CD8 T-cell responses to superinfection upon MCMV infection.

<p>(A) groups of 3 (young) and 12 (old) month old mice were primed with 10⁵ PFU of MCMV, 10⁶ PFU of VACV-WR or PBS (MOCK), and challenged 5 months later with 300 EID of influenza virus, PR/8 strain, intranasally (i.n.). 7 days post challenge blood lymphocytes were tested by ICCS for IFNγ responses to a 9 h in vitro stimulation with the NP³⁶⁶ peptide in the presence of BrefeldinA. Mean IFNγ responses in animal groups (n = 5/group)+SD is shown on the y axis. (B) 12 month-old mice were primed as indicated and challenged 5 months later with Influenza. The frequency of peptide specific cells in the blood CD8 pool was defined by peptide restimulation and ICCS. Group average (+SD) values for either peptide are indicated on the y axis. (C) Mice were primed with 10⁵ PFU of MCMV or 10⁶ PFU of VACV-IE1 at 2 months of age, and i.p. challenged with 50 PFU of West Nile Virus at 8 months of age. 7 days later, blood lymphocytes were in vitro stimulated with a pool of two immunodominant H^2d restricted WNV peptides and analyzed by ICCS. The frequency of IFNγ expressing T-cells in the CD8 pool of individual mice is displayed on the y axis. Horizontal lines indicate medians. (D) C57BL6/DBA2 F1 mice were primed with MCMV or VACV-IE1 at 3 months of age, challenged with HSV-1 at 8 months of age and frequencies of HSV-1 specific responses were determined 7 months later by pMHC tetramer staining and FCM. Each mouse is indicated with a symbol, horizontal lines are means, the dashed line shows the detection threshold. The p value for Mann-Whitney analysis is shown. (E, F) Mice were primed with MCMV or VACV-IE1 at 6, 12, 16 or 20 months of age, and assayed for responses to WNV challenge at 22 months of age (16, 10, 6 or 2 months post prime, as indicated below axis) using the protocol as in panel C. Each mouse is indicated with a symbol, horizontal lines are means. (E) % of CD8 cells responding to peptide stimulation in ICCS. (F) Leukocytes were in parallel assayed by polyclonal stimulation with anti-CD3 antibodies, and the peptide specific response was normalized to the CD3 (Max) response. Values in panels A, C, E and F were compared by 1-way ANOVA followed by Bonferroni comparison of individual columns, and significance is indicated (n.s. - p>0.05, *- p<0.05, ** - p<0.01, *** - p<0.001).