ORCID

http://orcid.org/0000-0002-2204-8676

Date of Award

Summer 8-15-2020

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Immunology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Classical dendritic cells (cDCs) are specialized antigen presenting cells that can be divided into distinct subsets based on the types of pathogens they respond to and the type of immune response they generate. The cDC1 subset is specialized in priming CD8 T cell responses through the process of cross-presentation. During cross-presentation, exogenous protein antigens are taken up by cDC1 and presented on MHCI molecules, allowing for the priming of CD8 T cells during conditions when DCs themselves are not directly infected. The ability to cross-present in vivo is unique to cDC1, and is essential for anti-viral responses and rejection of immunogenic tumors. The molecular mechanisms of cross-presentation remain incompletely understood, due to limited biochemical analysis of rare cDC1 cells, difficulty in their genetic manipulation, and reliance on in vitro systems based on monocyte-derived dendritic cells.

Through gene expression analysis of cDC1 versus cDC2, we found the vesicular transport gene Rab43 to be highly and specifically expressed by cDC1. Using a mouse line deficient in Rab43, we found that cDC1 lacking Rab43 were not able to cross-present to the same efficiency as wild type cDC1. However, monocyte-derived dendritic cells from these mice were normal in their ability to cross-present, calling into question the published data using these cells to study the cross-presentation pathway. Therefore, we developed a CRISPR-Cas9 screening system to discover additional members of the in vivo cross-presentation pathway. Through this process, we discovered Wdfy4, a gene specific to both cDC subsets and B cells that is essential for in vivo cross-presentation. Wdfy4 knockout mice are the first known genetic model to specifically lack the cross-presentation pathway without any observable deficiencies in other antigen presentation pathways or cell development. Through the use of these mice, we were able to show that cross-presentation is essential for the generation of CD8 T cell responses to viruses, the clearance of immunogenic tumors, and rejection of minor-mismatched antigen grafts but not for susceptibility to Listeria monocytogenes or protection against Toxoplasma gondii (both of which involve other functions of cDC1 which remained intact without Wdfy4). Through mass spectrometry, we determined that WDFY4 likely interacts with vesicular transport machinery after clathrin-mediated endocytosis and may be involved in vesicle sorting or recycling. These advances give new insight into the proteins involved in performing cross-presentation in vivo and improve our understanding of how the immune system responds to viruses and tumors.

Language

English (en)

Chair and Committee

Kenneth M. Murphy

Committee Members

Brian T. Edelson, Daved H. Fremont, Nima Mosammaparast, Steven J. Van Dyken,

Download

Share

COinS