Novel Technologies for Investigation of Self-reactive T cells in MS and Relevant

该核心将为PPG的所有项目提供重要资源。纳米井技术

This core will make important resources available to all Projects of this PPG. The nanowell technology represents a powerful tool to study the functional consequences of interactions between T cells and APC. The technology will enable in-depth investigation of the impact of different types of APCs on the functional programs of self-reactive T cells, which is of central importance to this PPG. A novel feature of this technology is that the functional consequences on both T cells and APC can be studied for interacting cell pairs, offering insights into the complex communication that programs self-reactive T cells with autoaggressive behavior. This core will enable integration of datasets from clinical specimens and experimental mouse models (Aim 1). It provides an important link between Projects 1 and 2, which both study myelin-specific T cells from patients with MS. The technology enables robotic isolation of cells of interest, such as myelin-specific T cells that produce combinations of pro-inflammatory cytokines (e.g. IL-17 + GM-CSF), for analysis of single-cell gene expression or clonal expansion. Furthermore, the technology will be made available for analysis of murine T cells, APCs and stromal cells (Projects 3 and 4). The core will also make recombinant proteins available to all Projects of this PPG, including experiments in the nanowell system (Aim 2), This core has provided such reagents to all Projects in the previous funding period and will continue to provide them for several efforts: 1. Studies by Dustin and Wucherpfennig have examined immunological synapse formation by human self-reactive T cells using the planar lipid bilayer system developed by the Dustin lab. Recent collaborative studies by the Love and Wucherpfennig labs have applied this approach to the nanowell system, enabling synapse structure to be related tp T cell function. These experiments require sets of human and murine recombinant proteins, including peptide-MHC complexes, ICAM-1 and CD80 (Projects 1 and 2). 2. Collaborative studies by the Kuchroo and Wucherpfennig labs have shown that tetramers of the MOG extracellular domain can be used to label MOG-specific B cells, and this reagent will be used in Project 3. 3. The turley, Kuchroo and Wucherpfennig labs have utilized Ig fusion proteins of podoplanin and CLEC-2 to study the function of these molecules in T cell - APC communication, which will be provided to Projects 3 and 4. This core will thus enable in-depth investigation of T cell - APC interactions in both humans and animal models to advance our understanding ofthe pathogenesis of MS.