2018 Fiscal Year Annual Research Report
キラーT細胞のエネルギー代謝機構の解析とPD-1阻害がん免疫治療の効果予測
| Project/Area Number |
18J15051
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| Research Institution | Kyoto University |
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Principal Investigator |
Kumar Alok 京都大学, 医学研究科, 特別研究員(DC2)
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| Project Period (FY) |
2018-04-25 – 2020-03-31
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| Keywords | biomarker / cancer immunotherapy |
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| Outline of Annual Research Achievements |
I classified many tumor cell lines into responsive and unresponsive groups in different backgrounds of mice (C57BL/6N and BALB/c). Further, we focused on studying effector T cells (CD8+ T cells) from the aspect of energy metabolism. Using our established strategy to measure the mitochondria respiration by Seahorse extracellular flux analyzer, we compared the energy metabolism of DLN CD8+ T cells from responsive and unresponsive tumor-bearing hosts that were underwent with or without PD-1 blockade therapy. Tumors were injected in mice and PD-1 blockade therapy were performed for two doses (on day 5 and 10) and sacrificed on day 12 for further analysis from mitochondrial energy metabolism aspect. CD8+ T cells were purified from DLN of treated mice and performed ‘mito-stress test’ to measure mitochondrial respiration using Seahorse extracellular flux analyzer. We found oxygen consumption rate (OCR) was significantly higher after PD-L1 blockade in responsive tumor (MC38) group over untreated group while in unresponsive tumor (LLC) group it was unchanged. We found similar observation for different background (BALB/c) where OCR was significantly higher in PD-L1 antibody treated group over untreated for responsive tumor (MethA) while it was unchanged for unresponsive tumor (Wehi3, CT26) group. Other parameters associated with OCR were also higher in PD-1 blockade over untreated in responsive group. The results indicate that the energy metabolism could tell the different effector response for responsive and unresponsive tumors to the PD-1 blockade treatment.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
On the basis of results from previous fiscal year, we can say that ‘OCR of total CD8’ could be a biomarker to identify the responder and non-responder cancer patients. Since in human cancer patients, we can get total CD8+ T cells from peripheral blood mononuclear cells (PBMCs), OCR measurement of total CD8+ T cells would be very beneficial for the clinical application. Before using OCR measurement for clinical application, it is important to confirm whether OCR of total CD8 is proportional to the mitochondrial activation status of tumor-reactive cytotoxic T cells (TRCTLs). In order to study mitochondrial activation status of TRCTLs, we need to establish a novel method to identify TRCTLs in mouse model. Further, we can compare the phenotype of TRCTLs for responsive and unresponsive tumor-bearing hosts.
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| Strategy for Future Research Activity |
It is very difficult to identify tumor-reactive cytotoxic T lymphocytes (TRCTLs) as PD-1 blockade activates a huge repertoire of TRCTLs that recognize the tumor antigens. There are no established methods to study TRCTLs so far. In this year a novel model will be established to detect TRCTLs where CellTrace-labeled CD45.1+ CD8+ T cells are adoptively transferred to CD45.2+ CD8-/- strain mice. I will examine the proliferation of the transferred CD8+ T cells and analyze the frequency of CellTracelow population which reacts with the tumor antigen in the host. I will examine the proliferation of transferred CD8+ T cells in the host with responsive or unresponsive tumor. After gating on highly proliferating CD8+ T cells several indicators of mitochondrial activation will be compared between PD-L1 mAb-treated and untreated groups for responsive and unresponsive tumor-bearing hosts. Different mitochondrial activation parameters will be compared by staining with MitoTracker Green, MitoTracker Deep Red and MitoSOX Red that stain mitochondrial mass, mitochondrial membrane potential, and mitochondrial ROS, respectively. In last, we will see whether mitochondrial activation parameters of TRCTLs correlate with the OCR of total CD8+ T cells.
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Research Products
(5 results)
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[Journal Article] Synergistically Triggers Reactive Oxygen Species and DNA Damage and Inhibits Salmonella enterica subsp. Enterica Serovar Typhi and Vibrio fluvialis2019
Author(s)
Ghosh, T., Srivastava, S. K. Gaurav, A., Kumar, A., Kumar, P., Yadav, A. S., Pathania,, R., Navani, N. K.
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Journal Title
Applied and Environmental Microbiology
Volume: 85 (4)
Pages: e02487-18
DOI
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