Multiplexed histology analyses for the phenotypic and spatial characterization of human innate lymphoid cells.

Innate lymphoid cells (ILCs) emerge in the last few years as important regulators of immune responses and biological processes. Although ILCs are mainly known as tissue-resident cells, their precise localization and interactions with the microenvironment are still unclear. Here we combine a multiplexed immunofluorescence technique and a customized computational, open-source analysis pipeline to unambiguously identify CD127+ ILCs in situ and characterize these cells and their microenvironments. Moreover, we reveal the transcription factor IRF4 as a marker for tonsillar ILC3, and identify conserved stromal landmarks characteristic for ILC localization. We also show that CD127+ ILCs share tissue niches with plasma cells in the tonsil. Our works thus provide a platform for multiparametric histological analysis of ILCs to improve our understanding of ILC biology.

PUL21a-Cyclin A2 interaction is required to protect human cytomegalovirus-infected cells from the deleterious consequences of mitotic entry.

Entry into mitosis is accompanied by dramatic changes in cellular architecture, metabolism and gene expression. Many viruses have evolved cell cycle arrest strategies to prevent mitotic entry, presumably to ensure sustained, uninterrupted viral replication. Here we show for human cytomegalovirus (HCMV) what happens if the viral cell cycle arrest mechanism is disabled and cells engaged in viral replication enter into unscheduled mitosis. We made use of an HCMV mutant that, due to a defective Cyclin A2 binding motif in its UL21a gene product (pUL21a), has lost its ability to down-regulate Cyclin A2 and, therefore, to arrest cells at the G1/S transition. Cyclin A2 up-regulation in infected cells not only triggered the onset of cellular DNA synthesis, but also promoted the accumulation and nuclear translocation of Cyclin B1-CDK1, premature chromatin condensation and mitotic entry. The infected cells were able to enter metaphase as shown by nuclear lamina disassembly and, often irregular, metaphase spindle formation. However, anaphase onset was blocked by the still intact anaphase promoting complex/cyclosome (APC/C) inhibitory function of pUL21a. Remarkably, the essential viral IE2, but not the related chromosome-associated IE1 protein, disappeared upon mitotic entry, suggesting an inherent instability of IE2 under mitotic conditions. Viral DNA synthesis was impaired in mitosis, as demonstrated by the abnormal morphology and strongly reduced BrdU incorporation rates of viral replication compartments. The prolonged metaphase arrest in infected cells coincided with precocious sister chromatid separation and progressive fragmentation of the chromosomal material. We conclude that the Cyclin A2-binding function of pUL21a contributes to the maintenance of a cell cycle state conducive for the completion of the HCMV replication cycle. Unscheduled mitotic entry during the course of the HCMV replication has fatal consequences, leading to abortive infection and cell death.

Inhibition of human cytomegalovirus immediate-early gene expression by cyclin A2-dependent kinase activity.

Human cytomegalovirus (HCMV) starts its lytic replication cycle only in the G(0)/G(1) phase of the cell division cycle. S/G(2) cells can be infected but block the onset of immediate-early (IE) gene expression. This block can be overcome by inhibition of cyclin-dependent kinases (CDKs), suggesting that cyclin A2, the only cyclin with an S/G(2)-specific activity profile, may act as a negative regulator of viral gene expression. To directly test this hypothesis, we generated derivatives of an HCMV-permissive glioblastoma cell line that express cyclin A2 in a constitutive, cell cycle-independent manner. We demonstrate that even moderate cyclin A2 overexpression in G(1) was sufficient to severely compromise the HCMV replicative cycle after high-multiplicity infection. This negative effect was composed of a strong but transient inhibition of IE gene transcription and a more sustained alteration of IE mRNA processing, resulting in reduced levels of UL37 and IE2, an essential transactivator of viral early gene expression. Consistently, cyclin A2-overexpressing cells showed a strong delay of viral early and late gene expression, as well as virus reproduction. All effects were dependent on CDK activity, as a cyclin A2 mutant deficient in CDK binding was unable to interfere with the HCMV infectious cycle. Interestingly, murine CMV, whose IE gene expression is known to be cell cycle independent, is not affected by cyclin A2. Instead, it upregulates cyclin A2-associated kinase activity upon infection. Understanding the mechanisms behind the HCMV-specific action of cyclin A2-CDK might reveal new targets for antiviral strategies.

General blockade of human cytomegalovirus immediate-early mRNA expression in the S/G2 phase by a nuclear, Daxx- and PML-independent mechanism.

