Browsing by Subject "T-cells"

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    Generating lightly immortalizing T-Cells to study the host immune response and immunotherapies
    (2023-04) Hoffman, Ashley S.; Umlauf, Benjamin J.
    Immortalized T-cell lines are key tools for studying disease response, immune pathways, and novel treatments. However, many of these lines are transformed into undefined cancerous cells to maintain unlimited growth potential, limiting their utility as an accurate model of host T-cells. This creates a need for T-cell lines that mimic healthy behavior, especially for testing T-cell-mediated therapies (e.g., CAR T-cell and checkpoint inhibitor) and T-cell exhaustion, a phenomenon observed in patients undergoing intense chronic therapies, that can still be shared amongst the scientific community. To this end, we have developed a method of lightly immortalizing T-cells from human donor peripheral blood monocytes (PBMCs), without producing a cancerous phenotype, by only expressing 2 genes: human telomerase reverse transcriptase (hTERT) and cyclin-dependent kinase 4 (Cdk4). Cdk4 and hTERT are key genes in driving cell cycle and survival. Over expression of these genes should create a slowly dividing T-cell population that is still capable of responding to immune cues and signals. Lightly immortalized T-cells will serve as a key resource for studying T-cell-mediated therapies such as chimeric antigen receptor (CAR) T-cells and effects of chronic treatment, including T-cell exhaustion. Here, we present a robust method for lightly immortalizing T-cells from PBMCs. Our protocol successfully extends the lifespan of PBMCs and selects for effector and helper T-cell populations in two donor PBMCs. To verify successful transfection and selection of T-cells hTERT and Cdk4 we used western blotting and FACs. We also observed typical T-cell behavior post-immortalization and achieved a resting population of healthy, actively dividing T-cells with measuring expression of CD3. In conclusion, this method provides a straightforward, reproducible, and shareable tool for understanding T-cell response to treatment and studying T-cell exhaustion in multiple patients.