O transplante de células-tronco hematopoéticas alogênico (alo-TCTH) é uma opção de tratamento para uma variedade de doenças neoplásicas e não neoplásicas, principalmente de origem hematológica sendo doença do enxerto-contra-hospedeiro (DECH) a sua principal complicação. As células Natural Killer (NK) são os primeiros linfócitos a se recuperarem após o TCTH. Além da capacidade de promover o efeito enxerto-versus-leucemia (EVL), as células NK do doador parecem capazes de promover a pega do enxerto e de prevenir o desenvolvimento da DECH. As células NK compreendem aproximadamente 10% dos linfócitos do sangue periférico e são caracterizadas fenotipicamente pela expressão do antígeno de superfície CD56 (CD, cluster of differentiation) e pela ausência de CD3 (CD56+CD3-). O subtipo de células NK CD56dim (baixa densidade do antígeno) é naturalmente mais citotóxico que o subtipo CD56bright (alta densidade do antígeno) o qual é caracterizado pela capacidade de produção de citocinas. Com base nisso, o objetivo do trabalho é avaliar a presença de células NK nos dias 7, 14, 21 e 28 após o TCTH alogênico e autólogo, caracterizando sua frequência, seu imunofenótipo e a sua capacidade de produzir fatores de crescimento hematopoético e citocinas relacionadas. / Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an option of treatment for a variety of neoplastic and non-neoplastic diseases and graft-versus-host disease (GVHD) is its main complication. Natural Killer cells (NK) are the first lymphocytes to recover after HSCT. In addition to the ability to promote graft versus leukemia effect (GVL), donor NK cells appear to be capable of promoting engraftment and preventing the development of GVHD. NK cells comprise approximately 10% of peripheral blood lymphocytes and are characterized phenotypically by the expression of the CD56 surface antigen and absence of CD3 (CD56 + CD3-). The CD56dim (low density of antigen) NK cell subtype is naturally more cytotoxic than the CD56bright (high density of antigen) subtype which is characterized by the ability to produce cytokines. Based on this, the objective of the study is to evaluate the presence of NK cells on days 7, 14, 21 and 28 after allogeneic and autologous HSCT, characterizing their frequency, their immunophenotype and their capacity to produce hematopoietic growth factors and related cytokines.

Cancer immunotherapy is an encompassing term referring to therapeutic strategies that aim to boost the immune system to fight cancer. These strategies include administering immune cells that have been altered to have greater anti-tumor activity or using biologics and small molecules that target immune components to also promote tumor clearance. Natural Killer (NK) cells are cells of the innate immune system that recognize and kill abnormal cells such as cancer cells and play an important role in the anti-tumor response. Because of their crucial role in tumor immunity, NK cells are prime targets for immunotherapies. Repurposing small molecule drugs is an attractive strategy to identify new immunotherapies from already approved drugs. Here, we screened 1,200 approved drugs from the Prestwick Chemical Library to identify drugs that increase NK cell cytotoxicity. We used a high-throughput luciferase-release cytotoxicity assay to measure the killing of the myeloid leukemia cell line, K562 cells expressing nano luciferase (NL) by NK92 cells, a human NK cell line. From the drug candidates identified from the screening assay, the antibiotic colistin sulfate increased cytotoxicity of the NK92 cell line and unstimulated human NK cells towards K562-NL cells. This increase in NK cytotoxicity was short-lived as pre-treating NK92 cells with colistin for 1 hour or 24 hours did not increase cytotoxicity. Also, we show pre-treating K562-NL target cells with colistin does not sensitize them to NK-mediated killing. Further studies are needed to uncover the mechanism of action of colistin, thus contributing to knowledge of fundamental NK cell biology regarding NK cell cytotoxicity which will aid in identifying additional small molecule drugs that enhance NK cell activity.

