Constitutive proteasomal processing is critical for the induction of inflationary CD8 T-cell responses.

<p>(<b>A</b>) IC-21 cells were infected with indicated viruses at an MOI of 0.2 with centrifugal enhancement. Splenocytes obtained from gBT-I.1 mice were used as effector cells at an E:T ratio of 3:1. Splenocytes were not restimulated upon isolation from the mice and used untouched for the assay. Co-culture was performed overnight (15h). Columns represent the mean percentage of IFNγ⁺ or TNFα⁺ cells from triplicate experiments, and error bars show the SEM. (<b>B</b>) 129/Sv mice were infected intraperitoneally (i.p.) with 2x10⁵ PFU of indicated MCMV recombinants and 10⁶ PFU of VACV^SL. Blood of mice was analysed for the fraction of CD8 T cells responding to <i>in vitro</i> re-stimulation with the SSIEFARL peptide for 6 hours, followed by intracellular staining for IFNγ. Grouped means +/- SEM of cells responding to the SSIEFARL peptide at 7, 14, 28, 60, 90, 120, 180 dpi are shown. The experiment was performed two times independently, at 5 mice per group in each experiment, and grouped averages from two experiments are shown. Difference in responses between groups infected with either MCMV^M45SL or MCMV^M45ASL was identified. Significance was assessed by Kruskal-Wallis test followed by Dunns post-analysis for MCMV^M45SL and MCMV^M45ASL infected mice (*<i>p</i><0.05, **<i>p</i><0.01). (<b>C</b>) Treatment with proteasomal inhibitors (but not protease inhibitors) impairs target cell recognition by HGIRNASFI-specific CTL. Target cells (LSECs) were pretreated for 5h with indicated inhibitors, washed twice with PBS and infected with MCMV^WT or MCMV^M45Cterm at an MOI 0.2 with centrifugal enhancement. Co-culture with HGIRNASFI-specific CTLs was performed at an E:T ratio 3:1 for 15h, upon which the T cells were collected and stained for intracellular IFNγ. The y-axis shows percentages of CTL responding by IFNγ to co-culture with target cells (mean +/- SEM from three experiments is shown). Labels below the x-axis show the deployed inhibitor and its concentration in μM; LC—lactacystin, MG—MG132, LP—leupeptin; Pos–positive control, target cells infected with indicated viruses without pretreatment with inhibitors; DMSO—infection in presence of the diluent for inhibitors Neg–negative control, untreated cells. (<b>D</b>) LMP7^-/- and C57BL/6 adult mice were infected i.v. with 10⁵ PFU of MCMV^WT (top panel) or MCMV^M45Cterm (bottom panel). The percentage of blood CD8 T cells stained by HGIRNASFI-D^b tetramers was measured at the indicated time points. The data show the mean values +/- SD from of pooled results from 2 independent experiments (in total 8 mice per each group) Significance on indicated time points was assessed by a Mann—Whitney <i>U</i> test. *<i>p</i> < 0.05. ***<i>p</i> < 0.0001, ns—not significant.