PROJECT SUMMARY An immense need for selective and antigen-specific immunotherapy without global immunosuppression exists for autoimmune diseases such as multiple sclerosis (MS) and a closely related condition known as myelin oligodendrocyte glycoprotein (MOG) antibody disease (MOGAD). This has prompted exploration of chimeric antigen receptor (CAR) T cell utilization to specifically eliminate autoreactive cells. We have created a unique version of CAR T cells in which peptide MHCII (pMHCII) was fused with signaling domains in order to recognize specific T cell receptors (TCRs). In preliminary studies we demonstrate that these pMHCII-CAR T cells specifically recognize a cognate TCR in vitro and can selectively kill antigen-specific CD4 T cells in vivo. Efficient depletion of high affinity MOG-specific CD4 T cells was associated with prevention of MOG-induced experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Modifications in pMHCII-CAR construction led to greater efficiency in eliminating lower-affinity MOG-reactive T cells which was associated with resolution of ongoing EAE. These data suggest an “activation energy” model of autoimmunity analogous to that of a chemical reaction, in which higher affinity self-reactive T cells are needed to provide the activation energy to initiate autoimmunity, but lower affinity T cells are capable of sustaining the autoimmune “reaction.” To address the hypothesis that CAR T cell technology can be used to eliminate auto-antigen-specific T cells and abrogate central nervous system (CNS) autoimmunity in mice and humans without global immunosuppression, we have formulated three specific aims. In Aim 1 we will improve the efficiency of low affinity T cell deletion in vivo. In Aim 2, we will test whether we can successfully target autoreactive CD4 T cells specific to all T cell epitopes of a protein in mice, as we predict that such an approach would be useful for treatment of human disease where the T cell autoantigen is identified by an autoantibody. Finally, in Aim 3, we will directly test whether MOGAD patients show an increased frequency of MOG-specific T cells using pMHCII-CARs for antigen discovery. In sum, our proposed studies will explore the “activation energy” model of autoimmunity and establish an optimal CAR T cell approach to eliminate low affinity autoreactive TCR specificities for the treatment of ongoing autoimmune disease. Finally, we will begin to translate these murine observations to human pMHCII-CAR T cells and assess their potential utility in a relevant human autoimmune CNS disease.