Author ORCID Identifier

0000-0001-5515-6510

Date of Graduation

12-2017

Document Type

Dissertation (PhD)

Program Affiliation

Clinical and Translational Sciences

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Eugenie S. Kleinerman, MD

Committee Member

Dean A. Lee, MD PhD

Committee Member

Michael A. Curran, PhD

Committee Member

Jordan Orange, MD PhD

Committee Member

Kimberly Schluns, PhD

Committee Member

Zahid H. Siddik, PhD

Abstract

Due to Natural Killer (NK) cells’ capacity to target tumor cells without prior sensitization, adoptive NK cell therapy represents a promising immunotherapy approach for pediatric cancer patients. Our laboratory has developed an NK cell expansion protocol that generates large quantities of NK cells for therapeutic infusion. Given that NK cells are heterogeneous, with variable receptor expression and potential to target tumor cells, the purpose of my study was to determine whether subpopulations of NK cells with enhanced anti-tumor potential could be identified for increased potency of the NK cell infusion product. In addition, we previously showed that our expanded NK cells secret 20 times more IFNγ than resting NK cells. Opposing effects have been reported for IFNγ on tumor sensitivity to NK-mediated lysis. Therefore, another aim of my study was to evaluate the effect of IFNγ on tumor sensitivity to NK cell mediated lysis, tumor expression of NK cell ligands, and NK:tumor interactions using a standardized panel of cell lines corresponding to 6 types of pediatric malignancies.

My results demonstrate the presence of unique NK cell subpopulations in the expanded product that are absent in primary NK cells. These subpopulations co-express higher levels of multiple activating receptors than primary NK cells. Moreover, compared to the NK cell subpopulations that are common to both primary and expanded NK cells, the unique subpopulations showed increased degranulation (CD107a) and IFNγ secretion in response to tumor cell encounter.

I also demonstrate that IFNγ has a variable impact on NK-mediated lysis of pediatric tumor cell lines, with some cell lines becoming more resistant to NK cells and others becoming more sensitive. Broad screening of NK cell ligands on these cell lines using mass cytometry, and flow cytometry, show that both exogenous and NK cell secreted IFNγ cause significant upregulation of PD-L1, ICAM-1, and MHC-class I, but this upregulation varies widely between the cell lines. Modeling of the data suggests that the effect of IFNγ on NK cell-mediated tumor lysis is mostly dependent on relative changes in MHC-class I and ICAM-1 expression. In cell lines with increased sensitivity after IFNγ treatment, ICAM-1 upregulation exceeded that of MHC-class I upregulation. This ICAM-1 upregulation resulted in increased conjugate formation between the NK cells and tumor cells. Timelapse imaging of neurobastoma cells with increased sensitivity revealed that IFNγ treatment also decreased the time for NK cell encounter of tumor cells (tseek). Blocking of ICAM-1 weakened the increased sensitivity observed after IFNγ treatment for selected cell lines and NK donors. Although ICAM-1 and MHC-class I were identified as key role players, the effects of MHC-class I and ICAM-1 are not always predictable.

This identification of hyperactive NK cell subpopulations with enhanced IFNγ secretion and a better understanding of the impact of IFNγ on NK:tumor interactions provides important information that can be used to further improve NK cell immunotherapy of cancer.

Keywords

NK cells, IFN gamma, ICAM-1, MHC, PD-L1, CyTOF, mass cytometry, pediatric cancer, immunotherapy

Available for download on Thursday, December 13, 2018

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