No abstract

After primary infection, human cytomegalovirus (HCMV) persists as a life-long latent infection, with host immunosuppression often resulting in clinical reactivation. During lytic infection, major changes in the expression of secreted cellular proteins (the secretome) occur that have profound effects on host–cell interactions, particularly at the level of the host immune response. In contrast, little is known about changes in the secretome that accompany latent infection, yet this is likely to be of major importance for the life-long carriage of this persistent human pathogen in the face of constant immunosurveillance. We have analyzed the secretome of cells carrying latent HCMV and have identified changes in several secreted cellular proteins known to be involved in regulation of the immune response and chemoattraction. Here, we show that a latency-associated increase in CC chemokine ligand (CCL)8 results in the recruitment of cluster of differentiation (CD)4 + T cells to supernatants from latently infected CD34 + cells but that these latent supernatants, also rich in immunosuppressive factors, inhibit cytokine secretion and cytotoxicity of HCMV-specific T-helper (Th)1 CD4 + T cells. These results identify a strategy by which sites of latent HCMV can firstly recruit CD4 + T cells and then inhibit their antiviral effector functions, thereby aiding the maintenance of latent infection in the face of the host immune response.

Regulatory T cells (Tregs) are an essential component of the cellular immune response, occupying a key role in maintaining immunological tolerance and present an attractive therapeutic target in a range of immunopathologies. Comprehensive analysis of the human Treg compartment has been restricted due to technical limitations. The advent of mass cytometry enables simultaneous assessment of vastly increased phenotypic parameters at single-cell resolution. In this study, we used mass cytometry to examine the complexity of human Tregs using an extensive panel of surface markers associated with Treg function and phenotype. We applied unsupervised clustering analysis, revealing 22 distinct subpopulations of Tregs, representing previously identified and novel subpopulations. Our data represent the most in-depth phenotypic description of the human Treg compartment at single-cell resolution and show a hitherto unrecognized degree of phenotypic complexity among cells of the regulatory lineage.

It is generally accepted that, following primary infection, human cytomegalovirus (HCMV) establishes lifelong latency in CD34؉ progenitor cells and other derivative cells of the myeloid lineage. In this study, we show that the viral UL144 gene is expressed during latent infection in two cell types of the myeloid lineage, CD34؉ and CD14 ؉ monocytes, and that the UL144 protein is functional in latently infected monocytes. However, this latency-associated expression of UL144 occurs only in certain isolates of HCMV and depends on the presence of functional GATA-2 transcription factor binding sites in the UL144 promoter, in contrast to the viral latency-associated gene LUNA, which we also show is regulated by GATA-2 but expressed uniformly during latent infection independent of the virus isolate. Taken together, these data suggest that the HCMV latency-associated transcriptome may be virus isolate specific and dependent on the repertoire of transcription factor binding sites in the promoters of latencyassociated genes.

Human cytomegalovirus (HCMV) is a widely prevalent human herpesvirus, which, after primary infection, persists in the host for life. In healthy individuals, the virus is well controlled by the HCMV-specific T cell response. A key feature of this persistence, in the face of a normally robust host immune response, is the establishment of viral latency. In contrast to lytic infection, which is characterised by extensive viral gene expression and virus production, long-term latency in cells of the myeloid lineage is characterised by highly restricted expression of viral genes, including UL138 and LUNA. Here we report that both UL138 and LUNA-specific T cells were detectable directly ex vivo in healthy HCMV seropositive subjects and that this response is principally CD4+ T cell mediated. These UL138-specific CD4+ T cells are able to mediate MHC class II restricted cytotoxicity and, importantly, show IFNγ effector function in the context of both lytic and latent infection. Furthermore, in contrast to CD4+ T cells specific to antigens expressed solely during lytic infection, both the UL138 and LUNA-specific CD4+ T cell responses included CD4+ T cells that secreted the immunosuppressive cytokine cIL-10. We also show that cIL-10 expressing CD4+ T-cells are directed against latently expressed US28 and UL111A. Taken together, our data show that latency-associated gene products of HCMV generate CD4+ T cell responses in vivo, which are able to elicit effector function in response to both lytic and latently infected cells. Importantly and in contrast to CD4+ T cell populations, which recognise antigens solely expressed during lytic infection, include a subset of cells that secrete the immunosuppressive cytokine cIL-10. This suggests that HCMV skews the T cell responses to latency-associated antigens to one that is overall suppressive in order to sustain latent carriage in vivo.

