SummaryAccording to in vitro assays, T cells are thought to kill rapidly and efficiently, but the efficacy and dynamics of cytotoxic T lymphocyte (CTL)-mediated killing of virus-infected cells in vivo remains elusive. We used two-photon microscopy to quantify CTL-mediated killing in mice infected with herpesviruses or poxviruses. On average, one CTL killed 2–16 virus-infected cells per day as determined by real-time imaging and by mathematical modeling. In contrast, upon virus-induced MHC class I downmodulation, CTLs failed to destroy their targets. During killing, CTLs remained migratory and formed motile kinapses rather than static synapses with targets. Viruses encoding the calcium sensor GCaMP6s revealed strong heterogeneity in individual CTL functional capacity. Furthermore, the probability of death of infected cells increased for those contacted by more than two CTLs, indicative of CTL cooperation. Thus, direct visualization of CTLs during killing of virus-infected cells reveals crucial parameters of CD8+ T cell immunity.

Neonates, including mice and humans, are highly susceptible to cytomegalovirus (CMV) infection. However, many aspects of neonatal CMV infections such as viral cell tropism, spatio-temporal distribution of the pathogen as well as genesis of antiviral immunity are unknown. With the use of reporter mutants of the murine cytomegalovirus (MCMV) we identified the lung as a primary target of mucosal infection in neonatal mice. Comparative analysis of neonatal and adult mice revealed a delayed control of virus replication in the neonatal lung mucosa explaining the pronounced systemic infection and disease in neonates. This phenomenon was supplemented by a delayed expansion of CD8+ T cell clones recognizing the viral protein M45 in neonates. We detected viral infection at the single-cell level and observed myeloid cells forming “nodular inflammatory foci” (NIF) in the neonatal lung. Co-localization of infected cells within NIFs was associated with their disruption and clearance of the infection. By 2-photon microscopy, we characterized how neonatal antigen-presenting cells (APC) interacted with T cells and induced mature adaptive immune responses within such NIFs. We thus define NIFs of the neonatal lung as niches for prolonged MCMV replication and T cell priming but also as sites of infection control.

The molecular mechanisms leading to reactivation of latent cytomegalovirus are not well understood. To study reactivation, the few cells in an organ tissue that give rise to reactivated virus need to be identified, ideally at the earliest possible time point in the process. To this end, mouse cytomegalovirus (MCMV) reporter mutants were designed to simultaneously express the red fluorescent protein mCherry and the secreted Gaussia luciferase (Gluc). Whereas Gluc can serve to assess infection at the level of individual mice by measuring luminescence in blood samples or by in vivo imaging, mCherry fluorescence offers the advatage of detection of infection at the single cell level. To visualize cells in which MCMV was being reactivated, precision-cut lung slices (PCLS) that preserve tissue microanatomy were prepared from the lungs of latently infected mice. By day 3 of cultivation of the PCLS, reactivation was revealed by Gluc expression, preceding the detection of infectious virus by approximately 4 days. Reactivation events in PCLS could be identified when they were still confined to single cells. Notably, using fractalkine receptor-GFP reporter mice, we never observed reactivation originating from CX3CR1 + monocytes or pulmonary dendritic cells derived therefrom. Furthermore, latent viral genome in the lungs was not enriched in sorted bone-marrow-derived cells expressing CD11b. Taken together, these complementary approaches suggest that CD11b + and CX3CR1 + subsets of the myeloid differentiation lineage are not the main reservoirs and cellular sites of MCMV latency and reactivation in the lungs. INTRODUCTIONHuman cytomegalovirus (HCMV) infection is highly prevalent in the human population (Mocarski et al., 2007). Primary infection often occurs in early childhood, usually without symptoms or with mild symptoms only. The acute infection is cleared by the immune system, but viral genomes remain in specific cells of the organism in a latent state. Cells of the myeloid lineage such as monocytes, which are the progenitors of dendritic cells and macrophages, but also other cell types such as endothelial cells, are considered to form the reservoir for latent HCMV genomes (reviewed by Jarvis & Nelson, 2002;Sinclair, 2008). Latency is operationally defined by the absence of detectable infectious virus and the capacity of the latent viral genomes to give rise to recurrent infection (Roizman & Sears, 1987). Reactivation of viral gene expression may be a relatively frequent event if viewed over long periods of time, however, the immune system in healthy individuals can recognize and terminate reactivation events even before virion assembly (Simon et al., 2006; reviewed by Reddehase et al., 2008). In addition, the immune system limits the spread of already reactivated virus, thus preventing recurrent viraemia and cytomegalovirus (CMV) disease (Polić et al., 1998). Consequently, HCMV reactivation is primarily a health risk for immunocompromised individuals such as transplant patients.The molecular events that lead to activati...

The immediate-early protein IE1 of human and mouse cytomegalovirus (MCMV) is one of the first proteins expressed during the productive infection cycle and upon reactivation from latency. The CMV IE1 proteins have been found to inhibit histone deacetylases, suggesting a role in the epigenetic regulation of viral gene expression. Consequently, the IE1 protein is considered to have a profound effect on reactivation, because small amounts of IE1 may be decisive for the switch to lytic replication. Here we asked if an MCMV ⌬ie1 mutant is able both to establish latency and to reactivate from the lungs of latently infected mice. Since the ⌬ie1 mutant was known to be attenuated during acute infection, we first defined conditions that led to comparable levels of viral genomes during latent infection with mutant and wild-type (wt) MCMV. Viral genome copy numbers dropped considerably at the onset of the latent infection but then remained steady for both viruses even after several months. Reactivation of the ⌬ie1 mutant and of wt MCMV from latency occurred with similar incidences in lung explant cultures at 4, 7, and 12 months postinfection. The increase in the frequency of a subset of MCMV-specific memory T cells, a possible indicator of frequent transcriptional reactivation events during latency, was in a comparable range for both viruses. Recurrence of the ⌬ie1 virus infection in vivo could also be induced by hematoablative treatment of latently infected mice. We conclude that the ie1 gene is not essential for the establishment of latency or for the reactivation of MCMV.

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