Chimeric T regulatory cells for type 1 diabetes treatment
Sanford Research, University of South DakotaDepartment of Medicine, Uniformed Services University
摘要：Type 1 Diabetes（ T1 D） typically presents in childhood with profound insulin deficiency due to the autoimmune destruction of β cells in pancreatic islets of Langerhans. In susceptible individuals, defective immune tolerance permits development of autoreactive T effector lymphocytes, which eliminate β cells directly. Thus, restoration of immune tolerance is the major prerequisite for any successful T1 D therapy. A major mechanism governing maintenance of immune homeostasis and peripheral tolerance is the suppression of autoreactive T effectors by regulatory T cells, Tregs. Impaired Tregs（both numerically and functionally） are widely believed to underlie the initiation and progression of T1 D auto-immunit y. Addressing this problem, we attempt to develop a novel Treg-based therapy for T1 D. Here we report engineering and in vitro testing of Islet-Specific Chimeric Antigen Receptor-bearing Tregs（ISCAR Tregs）. We designed the recognition part of IS-CAR consisting of a single-chain fragment（scFv） from an antibody specific for the human pancreatic endocrine marker HPi2, which we confirmed to recognize human, but not mouse β cells by FACS and immunofluorescence. This scFv was fused to a flexible linker, a trans-membrane domain, CD28 co-stimulatory, and CD3ζ activation domains. The resulted construct was cloned into retroviral vector pRetroX-IRES-ZsGreen1, which was used for transfection of different subpopulations of human T cells（total CD3+, CD4+, CD8+, and CD4+, CD25+, GITR+ Tregs） pre-sorted from the peripheral blood of healthy human donors. Our characterization of expanded IS-CAR-bearing T cells show that they are readily activated and tend to proliferate in co-cultures with human（βLox5） but not mouse（NIT-1） islet cells, thus, confirming the specificity of our IS-CAR. Additionally, we show that the number of Granzyme B+ T cells increase after co-culturing of IS-CAR-transduced total CD3+ cells with βLox5. Furthermore, we tested the ability of our IS-CAR to activate a functional response in IS-CAR+CD8+ T cells in a degranulation assay observing an increase of LAMP-1 levels after co-culturing with human islet cells. In sorted CD4+CD25+ Tregs we observed an increase of Foxp3, IL-10 and TGFβ levels in IS-CAR+ when compared to Mock（empty vector） transduced or IS-CAR-cells. Overall, our results demonstrate specificity of our IS-CAR construct toward islet cells, and provide in vitro evidence of sustained functionality of human IS-CAR bearing Tregs.
Grand Challenges in Immunology: Immunotherapy for Cancer and Beyond——The 2nd CMI-NI Joint Conference