Cytomegalovirus IE1 Research Articles

Polyfunctional T-Cell Signatures to Predict Protection from Cytomegalovirus after Lung Transplantation.

Cytomegalovirus (CMV), which is one of the most common infections after lung transplantation, is associated with chronic lung allograft dysfunction and worse post-transplantation survival. Current approaches for at-risk patients include a fixed duration of antiviral prophylaxis despite the associated cost and side effects. We sought to identify a specific immunologic signature that predicted protection from subsequent CMV. CMV-seropositive lung transplantation recipients were included in the discovery (n = 43) and validation (n = 28) cohorts. Polyfunctional CMV-specific immunity was assessed by stimulating peripheral blood mononuclear cells with CMV pp65 or IE-1 peptide pools and then by measuring T-cell expression of CD107a, IFN-γ, tumor necrosis factor-α (TNF-α), and IL-2. Recipients were prospectively monitored for subsequent viremia. A Cox proportional hazards regression model that considered cytokine responses individually and in combination was used to create a predictive model for protection from CMV reactivation. This model was then applied to the validation cohort. Using the discovery cohort, we identified a specific combination of polyfunctional T-cell subsets to pp65 that predicted protection from subsequent CMV viremia (concordance index 0.88 [SE, 0.087]). The model included both protective (CD107a(-)/IFN-γ(+)/IL-2(+)/TNF-α(+) CD4(+) T cells, CD107a(-)/IFN-γ(+)/IL-2(+)/TNF-α(+) CD8(+) T cells) and detrimental (CD107a(+)/IFN-γ(+)/IL-2(-)/TNF-α(-) CD8(+) T cells) subsets. The model was robust in the validation cohort (concordance index 0.81 [SE, 0.103]). We identified and validated a specific T-cell polyfunctional response to CMV antigen stimulation that provides a clinically useful prediction of subsequent cytomegalovirus risk. This novel diagnostic approach could inform the optimal duration of individual prophylaxis.

The immunological footprint of CMV in HIV-1 patients stable on long-term ART.

BackgroundMost HIV-infected persons are cytomegalovirus (CMV) seropositive and retain latent virus that can be reactivated by immune activation. Their T cell populations express markers reflecting a late stage of differentiation, but the contributions of HIV and CMV to this profile are unclear. We investigated the immunological “footprint” of CMV in HIV patients who had a history of extreme immunodeficiency but were now stable on antiretroviral therapy (ART).ResultsTwenty CMV seropositive HIV patients >50 years old with nadir CD4 T-cell counts <200 cells/μl were studied after >12 years on ART. 16 CMV seropositive and 9 CMV seronegative healthy controls were included. CMV antibody titres were higher in HIV patients than controls (P < 0.001-0.003). Levels of soluble B-cell activating factor (sBAFF) were elevated in patients (P = 0.002) and correlated with levels of CMV antibodies (P = 0.03-0.002), with no clear relationship in controls. CD8 T-cell IFNγ responses to the IE1 peptide (VLE) remained elevated in HIV patients (P = 0.005). The CD57+CD45RA+CD27− phenotype of CD8 T-cells correlated with age (r = 0.60, P = 0.006), antibodies against CMV IE1 protein (r = 0.44, P = 0.06) and CD4 T-cell IFNγ response to CMV lysate (r = 0.45, P = 0.05).ConclusionsHumoral and T-cell responses to CMV remained elevated in HIV patients after >12 years on ART. Age and presence of CMV disease influenced CD8 T-cell phenotypes. Elevated levels of sBAFF may be a consequence of HIV disease and contribute to high titres of CMV antibody.Electronic supplementary materialThe online version of this article (doi:10.1186/s12979-015-0041-0) contains supplementary material, which is available to authorized users.

Diversified phenotype of antigen specific CD8+ T cells responding to the immunodominant epitopes of IE and pp65 antigens of human cytomegalovirus

To study the cytomegalovirus (CMV)-specific CD8+ T cells in individuals with HLA A∗1101, A∗0201 and A∗2402, our findings showed that peptide SK-10-2, KI-10 and KV-10 of CMV IE and pp65 antigens were immunodominant in 198 individuals with HLA A∗1101, A∗0201 and A∗2402, the most frequent genotypes in Chinese. Interestingly, SK-10-2 induced the strongest T cell response to produce IFN-γ whereas the others did not induce prominent IFN-γ production despite they all induced remarkable T cell proliferation. The peptides induced different phenotypes including IFN-γhighTNF-αlow and TNF-αlowFoxp3low. It suggests that only some of CMV-reactive CD8+ T cells are real protective IFN-γhigh cytotoxic T cells.

