Functional visualization of NK Cell-mediated killing of metastatic single tumor cells

ナチュラルキラー（NK）細胞による転移がん細胞殺傷の可視化 --NK細胞とがん細胞の肺毛細血管上での戦いを実況中継する--. 京都大学プレスリリース. 2022-02-07.Natural killer (NK) cells lyse invading tumor cells to limit metastatic growth in the lung, but how some cancers evade this host protective mechanism to establish a growing lesion is unknown. Here we have combined ultra-sensitive bioluminescence imaging with intravital two-photon microscopy involving genetically-encoded biosensors to examine this question. NK cells eliminated disseminated tumor cells from the lung within 24 hrs of arrival, but not thereafter. Intravital dynamic imaging revealed that 50% of NK-tumor cell encounters lead to tumor cell death in the first 4 hrs after tumor cell arrival, but after 24 hrs of arrival, nearly 100% of the interactions result in the survival of the tumor cell. During this 24 hrs period, the probability of ERK activation in NK cells upon encountering the tumor cells was decreased from 68% to 8%, which correlated with the loss of the activating ligand CD155/PVR/Necl5 from the tumor cell surface. Thus, by quantitatively visualizing the NK-tumor cell interaction at the early stage of metastasis, we have revealed the crucial parameters of NK cell immune surveillance in the lung

Cytotoxic T Lymphocytes Regenerated from iPS Cells Have Therapeutic Efficacy in a Patient-Derived Xenograft Solid Tumor Model

Current adoptive T cell therapies conducted in an autologous setting are costly, time consuming, and depend on the quality of the patient's T cells. To address these issues, we developed a strategy in which cytotoxic T lymphocytes (CTLs) are regenerated from iPSCs that were originally derived from T cells and succeeded in regenerating CTLs specific for the WT1 antigen, which exhibited therapeutic efficacy in a xenograft model of leukemia. In this study, we extended our strategy to solid tumors. The regenerated WT1-specific CTLs had a strong therapeutic effect in orthotopic xenograft model using a renal cell carcinoma (RCC) cell line. To make our method more generally applicable, we developed an allogeneic approach by transducing HLA-haplotype homozygous iPSCs with WT1-specific TCR α/β genes that had been tested clinically. The regenerated CTLs antigen-specifically suppressed tumor growth in a patient-derived xenograft model of RCC, demonstrating the feasibility of our strategy against solid tumors

HLAハプロタイプホモ接合型iPS細胞に由来するKIRリガンド不適合HLA半合致組織に対するNK細胞のアロ反応性

京都大学0048新制・課程博士博士(医科学)甲第20758号医科博第81号新制||医科||6(附属図書館)京都大学大学院医学研究科医科学専攻(主査)教授 江藤 浩之, 教授 三森 経世, 教授 杉田 昌彦学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

A Dual Promoter System to Monitor IFN-γ Signaling in vivo at Single-cell Resolution

IFN-γ secreted from immune cells exerts pleiotropic effects on tumor cells, including induction of immune checkpoint and antigen presentation, growth inhibition, and apoptosis induction. We combined a dual promoter system with an IFN-γ signaling responsive promoter to generate a reporter named the interferon sensing probe (ISP), which quantitates the response to IFN-γ by means of fluorescence and bioluminescence. The integration site effect of the transgene is compensated for by the PGK promoter-driven expression of a fluorescent protein. Among five potential IFN-γ-responsive elements, we found that the interferon γ-activated sequence (GAS) exhibited the best performance. When ISP-G₅AS was introduced into four cell lines and subjected to IFN-γ stimulation, dose-dependency was observed with an EC₅₀ ranging from 0.2 to 0.9 ng/mL, indicating that ISP-GAS can be generally used as a sensitive biosensor of IFN-γ response. In a syngeneic transplantation model, the ISP-GAS-expressing cancer cells exhibited bioluminescence and fluorescence signals in an IFN-γ receptor-dependent manner. Thus, ISP-GAS could be used to quantitatively monitor the IFN-γ response both in vitro and in vivo

Visual evaluation of kinetic characteristics of PET probe for neuroreceptors using a two-phase graphic plot analysis

Objectives: In PET studies for neuroreceptors, tracer kinetics are described by the two-tissue compartment model (2-TCM), and binding parameters including the total distribution volume (VT), nondisplaceable distribution volume (VND), and binding potential (BPND) can be determined from model parameters estimated by kinetic analysis. The stability of binding parameter estimates depends on the kinetic characteristics of radioligands. To describe these kinetic characteristics, we previously developed a Two-phase graphic plot analysis in which VND and VT can be estimated from the x-intercept of regression lines for early and delayed phase, respectively. In this study, we applied this graphic plot analysis to visual evaluation of the kinetic characteristics of radioligands for neuroreceptors, and investigated a relationship between the shape of these graphic plots and the stability of binding parameters estimated by the kinetic analysis with 2-TCM in simulated brain tissue time-activity curves (TACs) with various binding parameters.Methods: 90-min TACs were generated with the arterial input function and assumed kinetic parameters according to 2-TCM. Graphic plot analysis was applied to these simulated TACs, and the curvature of the plot for each TAC was evaluated visually. TACs with several noise levels were also generated with various kinetic parameters, and the bias and variation of binding parameters estimated by kinetic analysis was calculated in each TAC. These bias and variation were compared with the shape of graphic plots.Results: The graphic plots showed larger curvature for TACs with higher specific binding and slower dissociation of specific binding. The quartile deviations of VND and BPND determined by kinetic analysis were smaller for radioligands with slow dissociation. Conclusions: The larger curvature of graphic plots for radioligands with slow dissociation might indicate a stable determination of VND and BPND by kinetic analysis. For investigation of the kinetics of radioligands, such kinetic characteristics should be considered