The onset of human cytomegalovirus (HCMV) lytic replication is strictly controlled by the host cell division cycle. Although viral entry of S/G2-phase cells is unperturbed expression of major immediate-early (MIE) genes IE1 and IE2 is tightly blocked in these cells. Besides the finding that cyclin-dependent kinase (CDK) activity is required for IE1/IE2 repression little is known about the nature of this cell cycle-dependent block. Here, we show that the block occurs after nuclear entry of viral DNA and prevents the accumulation of IE1/IE2 mRNAs, suggesting an inhibition of transcription. Remarkably, the presence of cis-regulatory regions of the MIE locus is neither sufficient nor necessary for IE1/IE2 repression in the S/G2 phase. Furthermore, the block of viral mRNA expression also affects other immediate-early transcribed regions, i.e. the US3 and UL36-38 gene loci. This suggests a mechanism of repression that acts in a general and not a gene-specific fashion. Such a nuclear, genome-wide repression of HCMV is typically mediated by the intrinsic immune defence at nuclear domain 10 (ND10) structures. However, we found that neither Daxx nor PML, the main players of ND10-based immunity, are required for the block to viral gene expression in the S/G2 phase. In addition, the viral tegument protein pp71 (pUL82), a major antagonist of the intrinsic immunity at pre-immediate-early times of infection, proved to be functional in S-phase cells. This suggests the existence of a yet undiscovered, CDK-dependent mechanism exerting higher-level control over immediate-early mRNA expression in HCMV-infected cells.

Human cytomegalovirus inactivates the G0/G1-APC/C ubiquitin ligase by Cdh1 dissociation.

The anaphase promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that targets regulators of the cell division cycle for degradation by the 26S proteasome. Discovered as a key regulator of mitosis, the APC/C has more recently been recognized to also play a limiting role in the control of G(0) maintenance, G(1)/S-transition and DNA-replication. Human cytomegalovirus (HCMV) has been shown to interfere with cell cycle regulation at different levels. It can induce an S phase-prone proliferation program in quiescent cells but at the same time this virus directly inhibits competitive cellular DNA replication. Here we show, that human cytomegalovirus (HCMV) inactivates the G(0)/G(1) APC/C rapidly after infection of quiescent fibroblasts, resulting in the untimely stabilization of APC/C substrates. APC/C inactivation is caused by the dissociation of its positive regulator, Cdh1. Surprisingly, this dissociation is independent from known Cdh1 inhibitors, Emi1 and Cyclin A, suggesting that APC/C-Cdh1 inhibition by HCMV is directly caused by a viral protein or an intermediate cellular factor distinct from Emi1 and Cyclin A. Thus, upon infection of quiescent cells HCMV not only activates the E2F-dependent G(1)/S transcription program but also facilitates protein accumulation of APC/C substrates by rapid Cdh1 dissociation.

Human cytomegalovirus immediate-early protein 2 (IE2)-mediated activation of cyclin E is cell-cycle-independent and forces S-phase entry in IE2-arrested cells.

In human cytomegalovirus (HCMV) infection, the isolated expression of the viral immediate-early protein 2 (IE2) 86 kDa regulatory protein coincides with an up-regulation of cyclin E gene expression, both in fibroblasts and U373 cells. Since IE2 also interferes with cell-cycle progression, it is unclear whether IE2 is a genuine activator of cyclin E or whether IE2-arrested cells contain elevated levels of cyclin E primarily as a consequence of them being arrested at the beginning of S phase. It is important to distinguish between these possibilities in order to define and analyse at a mechanistic level the proliferative and anti-proliferative capacities of IE2. Here we have shown that IE2 can activate cyclin E independent of the cell-cycle state and can therefore function as a genuine activator of cyclin E gene expression. A mutant of IE2 that failed to activate cyclin E also failed to promote G1/S transition. Instead, cells became arrested in G1. S-phase entry could be rescued in these cells by co-expression of cyclin E, but these cells still arrested in early S phase, as is the case with wild-type IE2. Our data demonstrate that IE2 can promote two independent cell-cycle functions at the same time: (i) the induction of G1/S transition via up-regulation of cyclin E, and (ii) a block in cell-cycle progression in early S phase. In G1, the proliferative activity of IE2 appears to be dominant over the anti-proliferative force, whereas after G1/S transition, this situation is reversed.

Human cytomegalovirus prevents replication licensing by inhibiting MCM loading onto chromatin.

To allow DNA replication only once per cell cycle, origins of replication are reactivated ('licensed') during each G1 phase. Licensing is facilitated by assembly of the pre-replicative complex (pre-RC) at origins that concludes with loading the mini-chromosome maintenance (MCM) complex onto chromatin. Here we show that a virus exploits pre-RC assembly to selectively inhibit cellular DNA replication. Infection of quiescent primary fibroblasts with human cytomegalovirus (HCMV) induces all pre-RC factors. Although this is sufficient to assemble the MCM-loading factors onto chromatin, as it is in serum-stimulated cells, the virus inhibits loading of the MCM complex itself, thereby prematurely abrogating replication licensing. This provides a new level of control in pre-RC assembly and a mechanistic rationale for the unusual HCMV-induced G1 arrest that occurs despite the activation of the cyclin E-dependent transcription programme. Thus, this particularly large virus might thereby secure the supply with essential replication factors but omit competitive cellular DNA replication.