Abstract
The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery.
Highlights
The efficient and comprehensive discovery of novel, relevant T cell antigens in human subjects and animal model systems is complicated by two factors
Overlapping minigenes are designed to encode all potential peptide epitopes derived from hundreds of selected proteins expressed in the tissue or pathogen of interest. Long oligonucleotides encoding these minigenes are synthesized in parallel on a programmable microarray, and released from the array as a single mixture [15] (Fig. 1a)
in vitro (IVT) products from each pool are transfected into irradiated autologous B cells in a 96-well format for use as antigen presenting cells (APC) (Fig. 1e)
Summary
The efficient and comprehensive discovery of novel, relevant T cell antigens in human subjects and animal model systems is complicated by two factors. The second challenge is that most tissues or pathogens express hundreds or thousands of proteins, each representing a potential T cell target antigen. The efficient expression of a single candidate antigen in large numbers of APC allows detection of rare T cell specificities within a mixed population [4,5,6]. Both of these approaches are limited – the former, in that only a small number of T cell specificities are assayed, and the latter, in that only a small number of potential target antigens are tested. These screens have identified two novel T cell epitopes targeted by subjects newly diagnosed with or at risk for type I diabetes
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
