The Role of STAT1 in IL-6 Mediated T Cell Homoeostasis

In the search for the mechanism leading to the loss of STAT1 phosphorylation in activated cells (Specific Aim 2) we have performed several experiments.  We sorted activated T cells from SEB-treated mice and found them to be unresponsive to another STAT1 activating cytokine, gammaIFN.  We have used both flow cytometry and western blots to show that the loss of gammaIFN-induced STAT1 phosphorylation in activated T cells is not due to loss of the receptor.  Therefore, our new data suggest that there is an inhibitor of STAT1 phosphorylation in activated T cells that is common to both IL-6 and gammaIFN receptor signaling.

Since recent publications have pointed to the SOCS proteins as inhibitors of cytokine signaling we have performed experiments to determine whether SOCS-1, -2, and -3 are upregulated in activated T cells.  We isolated RNA from resting and in vivo activated T cells and performed real-time PCR experiments to assess SOCS mRNA expression.  Our preliminary results show no increase in SOCS-1, -2, or -3 mRNA.  In fact we found a surprising drop in SOCS-1 and SOCS-3 mRNA following T cell activation.  Therefore, SOCS-1, -2, and -3 are unlikely to be involved in the failure of activated T cells to respond to IL-6 survival signals.

In order to assess whether there is a difference in gp130 phosphorylation and recruitment of STAT proteins in activated vs. resting T cells (Specific Aim 2), we first needed to make GST-STAT SH2 domain fusion proteins.  Thus, we have generated by RT-PCR the SH2 domains of STAT1 and STAT3.  The cDNAs have been inserted into the pGEX vector and the constructs have been sequenced to confirm that the SH2 domains are in frame with the GST moiety.  From transformed bacteria, we have optimized conditions for induction and purification of the GST-fusion proteins.  We have generated sufficient material to begin preliminary experiments on the receptor pull-down assays.