Cytotoxic T lymphocytes (CTLs) eliminate target cells primarily through polarized secretion of the contents of membrane-enclosed cytolytic granules, specialized secretory lysosomes loaded with the pore-forming protein perforin and the serine proteases granzymes. When a CTL recognizes its target, the contact area between the CTL and the target cell forms a highly organized and stable structure known as the immunological synapse. Subsequently, cytolytic granules migrate from their dispersed locations in the cytosol toward the immunological synapse, where they dock and fuse with the plasma membrane to secrete their contents. Released cytolytic molecules then enter the target cell and initiate programmed cell death (apoptosis). The regulation of cytolytic granule secretion is still poorly understood at the molecular level. The major goal of this pilot project is to dissect cytolytic granule secretion in CTLs using unbiased genome-wide CRISPR-Cas9 genetic screens. In our preliminary studies, we developed novel platforms to genetically dissect mammalian vesicle transport pathways using genome-wide CRISPR screens. Furthermore, we developed assays to measure cytolytic granule secretion in CTLs. Here, we will take strategic advantage of these systems to dissect cytolytic granule secretion using an unbiased CRISPR genetic screen. We will then validate the identified genes using a pooled secondary screen as well as individual gene editing. Finally, we will characterize the knockout phenotype of selected candidate genes in CTLs. If successfully accomplished, this proposed research will provide the first genome-scale view of cytolytic granule secretion, and will substantially expand our knowledge of how CTLs destroy target cells. Insights gleaned from these studies will help improve the efficacy and safety of cancer immunotherapy, and will facilitate the development of new therapeutics for immune disorders caused by defective cytolytic granule secretion.

Public Health Relevance

Cytotoxic T lymphocytes (CTLs) eliminate target cells primarily through polarized secretion of the contents of membrane-enclosed cytolytic granules. The major goal of this pilot project is to dissect cytolytic granule secretion in CTLs using unbiased genome-wide CRISPR-Cas9 genetic screens. Our findings will substantially expand our knowledge of how CTLs destroy target cells, and will facilitate the development of new therapeutics for immune disorders caused by defective cytolytic granule secretion.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Small Research Grants (R03)
Project #
5R03AI135473-02
Application #
9694160
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Jiang, Chao
Project Start
2018-05-03
Project End
2020-04-30
Budget Start
2019-05-01
Budget End
2020-04-30
Support Year
2
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Colorado at Boulder
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80303
Shen, Chong; Liu, Yinghui; Yu, Haijia et al. (2018) The N-peptide-binding mode is critical to Munc18-1 function in synaptic exocytosis. J Biol Chem 293:18309-18317