Our goal is to determine the molecular mechanisms by which reactive oxygen intermediates (ROI) control the activation, proliferation and differentiation of naive CD8+ T cells. During naive CD8+ T cell activation there is a rapid increase in tyrosine phosphorylation and calcium influx. At the same time there is also an increase in ROI. This increase in ROI is thought to be critical to normal T cell activation as co-incubation with anti-oxidants decreases the expansion of CD8+ T cells in vitro and in vivo during viral infection. The specific hypothesis that is being addressed in this proposal is that naive CD8+ T cell activation, proliferation and differentiation are controlled by ROI through the controlled modification of cysteines in signaling proteins to sulfenic acid.
Specific Aim 1 will test the effects of modulating ROI on naive CD8+ T cell activation, proliferation and differentiation. P14 TCR transgenic mice that overexpress both catalase and Cu superoxide dismutase (CAT/SOD) or lack peroxiredoxin II (Prx II -/-) will be used. Purified naive CD8+ T cells from these mice will be activated in vitro by polyclonal stimuli or after interaction with peptide coated bone marrow derived dendritic cells. The in vivo effects of ROI modulation on CD8+ T cell activation, proliferation and differentiation will be determined by adoptive transfer into naive recipients followed by LCMV infection.
In Specific Aim 2 we will determine the sulfenic acid modification of the protein tyrosine phosphatases SHP-1 and SHP-2 during CD8+ T cell activation, proliferation and differentiation. This will be accomplished through the use of our novel labeling reagents which allow the isolation and identification of sulfenic acid containing proteins. In the first part of Specific Aim 2, we will determine the modification of these PTPs in purified naive CD8+ T cells activated by polyclonal stimuli. In the second part of aim 2, we will determine how PTPs are modified in T cells activated by peptide coated dendritic cells.
Understanding how CD8+ T cells become activated, proliferate and differentiate is critical to developing treatments to increase the number of antigen-specific cells during vaccination or cancer therapy and it is also important to reducing unneeded cells during organ transplant or autoimmunity.
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