Chronic STAT3 signaling in STAT3 GOF causes CD8 T cell dysfunction via CD39 upregulation - PROJECT ABSTRACT STAT3 gain-of-function (GOF) is a rare, childhood-onset monogenic disease that amplifies inflammatory cytokine signaling. In this rare, inflammatory disease there is a paradoxical increase in both viral infections and in the incidence of autoimmunity. In STAT3 GOF patients we find impaired cytokine production and proliferation in CD8 T cells. Seeking mechanistic insight, we have found increased levels of CD39 protein, which is encoded by the STAT3 target gene ENTPD1, in CD8 T cells. CD39, an ectoenzyme that partners with CD73 to hydrolyze extracellular ATP to adenosine, is a negative regulator of immune activation. Adenosine in turn binds to the adenosine 2A receptor (A2AR) on T cells, potently inhibiting proximal TCR signaling and effector cytokine production. In the setting of chronic STAT3 signaling, and the resulting inflammation and autoimmunity, we propose that the CD39/axis serves as a rheostat for reducing inflammation. Our central hypothesis is that amplified STAT3 signaling drives CD39 expression, which increases ATP hydrolysis, thus increasing extracellular adenosine and impairing CD8 T cell function by binding to the adenosine 2A receptor (A2AR), thereby impairing both potential anti-viral and actively pathogenic autoreactive CD8 T cells in the setting of chronic STAT3 signaling. In Aim 1, we will test whether increased CD39 expression in CD8 T cells from STAT3 GOF patients and mice drives immune suppressive adenosine signaling to impair CD8 T cell function. These studies will also determine the impact of JAK inhibition (upstream of STAT3, the best available therapy for STAT3 GOF patients) in both STAT3 GOF samples from patients and mice, as well as perturbations of the CD39/adenosine axis in a mouse model of STAT3 GOF. In each case, we will assess the impact of chronic STAT3 signaling on CD8 T cell effector function, purinergic metabolite levels and the transcriptional impact on STAT3 target genes, CD8 T cell effector and adenosine signaling networks by directly comparing sorted CD39+ CD8 T cells vs. CD39- CD8 T cells and their impact on co-cultured “responder” bulk CD8 T cells. In Aim 2, we will determine the impact of inhibiting CD39 or A2AR on the susceptibility to autoimmunity and viral infection in STAT3 GOF. Specifically, we will assess the impact of inhibiting the CD39/adenosine axis in the NOD mouse model of type 1 diabetes (T1D), focusing on the impact on function when the STAT3 GOF patient variant STAT3+/K392R is exclusively expressed in islet-antigen specific CD8 T cells. In addition, we will assess the impact of inhibiting the CD39/adenosine axis in acute LCMV infection in B6 mice with co-transfer of STAT3+/K392R antigen specific CD8 T cells and STAT3+/+ antigen specific CD8 T cells. Defining the role of the CD39/adenosine axis could lead to novel therapeutic options for the many disease states rooted in chronic inflammation.
