To introduce a mutation in the mouse XLP locus, we engineered a construct based on a splice-site mutation that truncated at the beginning of the third exon, fusing GFP in frame to follow expression of the mutant protein
To introduce a mutation in the mouse XLP locus, we engineered a construct based on a splice-site mutation that truncated at the beginning of the third exon, fusing GFP in frame to follow expression of the mutant protein. activation and production of IFN- irrespective of their T helper phenotype (19). Similarly, ligation of SLAM on murine Th1 clones results in increased IFN- production (20). However, the contribution of cytokine regulation to the pathogenesis of XLP remains unclear. Because XLP is rare, there are few preinfection studies and their findings are quite variable. Nonetheless, the existence of phenotypes before apparent EBV infection raises the possibility of a more basic immune dysfunction, independent of viral infection (21C23). To develop a model that may present insights into the pathogenesis of this disease, we mutated the XLP locus in mice. SAP-mutant mice were viable, fertile, and transmitted the gene in Mendelian fashion. However, challenge with the infectious agents and lymphocyte choriomeningitis virus (LCMV) revealed abnormal immune responses and evidence of T cell hyperactivation with increased IFN- production, decreased B cell function, and, in a model of chronic infection, increased morbidity and mortality. Furthermore, splenocytes cultured from uninfected mice also exhibited a bias toward production of Th1 cytokines. The initial characterization of these mice reveals a phenotype with characteristics of XLP and suggests that cytokine misregulation may contribute to the phenotype of this genetic disease. Materials and Methods Antibodies. Anti-SAP C-terminal serum was raised against peptide GRGPQAPTGRRDSDI in rabbit. Anti-SAP N-terminal and anti-goat Ig HRP were obtained from Santa Cruz Biotechnology; anti-mouse CD3?, CD4, CD8, TCR, CD19, IFN-, Fc Block, and DX5 were obtained from Becton Dickinson/PharMingen; anti-green fluorescent protein (GFP), anti-mouse Ig HRP, and anti-rabbit Ig horseradish peroxidase (HRP) were obtained from Roche Molecular Biochemicals; anti-mouse Ig isotype HRP was obtained from Zymed; anti-IL-4 was obtained from BioSource International (Camarillo, CA); anti-tubulin was obtained from ICN; anti-CD28 was a gift from J. Powell and R. Schwartz (National Institute of JD-5037 Allergy and Infectious Disease); and anti-SLAM was a gift from A. O’Garra (DNAX). Expression Plasmids. pSX-Lck and pCEFL were gifts from J. S. Gutkind (National Institute of Dental Research, Bethesda); pEBG-SLAM and pEBG-CD84 are described in ref. 9. Full-length was JD-5037 generated by PCR from a mouse embryo day-15.5 Gene-Trapper library (using a 3 primer that mutated the stop codon to encode a Thr), sequenced, and subcloned into pEGFP-N1 (CLONTECH). Truncated SAP-GFP was generated by PCR (incorporating the T68I mutation in the 3 primer), sequenced, and ligated into pCEFL followed by a GFP variant (24). Generation of Mutant Mice. The targeting construct was derived from a 129Sv/Ev Lambda JD-5037 phage library (Stratagene) screened with EST Image Clone 719479 (Research Genetics, Huntsville, AL). The 5 arm of the construct was generated by PCR using a 3 primer to incorporate the T68I mutation (atgtactCtagatgctatctggaa). Genomic DNA arms and a Rabbit Polyclonal to TTF2 variant of GFP (24) were inserted sequentially into pPNT (25), and introduced into TC1 ES cells. Homologous recombinants were screened by Southern blots of genomic DNA digested with and GFP. Glutathione Polarization. Purified CD4+ or sorted naive CD4+62L+ cells from wild-type (WT) and SAP? mice were stimulated with anti-CD3 and anti-CD28 plus irradiated T-depleted WT or IL-4 JD-5037 deficient splenocytes under Th1 (IL-12 plus anti-IL-4), Th2 (IL-4 plus anti-IL-12), or null (anti-IL-4, anti-IL-12, plus anti-IFN-) conditions (29). Cells were restimulated with anti-CD3 and CD28 and cell culture supernatants were analyzed at 48 h for IL-4 and 72 h for IFN-. Infections. Mice were injected i.p. with 20 cysts (ME49 strain) and serum, spleens, and brains were harvested at 31 days postinfection (pi) for analysis (28). LCMV Infections. Mice (4C5 months old) were infected with LCMV Armstrong strain [2 105 plaque-forming units (pfu) i.p.] or Clone 13 (2C5 106 pfu i.v.) and killed for analyses at 8, 30, or 37 days pi (26). Cytotoxic activity of splenocytes was tested in.