Date of Award

2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Zihai Li

Second Advisor

Philip Howe

Third Advisor

Mark P. Rubinstein

Fourth Advisor

Stephen Tomlinson

Fifth Advisor

Xue-Zhong Yu

Abstract

GARP encoded by the Lrrc32 gene is the cell surface docking receptor for latent TGF-β1 mostly expressed on regulatory T cells (Treg) and platelets. Although GARP has been extensively studied for the ability to enhance latent TGF- β1 activation in the context of Treg, the expression and relevant functions on cancer cells and platelets had not been explored when this work started 4 years ago. In addition, a soluble form of GARP has been described as shed from the Treg cell surface; however, the mechanism to explain the soluble molecule formation and how it becomes biologically active remains elusive. The results contained in this dissertation cover several unknown aspects of GARP biology and shed light on GARP as a potential therapeutic target in cancer treatment and prognosis. The first part of the results section focuses on the mechanism behind the formation and the biological activity of soluble GARP. Here we describe two putative mechanisms that explain the generation of the soluble protein. The first mechanism is mediated by thrombin that enzymatically cleaves surface GARP to generate two cleaved products. The second mechanism is mediated by extracellular vesicles (EVs) secreted by cells, specifically exosomes, which include GARP/latent TGF-β as a complex. Importantly, this part of the thesis dissertation demonstrated the importance of integrins belonging to the alpha V family that mediate the endocytosis of soluble GARP (sGARP) in epithelial cells. In the second chapter of the results section, I report that GARP exerts oncogenic effects, promoting immune tolerance by enriching and activating latent TGF-β1 in the TME. In collaboration with other members in Dr. Zihai Li’s laboratory, I found that human breast, lung, colon, and prostate cancers expressed GARP aberrantly. In genetic studies utilizing normal mammary gland epithelial and carcinoma cells, GARP expression increased TGF-β bioactivity and promoted malignant transformation in immunodeficient mice. In immunocompetent breast carcinoma– bearing mice, GARP overexpression promoted Foxp3+ Treg cell activity, which in turn contributed in enhancing cancer progression and metastasis. Notably, administration of a GARP-specific monoclonal antibody, made by Dr. Zihai Li’s laboratory, limited metastasis in an orthotopic model of human breast cancer. Overall, these results define the oncogenic effects of the GARP–TGFβ1 axis in the TME and suggest mechanisms that might be exploited for diagnostic and therapeutic purposes. These results have been summarized and published in the journal Cancer Research in a paper entitled “Expression of TGF-β Docking Receptor GARP Promotes Oncogenesis and Immune Tolerance in Breast Cancer”1. In the third part of the result section, I focused on the role of platelet GARP. Here, I hypothesized that constitutive GARP expression on platelets enhances the activation of latent TGF-β1 released by platelets. This phenomenon is critical in the cross-talk between platelets and cancer cells where GARP promotes malignancy and resistance to therapy. Indeed, platelet-specific deletion of GARP-encoding gene Lrrc32 blunted TGF-β1 activity at the tumor site and potentiated protective immunity against both melanoma and colon cancer. This work was recently published in Science Immunology in an article entitled: “Platelets Subvert T Cell Immunity Against Cancer via GARP/TGF-β Axis”2. Overall, the results included in this thesis demonstrate that 1) the formation of soluble GARP is mediated by two novel mechanisms that can be exploited for cancer immunotherapy; 2) GARP is expressed on cancer cells and has oncogenic properties; and 3) GARP/TGF-β axis on platelets reduces the efficacy of the anti- tumor immunity by blunting anti-tumor T cell activity.

Rights

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