Date of Award

2019

Embargo Period

8-8-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Zihai Li

Second Advisor

Bei Liu

Third Advisor

Mark Rubinstein

Fourth Advisor

Philip Howe

Fifth Advisor

Steven Rosenzweig

Abstract

Monoclonal antibodies targeting the Programmed Death Receptor 1/Ligand 1 (PD-1/PD-L1) pathway have been approved in over 15 types cancer. However, as clinical data continues to be accumulated surrounding the use of these drugs, it has become increasingly clear that monotherapy, irrespective of tumor type, does not result in uniform, long lasting benefit. As such, the most pressing question surrounding the use of PD-1/PD-L1 blockade for the treatment of cancer is how to improve response rates in order to benefit more people. One avenue for improving patient outcomes is a combination-based intervention strategy where a PD-1/PD-L1 blocking antibody is combined with another treatment modality. Exposure to immunosuppressive cell types such as macrophages or cytokines like TGFβ1 in the tumor microenvironment, in conjunction with the PD-1/PD-L1 axis, can coordinate suppression via non-redundant pathways. This increases the likelihood of tumor survival even in the presence of PD-1 blockade. Thus, strategies that modulate the tumor microenvironment are of extreme interest given their potential for combination with a PD-1/PD-L1 backbone. Previous work from our lab highlighted platelets as an unexpected source of immune suppression in cancer. In vitro studies demonstrated soluble factors, predominantly TGFβ1 and lactic acid, released during the course of platelet activation were capable of suppressing T cell blastogenesis and function. We hypothesized that platelets, specifically platelet activation and function, represent a secondary mechanism of immune suppression in the tumor microenvironment by supplying excess TGFβ1. We tested this hypothesis in two specific aims that probed 1) how platelets shape the tumor microenvironment, as well as their potential as a target for therapeutic intervention in combination with PD-1 blockade and 2) the role of soluble platelet-derived TGFβ1 in immune surveillance and anti-cancer immunity. We found that enhanced tumor control in the absence of functional platelets was associated with increased frequency and function of tumor infiltrating T cells and decreased CD11b+ myeloid cells, specifically F4/80+ macrophages. Superior T cell function was not observed in the draining lymph nodes suggesting the suppressive effect of platelets was restricted to the tumor microenvironment. Multispectral histology and quantitative imaging analysis revealed genetic dampening of platelet function decreased TGFβ1 signaling despite showing no differences in impeding platelet extravasation into the tumor bed. Pharmacologic inhibition of platelet function or antibody mediated platelet depletion in mice with established tumors significantly increased CD4+ and CD8+ T cell infiltration and decreased CD11b+ myeloid cell frequency in tumors. Finally, pretreatment with antiplatelet inhibitors aspirin and clopidogrel synergized with PD-1 blockade to improve tumor control. These findings validate platelets as a secondary mechanism of anti-tumor immune suppression and represent a novel target to improve PD-1 blockade. Next, we developed a mouse model in which soluble TGFβ1 was specifically deleted from the megakaryocyte and platelet compartment (Plt-Tgfb1 KO). Serum analysis demonstrated an approximately 90% reduction in systemic TGFβ1 concentration, validating the platelet as a predominant source of TGFβ1 in vivo. Immune phenotyping found Plt-Tgfb1 KO mice had subclinical signs of autoimmunity which became more prominent with age, indicating platelets participate in peripheral immune tolerance via TGFβ1. Surprisingly, enhanced immune function did not translate into increased immune surveillance as Plt-Tgfb1 KO mice displayed accelerated tumor development in a chemically induced carcinogenesis model. This was associated with an accumulation of a highly activated, terminally differentiated CD8+ T cell population that had dual expression of PD-1 and Tim-3. We were able to recapitulate the terminally differentiated tumor infiltrating CD8+ T cell population using the syngeneic MC-38 colon cancer model. Interestingly, MC-38 bearing Plt-Tgfb1 KO mice were non-responsive to PD-1 blockade compared to wild type controls, which demonstrated complete tumor eradication in 50% of mice. These findings suggest a potentially paradoxical role for platelet-derived TGFβ1 in anti-cancer immunity.

Rights

All rights reserved. Copyright is held by the author.

Download

Share

COinS