Human cytomegalovirus (HCMV) infection and periodic reactivation are generally well controlled by the HCMV-specific T cell response in healthy people. While the CD8 ϩ T cell response to HCMV has been extensively studied, the HCMVspecific CD4 ϩ T cell effector response is not as well understood, especially in the context of direct interactions with HCMV-infected cells. We screened the gamma interferon (IFN-␥) and interleukin-10 (IL-10) responses to 6 HCMV peptide pools (pp65, pp71, IE1, IE2, gB, and US3, selected because they were the peptides most frequently responded to in our previous studies) in 84 donors aged 23 to 74 years. The HCMV-specific CD4 ϩ T cell response to pp65, IE1, IE2, and gB was predominantly Th1 biased, with neither the loss nor the accumulation of these responses occurring with increasing age. A larger proportion of donors produced an IL-10 response to pp71 and US3, but the IFN-␥ response was still dominant. CD4 ϩ T cells specific to the HCMV proteins studied were predominantly effector memory cells and produced both cytotoxic (CD107a expression) and cytokine (macrophage inflammatory protein 1␤ secretion) effector responses. Importantly, when we measured the CD4 ϩ T cell response to cytomegalovirus (CMV)-infected dendritic cells in vitro, we observed that the CD4 ϩ T cells produced a range of cytotoxic and secretory effector functions, despite the presence of CMV-encoded immune evasion molecules. CD4 ϩ T cell responses to HCMV-infected dendritic cells were sufficient to control the dissemination of virus in an in vitro assay. Together, the results show that HCMV-specific CD4 ϩ T cell responses, even those from elderly individuals, are highly functional and are directly antiviral.IMPORTANCE Human cytomegalovirus (HCMV) infection is carried for a lifetime and in healthy people is kept under control by the immune system. HCMV has evolved many mechanisms to evade the immune response, possibly explaining why the virus is never eliminated during the host's lifetime. The dysfunction of immune cells associated with the long-term carriage of HCMV has been linked with poor responses to new pathogens and vaccines when people are older. In this study, we investigated the response of a subset of immune cells (CD4 ϩ T cells) to HCMV proteins in healthy donors of all ages, and we demonstrate that the functionality of CD4 ϩ T cells is maintained. We also show that CD4 ϩ T cells produce effector functions in response to HCMV-infected cells and can prevent virus spread. Our work demonstrates that these HCMV-specific immune cells retain many important functions and help to prevent deleterious HCMV disease in healthy older people.

CD8؉ T cells specific for pp65, IE1, and IE2 are present at high frequencies in human cytomegalovirus (HCMV)-seropositive individuals, and these have been shown to have phenotypes associated with terminal differentiation, as well as both cytokine and proliferative dysfunctions, especially in the elderly. However, more recently, T cell responses to many other HCMV proteins have been described, but little is known about their phenotypes and functions. Consequently, in this study, we chose to determine the diversity of HCMVspecific CD8؉ T cell responses to the products of 11 HCMV open reading frames (ORFs) in a cohort of donors aged 20 to 80 years old as well as the ability of the T cells to secrete gamma interferon (IFN-␥). Finally, we also tested their functional antiviral capacity using a novel viral dissemination assay. We identified substantial CD8 ؉ T cell responses by IFN-␥ enzyme-linked immunospot (ELISPOT) assays to all 11 of these HCMV proteins, and across the cohort, individuals displayed a range of responses, from tightly focused to highly diverse, which were stable over time. CD8؉ T cell responses to the HCMV ORFs were highly differentiated and predominantly CD45RA ؉ , CD57 ؉ , and CD28 ؊ , across the cohort. These highly differentiated cells had the ability to inhibit viral spread even following direct ex vivo isolation. Taken together, our data argue that HCMV-specific CD8 ؉ T cells have effective antiviral activity irrespective of the viral protein recognized across the whole cohort and despite viral immune evasion. IMPORTANCEHuman cytomegalovirus (HCMV) is normally carried without clinical symptoms and is widely prevalent in the population; however, it often causes severe clinical disease in individuals with compromised immune responses. HCMV is never cleared after primary infection but persists in the host for life. In HCMV carriers, the immune response to HCMV includes large numbers of virus-specific immune cells, and the virus has evolved many mechanisms to evade the immune response. While this immune response seems to protect healthy people from subsequent disease, the virus is never eliminated. It has been suggested that this continuous surveillance by the immune system may have deleterious effects in later life. The study presented in this paper examined immune responses from a cohort of donors and shows that these immune cells are effective at controlling the virus and can overcome the virus' lytic cycle immune evasion mechanisms.

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.