The Position Beta57 of IAg7 Controls the Early Anti-Insulin Response and Onset of Diabetes in NOD mice to Link MHC and Disease [RT-PCR]

Abstract- The class II region of the Major Histocompatibility locus is the main contributor to the genetic susceptibility to type 1 diabetes (T1D). The loss of an aspartic acid at position 57 of diabetogenic HLA-DQ8 chains supports this association; it influences the recognition of peptides in the context of HLA-DQ8, and I-Ag7 using a mechanism termed the P9 switch. Here, we built register-specific insulin MHC tetramers, Ins12-20 and Ins13-21, to examine anti-insulin CD4 T cell responses during the early pre-diabetic phase of disease in mice. A single cell gene expression analysis and single cell T cell receptor (TCR) sequencing of anti-insulin CD4 T cells performed in 6 and 12-week-old NOD mice, revealed tissue-specific gene expression signatures. TCR signaling and clonal expansion were found only in the islets of Langerhans. and produced either classical Th1 differentiation or an unusual Treg phenotype, independent of TCR usage. The early phase of the anti-insulin response was dominated by cells specific for Ins12-20, the register that supports a P9 switch mode of recognition. The presence of the switch was demonstrated by TCR sequencing, re-expression, mutagenesis, and functional testing of alpha/beta TCR pairs in vitro. The genetic correction of the beta57 mutation resulted in the disappearance of D/E residues in the CDR3beta of anti-Ins12-20 T cells, and inability of cells normally activated by a switch to recognize the Ins9-23 peptide. These results provide the first molecular mechanistic explanation that links the unique MHC class II polymorphism of T1D with the recognition of islet antigens and disease onset. Murine CD4+ T cells, isolated from different tissues, were stained with either IAg7-1220 or IAg7-1321; both tetramers were labelled with the fluorscent moiety PE. Cells stained with either tetramer were single cell sorted into single wells of 96 well plates. Single cell cDNA libraries were made according to the Fluidigm BioMark "Two Step Single Cell Gene Expression Using EvaGreen" protocol. Then gene expression of 96 genes was assessed utilizing the Fluidigm Dynamic Array 96x96 IFC and the "GE Fast 96x96 PCR+Melt v2.pcl" cycling conditions. Data was pre-processed via the SINGuLAR Analysis Toolset and then futhered analyzed using custom R scripts that incorporated tSNE, clustering via k.means and differential gene expression analysis. The protocol can be found using PMID 29224077 (Holt et al).