Neuroblastoma is an aggressive solid tumor that develops from immature cells of the nervous system and is almost exclusively diagnosed in infants and young children. Over the past decade a multitude of immune based therapies have been explored as therapeutic candidates for patients with neuroblastoma. The anti-GD2 monoclonal antibody, 3F8, and more recently, natural kill (NK) cell based therapies have been accepted as hopeful therapeutic options for patients with Neuroblastoma. These options however have many drawbacks including dose limiting pain, the development of tolerance, reliance on MHC mismatch and possible reliance on the invariant NK (iNK) cells population. Gamma Delta T cells, a subpopulation of T cells composed of a T cell receptor (TCR) with a gamma and a delta chain instead of an alpha and a beta; chain, have been shown to recruit a more robust immune response then both 3F8 and NK cells through their activation of antigen presenting cells (APCs) and non-reliance on MHC mismatch. Gamma Delta T cells are also able to recruit NK cells as well as other cytotoxic lymphocytes. For these reasons, it is believed that Gamma Delta T cell based treatment alone or in combination with an anti-GD2 monoclonal antibody may have a greater efficacy than either NK cells or an anti-GD2 monoclonal antibody alone. The intent of this thesis is to explore and evaluate the current state of Gamma Delta T cell based immunotherapy against the backdrop of NK cell based immunotherapy for neuroblastoma.

Surgery precipitates a hypercoagulable state and has been shown to increase the development of cancer metastases in animal models, however mechanism(s) responsible for this are largely unknown. We hypothesize that the prometastatic effect of surgery may be secondary to postoperative hypercoagulable state. Surgical stress was induced in mice by partial hepatectomy or nephrectomy, preceded by intravenous injection of CT26-LacZ or B16F10-LacZ cells to establish pulmonary metastases with or without perioperative anticoagulation and their lung tumor cell emboli (TCE) were quantified. Fibrinogen and platelets were fluorescently labeled prior to surgical stress to evaluate TCE-associated fibrin and platelet clots. Surgery significantly increased metastases while anticoagulation with five different agents attenuated this effect. Fibrin and platelet clots were associated with TCE significantly more frequently in surgically stressed mice. Surgery promotes the formation of fibrin and platelet clots around TCE and this appears to be the mechanism for the increase in metastases seen following surgery.

Natural killer (NK) cells are lymphocytes of the innate immune system that play a major role in the destruction of both tumours and virally-infected cells. The cytotoxicity of NK cells is tightly controlled by signals received through activating and inhibitory receptors. NK cells express a variety of inhibitory receptors such as Ly49 receptors. Ly49 receptors bind to class I MHC molecules that expressed on normal cells. Using Ly49-deficient (NKCKD) mice we show that Ly49-KD NK cells successfully recognize and kill influenza virus-infected cells and that NKCKD mice exhibit better survival than wild-type mice. Moreover, influenza virus infection has a propensity to upregulate cell surface expression of MHC-I on murine lung epithelial cells in vivo. Significantly, we demonstrate increased lung damage of WT-mice versus NKCKD mice after influenza virus infection as determined by histological analyses. This data indicated that absence of Ly49 inhibitory NK receptors greatly enhances survival of infected mice.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22371号 / 医博第4612号 / 新制||医||1043(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 松田 文彦, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Rag1-/- mutant zebrafish lack lymphocytes and were used to study the basis of acquired protective immunity in the absence of lymphocytes to the intracellular bacterium Edwardsiella ictaluri. This study morphologically identified and quantified lymphocyte like cells (LLCs) present in the liver, kidney and spleen of these fish. LLCs included Natural Killer (NK) cells and non-specific cytotoxic cells (NCCs) and were discriminated by size, and by the presence of cytoplasmic granules. The antibodies anti-NITR9, anti-NCCRP-1 (5C6) and anti-MPEG-1 were used to evaluate these cell populations by flow cytometry. Gene expression profiles in these tissues were evaluated after the Rag1-/- mutants were intra coelomically injected with the toll like receptor (TLR)-2 ligand, β glucan, TLR3 ligand, Poly I:C, or TLR 7/8 ligand, R848. The genes interferon y (infγ), expressed by activated NK cells and macrophages, tumor necrosis factor α (tnfα), expressed by activated macrophages, myxovirus resistance (mx) expressed by cells induced by IFNα, T-cell transcription factor (t-bet) expressed by NK cells and novel immune type-receptor 9 (nitr-9) expressed by NK cells were evaluated. The TLR ligands induced different patterns of expression and stimulated both macrophages and NK cells. Then fish were vaccinated with an attenuated mutant of E. ictaluri (RE33®) with or without the TLR ligands then challenged with WT E. ictaluri to evaluate protection. RE33® alone and each TLR ligand alone provided protection. Coministration of β glucan and RE33® or R848 and RE33® resulted in survival higher than RE33® alone showing an adjuvant effect. Tissue specific gene expression of ifnγ, t-bet, nitr9, NK cell lysin a (nkla), nklb, nklc and nkld were correlated to protection. The final component of this study was the development of tools to discriminate NK cell populations and evaluate the contribution of macrophages. Rag1-/- zebrafish were modified to express cherry red in lymphocyte like cells using the Lymphocyte specific tyrosine kinase (lck) promotor. Also, rag1-/- zebrafish were modified so that the gene encoding the proto-oncogene serine/threonine-protein kinase that is involved in macrophage training (raf1) is disrupted. This study indicated that the acquired protection in the absence of lymphocytes likely involves NK cells with possible contribution by macrophages.

