Category: Diabetes and other autoimmune endocrine diseases
CD8+ effector T cells largely contribute in the destruction of human pancreatic beta cells suggesting that MHC class I is abundant in the inflamed islets and can be employed to redirect regulatory T cells (Tregs) specificities to islets.
We developed novel methods allowing: (1) efficient Tregs expansion in which the endogenous TCR is removed using a non-viral CRISPR-based approach combined with lentiviral transduction for the insertion of an engineered TCR, (2) non-viral substitution of the CD4 co-receptor with CD8α chain, (3) human islets transplantation into the spleen of NSG mice to increase the interaction with transferred human T cells.
Nine day after electroporation, mean efficiency of TCR knockout (KO) in Tregs was 74% for TRAC (n=3) and 79% for TRBC (n=5), which significantly impaired TCR-stimulated suppression of Tregs in vitro. An HLA-A2 restricted TCR specific for preproinsulin (PPI15-24) was cloned into lentivirus and transduced in TCRKO T cells restoring up to 100% TCR expression. Importantly PPI15-24 tetramer staining was detected only on CD8 T cells demonstrating that antigen engagement by this TCR is CD8 co-receptor dependent. Using a non-viral approach, we inserted the CD8α chain into the CD4 locus with up to 25% efficiency. Finally, we transplanted 4000 human islets equivalent into spleens of NSG mice which reverted streptozotocin-induced diabetes in 6/9 mice up to 100 days.
Altogether, we generated the tools to engineer Tregs with HLA-class I restricted specificity. Preclinical testing of Tregs suppression in humanized mouse model for type 1 diabetes is currently under investigation
Yannick Mueller– UCSF
Roxxana Valencia– UCSF
Shen Dong– UCSF
Theo Roth– UCSF
David Nguyen– UCSF
Emilie Ronin– UCSF
Alexander Marson– University of California San Francisco
Jeffrey Bluestone– Professor, UCSF
Qizhi Tang– Professor, University of California San Francisco