Hepatitis C Virus (HCV) is a small RNA virus that progresses to chronicity in 50-80% of infected individuals. Direct-acting antivirals (DAAs) are revolutionary treatments for HCV with 90-98% cure rates. However, over time, chronic HCV infections can result in advanced liver disease, including cirrhosis. Patients with advanced fibrosis experience a poor response to vaccination, recurrent infections and increased risk for hepatocellular carcinoma (HCC). These outcomes are, in part, a consequence of immune dysfunction. Increased inhibitory receptor and Galectin-9 (GAL-9) expression is a possible mechanism promoting lymphocyte dysfunction. In this study, blood samples were collected from chronic HCV patients with different degrees of liver fibrosis. I conducted a 13-parameter flow stain on the peripheral blood mononuclear cells (PBMC) of these patients. Next, I measured the expression of inhibitory receptors (PD-1, CTLA-4, LAG-3, TIGIT and TIM-3) and GAL-9 on bulk T cell and NK cells of 15 chronic HCV patients with no to moderate fibrosis (F0-F2) and 15 with advanced fibrosis (F3-F4). To analyze receptor co-expression, I employed t-distributed stochastic neighbor embedding (t-SNE) analysis to dimensionally reduce the multi-parametric data. Notably, I found that F3-F4 patients had higher frequencies of >3 inhibitory receptor co-expression on NK cells. Moreover, t-SNE analysis of bulk T cells revealed that F3-F4 patients manifest a higher frequency of cells in the clusters with CD25+TIGITmed-hi CD4+ T cells and PD-1medLAG-3med-hiGAL-9med-hi CD4+ T cells. t-SNE analysis of NK cells also showed that F3-F4 patients manifest a higher frequency of cells in the cluster with CD25+TIGITmed-hiTIM-3med-hi CD56Dim NK cells and CCR7+ PD-1medLAG-3med-hiGAL-9med-hi CD56Dim NK cells. Lastly, the frequency of cells in these clusters was found to positively correlate with patient’s extent of liver damage. In conclusion, I identified phenotypes of immune dysregulation that could explain the increased susceptibility to infection and HCC in chronic HCV patients with advanced fibrosis. These phenotypes could identify targets for combinatorial checkpoint blockade therapy to potentially improve immune function in these patients.

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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 Science / Kyoto University / DFAM

Surgery-induced myeloid derived suppressor cells (sxMDSC)s mediate postoperative suppression of Natural Killer (NK) cells, which enables postoperative cancer recurrence and metastases. Currently, no therapeutics against sxMDSCs have been developed. Recent research has identified that the myeloid-restricted PI3K isoform (PI3Kγ) mediates MDSC activity. I targeted PI3Kγ in sxMDSCs as a therapeutic to reduce postoperative NK cell suppression and metastatic burden. Additionally, I investigated the efficacy of a sxMDSC-specific antibody-drug conjugate (ADC) with a PI3Kγ inhibitor payload. Pharmacological inhibition of PI3Kγ in sxMDSCs led to reduced AKT phosphorylation and reduced suppression of NK cytotoxicity in human and murine models. PI3Kγ inhibition also reduced postoperative metastatic burden. Despite the novelty of the sxMDSC-specific ADC, it didn’t provide considerable benefits in reducing NK cell suppression compared to the unconjugated PI3Kγ inhibitor. However, this is a “first iteration” in what could be a powerful approach to targeting sxMDSCs, thereby preventing postoperative metastatic burden.

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京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24885号 / 医科博第152号 / 新制||医科||10(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 河本 宏, 教授 濵﨑 洋子, 教授 上野 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Natural Killer (NK) cells can recognize and lyse a large variety of tumor cells and have been of interest as a potential cancer treatment option. Our group has developed a particle-based NK cell expansion method that utilizes plasma membrane particles (PM-particles) derived from K562 cells genetically engineered to express membrane bound IL21 and 41BBL(K562-mbIL21-41BBL), two proteins that stimulate growth and activity of NK cells. This method selectively expands highly cytotoxic NK cells > 400-fold in 14 days of culture. Currently NK cells are expanded in vitro using Fetal Bovine Serum (FBS) as a serum-supplement to promote cell growth. While effective, the use of animal products is not preferred in cell cultures grown for clinical purposes. This project tested Human Platelet Lysates (HPL) as a potential replacement for FBS in NK cell culture. NK cells were expanded using PM21-particle based expansion method with either FBS or HPL as supplements. Their growth characteristics, phenotype and functionality were assessed and compared. Results of this study determined that HPL is a viable option to replace FBS in NK cell culture for clinical applications, as there was no significant difference between the two serum supplements.