Signal specificity amongst STAT1- and STAT3- inducing cytokines in the context of Th17 differentiation

Abstract

The adaptive immune response is very important for our survival in that it gives us the capability of detecting a wide variety of foreign material, allows for the elimination of pathogens, and provides memory to protect against future attacks by the same pathogen. Key mediators of the adaptive immune response are CD4+ T cells, which depending on the cytokine milieu, and the activating conditions during antigen recognition, can differentiate into different effector T cells. One particular type of effector T cell, Th 17, is highly inflammatory and has been implicated in various autoimmune diseases, such as Multiple Sclerosis. Three chapters within this thesis investigate the conditions which lead to Th17 differentiation and the mechanisms involved in their regulation. Th 17 cells can be obtained in vitro by culturing naive CD4+ T cells with IL-6 and TGF-p under activating conditions. IL-6 was thought to primarily activate the transcription factor STAT3, which has been shown to be necessary for ThI7 differentiation. Numerous cytokines activate STAT3, but IL-6 is the most potent inducer of TH17 cells, so we sought to find out what is special about IL-6's induction of STAT3. In the first of these three chapters, we propose a simple genetic network which is capable of translating IL-6's high amplitude, transient STAT3 signal into a pro-inflammatory response and IL-10's low amplitude, sustained STAT3 signal into an antiinflammatory response. This network is able to predict that IL-6 and IL-10 would induce an indistinguishable anti-inflammatory response in SOCS3-/- cells where IL-6's STAT3 signal is sustained. In the second of these chapters, we continue our research into the origin of signal specificity in cytokine signaling by systematically characterizing the activation of STAT1 and STAT3 by IL-6, IL-10, IL-21, IL- 27, and different combinations of cytokines in CD4+ T cells. In this analysis we find that the ratio of STAT3 to STAT1 activated is the important quantity in determining whether or not a cytokine will be an inducer of TH17 differentiation (IL-6, IL-21) or an inhibitor (IL-27). We show that in the absence of STAT1, that IL-6 and IL-27 are both potent inducers of TH17 differentiation since they have similar STAT3 activation profiles. In the third of these chapters, we develop a simple algorithm for clustering gene activation profiles for intermediate numbers of genes measured (10-50) and use it to analyze a 96- hour time course of gene activation during Th 17 differentiation for a number of genes of interest. In order for T cells to differentiate into effector cells, they must first recognize antigen which is presented on the surface of an antigen presenting cell by a membrane-bound extracellular complex called MHC. The MHC have a groove which peptide fragments (antigen) are bound in. Without peptide loaded in the pocket, the MHC are quite unstable so they are synthesized with a generic peptide fragment loaded. A protein, DM, is responsible for stabilizing the MHC while the generic peptide is ejected and the peptide fragment of interested is loaded. Two chapters within this thesis investigate the role of DM in peptide loading / unloading and attempt to characterize the interaction of DM with MHC.