Positive role of promyelocytic leukemia protein in type I interferon response and its regulation by human cytomegalovirus.

Promyelocytic leukemia protein (PML), a major component of PML nuclear bodies (also known as nuclear domain 10), is involved in diverse cellular processes such as cell proliferation, apoptosis, gene regulation, and DNA damage response. PML also acts as a restriction factor that suppresses incoming viral genomes, therefore playing an important role in intrinsic defense. Here, we show that PML positively regulates type I interferon response by promoting transcription of interferon-stimulated genes (ISGs) and that this regulation by PML is counteracted by human cytomegalovirus (HCMV) IE1 protein. Small hairpin RNA-mediated PML knockdown in human fibroblasts reduced ISG induction by treatment of interferon-β or infection with UV-inactivated HCMV. PML was required for accumulation of activated STAT1 and STAT2, interacted with them and HDAC1 and HDAC2, and was associated with ISG promoters after HCMV infection. During HCMV infection, viral IE1 protein interacted with PML, STAT1, STAT2, and HDACs. Analysis of IE1 mutant viruses revealed that, in addition to the STAT2-binding domain, the PML-binding domain of IE1 was necessary for suppression of interferon-β-mediated ISG transcription, and that IE1 inhibited ISG transcription by sequestering interferon-stimulated gene factor 3 (ISGF3) in a manner requiring its binding of PML and STAT2, but not of HDACs. In conclusion, our results demonstrate that PML participates in type I interferon-induced ISG expression by regulating ISGF3, and that this regulation by PML is counteracted by HCMV IE1, highlighting a widely shared viral strategy targeting PML to evade intrinsic and innate defense mechanisms.

Analysis of the Functional Interchange between the IE1 and pp71 Proteins of Human Cytomegalovirus and ICP0 of Herpes Simplex Virus 1

Human cytomegalovirus (HCMV) immediate early protein IE1 and the tegument protein pp71 are required for efficient infection. These proteins have some functional similarities with herpes simplex virus 1 (HSV-1) immediate early protein ICP0, which stimulates lytic HSV-1 infection and derepresses quiescent HSV-1 genomes. All three proteins counteract antiviral restriction mediated by one or more components of promyelocytic leukemia (PML) nuclear bodies, and IE1 and pp71, acting together, almost completely complement ICP0 null mutant HSV-1. Here, we investigated whether ICP0 might substitute for IE1 or pp71 during HCMV infection. Using human fibroblasts that express ICP0, IE1, or pp71 in an inducible manner, we found that ICP0 stimulated replication of both wild-type (wt) and pp71 mutant HCMV while IE1 increased wt HCMV plaque formation and completely complemented the IE1 mutant. Although ICP0 stimulated IE2 expression from IE1 mutant HCMV and increased the number of IE2-positive cells, it could not compensate for IE1 in full lytic replication. These results are consistent with previous evidence that both IE1 and IE2 are required for efficient HCMV gene expression, but they also imply that IE2 functionality is influenced specifically by IE1, either directly or indirectly, and that IE1 may include sequences that have HCMV-specific functions. We discovered a mutant form of IE1 (YL2) that fails to stimulate HCMV infection while retaining 30 to 80% of the activity of the wt protein in complementing ICP0 null mutant HSV-1. It is intriguing that the YL2 mutation is situated in the region of IE1 that is shared with IE2 and which is highly conserved among primate cytomegaloviruses. Herpesvirus gene expression can be repressed by cellular restriction factors, one group of which is associated with structures known as ND10 or PML nuclear bodies (PML NBs). Regulatory proteins of several herpesviruses interfere with PML NB-mediated repression, and in some cases their activities are transferrable between different viruses. For example, the requirement for ICP0 during herpes simplex virus 1 (HSV-1) infection can be largely replaced by ICP0-related proteins expressed by other alphaherpesviruses and even by a combination of the unrelated IE1 and pp71 proteins of human cytomegalovirus (HCMV). Here, we report that ICP0 stimulates gene expression and replication of wt HCMV but cannot replace the need for IE1 during infection by IE1-defective HCMV mutants. Therefore, IE1 includes HCMV-specific functions that cannot be replaced by ICP0.

