Control of primary mouse cytomegalovirus infection in lung nodular inflammatory foci by cooperation of interferon-gamma expressing CD4 and CD8 T cells.

Human cytomegalovirus (CMV) and mouse cytomegalovirus (MCMV) infection share many characteristics. Therefore infection of mice with MCMV is an important tool to understand immune responses and to design vaccines and therapies for patients at the risk of severe CMV disease. In this study, we investigated the immune response in the lungs following acute infection with MCMV. We used multi-color fluorescence microscopy to visualize single infected and immune cells in nodular inflammatory foci (NIFs) that formed around infected cells in the lungs. These NIFs consisted mainly of myeloid cells, T cells, and some NK cells. We found that the formation of NIFs was essential to reduce the number of infected cells in the lung tissue, showing that NIFs were sites of infection as well as sites of immune response. Comparing mice deficient for several leukocyte subsets, we identified T cells to be of prime importance for restricting MCMV infection in the lung. Moreover, T cells had to be present in NIFs in high numbers, and CD4 as well as CD8 T cells supported each other to efficiently control virus spread. Additionally, we investigated the effects of perforin and interferon-gamma (IFNγ) on the virus infection and found important roles for both mechanisms. NK cells and T cells were the major source for IFNγ in the lung and in in vitro assays we found that IFNγ had the potential to reduce plaque growth on primary lung stromal cells. Notably, the T cell-mediated control was shown to be perforin-independent but IFNγ-dependent. In total, this study systematically identifies crucial antiviral factors present in lung NIFs for early containment of a local MCMV infection at the single cell level.

In Vivo Killing Capacity of Cytotoxic T Cells Is Limited and Involves Dynamic Interactions and T Cell Cooperativity.

According 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.

Nodular inflammatory foci are sites of T cell priming and control of murine cytomegalovirus infection in the neonatal lung.

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.