Daptomycin 103060-53-3 had been incubated with the synthetic peptide

Inoculated via the intraperitoneal route Daptomycin 103060-53-3 with 500 l PBS containing 106 PFU. Following infections, mice were observed daily for signs of disease and imminent death. Antiviral treatment was administered systemically via the i.p. route with 500 l PBS containing 400 g of cidofovir. Flow cytometry. T cell responses were detected as described previously. Briefly, for T cell responses, lymphocytes were obtained from mice and were made into single cell suspensions. Following osmotic lysis of red blood cells with 0.84% NH4 Cl, cells were washed, 2 106 cells were added to the wells of 96 well plates, and supernatants of hybridoma 2.4G2 were added to block nonspecific binding of the labeled antibody to Fc receptors. The cells were stained for cell surface molecules and were then fixed with 0.5% paraformaldehyde in PBS. The following Abs and staining reagents were used: anti CD4, anti CD14, anti CD16, and anti CD19 conjugated with fluorescein isothiocyanate for a dump gate, phycoerythrin conjugated anti CD8 , and an H 2Kb Ig recombinant fusion protein, which had been incubated with the synthetic peptide TSYKFESV, ITYRFYLI, STLNFNNL, KSYNYMLL, or SIFRFLNI and used as recommended by the manufacturer. At least 100,000 cells were analyzed by flow cytometry using an LSR II system. Data analysis and statistics. Unless otherwise indicated, all data presented correspond to one experiment representative of at least two similar experiments with groups of four to five mice. Spleens were analyzed individually except for the experiment for which results are shown in Fig. 1, where data correspond to pooled spleens and four independent experiments. Statistical analysis was performed using GraphPad Prism software. All statistical analyses were performed using an unpaired two tailed t test. When applicable, data are displayed as means standard errors of the means with P values.
RESULTS IFN / and Prf/ mice mount adaptive immune responses with increased frequencies of anti VACV memory CD8 T cells. To examine the roles of IFN and Prf in anti VACV CD8 T cell responses, we infected B6, IFN /, and Prf/ mice with 106 PFU VACV. All the B6 and Prf/ mice and most IFN / mice survived the infection without overt symptoms of disease. In four experiments where we infected a total of 40 IFN / mice, 6 animals became very sick 7 to 10 days postinfection and had to be euthanized. None of the other 34 animals showed signs of disease. This is consistent with a previous report showing that IFN / mice control VACV following infection by tail scarification. Sixty days postinfection, the anti VACV CD8 T cell responses were analyzed. Compared with those in B6 mice, both Prf/ and IFN / mice had significantly higher frequencies of Kb restricted memory CD8 T cells specific for the dominant Kb restricted B8R peptide and four other subdominant Kb restricted peptides. However, the hierarchy raltegravir 871038-72-1 of immunodominance for the five determinants remained unchanged. The increased frequencies of memory CD8 T cells in IFN / and Prf/ mice were not due to virus persistence, because no virus was detectable in the spleen, liver, and ovaries 30 dpi, the memory cells were GzB negative, indicating that they were resting, and anti VACV memory CD8 T cells from VACV immune B6 mice did not expand when transferred to VACV.