Type 1 diabetes is mediated by T cell entry into pancreatic islets and the subsequent destruction of insulinproducing ? 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 ?self-cleaving? 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 ?bystander? T cells do not gain entry into islets, suggesting that access may be tightly regulated. The simplest hypothesis is that ?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?. Four related questions will be addressed. (A) Can non-infiltrating T cells enter islets in (a) the presence of ?driver? 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 ?diabetogenic T cells have a unique migratory and/or molecular signature which defines their pathogenicity?. 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.
| Bettini, Matthew L; Bettini, Maria; Vignali, Dario A A (2012) T-cell receptor retrogenic mice: a rapid, flexible alternative to T-cell receptor transgenic mice. Immunology 136:265-72 |
| Bettini, Maria; Castellaw, Ashley H; Lennon, Greig P et al. (2012) Prevention of autoimmune diabetes by ectopic pancreatic ?-cell expression of interleukin-35. Diabetes 61:1519-26 |
| Collison, Lauren W; Vignali, Dario A A (2011) In vitro Treg suppression assays. Methods Mol Biol 707:21-37 |
| Workman, Creg J; Collison, Lauren W; Bettini, Maria et al. (2011) In vivo Treg suppression assays. Methods Mol Biol 707:119-56 |
| Burton, Amanda R; Baquet, Zachary; Eisenbarth, George S et al. (2010) Central nervous system destruction mediated by glutamic acid decarboxylase-specific CD4+ T cells. J Immunol 184:4863-70 |
| Bettini, Matthew L; Vignali, Dario A A (2010) Development of thymically derived natural regulatory T cells. Ann N Y Acad Sci 1183:1-12 |
| Bettini, Maria; Vignali, Dario A A (2009) Regulatory T cells and inhibitory cytokines in autoimmunity. Curr Opin Immunol 21:612-8 |
| Lennon, Greig P; Bettini, Maria; Burton, Amanda R et al. (2009) T cell islet accumulation in type 1 diabetes is a tightly regulated, cell-autonomous event. Immunity 31:643-53 |
| Vignali, Dario A A; Collison, Lauren W; Workman, Creg J (2008) How regulatory T cells work. Nat Rev Immunol 8:523-32 |
