Fulvestrant ICI 182780 was partly dependent on increased virus loads

Sed the role of IFN or Prf in immunodominance. Here we examined Fulvestrant ICI 182780 the roles of IFN and Prf in shaping the anti VACV CD8 T cell response. We found that IFN deficiency resulted in an increased number of anti VACV effector and memory CD8 T cells, which was partly dependent on increased virus loads during the acute phase of the infection. On the other hand, Prf deficiency resulted in an increased number of VACV specific memory CD8 T cells only during the memory phase. Remarkably, neither Prf deficiency nor IFN deficiency had any effect on the immunodominance hierarchy of B8R and four of the most prominent Kb restricted subdominant CD8 T cell determinants either during acute infection or after recovery. MATERIALS AND METHODS Viruses. Initial stocks of VACV Western Reserve were obtained from Bernard Moss and were amplified in HeLa S3 cells as described previously. Briefly, HeLa S3 cells in T150 flasks were infected with 0.1 PFU/cell VACV. After 3 or 4 days, cells were collected, resuspended in phosphate buffered saline, frozen and thawed three times, and stored in aliquots at 80 as virus stocks. Virus titers in VACV stocks were determined by plaque assays on confluent BSC 1 cells using 10 fold serial dilutions of the stocks in 0.5 ml RPMI 2.5 medium in six well plates for 1 h. A 2 ml volume of fresh RPMI 2.5 medium was added, and the cells were incubated at 37 for 3 days. Next, the medium was aspirated, and the cells were fixed and stained for 10 min with 0.1% crystal violet in 20% ethanol. The fix/stain solution was subsequently aspirated, the cells air dried, the plaques counted, and the PFU/ml in stocks calculated accordingly. For the determination of the virus titers in ovaries, both ovaries were homogenized in a medium using a TissueLyser. The virus titers were calculated as PFU/ovaries. To determine virus titers in spleens, the spleens were made into a single cell suspension between two frosted slides and were resuspended in 10 ml complete RPMI medium. A 1 ml volume of the cell suspension was frozen and thawed three times, and titers were determined in 10 fold serial dilutions of the cell lysates as described above.
Virus titers were calculated as PFU/spleen. To determine the virus titers in the liver, a portion of the liver was weighed and homogenized in the medium using a TissueLyser. The virus titers were calculated as PFU/g. Mice and infections. The Fox Chase Cancer Center Institutional Animal Care and Use Committee approved the experimental protocols involving animals. C57BL/6J and C57BL/6NTac mice Telaprevir 402957-28-2 were purchased from Taconic or the Jackson Laboratory, respectively, when they were 8 to 10 weeks of age. The mice were allowed to rest for at least 1 week before use in experiments. Experiments demonstrated that the VACV specific CD8 T cell responses in B6 mice from Taconic and the Jackson Laboratory were not significantly different. All the figures in this report show the results of experiments carried out with Taconic B6 mice. B6.129S7 Ifngtm1Ts/J and C57BL/6 Prf1tm1Sdz/J mice were initially purchased from the Jackson Laboratory and were bred in the Fox Chase Cancer Center Laboratory Animal Facility. IFN / and Prf/ mice were genotyped using the standard PCR protocols described by the Jackson Laboratory. Unless otherwise indicated, VACV was.