Generation of Antigen-Specific Cytotoxic T Lymphocytes with Peripheral B Cells Activated By α-Galactosylceramide

Dendritic cells (DCs) are well known as the most potent professional antigen presenting cells (APCs). Nonetheless, the use of these cells in immunotherapy has been limited due to the time consuming and laborious steps required to generate DCs from monocytes in vitro. Therefore, alternative APCs has drawn much attention because of their relative convenience in manipulation.In this study, the efficacy of B cells as APCs, as compared to DCs, in induction of cytotoxic T lymphocytes (CTLs) against cytomegalovirus (CMV)-specific antigens was evaluated. B cells were isolated by depletion of peripheral blood mononuclear cell (PBMCs) from healthy individuals with MACS system, loaded with α-galactosylceramide (α-GalCer) for inducing B cell activation, and nucleofected with CMV-antigen coding plasmid DNA, pCK-pp65-IRES-IE1. As other APCs, monocyte-derived DCs were induced with various cytokines (GM-CSF, IL-4, IL-1b, TNF-a), for 6 days and nucleofected with the same plasmid DNA. Ag-nucleofected B cells or DCs were cocultured with T cells for 14 days in vitro. The cells were harvested and subsequently immunoassayed.Proliferation of cells was more expanded by about 25~32% in CMV-CTLs induced by DCs compared to of B cells, but there was no significant difference in immunogenicity between CMV-CTLs induced with B cells and DCs. Compared to CMV-CTLs induced by DCs, the CTLs induced by α-GalCer-loaded B cells induced similar cytotoxicity against CMV antigen (Ag) in vitro. The CMV-CTLs by α-GalCer-loaded B cells recognized CMV antigen pp65 (median 88 SFC/105) and IE-1 (median 86 SFC/105) in donor 1, and CMV antigen pp65 (median 31 SFC/105) and IE-1 (median 37 SFC/105) in donor 2. Similarly, the CMV-CTLs by DCs recognized CMV antigen pp65 (median 133 SFC/105) and IE-1 (median 32 SFC/105) in donor 1, and CMV antigen pp65 (median 37 SFC/105) and IE-1 (median 43 SFC/105) in donor 2.Immunogenicities of both CTLs were similar not only on IFN-γ ELISPOT (Enzyme-linked immunospot) assay but also on cytotoxicity assay. The CMV-CTLs by α-GalCer-loaded B cells have killing activity against CMV antigen pp65 (100%, at E:T ratio 10:1) and IE1 (85%, at E:T ratio 10:1) in donor 1, and CMV antigen pp65 (69%, at E:T ratio 10:1) and IE1 (27%, at E:T ratio 10:1) in donor 2. Also, the CMV-CTLs by DCs show killing activity against CMV antigen pp65 (100%, at E:T ratio 10:1) and IE1 (42%, at E:T ratio 10:1) in donor 1, and CMV antigen pp65 (88%, at E:T ratio 10:1) and IE1 (64%, at E:T ratio 10:1) in donor 2.These observations suggest that α-GalCer-loaded B cells could be used in general as APCs instead of DCs. Using the B cells as APCs have several benefits such as cost-effectiveness, less time-consuming, and less laborious compared to when DCs are used. Furthermore, nucleofection technique might be useful in delivering antigen-coding DNA, not only for virus antigens but also for tumor antigens, directly into the nucleus. Our results demonstrate that α-GalCer-loaded B cells could be potent APCs in generating antigen-specific CTLs for cellular vaccines and adoptive immunotherapy.Acknowledgment: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2012R1A1A2008316) and we thank Ann M. Leen, Helen E. Heslop and Malcomn K. Brenner from Center for Cell and Gene Therapy, Baylor College of Medicine Center for their kind help. DisclosuresNo relevant conflicts of interest to declare.

Crystal structure of cytomegalovirus IE1 protein reveals targeting of TRIM family member PML via coiled-coil interactions.

