki Hyodo¹, Nobuyuki Kuribayashi², Ryouta Kamimura¹, Yuta Sawatani¹, Tomonori Hasegawa¹, Chonji Fukumoto¹,
Koh-Ichi Nakashiro², Daisuke Uchida², Hitoshi Kawamata¹
Affiliation(s)：: ¹Department of Oral and Maxillofacial Surgery, Dokkyo Medical University, ²Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Ehime University

p53 is one of the most investigated tumor suppressor genes, and referred to as the "guardian of the genome". We have demonstrated that missense mutation of the p53 gene may act as an oncogene depending on the mutation site and may be a predictor of therapeutic response. In this study, we examined whole-exome sequencing (WES) of p53 gene in the clinical materials of oral squamous cell carcinoma (SCC) and the association of the mutated p53 gene, other driver gene mutations and biological characteristics.
Genomic DNA was extracted from biopsy or surgical materials, a total of 67 patients with oral SCC, who visited the Department of Oral and Maxillofacial surgery at Dokkyo medical university and Ehime university. Mutation analysis of the coding entire exons of TP53, NOTCH1, CDKN2A and FBXW7, and exons 10 and 21 of PIK3CA, and exons 2 and 3 of HRAS gene were analyzed using a next-generation sequencer. The mutation rate of p53 gene was 80.6% (54/67). We detected 32 cases of missense mutations, 9 cases of frameshift mutations, 10 cases of nonsense mutations, 3 cases of splicing site mutations and 2 cases of in-frame deletion. Although most of the mutations or deletions were detected in the DNA-binding domain, so-called hot spot, the mutations were partially detected in the transactivation domain or oligomerization domain. We could confirm the false negative results of p53 negative staining in immunohistochemistry as truncated p53 mutation by WES. Moreover, we observed a correlation between the mutated p53 gene and clinical outcomes.
These results indicated that WES of p53 gene may help to determine the optimal therapeutic strategy for individual patients with oral SCC.