Discovery of T Cell Antigens by High-Throughput Screening of Synthetic Minigene Libraries

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.

A T-cell response to a liver-stagePlasmodiumantigen is not boosted by repeated sporozoite immunizations

“…Design, synthesis, pooling, and use of a minigene library containing 1,988 components predicted to bind BALB/c MHC is in SI Materials and Methods. The technique was as described (32).…”

“…To identify novel malaria vaccine candidates, we developed a HTS approach based on massively parallel synthesis of thousands of oligonucleotides that encode candidate peptides (32) (Fig. S1).…”

“…In lieu of peptides, plasmidencoded parasite genes can be transfected into antigen-presenting cells (APCs) (31), but this approach is low throughput and difficult because of the A/T-richness of Plasmodial genes. Instead here, we used a minigene-based high-throughput screen (HTS) for CTLs (32) to study a library of Plasmodium yoelii-derived antigens in sporozoite-vaccinated mice. Our approach identified a unique CTL target antigen that provided surprising insights about CTL responses in multiply-immunized mice.…”

Treating Human Autoimmunity: Current Practice and Future Prospects

“…Thus, discovery of the major antigens targeted in human auto-immune diseases will greatly enhance our ability to reestablish antigen-specific immune tolerance in both antigen-SIT and all other current tolerance-promoting strategies. Recent advances in high-throughput screening approaches with self-protein libraries and autologous T cells from autoimmune patients will undoubtedly enhance our ability to do so (55). It is imperative that sufficient research initiative and financial resources are allocated to these lines of investigation.…”

A functional framework for interpretation of genetic associations in T1D

“…There are a variety of potential antigenic targets in T1D, including T cell immunity to islet proteins, such as proinsulin, GAD, IA2, IGRP, ZnT8, and others [14;15]. It is not known whether this trimolecular model for HLA avidity skewing applies to all, or whether there are differences during the early stages of disease initiation compared to later during disease progression, following antigenic determinant spreading of the immune response.…”