Two-Domain MHC Class II Molecules Form Stable Complexes with Myelin Basic Protein 69 – 89 Peptide That Detect and Inhibit Rat Encephalitogenic T Cells and Treat Experimental Autoimmune Encephalomyelitis Gregory G. Burrows, 1 * ²‡ Bruce F. Bebo, Jr.,* ² Kirsten L. Adlard,* Arthur A. Vandenbark,* ²§ and Halina Offner* ² We designed and expressed in bacteria a single-chain two-domain MHC class II molecule capable of binding and forming stable complexes with antigenic peptide. The prototype “ 1  1 ” molecule included the  1 domain of the rat RT1.B class II molecule covalently linked to the amino terminus of the  1 domain. In association with the encephalitogenic myelin basic protein (MBP) 69 – 89 peptide recognized by Lewis rat T cells, the  1  1 /MBP-69 – 89 complex specifically labeled and inhibited activation of MBP-69 – 89 reactive T cells in an IL-2-reversible manner. Moreover, this complex both suppressed and treated clinical signs of experimental autoimmune encephalomyelitis and inhibited delayed-type hypersensitivity reactions and lymphocyte proliferation in an Ag-specific manner. These data indicate that the  1  1 /MBP-69 – 89 complex functions as a simplified natural TCR ligand with potent inhibitory activity that does not require additional signaling from the  2 and  2 domains. This new class of small soluble polypeptide may provide a template for designing human homologues useful in detecting and regulating potentially autopathogenic T cells. The Journal of Immunology, 1998, 161: 5987–5996. A ntigen-specific CD4 + T cells appear to be a central com- ponent in the pathogenesis of a variety of human dis- eases including multiple sclerosis (MS) 2 , rheumatoid ar- thritis, sarcoidosis, autoimmune uveitis, transplant rejection, and graft-vs-host disease (1–5). Such T cells home to the target tissue where autoantigen is present, and, after local activation, selectively produce Th1 lymphokines (6). This cascade of events leads to the recruitment and activation of lymphocytes and monocytes that ul- timately destroy the target tissue (7). Ag-driven activation of CD4 + T cells is a multistep process initiated by coligation of the TCR and CD4 by the MHC class II/peptide complex present on APC (signal 1), as well as costimu- lation through additional T cell surface molecules such as CD28 or OX40 (signal 2). The classic experiment by Quill and Schwartz (8) demonstrated that stimulation through the TCR by cell-associated MHC class II/peptide in the absence of costimulation, rather than being a neutral event, induced a state of unresponsiveness to sub- sequent optimal Ag presentation, a phenomenon termed anergy (8, 9). Thus, a direct approach toward Ag-driven immunosuppression is to present the complete ligand, Ag plus MHC, in the absence of costimulatory signals that are normally provided by specialized APCs. MHC class II molecules are heterodimeric membrane-bound glycoproteins made up of noncovalently associated - and -polypeptide subunits. Each subunit consists of a short cytoplas- mic tail, a single membrane-spanning sequence, and two extracel- lular domains. X-ray crystallographic studies have demonstrated that peptides from processed Ag bind to MHC molecules in the membrane distal pocket formed by the  1 and  1 domains (10, 11). Moreover, the  2 domain contains a CD4-binding site that coli- gates CD4 when the  1  1 domains with associated antigenic pep- tide interact with the TCR - and -chains (12–15), whereas the  2 domain appears to contribute to ordered oligomerization in T cell activation (16). Complexes of MHC/Ag have been purified as de- tergent extracts of lymphocyte membranes (17) and as associated recombinant proteins using baculovirus and bacterial expression systems (18 –22). These two-chain, four-domain molecular com- plexes, after loading with selected peptide epitopes, have been demonstrated to interact with T cells in an Ag-specific manner (20, 23–27). To develop a simple and effective agent that could bind selec- tively to the TCR, we have designed molecules consisting of the  1 and  1 domains of the rat RT1.B MHC class II molecule geneti- cally linked into a single polypeptide chain. Molecules with this design would be useful for studying binding specificity in vitro for exploring primary TCR signaling events independent of costimu- latory input associated with the MHC II  2  2 domains or with other molecules expressed by APCs and for treating CD4 + T cell- mediated autoimmune disease in an MHC II/epitope-specific man- ner. Experimental autoimmune encephalomyelitis (EAE) is a para- lytic, inflammatory, and sometimes demyelinating disease medi- ated by CD4 + T cells specific for central nervous system (CNS) myelin components including myelin basic protein (MBP). EAE shares a number of immunologic abnormalities with the human *Neuroimmunology Research, Veterans Affairs Medical Center, Portland, OR 97201; and ² Department of Neurology, ‡ Department of Biochemistry and Molecular Biology, and § Department of Molecular Microbiology and Immunology, Oregon Health Sci- ences University, Portland, OR 97201 Received for publication March 16, 1998. Accepted for publication August 6, 1998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Correspondence should be addressed to Dr. Gregory G. Burrows, Neuroimmunol- ogy Research R&D-31, Veterans Affairs Medical Center, Portland, OR 97201; E-mail address: ggb@ohsu.edu. 2 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental auto- immune encephalomylitis; CNS, central nervous system; MBP, myelin basic protein; PE, phycoerythrin;; DTH, delayed-type hypersensitivity; PPD, purified protein deriv- ative; MFI, mean fluorescence intensity; LN, lymph node. Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00