Targeting Rare Populations of Murine Antigen-Specific T Lymphocytes by Retroviral Transduction for Potential Application in Gene Therapy for Autoimmune Disease 1 Gina L. Costa,* Jacqueline M. Benson,* Christine M. Seroogy,* Philip Achacoso, ² C. Garrison Fathman, 2 * and Garry P. Nolan ²‡ CD4  T cells are important mediators in the pathogenesis of autoimmunity and would therefore provide ideal candidates for lymphocyte-based gene therapy. However, the number of Ag-specific T cells in any single lesion of autoimmunity may be quite low. Successful gene transfer into autoantigen-specific CD4  T cells would serve as an ideal vehicle for site-targeted gene therapy if it were possible to transduce preferentially the small number of autoantigen-specific T cells. In this study we have demonstrated that retroviral infection of CD4  lymphocytes from either autoantigen-stimulated TCR transgenic mice, or Ag-activated immunized nontransgenic mice, with a retroviral vector (pGCIRES), resulted in the transduction of only the limited number of Ag-reactive CD4  T cells. In contrast, polyclonal activation of the same cultures resulted in transduction of non-antigen-specific lymphocytes. Transduction of Ag-reactive CD4  T cells with pGCIRES retrovirus encoding the regulatory genes IL-4 (IL4) and soluble TNF receptor (STNFR) resulted in stable integration and long-term expression of recombinant gene products. Moreover, expression of the pGCIRES marker protein, GFP, directly correlated with the expression of the upstream regulatory gene. Retroviral trans- duction of CD4  T cells targeted specifically Ag-reactive cells and was cell cycle-dependent and evident only during the mitosis phase. These studies suggest that retroviral transduction of autoantigen-specific murine CD4  T cells, using the pGCIRES retroviral vector, may provide a potential method to target and isolate the low frequency of autoantigen-specific murine CD4  T cells, and provides a rational approach to gene therapy in animal models of autoimmunity. The Journal of Immunology, 2000, 164: 3581–3590. A utoantigen-specific CD4 + T lymphocytes have been im- plicated in the pathogenesis of autoimmune diseases (1– 6). Tissue-specific homing properties of autoantigen- specific CD4 + T cells suggested that these cells might be ideal vehicles for delivery of retroviral-encoded regulatory proteins in a site-specific manner as a therapy for autoimmune diseases. In many autoimmune diseases, disease pathogenesis is mediated by Ag-specific CD4 + T cells that secrete Th1 pro-inflammatory cy- tokines (7, 8). Systemic administration of Th2 “regulatory” cyto- kines, which serve to counter the effects of the pro-inflammatory Th1 cytokines, has previously been shown to ameliorate autoim- mune diseases (9 –11). However, the inherent problems of non- specific toxicity limit the usefulness of systemic cytokine delivery as a potential therapy. Therefore, a rational approach to treating autoimmune diseases might include manipulation of autoantigen- specific T cells for the delivery of regulatory (Th2-type) cytokines to autoimmune lesions (12–17). Varied approaches to alter or abrogate inflammatory T cell re- sponses in autoimmune disease states have been explored. Among the most promising are those that exploit T cells for delivery of therapeutic proteins (18 –21). Early studies from our laboratory, using an animal model of the human disease multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), demonstrated that autoantigen-specific T cell hybridomas, transduced using ret- roviral vectors containing genes encoded for regulatory cytokines were capable of delivering cytokines to the autoimmune lesions in the CNS (22, 23). Disease could be ameliorated or exacerbated depending on the cytokine that was delivered. Transduced T cell hybridomas, which lacked the tissue-specific TCR for Ag but se- creted the same anti-inflammatory cytokines, were ineffective. Thus, tissue-specific homing and/or retention was required for therapeutic effect. More recent studies have used transduced au- toantigen-specific T cell clones to deliver immunosuppressive pro- teins to autoimmune lesions of EAE (24). These studies demon- strated that expression of retroviral-encoded regulatory cytokines did not alter the homing abilities of transduced T cell hybridomas or T cell lines, and, importantly, demonstrated that T cells, trans- duced by retrovirus for the expression of regulatory genes, had the potential to deliver these proteins in a site-specific manner. If non- transformed, primary T cells could be used to replace T cell hy- bridomas and T cell lines, then transduction of autoantigen-specific T cells would represent a viable therapeutic strategy for the treat- ment of autoimmune disease. Primary murine CD4 + T cells have been extremely difficult to infect with retroviruses, and are more often refractory to retroviral transduction. However, data presented in this paper demonstrate *Department of Medicine, Division of Immunology, ² Department of Molecular Phar- macology, and ‡ Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford CA 94305 Received for publication October 25, 1999. Accepted for publication January 27, 2000. 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 G.L.C. was supported by Molecular and Cellular Immunobiology National Institutes of Health Training Grant 5T32-AI-07290. This work was supported in part by Na- tional Institutes of Health Grants AI 39646 (C.G.F. and G.P.N.), AI 36535 (C.G.F.), and AI 35304 (G.P.N.), and National Institutes of Health Contract NO1-AR-6-2227 (C.G.F. and G.P.N.). 2 Address correspondence and reprint requests to Dr. C. Garrison Fathman, Depart- ment of Medicine, Division of Immunology and Rheumatology, Room S-021, Stan- ford University School of Medicine, Stanford, CA 94305-5111. E-mail addresses: cfathman@leland.stanford.edu or ginac@leland.stanford.edu Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00