Human cytomegalovirus (HCMV) encodes four viral Fc-gamma receptors (vFcγRs) that counteract antibody-mediated activation in vitro, but their role in infection and pathogenesis is unknown. To examine their in vivo function in an animal model evolutionarily closely related to humans, we identified and characterized Rh05, Rh152/151 and Rh173 as the complete set of vFcγRs encoded by rhesus CMV (RhCMV). Each one of these proteins displays functional similarities to their prospective HCMV orthologs with respect to antagonizing host FcγR activation in vitro. When RhCMV-naïve male rhesus macaques were infected with vFcγR-deleted RhCMV, peak plasma DNAemia levels and anti-RhCMV antibody responses were comparable to wildtype infections of both male and female animals. However, the duration of plasma DNAemia was significantly shortened in immunocompetent, but not in CD4 + T cell-depleted animals. Since vFcγRs were not required for superinfection of rhesus macaques, we conclude that these proteins can prolong lytic replication during primary infection by evading virus-specific adaptive immune responses, particularly antibodies. The role of viral Fc-gamma receptors in rhesus cytomegalovirus (RhCMV) infection is unclear. Here, the authors characterized RhCMV vFcγRs and report that their deletion did not affect virus replication, tropism or superinfection in rhesus macaques but increased susceptibility of the virus to antibody control.

ABSTRACTThe signal sequences of the human cytomegalovirus (CMV) UL40 protein, and its rhesus CMV counterpart, Rh67, contain a peptide (VMAPRT[L/V][F/I/L/V]L, VL9) that can be presented by Major Histocompatibility Complex (MHC) antigen E. The CMV VL9 peptides replace VL9 peptides derived from classical MHC (Ia) signal sequences, which are lost when CMV disrupts MHC Ia expression, as well as antigen processing and presentation. This allows infected cells to maintain surface expression of MHC-E and escape killing by NK cells expressing the inhibitory NKG2A/CD94 receptor. We demonstrate that processing of the Rh67 VL9 peptide mirrors that of UL40, despite the lack of sequence conservation elsewhere in the two proteins. As previously shown for UL40, up-regulation of MHC-E expression by Rh67 only requires its signal sequence, with sequences upstream of VL9 critical for conferring independence from TAP, the Transporter Associated with Antigen Processing. Additionally, we show that processing of both VL9 peptides depends on cleavage of the signal sequences by the host protease Signal Peptide Peptidase. Notably, our results also reveal that the mature UL40 and Rh67 proteins contribute to CMV immune evasion by down-regulating surface expression of MHC Ia. Unexpectedly, while the Rh67 VL9 peptide is resistant to the effects of the Rh67 protein, the UL40 protein is able to counteract up-regulation of MHC-E expression mediated by its own VL9 peptide. This suggests differences in the mechanisms by which the two VL9 peptides up-regulate MHC-E, which may have implications for translating a RhCMV-vectored SIV vaccine to HIV-1 using HCMV as a vector.IMPORTANCECell surface MHC-E expression, which requires a peptide (VMAPRT[L/V][F/I/L/V]L, “VL9”) from the signal sequences of other MHC class I proteins, prevents cells from being killed by CD94/NKG2A-expressing Natural Killer cells. In cells infected with human CMV, the endogenous VL9 peptide is replaced by one from the signal sequence of the HCMV UL40 protein. We show that processing of the VL9 peptide of Rh67, the rhesus CMV equivalent of UL40, mirrors that of UL40, despite a lack of sequence homology between the two proteins. Of note, we also show that the mature UL40 and Rh67 proteins, which have no previously described function, contribute to CMV immune evasion by reducing classical MHC class I surface expression. Importantly, the mature UL40 protein, but not the mature Rh67 protein, can decrease the up-regulation of MHC-E mediated by its signal sequence VL9 peptide, which may have implications for HCMV as a vaccine vector.

