Characterization of Jarid1b Expression During CD8 T Cell Differentiation
Open Access
- Author:
- Mattia, Michael Joseph
- Area of Honors:
- Veterinary and Biomedical Sciences
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Dr. Vandana Kalia, Thesis Supervisor
Dr. Lester C Griel Jr., Thesis Honors Advisor - Keywords:
- Jarid1b
histone
demethylase - Abstract:
- The overall goal of this project was to elucidate the role of the Jarid1b histone demethylase in the maturation and differentiation of CD8+ T cells. It is known that Jarid1b downregulates the oncogenic miR-17-92 cluster, which in turn drives terminal effector differentiation and compromises memory potential by promoting excessive proliferation during acute infection. Thus, our hypothesis was that Jarid1b is rapidly downregulated during effector differentiation to promote expansion of antigen-specific CD8+ T cells, but it is preferentially expressed in memory-fated CD8+ T cell subsets to promote memory function and longevity. The first aim of the experiment was to establish a western blot protocol to detect murine Jarid1b. Bone marrow cells were selected for this initial step as they are enriched in stem cells that are known to express higher levels of Jarid1b. The next aim of this study was to assess expression levels of Jarid1b in mature CD8+ T cells at the naïve, effector, and memory differentiation states. After establishing these levels, the subsequent steps involved creating retroviral constructs that possessed the ability to knock down the JARID1B gene through RNA interference. The most efficient of these constructs would be selected and used to transfect lymphocytes to knock down the JARID1B gene, inject these lymphocytes into P14 mouse models and observe their immunological fates following stimulation with LCMV. Western blot analysis revealed that Jarid1b was downregulated at day 2 of splenocyte maturation and continued through to day 8 of maturation, where the downregulation was most pronounced. Flow cytometry was run to characterize the splenocytes and determine their true state of immunological development. Following the preparation of five Jarid1b-knockdown retroviral constructs, western blot analysis was used to determine that construct #2 most effectively knocked down Jarid1b and was selected for use in the transfection of the 293T cell line. Future studies which will involve in vivo knockdown of Jarid1b in antigen-specific CD8 T cells to determine the functional significance of Jarid1b during effector and memory CD8 T cell differentiation.