Discipline: Biological Sciences
Subcategory: Cancer Research
Torhera Durand - University of the Virgin Islands
Co-Author(s): Courtney Smith, Yale University, New Haven, CT; Dr. Mark Lemmon, Yale University, New Haven, CT
T cell immunoglobulin and mucin protein 3 (TIM3) is a cell surface protein expressed on T helper type 1 cells and is proposed to be a regulator of human T cell exhaustion. In the presence of chronic disease and chronic infection, normally functioning T cells become “exhausted”, a state characterized by the expression of inhibitory receptor molecules. TIM3 acts as an exhaustion marker and is upregulated in exhausted T cells. TIM3 interacts with cellular ligands by functioning as an inhibitory protein to limit normal T cell function, thus enhancing cellular exhaustion. It has been suggested in published literature that the beta-galactoside binding protein, galectin-9, acts on T cells to induce apoptosis, but whether this induced cell death is dependent on TIM3 remains an area of active debate and conflicting results. The goal of this project was to investigate the effects of galectin-9 and resolve the role of TIM3. Understanding the role of T cell inhibitory receptors, such as TIM3, and their associated ligands has become the forefront of immuno-oncology research. By understanding the mechanisms of cancer cell activation of T cell inhibitory receptors like TIM3, researchers can devise breakthrough immunotherapy treatments for cancer patients by preventing immune cell inhibition through the blockade of inhibitory receptors. We hypothesized that Galectin-9 induced apoptosis of exhausted Jurkat T lymphocytes is dependent on T Cell Immunoglobulin and Mucin Protein 3 (TIM3). Galectin-9 protein ability to induce apoptosis was investigated using Jurkat T lymphocytes placed under an untreated negative control, a positive control of staurosporine, and 50, 100, or 200Nm of galectin-9 for 4, 12, and 18 hours. Across the concentration range of 50, 100, and 200 nM, and time range of 4, 12, and 18 hours, commercial galectin-9 does not increase the caspase activity relative to untreated cells. CRISPR-Cas9 knockout was used to inactivate TIM3 in Jurkat T lymphocytes and in vitro CRISPR-Cas9 digest was performed to determine the efficiency of each CRISPR guide RNA used during the CRISPR-Cas9 knockout. By using these methods to determine if galecin-9 induced apoptosis is dependent upon the T cell inhibitory receptor TIM3, researchers can develop TIM3 antibodies that can be used to block immune cell inhibition by cancer cells.Not Submitted
Funder Acknowledgement(s): This research was funded by the Yale University Summer Undergraduate Research Fellowship
Faculty Advisor: Dr. Mark Lemmon, firstname.lastname@example.org
Role: I was responsible for treating and splitting all of the Jurkat cells used for the experiments, conducting caspase and tunnel assays to determine levels of apoptosis, determining the puromycin kill curve, performing CRISPR Cas9 gene knockout on Jurkat cells through insertion of puromycin resistance and CRISPR guide RNA.