The nonpolymorphic major histocompatibility complex E (MHC-E) molecule is up-regulated on many cancer cells, thus contributing to immune evasion by engaging inhibitory NKG2A/CD94 receptors on NK cells and tumor-infiltrating T cells. To investigate whether MHC-E expression by cancer cells can be targeted for MHC-E–restricted T cell control, we immunized rhesus macaques (RM) with rhesus cytomegalovirus (RhCMV) vectors genetically programmed to elicit MHC-E–restricted CD8 + T cells and to express established tumor-associated antigens (TAAs) including prostatic acidic phosphatase (PAP), Wilms tumor-1 protein, or Mesothelin. T cell responses to all three tumor antigens were comparable to viral antigen-specific responses with respect to frequency, duration, phenotype, epitope density, and MHC restriction. Thus, CMV-vectored cancer vaccines can bypass central tolerance by eliciting T cells to noncanonical epitopes. We further demonstrate that PAP-specific, MHC-E–restricted CD8 + T cells from RhCMV/PAP-immunized RM respond to PAP-expressing HLA-E + prostate cancer cells, suggesting that the HLA-E/NKG2A immune checkpoint can be exploited for CD8 + T cell–based immunotherapies.

The commonly used antibodies 3D12 and 4D12 recognise the human leukocyte antigen E (HLA-E) protein. These antibodies bind distinct epitopes on HLA-E and differ in their ability to bind alleles of the major histocompatibility complex E (MHC-E) proteins of rhesus and cynomolgus macaques. We confirmed that neither antibody cross-reacts with classical HLA alleles, and used hybrids of different MHC-E alleles to map the regions that are critical for their binding. 3D12 recognises a region on the alpha 3 domain, with its specificity for HLA-E resulting from the amino acids present at three key positions (219, 223 and 224) that are unique to HLA-E, while 4D12 binds to the start of the alpha 2 domain, adjacent to the C terminus of the presented peptide. 3D12 staining is increased by incubation of cells at 27°C, and by addition of the canonical signal sequence peptide presented by HLA-E peptide (VL9, VMAPRTLVL). This suggests that 3D12 may bind peptide-free forms of HLA-E, which would be expected to accumulate at the cell surface when cells are incubated at lower temperatures, as well as HLA-E with peptide. Therefore, additional studies are required to determine exactly what forms of HLA-E can be recognised by 3D12. In contrast, while staining with 4D12 was also increased when cells were incubated at 27°C, it was decreased when the VL9 peptide was added. We conclude that 4D12 preferentially binds to peptide-free HLA-E, and, although not suitable for measuring the total cell surface levels of MHC-E, may putatively identify peptide-receptive forms.

Strain 68-1 rhesus cytomegalovirus (RhCMV) vectors expressing simian immunodeficiency virus (SIV) antigens elicit CD8(+) T cells recognizing epitopes presented by major histocompatibility complex II (MHC-II) and MHC-E but not MHC-Ia. These immune responses mediate replication arrest of SIV in 50 to 60% of monkeys. We show that the peptide VMAPRTLLL (VL9) embedded within the RhCMV protein Rh67 promotes intracellular MHC-E transport and recognition of RhCMV-infected fibroblasts by MHC-E-restricted CD8(+) T cells. Deletion or mutation of viral VL9 abrogated MHC-E-restricted CD8(+) T cell priming, resulting in CD8(+) T cell responses exclusively targeting MHC-II-restricted epitopes. These responses were comparable in magnitude and differentiation to responses elicited by 68-1 vectors but did not protect against SIV. Thus, Rh67-enabled direct priming of MHC-E-restricted T cells is crucial for RhCMV/SIV vaccine efficacy.

