Streamlined protocol for TCR sequencing, cloning, and in vivo functional analysis of islet antigen reactive human T cells (BA11P.131)

Abstract

Abstract Type 1 diabetes (T1D) is an autoimmune disease characterized by the selective destruction of insulin secreting beta cells present in the islets of Langerhans. A large body of evidence has implicated T cells as the predominant mediators of beta cell destruction leading to T1D development. The goal of our research is to develop a system for isolation and characterization of diabetogenic CD4+ T cells from the peripheral blood of early onset T1D patients. We were able to obtain a blood sample from a treatment-naïve T1D patient at the time of diagnosis. Through a novel multiplex-nest PCR protocol, we cloned corresponding alpha and beta TCR chains from individual islet antigen-specific T cells and tested them in vitro for antigenic specificity and HLA restriction. Utilizing a humanized TCR/HLA retrogenic mouse system, we are testing human islet antigen reactive TCRs for their in vivo functionality and diabetogenic potential.