Author ORCID Identifier

0000-0001-9158-3464

Date of Graduation

8-2017

Document Type

Dissertation (PhD)

Program Affiliation

Immunology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Dean Lee

Committee Member

Eugenie Kleinerman

Committee Member

Stephen Ullrich

Committee Member

Silke Paust

Committee Member

Yoshihiro Komatsu

Abstract

Osteosarcoma (OS) is the most common primary bone tumor. Despite new treatment options, 5-year survival for metastatic OS has remained at only 30% for the last 30 years. Adoptive transfer of Natural Killer (NK) cells holds promise for a new, non-toxic therapy for OS. NK cells are part of the innate immune system and readily kill metastatic and chemotherapy-resistant OS in vitro and in murine models. However, there is little data regarding their efficacy in animal models with an intact immune system. In addition, the OS tumor microenvironment is highly suppressive, producing TGFβ which impedes NK cell killing of solid tumors. We set out to overcome these hurdles by characterizing NK cells in the canine model, which spontaneously develops OS, has a complete tumor microenvironment, an intact immune system, and is an established animal model with proven translatability to human OS. However, little is known about canine NK, preventing the testing of NK cell therapy in canine OS. We also set out to improve NK cell therapy by generating NK cells resistant to TGFβ suppression. To this end, we generated a novel anti-canine NKp46 antibody and characterized canine NK cells as CD3-/NKp46+. Canine NK cells have a median 3-week expansion of 20,000-fold on K562 feeder cells expressing membrane-bound IL-21. Canine NK cells efficiently kill OS, secrete IFNγ and TNFα, and express genes for NK cell receptors. Using a novel non-genetic approach, we generated human NK with reduced sensitivity to TGFβ- Resistant NK (ReNK). ReNK retain high cytolytic activity and produce remarkably more IL-6, IFNγ, and TNFα plus and minus TGFβ treatment compared to Standard NK cells. We characterized the TGFβ pathway in ReNK and found a near complete loss in SMAD3 protein and decreased TGFBR3 expression. ReNK also have enhanced STAT3 activation. In summary, we characterized canine NK cells, establishing the necessary tools to test NK cell therapy in canines to inform the use of NK cells in both human and canine OS. We also generated NK cells with enhanced anti-tumor activity that represent a promising therapeutic option for tumors with high TGFβ production such as OS and brain tumors.

Keywords

NK cells, natural killer cells, immunotherapy, TGF-beta, canine, comparative oncology, osteosarcoma

Available for download on Thursday, June 14, 2018

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