Date of Award

1-1-2018

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Kevan C. Herold

Abstract

The receptor for advanced glycation endproducts (RAGE) functions as part of an innate immune signaling pathway and can be found within adaptive immune cells; however its role is not understood. We hypothesized that if we knocked down or knocked out RAGE from RAGE containing T cells, the cells would decrease the amplitude of intracellular signaling proteins associated with the T Cell Receptor cascade (TCR) and decrease the production of inflammatory cytokines. We used electroporation, siRNA knockdowns, Crispr/Cas9 knockouts, western blotting, flow and phospho-flow cytometry, and Luminex assays to delineate how RAGE and the TCR interact.

We assessed both external and internal cellular proteins from the RAGE and TCR signaling cascades. Jurkat cells expressed RAGE. Neither the pooled siRNA knockdowns nor Crispr/Cas9 knockouts of RAGE changed the expression of surface CD3 but they did decrease ratios of phosphorylated to total zeta chain-associated protein kinase 70, the next step in the TCR cascade. Further down the cascade knockdowns and knockouts reduced the levels of phosphorylated extracellular regulated kinases, proteins more proximal to the initiation of proliferation and inflammation, common to both the TCR and RAGE pathways. Endogenous high mobility group box 1 protein, a known RAGE ligand, decreased with initiation of TCR signaling by stimulation with anti-CD3/anti-CD28 in Jurkats more than in RAGE knockouts. Both knockdowns and knockouts reduced internal levels of diaphanous1, the immediate downstream signaling protein from RAGE, which mediates cascades relating to proliferation and inflammation.

Additionally knockouts produced less interleukin-2 and tumor necrosis factor-α than wild type Jurkat cells.

Through cytokine release and increased phosphorylation of intracellular signaling proteins in the TCR cascade, RAGE containing cells may enhance the response to a TCR stimulus driving cell proliferation and tissue inflammation by connecting signaling of the innate and adaptive immune systems.

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