P3D317: Juvenile Idiopathic Arthritis: The Effects of Wild-type GATA3 on Cytokine Expression in Peripheral Blood Mononuclear Cells

Background: Juvenile Idiopathic Arthritis (JIA) is the most common autoimmune arthritis in children. JIA has no cure, and its pathogenesis is poorly understood. In JIA patient synovial fluid from joints with active arthritis, there are increased amounts of inflammatory T helper 1 (Th1) cells and the cytokine they produce, interferon gamma (IFNg). In Th cells, GATA3 increases the generation of anti-inflammatory Th2 cells and decreases the generation of inflammatory Th1 cells. We sought to introduce a mutated GATA3 gene identified from a JIA patient into T cells to test if it would cause an increase in the production of IFNg. This project aimed to take the first step towards testing this hypothesis by determining if expression of wild-type GATA3 in T cells changes IFNg expression.

Methods: We introduced a plasmid expressing wild-type, FLAG-tagged GATA3 into peripheral blood mononuclear cells (PBMCs) using the Lonza Amaxa Human T cell Nucleofector Kit. The FLAG tag is an extra set of amino acids included at the N-terminus of the GATA3 protein that was used to identify the expressed protein during flow cytometry. After nucleofection, the PBMCs underwent T cell activation with anti-CD3 and anti-CD28 and incubation. After a one-day incubation, cells were stimulated for 4 hours with a cocktail to enhance cytokine production and prevent cytokine release from the cell. Cells were stained for flow cytometry using live/dead and fluorophore-conjugated CD3, CD8, CD4, FLAG, and IFNg antibodies. Cells were analyzed by flow cytometry, and the types of T cells expressing FLAG and IFNg were determined.

Results: Live cells expressing FLAG-GATA3 and IFNg were identified by flow cytometry for PBMCs and FLAG-GATA3 nucleofected PBMCs that were unstimulated or stimulated. A flow cytometry plot depicts FLAG-GATA3 and IFNg expression in these cells (Figure 1A). The percent of each sample expressing IFNg was determined (Figure 1B). The data show that, when not stimulated, PBMCs and FLAG-GATA3 PBMCs produce low numbers of IFNg-producing cells. When stimulated, PBMCs had dramatically enhanced IFNg production, whereas FLAG-GATA3 PBMCs exhibited a lessened IFNg production.

Conclusion: We are able to introduce a protein of choice into T cells to study the protein impact on T cell cytokine production. T cells expressing FLAG-GATA3 exhibited a reduced production of IFNg. Using these techniques in future experiments, we will study how introduction of a mutant GATA3 affects T cell cytokine production of IFNg. With this research, we aim to learn how genetic mutations, such as the mutation in GATA3, contribute to the pathogenesis of JIA by impacting inflammatory cytokine production.

This work was supported by the Vanderbilt-Meharry James Puckette Carter Summer Scholarship Program; the Vanderbilt University Medical Center Pediatric Rheumatology Research Department (K08AR081405).