Eradication of surface MHC class Ia expression in an insulinoma cell clone by TALEN-mediated gene deletion prevents recognition by diabetogenic CD8+ T cells (THER5P.843)

Abstract

Abstract In type 1 diabetes, islet transplantation to replace β cells killed by pathogenic T cells is a promising means of attaining insulin independence; however, graft durability is limited by auto- & alloreactive responses. While autologous stem cells ultimately may circumvent the latter obstacle, the inciting CD8+ T-cell effectors are a threat to long-term engraftment. Islet cell manipulation to prevent expression of class Ia molecules could render them “invisible” to diabetogenic CTL, & we hypothesized that transcription activator-like effector nucleases (TALENs) could be used to engineer this modification. TALENs designed to flank a Bgl1 site in exon 1 of H2-Kd/Db were transfected into the NOD insulinoma line NIT-1, & cells with high nuclease activity were sorted using a GFP reporter plasmid encoding the target sequence. A clone isolated by limiting dilution (KG) was negative for H2-Kd/Db surface expression, & PCR amplicons were resistant to Bgl1 digestion. Genomic DNA sequencing showed biallelic modifications of both genes, with one Db sequence containing a 4 bp insertion. The remaining 3 alleles appeared to be repaired by gene conversion, causing frameshift mutations. When pulsed with cognate peptide, KG failed to stimulate diabetogenic CD8+ T cells purified from TCR-transgenic NOD.8.3 mice, as measured by CFSE dilution & IFN-γ production. These data demonstrate that TALEN-mediated genomic modification of insulin-producing cells can prevent recognition by autoreactive CTL.