- Authors: Parras D. et al.
- Year: 2021
- Journal: Front Immunol 12 737428
- Applications: in vitro / DNA / jetOPTIMUS
- Cell type: HEK-293T
Description: Human embryonic kidney Fibroblast
Known as: HEK293T, 293T
HEK-293T–I-Ag7 cells (2.5 × 104) plated in a flat-bottom 96-well plate were transfected with 100 ng/well of pCMV-Ii1-80-β-cell autoantigen-BDC2.5mi DNA, using jetOPTIMUS (Polyplus). After 24 h, 105 4.1- or BDC2.5-JurMA cells were added and co-cultured for 48 h followed by luciferase activity measurement.
The mechanisms underlying the major histocompatibility complex class II (MHCII) type 1 diabetes (T1D) association remain incompletely understood. We have previously shown that thymocytes expressing the highly diabetogenic, I-Ag7-restricted 4.1-T-cell receptor (TCR) are MHCII-promiscuous, and that, in MHCII-heterozygous mice, they sequentially undergo positive and negative selection/Treg deviation by recognizing pro- and anti-diabetogenic MHCII molecules on cortical thymic epithelial cells and medullary hematopoietic antigen-presenting cells (APCs), respectively. Here, we use a novel autoantigen discovery approach to define the antigenic specificity of this TCR in the context of I-Ag7. This was done by screening the ability of random epitope-GS linker-I- Aβg7 chain fusion pools to form agonistic peptide-MHCII complexes on the surface of I- Aαd chain-transgenic artificial APCs. Pool deconvolution, I-Ag7-binding register-fixing, TCR contact residue mapping, and alanine scanning mutagenesis resulted in the identification of a 4.1-TCR recognition motif XL(G/A)XEXE(D/E)X that was shared by seven agonistic hybrid insulin peptides (HIPs) resulting from the fusion of several different chromogranin A and/or insulin C fragments, including post-translationally modified variants. These data validate a novel, highly sensitive MHCII-restricted epitope discovery approach for orphan TCRs and suggest thymic selection of autoantigen-promiscuous TCRs as a mechanism for the murine T1D-I-Ag7-association.