Date

May 2009

Document Type

Thesis

Degree Name

Ph.D.

Department

Dept. of Cell and Developmental Biology

Institution

Oregon Health & Science University

Abstract

We sought to determine the molecular mechanisms by which OX40 stimulation enhances T cell function. Gene array analysis of OX40-activated Ag-specific T cells suggested a potential role for the transcription factor c-Myc and the antagonist Mad family proteins. Our model system was comprised of OVA-specific D011.10 T cells which were adoptively transferred into BALB/c recipients, which were then immunized with soluble OVA and an agonist anti-OX40 antibody or rat Ig control. Using this model system, we previously showed that the OX40 agonist antibody enhanced Ag-specific T cell proliferation and subsequent survival. Western blot analysis of Ag-specific T cells purified ex vivo from draining lymph nodes (DLNs) revealed that Mad4, Mnt and c-Myc proteins were all up-regulated at day 3 after OX40 engagement. Mad4 and Mnt protein levels peaked at 3-4 days after immunization and anti-OX40 engagement then decreased later as the cells contracted in size. In contrast, c-Myc protein levels remained unchanged over time following anti-OX40 stimulation. Anti-OX40 stimulation stabilized both Mad4 and Mnt proteins against post-translational degradation, and we demonstrated the presence of a serine residue in Mad4 that was essential for mediating accelerated protein degradation. We hypothesize that the up-regulation of c-Myc in activated T cells drives proliferation and subsequent activation-induced cell death, which is counteracted by Mad4 and Mnt proteins after anti-OX40 engagement. This increase in Mad4 and Mnt protein levels allowed for T cells to survive the “blast crisis” phase. Consistent with this hypothesis, siRNA knockdown of Mad4 and Mnt proteins led to decreased survival of anti-OX40-activated T cells. These data provide evidence that Mad4 and Mnt most likely play a role to enhanced memory T cell survival following anti-OX40 engagement.

Identifier

doi:10.6083/M4M61H76

School

School of Medicine

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