PML nuclear bodies (PML-NBs) are enigmatic structures of the cell nucleus that act as key mediators of intrinsic immunity against viral pathogens. PML itself is a member of the E3-ligase TRIM family of proteins that regulates a variety of innate immune signaling pathways. Consequently, viruses have evolved effector proteins to modify PML-NBs; however, little is known concerning structure-function relationships of viral antagonists. The herpesvirus human cytomegalovirus (HCMV) expresses the abundant immediate-early protein IE1 that colocalizes with PML-NBs and induces their dispersal, which correlates with the antagonization of NB-mediated intrinsic immunity. Here, we delineate the molecular basis for this antagonization by presenting the first crystal structure for the evolutionary conserved primate cytomegalovirus IE1 proteins. We show that IE1 consists of a globular core (IE1CORE) flanked by intrinsically disordered regions. The 2.3 Å crystal structure of IE1CORE displays an all α-helical, femur-shaped fold, which lacks overall fold similarity with known protein structures, but shares secondary structure features recently observed in the coiled-coil domain of TRIM proteins. Yeast two-hybrid and coimmunoprecipitation experiments demonstrate that IE1CORE binds efficiently to the TRIM family member PML, and is able to induce PML deSUMOylation. Intriguingly, this results in the release of NB-associated proteins into the nucleoplasm, but not of PML itself. Importantly, we show that PML deSUMOylation by IE1CORE is sufficient to antagonize PML-NB-instituted intrinsic immunity. Moreover, co-immunoprecipitation experiments demonstrate that IE1CORE binds via the coiled-coil domain to PML and also interacts with TRIM5α We propose that IE1CORE sequesters PML and possibly other TRIM family members via structural mimicry using an extended binding surface formed by the coiled-coil region. This mode of interaction might render the antagonizing activity less susceptible to mutational escape.

177. A single bout of exercise augments the expansion of multi-virus specific T-cells in healthy humans

Viral infections are a major cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). The adoptive transfer of multi-virus specific cytotoxic T-cell lines (CTLs) expanded from a suitably matched donor can reproducibly control infections after HSCT. However, the failure to generate sufficient numbers of virus-specific CTLs ex vivo can often result in inadequate restoration of antiviral immunity and poor prognosis. Because exercise mobilizes large numbers of viral specific T-cells into the bloodstream, we hypothesized that a single bout of exercise would increase the expansion of multi-viral-specific CTLs in would be HSCT donors. Healthy CMV and EBV seropositive subjects performed 30-min of cycling exercise at +15% of the individual blood lactate threshold. A fixed number of PBMCs isolated before and immediately after exercise were pulsed with overlapping peptides spanning all target viral antigens and expanded with IL-4, IL-7 and IL-15 over 9-days. Exercise remarkably increased the expansion of cytomegalovirus (CMV) pp65 (∼3.8-fold), CMV IE-1 (∼4.7-fold) and Epstein-Barr virus (EBV) BMLF-1 (∼2.8-fold) specific CTLs over 9-days, although did not reliably increase the expansion of EBV LMP-2 specific CTLs. All virus-reacting cells were of a CD62L- memory phenotype and remained elevated after exercise even when adjusted for input T-cell numbers. We conclude that exercise may serve as a simple strategy to reliably augment the ex vivo expansion of viral-specific CTLs for adoptive transfer immunotherapy after HSCT.

Pretransplant CD8 T-Cell Response to IE-1 Discriminates Seropositive Kidney Recipients at Risk of Developing CMV Infection Posttransplant

Cytomegalovirus (CMV) infection is an ongoing clinical problem in solid-organ transplantation (SOT). Pretransplant CMV serology is currently the only tool for assessing the risk of CMV infection, although cellular immune responses driven by CMV-specific CD4 and CD8 T lymphocytes are important for controlling viral replication. Therefore, the analysis of CMV-specific T cells may be useful for estimating the risk of infection. This is a prospective study of patients with kidney transplants and no prophylactic treatment for CMV replication. CD4 and CD8 T-cell responses to the major CMV pp65 and IE-1 antigens in 15 seropositive patients at intermediate risk of CMV infection were investigated, according to current algorithms. Intracellular flow cytometry was employed to determine IFN-γ production as a functional readout. The response was analyzed in pretransplant samples and prospectively at 1 and 6 months and at 1 year posttransplant. It was observed that the CD8 responses to IE-1 antigen were practically absent pretransplant in patients who developed CMV infection posttransplant. Within the group of patients free of infection, CD8 responses to IE-1 were detected more frequently and were significantly higher (P=0.0083). In a receiver operating characteristics curve analysis (AUC=0.929; P=0.010; 95% CI: 0.078-1.0), low CD8 responses to IE-1 (≤0.05%) pretransplant predicted the development of CMV infection under the immunosuppressive regime after transplant with 100% specificity and 85.7% sensitivity. Assessment of IE-1-specific CD8 T-cell frequencies pretransplant may be a useful tool for identifying seropositive SOT patients at risk of developing CMV infection posttransplant.

