(19, 27), in vivo treatment with IL-12 and/or CTLA4-Ig was performed at this time period. to determine how costimulation affects infection-induced pathology in IL-10 KO mice. Recent studies from this laboratory exhibited that in the absence of IL-10, inhibition of the CD40/CD40L or CD28/B7 pathways does not impact survival; however, simultaneous blockade of these pathways resulted in decreased production of IFN- and survival of IL-10 KO mice (63). These findings suggested that this CD28/B7 and CD40/CD40L interactions are parallel pathways that contribute to the CD4+-T-cell-mediated pathology in IL-10 KO mice. In addition, these studies raised fundamental questions about whether B7.1 or B7.2 played a preferential role in the regulation of T-cell activation in this model and whether the CD40/CD40L conversation directly regulates IL-12 production or T-cell activation. To address these questions, a series of studies were performed in which infected IL-10 KO mice were treated with numerous combinations of antibodies to antagonize IL-12, as well as the CD28/B7 and CD40/CD40L interactions, and the effects on Etodolac (AY-24236) survival, cytokine production, and pathology were monitored. Together with the findings of our previous studies, these data reveal a complex hierarchy between the costimulatory pathways and show that the CD28/B7 interaction has a unique contribution to the development of a pathological T-cell response, which is usually unique from your role of the CD40/CD40L and IL-12 pathways. MATERIALS AND METHODS Mice. Female CBA/CaJ and Swiss Webster mice were obtained from The Jackson Laboratories (Bar Habor, Maine). IL-10 KO mice, originally provided by DNAX (38), were generated by backcrossing C57BL/6-129/OLA IL-10 KO mice onto the C57BL/6 background for seven generations. IL-10 KO mice were genotyped by PCR (protocol was provided by D. Rennick, DNAX) and bred and managed in Thoren Unit cages in the Gene Therapy Animal Facility at the University or college of Pennsylvania. Experiments were performed with 4- to 8-week-old female IL-10 KO mice. Parasites. The Me49 strain of was managed in infected Swiss Webster and Etodolac (AY-24236) CBA/CaJ mice. Me49 cysts were prepared from brains of donor mice as previously explained (6, 7). Mice were infected with 20 cysts by intraperitoneal (i.p.) injection in a volume of 200 l. Reagents. Complete RPMI 1640 (Life Technologies, Gaithersburg, Md.) medium was supplemented with 10% heat-inactivated fetal calf serum, 1% sodium pyruvate, 1% nonessential amino acids, 0.1% -mercaptoethanol, 100 U of penicillin/ml, and 100 g of streptomycin/ml. HuCTLA4-Ig, a fusion protein comprised of the human CTLA4 extracellular domain name and the Fc portion of human immunoglobulin G (IgG), was supplied by Bristol Myers Squibb Research Institute (Princeton, N.J.). IL-12 was purified from ascites by ammonium sulfate precipitation, and CD40L (MR1) was obtained from TSD Bioscience (Newark, Del.). B7.1 and B7.2 were provided by the Etodolac (AY-24236) Genetics Institute (Andover, Mass.). Human chimeric L6 (ChiL6; Bristol Myers Squibb), Etodolac (AY-24236) rat IgG, and hamIgG (Sigma, St. Louis, Mo.) were used as control antibodies. IL-12 and CD40L (or the control antibodies ratIgG and hamIgG, respectively) were used at a concentration of 200 g Etodolac (AY-24236) per treatment per mouse, and B7.1, B7.2, and CTLA4-Ig (or the control antibodies ratIgG and ChiL6, respectively) were used at a concentration of 300 g per treatment per mouse. IL-10 KO mice that had been infected with Me49 were given the blocking antibodies or the control antibodies at days 5 and 7 postinfection (p.i.). Sera from these mice were collected at day 8 p.i. and analyzed for cytokine production by enzyme-linked immunosorbent assay. For antigen-specific recall responses, antibodies were used at a concentration of 20 g/ml. Cytokine assays. IL-12 p40 levels were measured by using monoclonal antibody C17.8 as a capture antibody and biotinylated C15.6 as a detecting antibody (hybridomas were provided by Giorgio Trinchieri, Wistar Institute, Philadelphia, Pa.). IFN- levels were measured by using Rabbit Polyclonal to SERPINB12 R46A2 (capture antibody) and biotinylated AN18 (detecting antibody). In vitro recall response. Spleen cells from infected mice were harvested at day 8 p.i. and dissociated into a single-cell suspension. Erythrocytes were depleted by using 0.83% ammonium chloride (10 min, 4C), cells were washed twice in complete RPMI medium and resuspended at a final concentration of 4 106/ml. A total of 4 105 cells were plated per well in a final volume of 200 l in 96-well plates (Costar, Cambridge, Mass.) and were stimulated with soluble lysate antigen (TLA). TLA was prepared from RH strain tachyzoites as previously explained (52), titrated to determine the.