The killing project : increasing the efficiency of cytotoxic T lymphocyte in killing cytomegalovirus-infected cells in an in vitro setting

    Item Description
    Linked Agent
    Creator (cre): Ferguson, Heather Amanda
    Advisor (adv): Golden, Kendra
    May 6, 2011
    Graduation Year

    Cytomegalovirus (CMV) is a fairly ubiquitous virus that is capable of remaining unseen by the body’s immune system until the immune system is compromised. The lack of immune response to CMV-infected cells is due to the virus’ ability to interfere with MHC class I and II. Cytotoxic T Lymphocytes (CTLs) kill cells presenting cognate peptide presented on MHC class I using perforin or granzyme. Though it is understood that these work as a part of the body’s acquired immune system and respond to specific peptides presented on major histocompatibility complexes, little is understood about the exact mechanisms by which CTLs are able to kill virus-infected cells. In fact most studies done on CTL killing have been done on peptide loaded, non-virus-infected cells. In order to explore the mechanisms of CTL killing of infected cells in vitro, we attempted to recreate and expand upon the results of previous lab research involving virus-infected mouse fibroblasts with a wild type (WT) and mutant strain of cytomegalovirus. In the mutant strain of CMV (TKO), we knocked out the three immune evasion genes that interfere with MHC class I presentation specifically. Chromium release assays were performed using T-cells harvested from murine OT1 cells lines. Using the original protocol, initial results indicated similar levels of killing among WT, TKO, and uninfected cells. When the protocol was altered and T-cells were grown in different cytokine conditions it was shown that cytokine IL12 had a particularly strong effect on CTL killing efficacy. Additional chromium release assays will determine whether killing of all cells in increased when CTLs are grown in IL12. Understanding the ideal environment for CTLs to kill target cells may provide a deeper understanding of what is important for an effective immune response.

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