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

(A) 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) 129/Sv mice were infected intraperitoneally (i.p.) with 2x105 PFU of indicated MCMV recombinants and 106 PFU of VACVSL. Blood of mice was analysed for the fraction of CD8 T cells responding to in vitro 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 MCMVM45SL or MCMVM45ASL was identified. Significance was assessed by Kruskal-Wallis test followed by Dunns post-analysis for MCMVM45SL and MCMVM45ASL infected mice (*p<0.05, **p<0.01). (C) 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 MCMVWT or MCMVM45Cterm 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. (D) LMP7-/- and C57BL/6 adult mice were infected i.v. with 105 PFU of MCMVWT (top panel) or MCMVM45Cterm (bottom panel). The percentage of blood CD8 T cells stained by HGIRNASFI-Db 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 U test. *p < 0.05. ***p < 0.0001, ns—not significant.