|Ph.D Student||Fishman-Turjeman Sigal|
|Subject||Developing a New Genetic Approach for the Immunotherapy of|
Type 1 Diabetes
|Department||Department of Medicine||Supervisors||PROF. Nathan Karin|
|ASSOCIATE PROF. Gidi Gross|
|Full Thesis text|
Type 1 diabetes (T1D) is an autoimmune disease that results from destruction of insulin-producing b cells in pancreatic islets by the patient's T cells. Accumulating findings in the Non Obese Diabetic (NOD) mouse model for T1D, and recently in T1D patients, ascribe a critical role to CD8 T cells in the initial b cell insult, as well as in later stages of the disease. The selective immunotargeting of diabetogenic CD8 T by either CD8 cytotoxic T lymphocytes (CTLs) or CD4 regulatory T cells (Tregs) emerges as a promising avenue in the immunotherapy of T1D.
Chimeric MHC molecules supplemented with TCR signaling motifs function as activation receptors and can redirect gene-modified T cells against pathogenic CD8 T cells. In previous studies we have shown that β2m operates as a universal signaling component of MHC-I molecules when fused with the CD3-ζ chain. Linking the H-2Kd-binding insulin B chain peptide InsB15-23 to the N-terminus of β2m/CD3-ζ redirected CD8 T cells against pathogenic NOD CD8 T cells in a peptide-specific manner. CD8 T cells from mice transgenic for InsB15-23/β2m/CD3-ζ blocked diabetes in NOD.SCID mice and suppressed disease in NOD mice.
In this work I exploit this genetic platform for redirecting both CD8 T cells and CD4 Tregs against pathogenic NOD CD8 T cells in an antigen-specific manner using electroporation of in-vitro-transcribed mRNA.
In the first part of this work I show that the chimeric polypeptide products paired with endogenous heavy chains and transmitted strong activation signals upon MHC-I cross-linking. An InsB15-23-H-2Kd-specific CD8 T cell hybrid activated T cells transfected with InsB15-23/β2m/CD3-ζ mRNA only when these expressed H-2Kd. Purified NOD CD8 T cells expressing either InsB15-23/β2m/CD3-ζ or IGRP206-214/β2m/CD3-ζ killed their respective autoreactive CD8 T cell targets. In-vivo, mRNA-transfected NOD CD8 T cells reduced insulitis and protected NOD mice from diabetes at a statistically significant level.
In the second arm of this study I assessed both the induction of Tregs via the ectopic expression of FoxP3 mRNA in naïve CD4 T cells and natural Tregs (nTregs) expanded ex-vivo. Polyclonal CD4 T cells from BALB/c or NOD mice co-transfected with mRNA encoding FoxP3 and InsB15-23/β2m/CD3-ζ suppressed activation of the T cell hybrid and the pathogenic NOD CD8 T cells G9. This effect was more pronounced than that exerted through FoxP3 alone or when InsB15-23/β2m was fused with an inert anchor rather than CD3-ζ. Similar peptide-specific suppression was also observed with ex-vivo-expanded nTregs.
Collectively, my findings demonstrate that mRNA encoding chimeric MHC-I receptors can redirect both effector CD8 and CD4 Tregs against diabetogenic CD8 T cells, offering of a new approach for the treatment of type 1 diabetes.