Increasing evidence suggests that cancer cells highjack developmental programs for disease initiation and progression. Melanoma arises from melanocytes that originate during development from neural crest stem cells (NCSCs). Here, we identified the transcription factor Yin Yang 1 (Yy1) as an NCSCs regulator. Conditional deletion of Yy1 in NCSCs resulted in stage-dependent hypoplasia of all major neural crest derivatives due to decreased proliferation and increased cell death. Moreover, conditional ablation of one Yy1 allele in a melanoma mouse model prevented tumorigenesis, indicating a particular susceptibility of melanoma cells to reduced Yy1 levels. Combined RNA sequencing (RNA-seq), chromatin immunoprecipitation (ChIP)-seq, and untargeted metabolomics demonstrated that YY1 governs multiple metabolic pathways and protein synthesis in both NCSCs and melanoma. In addition to directly regulating a metabolic gene set, YY1 can act upstream of MITF/c-MYC as part of a gene regulatory network controlling metabolism. Thus, both NCSC development and melanoma formation depend on an intricate YY1-controlled metabolic program.

Human cytomegalovirus (HCMV) depends on and modulates multiple host cell membrane proteins during each stage of the viral life cycle. To gain a global view of the impact of HCMV-infection on membrane proteins, we analyzed HCMV-induced changes in the abundance of membrane proteins in fibroblasts using stable isotope labeling with amino acids (SILAC), membrane fractionation and protein identification by two-dimensional liquid chromatography and tandem mass spectrometry. This systematic approach revealed that CD81, CD44, CD98, caveolin-1 and catenin delta-1 were down-regulated during infection whereas GRP-78 was up-regulated. Since CD81 downregulation was also observed during infection with UV-inactivated virus we hypothesized that this tetraspanin is part of the viral entry process. Interestingly, additional members of the tetraspanin family, CD9 and CD151, were also downregulated during HCMV-entry. Since tetraspanin-enriched microdomains (TEM) cluster host cell membrane proteins including known CMV receptors such as integrins, we studied whether TEMs are required for viral entry. When TEMs were disrupted with the cholesterol chelator methyl-β-cylcodextrin, viral entry was inhibited and this inhibition correlated with reduced surface levels of CD81, CD9 and CD151, whereas integrin levels remained unchanged. Furthermore, simultaneous siRNA-mediated knockdown of multiple tetraspanins inhibited viral entry whereas individual knockdown had little effect suggesting essential, but redundant roles for individual tetraspanins during entry. Taken together, our data suggest that TEM act as platforms for receptors utilized by HCMV for entry into cells.

The neural crest (NC) is an embryonic stem/progenitor cell population that generates a diverse array of cell lineages, including peripheral neurons, myelinating Schwann cells, and melanocytes, among others. However, there is a long-standing controversy as to whether this broad developmental perspective reflects in vivo multipotency of individual NC cells or whether the NC is comprised of a heterogeneous mixture of lineage-restricted progenitors. Here, we resolve this controversy by performing in vivo fate mapping of single trunk NC cells both at premigratory and migratory stages using the R26R-Confetti mouse model. By combining quantitative clonal analyses with definitive markers of differentiation, we demonstrate that the vast majority of individual NC cells are multipotent, with only few clones contributing to single derivatives. Intriguingly, multipotency is maintained in migratory NC cells. Thus, our findings provide definitive evidence for the in vivo multipotency of both premigratory and migrating NC cells in the mouse.