The Human Cytomegalovirus IE1 Protein: Past and Present Developments

ABSTRACT: Human cytomegalovirus (HCMV), a member of the β-herpesvirus subfamily, is an important pathogen that infects the majority of the human population. The evolutionary success of HCMV largely depends on its ability to evade host defense systems and establish a lifelong persistence after primary infection. In fact, HCMV has dedicated a considerable part of its gene products to manipulate or disable immune effector processes. This review focuses on the major immediate–early protein IE1 – a multifunctional key regulator that has the capacity to counteract the first host defense activities. We summarize the known structural and mechanistic features by which IE1 modulates innate immune mechanisms as well as other cellular processes, and discuss how the individual functions of IE1 contribute to the success of a lytic HCMV infection.

Cytomegalovirus Vaccines

An effective cytomegalovirus (CMV) vaccine could prevent the majority of birth defects caused by congenital CMV infections. Candidate vaccines in clinical evaluation include live attenuated, protein subunit, DNA, and viral-vectored approaches. Subunit approaches have focused on the CMV proteins pp65 and IE1 as important inducers of cytotoxic T cells and glycoprotein B (gB) as an important inducer of neutralizing antibodies. A vaccine comprised of recombinant gB protein with MF59 adjuvant reduced the incidence of primary infection by 50%. Recent revelations regarding CMV entry pathways into different cell types suggest a possible course for improvement. A 5-subunit pentameric complex is uniquely required for endothelial and epithelial cell entry. Sera from naturally infected subjects contain high-potency neutralizing activities specific for this complex, whereas the gB/MF59 vaccine fails to induce comparable neutralizing activities. A vaccine's ability to induce salivary antibodies that neutralize epithelial cell entry may be especially important for preventing oral transmission as the first cells infected are presumably epithelial cells of the oral mucosa. In addition, recent evidence suggests that antibodies can inhibit postentry CMV spread between endothelial and epithelial cells. Such activities may serve to limit viral replication in tissues or impair dissemination to the placenta and fetus. Thus, inclusion of epitopes derived from the pentameric complex may provide enhanced efficacy by inducing potent neutralizing/spread-inhibiting antibodies that target virus replication in a broad spectrum of cell types. Next-generation vaccine candidates in preclinical development incorporate peptides, subunits, or multisubunit complexes representing parts or all of the pentameric complex. Approaches include peptides, recombinant proteins, DNA, replication-defective viral vectors, genetically disabled CMV, and inactivated CMV virions. The diversity of novel strategies under development engenders optimism that a successful candidate will emerge.

Genetically engineered fixed K562 cells: potent “off-the-shelf” antigen-presenting cells for generating virus-specific T cells

Background aimsThe human leukemia cell line K562 represents an attractive platform for creating artificial antigen-presenting cells (aAPC). It is readily expandable, does not express human leukocyte antigen (HLA) class I and II and can be stably transduced with various genes. MethodsIn order to generate cytomegalovirus (CMV) antigen-specific T cells for adoptive immunotherapy, we transduced K562 with HLA-A∗0201 in combination with co-stimulatory molecules. ResultsIn preliminary experiments, irradiated K562 expressing HLA-A∗0201 and 4-1BBL pulsed with CMV pp65 and IE-1 peptide libraries failed to elicit antigen-specific CD8+ T cells in HLA-A∗0201+ peripheral blood mononuclear cells (PBMC) or isolated T cells. Both wild-type K562 and aAPC strongly inhibited T cell proliferation to the bacterial superantigen staphylococcal enterotoxin B (SEB) and OKT3 and in mixed lymphocyte reaction (MLR). Transwell experiments suggested that suppression was mediated by a soluble factor; however, MLR inhibition was not reversed using transforming growth factor-β blocking antibody or prostaglandin E2 inhibitors. Full abrogation of the suppressive activity of K562 on MLR, SEB and OKT3 stimulation was only achieved by brief fixation with 0.1% formaldehyde. Fixed, pp65 and IE-1 peptide-loaded aAPC induced robust expansion of CMV-specific T cells. ConclusionsFixed gene-modified K562 can serve as effective aAPC to expand CMV-specific cytotoxic T lymphocytes for therapeutic use in patients after stem cell transplantation. Our findings have implications for broader understanding of the immune evasion mechanisms used by leukemia and other tumors.

Abstract 4777: Cytomegalovirus infection leads to pleomorphic rhabdomyosarcomas in Trp53 +/- mice.

