Project 6

CD4+CD25+ regulatory T cells (TR) are implicated in the pathogenesis of diabetes and the transfer of TR specific for islet antigens is an effective treatment for diabetes in animal models raising the possibility that islet specific TR could be used therapeutically to prevent or cure T1D. However in order to realize this goal, methods must be developed to isolate or produce human TR targeted to the pancreatic islet cells in adequate numbers for therapeutic infusion, or alternatively, to enhance the formation of such TR in vivo. In this pilot project we propose to focus on the former approach. Specifically, we will focus on the in vitro generation and expansion of CD4+CD25+FoxP3+ TR which arise de novo as a result of activation of CD4+CD25-FoxP3- T cells. We refer to these cells as de novo generated TR (gTR) to differentiate them from TR which are directly isolated from the blood, or those expanded from CD4+CD25+ precursor cells. Our laboratory has demonstrated that activation of CD4+CD25- T cells with APC and peptide followed by isolation utilizing MHC class II tetramers results in gTR of a chosen MHC-peptide specificity. These gTR, like those directly isolated from the blood, will suppress bystander T cell responses in a cell contact dependent manner once activated with their cognate antigen. Thus such antigen specific gTR have the potential for therapeutic application in T1D. This pilot program is designed to extend these findings by developing methods to enhance the generation, isolation and expansion of gTR specific for islet antigens with the purpose of adapting this approach for therapeutic application.

1. We first aim to optimize the conditions under which antigen specific gTR can be generated de novo from CD4+CD25- T cells. Initially, isolation and generation of gTR will be optimized by using the foreign antigen HA (306-319). We will then apply these techniques to islet antigens, adjusting conditions to favor the generation of gTR specific for these self antigens. Outcomes will be assessed based on the number, specificity and function of the resulting gTR, as well as by examining FoxP3 expression and cell surface markers of the newly generated gTR.
2. We will then aim to determine the optimal activation and culture conditions for the survival and expansion of the de novo generated, islet specific TR. gTRs isolated as per protocols developed in Aim 1 will be expanded in vitro using high dose IL-2, irradiated APC and magnetic beads coated with either αCD3/αCD28 or MHC-peptide monomer/ αCD28. The resulting purity, number, phenotype and suppressive function will be measured to establish the ideal expansion protocol and QC protocols. Protocols developed by the Bluestone and June laboratories in their work to expand TR obtained directly from the blood will complement the studies in this aim and will be applied to the expansion of de novo generated gTR when appropriate.