Schwann cells, the myelinating glia of the peripheral nervous system (PNS), originate from multipotent neural crest cells that also give rise to other cells, including neurons, melanocytes, chondrocytes, and smooth muscle cells. The transcription factor Sox10 is required for peripheral glia specification. However, all neural crest cells express Sox10 and the mechanisms directing neural crest cells into a specific lineage are poorly understood. We show here that histone deacetylases 1 and 2 (HDAC1/2) are essential for the specification of neural crest cells into Schwann cell precursors and satellite glia, which express the early determinants of their lineage myelin protein zero (P0) and/or fatty acid binding protein 7 (Fabp7). In neural crest cells, HDAC1/2 induced expression of the transcription factor Pax3 by binding and activating the Pax3 promoter. In turn, Pax3 was required to maintain high Sox10 levels and to trigger expression of Fabp7. In addition, HDAC1/2 were bound to the P0 promoter and activated P0 transcription. Consistently, in vivo genetic deletion of HDAC1/2 in mouse neural crest cells led to strongly decreased Sox10 expression, no detectable Pax3, virtually no satellite glia, and no Schwann cell precursors in dorsal root ganglia and peripheral nerves. Similarly, in vivo ablation of Pax3 in the mouse neural crest resulted in strongly reduced expression of Sox10 and Fabp7. Therefore, by controlling the expression of Pax3 and the concerted action of Pax3 and Sox10 on their target genes, HDAC1/2 direct the specification of neural crest cells into peripheral glia.

During vertebrate development, neural crest stem cells (NCSCs) give rise to neural cells of the peripheral nervous system and to a variety of mesenchymal cell types, including smooth muscle, craniofacial chondrocytes, and osteocytes. Consistently, mesenchymal stem cells (MSCs) have recently been shown to derive in part from the neural crest (NC), although the mechanisms underlying MSC generation remains to be identified. Here, we show that transforming growth factor β (TGFβ)-mediated suppression of the NCSC transcription factor Sox10 induces a switch in neural to mesenchymal potential in NCSCs. In vitro and in vivo, TGFβ signal inactivation results in persistent Sox10 expression, decreased cell cycle exit, and perturbed generation of mesenchymal derivatives, which eventually leads to defective morphogenesis. In contrast, TGFβ-mediated downregulation of Sox10 or its genetic inactivation suppresses neural potential, confers mesenchymal potential to NC cells in vitro, and promotes cell cycle exit and precocious mesenchymal differentiation in vivo. Thus, negative regulation of Sox10 by TGFβ signaling promotes the generation of mesenchymal progenitors from NCSCs. Our study might lay the grounds for future applications demanding defined populations of MSCs for regenerative medicine.

The embryonic head development, including the formation of dental structures, is a complex and delicate process guided by specific genetic programs. Genetic changes and environmental factors can disturb the execution of these programs and result in abnormalities in orofacial and dental structures. Orofacial clefts and hypodontia/ oligodontia are examples of such abnormalities frequently seen in dental clinics. An insight into the mechanisms and genes involved in the formation of orofacial and dental structures has been gradually gained by genetic analysis of families and by the use of experimental vertebrate models such as the mouse and chick models. The development of novel clinical therapies for orofacial and dental pathological conditions depends very much on a detailed knowledge of the molecular and cellular processes that are involved in head formation.

During vertebrate development, neural crest cells are exposed to multiple extracellular cues that drive their differentiation into neural and non-neural cell lineages. Insights into the signals potentially involved in neural crest cell fate decisions in vivo have been gained by cell culture experiments that have allowed the identification of instructive growth factors promoting either proliferation of multipotent neural crest cells or acquisition of specific fates. For instance, members of the TGFbeta factor family induce neurogenesis and smooth muscle cell formation at the expense of other fates in culture. In vivo, conditional ablation of various TGFbeta signaling components resulted in malformations of non-neural derivatives of the neural crest, but it is unclear whether these phenotypes involved aberrant fate decisions. Moreover, it remains to be shown whether neuronal determination indeed requires TGFbeta factor activity in vivo. To address these issues, we conditionally deleted Smad4 in the neural crest, thus inactivating all canonical TGFbeta factor signaling. Surprisingly, neural crest cell fates were not affected in these mutants, with the exception of sensory neurogenesis in trigeminal ganglia. Rather, Smad4 regulates survival of smooth muscle and proliferation of autonomic and ENS neuronal progenitor cells. Thus, Smad signaling plays multiple, lineage-specific roles in vivo, many of which are elicited only after neural crest cell fate decision.