Analysis of Ox40 Expression as Function of T Cell Activation

Fine Song¹, Robert H. Arch², Biology Department, Washington University, St. Louis, MO¹, Departments of Internal Medicine and Pathology & Immunology, Washington University School of Medicine²

T cell activation is a critical step in the immune response needed for the survival of higher eukaryotes. T cells are activated when the T cell receptor (TCR) on the cell surface recognizes foreign antigen in the context of the major histocompatibility complex (MHC) presented by antigen-presenting cells (APCs). TCR stimulation in conjunction with costimulatory signals results in enlargement and rapid proliferation of T cells that can then target invading pathogens and foreign antigens. However, after eliminating the stimulating agent, T cells must undergo apoptosis (programmed cell death) to prevent the accumulation of unwanted T cells. To regulate the balance between survival and death of T cells, intracellular signal transduction pathways integrate signals triggered by extracellular signals received by cell surface receptors.

Members of the Tumor Necrosis Factor Receptor (TNFR) family of cell surface receptors are crucial in regulating cell survival and apoptosis. They can be categorized into two subfamilies depending on the presence or absence of a death domain (DD) in the cytoplasmic domain of the receptors. Receptors with a DD are thought to trigger apoptosis whereas receptors lacking a DD can promote cell survival. Ox40 is a TNFR-related protein that is upregulated during T cell activation. In our lab we sought to establish whether alternative splicing is a regulatory mechanism of Ox40 expression. To this end, we isolated mouse splenocytes and analyzed changes in surface expression of Ox40 and other surface receptors over time by Fluorescence Activated Cell Scanning (FACS) analysis after activating T cells with antigen in vitro. We also defined changes in Ox40 expression by Polymerase Chain Reaction (PCR) using cDNA templates synthesized by reverse transcription of RNA isolated from the in vitro activated T cells. For these experiments, we used two sets of primers to amplify nucleotides encoding the extracellular/transmembrane domains (ECD/TMD) and transmembrane/intracellular domains (TMD/ICD) of Ox40, respectively.

Our results indicate that Ox40 is not alternatively spliced. Peaks in protein surface expression (as measured by FACS) correlated to mRNA levels (as analyzed by PCR). Furthermore, semiquantitative PCRs with the two primer pairs revealed similar levels of mRNA encoding for the ECD/TMD and TMD/ICD of Ox40, respectively. We will continue to study the functions of Ox40 during T cell activation and utilize the receptor as a tool to better control T cell survival and apoptosis. This will lead to the development of novel therapeutic approaches targeting T cell mediated disorders including autoimmune (type 1) diabetes and asthma.