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Background: Life-prolonging tumour removal surgery is associated with increased metastasis and disease recurrence. Natural Killer (NK) cells are critical for the anti-tumour immune response. Postoperatively, NK cell cytotoxicity and interferon-gamma (IFNγ) production are profoundly suppressed and this dysfunction has been linked to increased metastases/poor patient outcomes. NK cell activity depends on the integration of signals through receptors and can be modulated by soluble factors, including transforming growth factor- beta (TGFβ). The postoperative period is characterized by the expansion of myeloid-derived suppressor cells (sxMDSCs), which inhibit NK cell effector functions. I hypothesize that impaired NK cell IFNγ production is due to altered signaling pathways caused by sxMDSC-derived TGFβ. Methods: Postoperative changes in NK cell receptor expression, receptor-dependent phosphorylation of downstream targets, and rIL-2/12-stimulated IFNγ production were assessed using newly developed whole blood assays utilizing peripheral blood samples from cancer surgery patients. Isolated healthy NK cells were incubated in the presence of healthy/baseline/postoperative day (POD) 1 plasma or isolated sxMDSCs and NK cell phenotype and function were assessed. NK cells were also cultured with plasma in the presence/absence of a TGFβ blocking monoclonal antibody (mAb) or a TGFβ RI small molecule inhibitor (smi). Single-cell RNA-sequencing was performed on six colorectal cancer surgery patients at baseline and on POD1. S6 phosphorylation was used as a proxy for mammalian target of rapamycin complex (mTORC) 1 activity to investigate the mechanism of TGFβ-mediated NK cell dysfunction. Results: Intracellular NK cell IFNγ, activating receptors CD132 (IL-2R), CD212 (IL-12R), NKG2D, and DNAM-1, and the phosphorylation of downstream targets STAT5, STAT4, p38 MAPK, and S6 were significantly reduced on POD1. TGFβ was increased in patient plasma on POD1. The dysfunctional phenotype could be phenocopied in healthy NK cells through the addition of rTGFβ1 or by incubation with POD1 plasma. This dysfunctional phenotype could be prevented with the addition of an anti-TGFβ mAb or a TGFβ RI smi in culture. RNA-sequencing revealed a reduction in transcripts associated with mTOR effector functions, suggesting an impairment in mTOR. S6 phosphorylation was maintained with the addition of TGFβ-specific therapies. The hyporesponsive NK cell phenotype was reproduced upon culture of healthy NK cells with sxMDSCs and sxMDSCs were shown to produce soluble TGFβ in culture. Conclusion: Surgically stressed NK cells display a dysfunctional phenotype, which could be prevented in vitro through the addition of TGFβ-specific blocking therapies. sxMDSCs produced TGFβ and co- incubation induced dysfunction in healthy NK cells. The recovery of impaired S6 phosphorylation with TGFβ-specific therapies suggests that TGFβ is inducing NK cell dysfunction via inhibition of mTORC1 activity. The perioperative period of immunosuppression presents a window of opportunity for novel therapeutics to prevent metastases and cancer recurrence among cancer surgery patients.