MHC class I binding by a latency-associated epitope impairs host colonization.

<p>C57BL/6 mice were infected i.n. with 10⁴ PFU of the indicated viruses. (A) The latent load in spleens was determined by explant co-culture assay (closed symbols) and pre-formed infectious virus was quantified by plaque assay (open symbols). Each point shows the titre of 1 mouse, horizontal lines arithmetic means and dashed horizontal line limit of detection of assay. At day 14, vOVA, vQ4, vV4, vG4, vR4 and vE1 latent loads were significantly below vWT (p<0.05, by two-tailed unpaired t-test). vA8 latency loads were not significantly different from vWT (p = 0.07). (B–C) Reciprocal frequencies of viral DNA⁺ cells in (B) total splenocytes or (C) GC B cells. Bars represent the frequency of viral DNA⁺ cells with 95% confidence intervals. (D) Representative spleen sections showing dark stained latently infected cells by <i>in situ</i> hybridization. (E) % tetramer positive CD8⁺ T cells at each time point from spleens (arithmetic mean +/− SEM of 3 independent assays). * p<0.05, ** p<0.01, **** p<0.0001; using a two-tailed unpaired t-test. (F) Functional capacity of splenic CTL determined by intracellular interferon-gamma staining after <i>ex vivo</i> stimulation. Data show % CD8⁺ T cells responding to each peptide (arithmetic mean +/− SEM of 3 independent assays). * p<0.05, ** p<0.01; using a two-tailed unpaired t-test. (G–H) <i>In vivo</i> CTL activity at 11 days post-infection. (G) At day 10 post-infection 50∶50 mixes consisting of 2×10⁶ unpulsed CD45.1⁺ CFSE^lo splenocytes and 2×10⁶ OVA-, E1- or A8-pulsed CD45.1⁺ CFSE^hi splenocytes were transferred intravenously into vOVA, vE1 or vA8 infected C57BL/6 mice. The same mix of cells was transferred into vWT infected mice C57BL/6 as internal control. In the next day, the proportion of CFSE^hi and CFSE^lo cells among CD45.1⁺ cells recovered from the spleen was analysed by FACS. Representative FACS plots showing % of unpulsed CD45.1⁺ CFSE^lo and OVA-, E1, or A8-pulsed CD45.1⁺ CFSE^hi splenocytes. (H) % target cell killing. Three to four mice were analyzed per group, and experiments repeated three times.