Engineered Dendritic Cell-Directed Concurrent Activation of Multiple T cell Inhibitory Pathways Induces Robust Immune Tolerance

Abstract

Inhibitory/repressor-receptors are upregulated significantly on activated T cells, and have been the molecules of attention as targets for inducing immune tolerance. Induction of effective antigen specific tolerance depends on concurrent engagement of the TCR and one or more of these inhibitory receptors. Here, we show, for the first time that dendritic cells (DCs) can be efficiently engineered to express multiple T cell inhibitory ligands, and enhanced engagement of T cell inhibitory receptors, upon antigen presentation, by these DCs can induce effective CD4+ T cell tolerance and suppress autoimmunity. Compared to control DCs, antigen presentation by DCs that ectopically express CTLA4, PD1 and BTLA selective ligands (B7.1wa, PD-L1, and HVEM-CRD1 respectively) individually (mono-ligand DCs) or in combination (multi-ligand DCs) causes an inhibition of CD4+ T cell proliferation and pro-inflammatory cytokine response, as well as increase in Foxp3+ Treg frequency and immune regulatory cytokine production.Administration of self-antigen (mouse thyroglobulin; mTg) loaded multi-ligand DCs caused hyporesponsiveness to mTg challenge, suppression of autoantibody production, and amelioration of experimental autoimmune thyroiditis. Overall, this study shows that engineered DC-directed enhanced concurrent activation of multiple T cell coinhibitory pathways is an effective way to induce self-antigen specific T cell tolerance to suppress ongoing autoimmunity.

Gastrin producing syngeneic mesenchymal stem cells protect non-obese diabetic mice from type 1 diabetes

“…The third-generation replication-incompetent lentiviral cDNA cloning vector (pCDH1-copGFP) and packaging plasmid were purchased from SBI and used as described in our previous report [47]. For cloning Gastrin cDNA, pCDH1-copGFP vector was digested using NheI (downstream of CMV promoter) and NotI restriction enzymes, and the restriction digestion fragment was replaced with PCR amplified and NheI and NotI digested mouse Gastrin cDNA.…”

“…Immunological assays were carried out as described in our previous studies [44,47]. LPS exposed bone marrow derived dendritic cells (BM DCs) were cultured in the presence of control-and Gastrin-MSCs for 24h and examined for surface expression levels of activation markers such as CD80, CD86, CD40 and MHC II by FACS.…”