Control of CD4+ T cell Islet Entry and Diabetogenicity

St. Jude Children'S Research Hospital, Memphis TN

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Abstract

roject Summary Type 1 diabetes is mediated by T cell entry into pancreatic islets and the subsequent destruction of insulinproducing [unreadable] cells. Determining the molecules and mechanisms that control these critical events is the focus of this application. We have developed an novel approach for the rapid generation of TCR transgenic mice [referred to as retrogenic (Rg) mice] using retroviral-mediated stem cell gene transfer and [unreadable]self-cleaving[unreadable] 2A peptide-linked multicistronic retroviral vectors that express both TCR chains and a fluorescent protein marker. We have selected a panel of 12 TCR that possess a very broad range of insulitogenic and diabetogenic potentials, generating T cells that (i) fail to enter islets, (ii) cause insulitis but not diabetes, (iii) cause insulitis and diabetes with expected kinetics (eg. BDC2.5), or (iv) induce extremely rapid diabetes. Aim 1: What governs CD4+ T cell islet entry and retention? Our preliminary studies suggest that [unreadable]bystander[unreadable] T cells do not gain entry into islets, suggesting that access may be tightly regulated. The simplest hypothesis is that [unreadable]appropriate activation in the draining pancreatic lymph node is required to induce expression of the chemokine and/or homing receptors required to mediate islet entry[unreadable]. Four related questions will be addressed. (A) Can non-infiltrating T cells enter islets in (a) the presence of [unreadable]driver[unreadable] T cells and/or (b) following peripheral activation? (B) Do infiltrating, non-diabetogenic T cells mediate enhanced insulitis and diabetes in the presence of diabetogenic T cells? (C) What is the mechanism that regulates T cell entry into islets? (D) How do T cells with differing insulitogenic potentials alter (or are altered by) the function of non-T cell populations? These questions will be addressed by generating TCR Rg mice carrying two T cell populations (eg. diabetogenic and bystander), in the presence or absence of a polyclonal T cell pool, and flow cytometry, immunohistochemistry and genetic profiling used to determine what governs and regulates T cell islet entry and retention, and how this is modulated by DCs, B cells and islet endothelium. Aim 2: What makes a CD4+ T cell diabetogenic and how is its function modulated? We have identified 6 TCR that all mediate T cell entry into islets but possess vastly different diabetogenic potentials leading us to hypothesize that [unreadable]diabetogenic T cells have a unique migratory and/or molecular signature which defines their pathogenicity[unreadable]. Four related questions will be addressed. (A) Does the kinetics of islet entry influence T cell diabetogenic potential? (B) How do T cells with differing diabetogenic potentials alter (or are altered by) the function of non-T cell populations? (C) What genes are responsible for T cell diabetogenicity? (D) What is the interrelationship between diabetogenic and regulatory T cells? This will be addressed by using TCR Rg mice, novel genetic mouse models, flow cytometry, genetic profiling etc. to track the kinetics of islet entry, the role of islet-resident DCs and B cells, and the contribution of Tregs in modulating T cell diabetogenic potential.