Project Summary Multiple sclerosis (MS) is an autoimmune disease of the central nervous system and remains a major cause of disability in both young and older populations. Experimental autoimmune encephalomyelitis (EAE) is the most commonly used animal model for MS, and is induced by immunization with disease-causative self-antigens such as myelin oligodendrocyte glycoprotein (MOG). It is generally believed that MS is mediated by immune responses, especially T cells, against myelin antigens, followed by neurological impairment. T cells are the major component of the immune system, and are regulated by stimulatory and inhibitory molecules. Among the T cell regulators, the B7 family members are of central importance. In the past several years, several new drugs that target the B7 family members including PD-L1/PD-1 and CTLA-4 have been approved by the FDA for the treatment of autoimmune disease and cancer. Butyrophilin (BTN) and BTN-like (BTNL) molecules share sequence, structural, and functional similarity with the B7 family, thereby being considered as extended B7 family members. BTNL2 is a member of the BTNL family. It has been reported that BTNL2 mutations are associated with inflammatory autoimmune diseases and that BTNL2 protein can inhibit T cell functions in vitro. The BTNL2 receptor is expressed on activated T cells, and seems to be distinct from the receptors for other B7 family members including CD28, CTLA4, ICOS, BTLA and PD-1. We have shown that recombinant murine BTNL2-IgG2 Fc (mBTNL2-Ig) fusion protein can inhibit the proliferation, activation and cytokine production of autoreactive T cells from NOD mice in vitro. We were the first to show that in vivo administration of mBTNL2-Ig attenuates type 1 diabetes and graft-versus-host disease in mice, which was associated with its ability to inhibit the proliferation, activation and cytokine production of effector T cells, but increase the generation of regulatory T cells. Previous studies of BTNL2 by us and other groups used a mouse form of protein. However, whether human BTNL2 (hBTNL2) has similar functions has not been determined. In this proposal, we will first produce hBTNL2-Ig protein, and determine its ability to inhibit both mouse and human T cell proliferation and activation in vitro. Since MS is a T cell-mediated autoimmune disease and mBTNL2-Ig can inhibit T cell functions, we hypothesize that administration of either mBTNL2-Ig or hBTNL-2 protein can ameliorate EAE in mice. This hypothesis will be tested in Aim1. Although the BTNL2 receptor is distinct from the receptors for existing B7 family members, the nature of the receptor remains unknown. In Aim 2, we plan to identify the receptor by new or traditional approaches. These exploratory studies, if successful, have the potential to lead to a new approach to prevent and treat MS. The identification of the BTNL2 receptor will allow for better understanding of how BTNL2 inhibits T cell functions, and facilitate the therapeutic dev...