NATURE M EDICINE • VO LUM E 4 • N UM BER 1 1 • N O VEM BER 1 9 9 8 1253 ARTICLES The CD40 molecule has a central role in the induction and reg- ulation of immune responses. It is expressed by B cells, hematopoietic progenitors, endothelial cells, professional anti- gen-presenting cells and epithelial cells (including those in the thymus) 1,2 , as well as a subset of mature T cells 3 . Interaction of this molecule with the CD40 ligand (CD40L, or CD154) usually leads to cell activation, differentiation or both 2,4–6 . In contrast to its receptor, CD40L is expressed mostly by activated T cells 7 . Its production is exquisitely regulated, occurring after T-cell ac- tivation and in concert with other receptor–counter-receptor pairs while the T cells and their targets are in intimate contact, usually within a specialized microenvironment 8 . Inherited defi- ciency of the CD40L molecule, the so-called X-linked hyper- IgM syndrome, is associated with severe immune impairment, characterized by failure of immunoglobulin isotype switching and defects of cell-mediated immunity 9,10 . Because expression of CD40L is normally closely synchro- nized with T-cell activation, fewer than 1% of circulating mononuclear cells are estimated to be CD40L+ at any given time (M.E. Conley, unpublished observations). Several clinical studies with other transgenes have shown that equivalent lev- els of expression can be attained in vivo after transduction of human hematopoietic progenitor cells, indicating that CD40L deficiency may be amenable to correction by gene transfer, even with vectors available now 11,12 . We therefore studied the therapeutic effect of CD40L transgene expression in a CD40L –/– murine model, whose features closely resemble those of the human disease 13 . Because the CD40L gene is normally tightly regulated, we also examined the consequences of constitutive expression of the transgene on the growth and development of the immune system 14–21 . Our results show that low-level expression of CD40L will cor- rect the major immunodeficiencies in CD40L –/– mice. However, with extended follow-up, approximately two-thirds of the mice developed T-cell lymphoproliferations, most of which gener- ated overt lymphoblastic lymphomas. Thus, our findings raise the possibility that use of gene transfer to treat monogenic dis- orders may have adverse long-term consequences, even when the transgene is expressed at apparently normal levels. Transfer of the murine CD40L gene Bone marrow from female CD40L –/– donor mice was trans- duced with the mCD40L gene and injected intravenously into male CD40L –/– recipient mice that had been ‘lethally irradi- ated’ (that is, given a dose of irradiation sufficient to ablate the host hematopoieitic system). The same gene was also trans- ferred to unirradiated CD40L –/– mice by transducing thymic cells from the female bone marrow donors with the mCD40L retroviral producer line in a transwell co-culture system. Control mice were injected with transduced bone marrow under identical conditions, except that the neomycin phos- photransferase gene was substituted for the mCD40L gene, whereas transduced thymic cells were injected subcutaneously into the axillae of male CD40L –/– mice. Expression of the trans- Thymic lymphoproliferative disease after successful correction of CD40 ligand deficiency by gene transfer in mice MICHAEL P. BRO W N 1 , DAVID J. TOPHAM 2 , MARK Y. SANGSTER 2 , JINGFENG ZH AO 1 , KIRSTEN J. FLYN N 2 , SH ERRI L. SURMAN 2 , DAVID L. W OODLAND 2 , PETER C. DOHERTY 2 , ANDREW G. FARR 3 , PAUL K. PATTENGALE 4 & MALC O LM K. BREN N ER 5 1 Cell and Gene Therapy Program and 2 Department of Immunology, St Jude Children’s Research Hospital, 332 N. Lauderdale St, Memphis, Tennessee 38105, USA 3 Departments of Biological Structure and Immunology, University of W ashington, Seattle, W ashington 98195, USA 4 Department of Pathology, Children’s Hospital Los Angeles, Los Angeles, California 90027, USA 5 Center for Cell and Gene Therapy, Baylor College of Medicine, 6621 Fannin St, MC3-3320, Houston, Texas 77030, USA Correspondence should be addressed to M.K.B. Inherited deficiency of the CD40 ligand (X-linked hyper-IgM syndrome) is characterized by fail- ure of immunoglobulin isotype switching and severe defects of cell-mediated immunity. To test the potential for gene transfer therapy to correct this disorder, we transduced murine bone mar- row or thymic cells with a retroviral vector containing the cDNA for the murine CD40 ligand (CD40L) and injected them into CD40L –/ – mice. Even low-level, constitutive expression of the transgene stimulated humoral and cellular immune functions in these mice. With extended fol- low-up, however, 12 of 19 treated mice developed T-lymphoproliferative disorders, ranging from polyclonal increases of lymphoblasts to overt monoclonal T-lymphoblastic lymphomas that involved multiple organs. Our findings show that constitutive (rather than tightly regulated), low-level expression of CD40L can produce abnormal proliferative responses in developing T lymphocytes, apparently through aberrant interaction between CD40L + and TCRαβ + CD40 + thy- mocytes. Current methods of gene therapy may prove inappropriate for disorders involving highly regulated genes in essential positions in proliferative cascades. 1998 Nature America Inc. • http://medicine.nature.com 1998 Nature America Inc. • http://medicine.nature.com