Abstract Despite not being considered oncogenic, cytomegalovirus (CMV) has been detected in an increasing number of malignancies. However, recent studies have suggested mechanisms through which CMV may modulate the tumor environment, encouraging its study as a positive modifier of tumorigenesis. In this study, we investigated the effects of CMV infection in Trp53 heterozygous mice. Animals were infected with murine CMV (MCMV) after birth at 2 days (P2) or 4 weeks of age via intraperitoneal injection and then monitored for tumor formation. Additional control cohorts were mock-infected (saline vehicle) or infected with HSV-1, a similar herpes virus. Following MCMV infection, widespread systemic infection ensued followed by immune clearance of the virus resulting in seroconversion. Mice infected at P2 developed tumors at a high frequency (43%) by 9 months of age. In contrast, only 3% of mock-infected or mice infected at 4 weeks developed tumors. The majority of tumors from P2 MCMV-infected mice were pleomorphic rhabdomyosarcomas (RMS) as evidenced by MyoD1, Desmin, and Myogenin staining. Importantly, MCMV-infected tumors contained MCMV DNA, RNA, and protein. An examination of clinical cases revealed that human RMS (embryonal, alveolar, and pleomorphic) harbored human CMV IE1 and pp65 protein as well as viral RNA. All of the cases (n=18) tested positive for CMV genetic material. CMV protein was found throughout the tumor including pleomorphic cells. Additionally, perivascular areas demonstrated intense staining consistent with other reports of linking CMV with angiogenesis. To confirm the selective presence of HCMV DNA in human RMS, quantitative PCR analysis showed that tumors expressed an average of 81.1 copies/ng of HCMV-UL83 and of 22.2 copies/ng of HCMV-UL146 compared to 0 and 0.49 copies/ng in normal muscle, respectively. The amplified products’ nucleotide sequence was greater than 95% identical to that of a clinical strain Taken together, our findings offer support for the hypothesis that CMV contributes to the development of pleomorphic RMS in the context of Trp53 mutation, a situation which occurs with high frequency in human RMS. Citation Format: Richard L. Price, Katherine Bingmer, Lualhati Harkins, Hans Iwenofu, Chang-Hyuk Kwon, Charles Cook, Christopher Pelloski, E Antonio Chiocca. Cytomegalovirus infection leads to pleomorphic rhabdomyosarcomas in Trp53 +/- mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4777. doi:10.1158/1538-7445.AM2013-4777

Cytomegalovirus Infection Leads to Pleomorphic Rhabdomyosarcomas in Trp53+/− Mice

Cytomegalovirus (CMV) has been detected in several human cancers, but it has not proven to be oncogenic. However, recent studies have suggested mechanisms through which cytomegalovirus may modulate the tumor environment, encouraging its study as a positive modifier of tumorigenesis. In this study, we investigated the effects of cytomegalovirus infection in Trp53 heterozygous mice. Animals were infected with murine cytomegalovirus (MCMV) after birth at 2 days (P2) or 4 weeks of age and then monitored for tumor formation. Mice injected at 2 days of age developed tumors at a high frequency (43%) by 9 months of age. In contrast, only 3% of mock-infected or mice infected at 4 weeks developed tumors. The majority of tumors from P2 MCMV-infected mice were pleomorphic rhabdomyosarcomas (RMS) harboring MCMV DNA, RNA, and protein. An examination of clinical cases revealed that human RMS (embryonal, alveolar, and pleomorphic) harbored human cytomegalovirus IE1 and pp65 protein as well as viral RNA. Taken together, our findings offer support for the hypothesis that cytomegalovirus contributes to the development of pleomorphic RMS in the context of Trp53 mutation, a situation that occurs with high frequency in human RMS.

Lower proportion of naïve peripheral CD8+ T cells and an unopposed pro-inflammatory response to human Cytomegalovirus proteins in vitro are associated with longer survival in very elderly people

The low percentages of naïve T cells commonly observed in elderly people are thought to be causally associated with mortality, primarily from infectious disease, and are taken as a hallmark of “immunosenescence”. Whether low levels of naive cells actually do associate with mortality has, however, not been tested in longitudinal studies. Here, we present correlations between peripheral T-cell phenotypes and 8-year survival in individuals from the population-based prospective Leiden 85-plus Study. Counter-intuitively, we found that a lower frequency of naïve CD8+ T cells (characterized as CD45RA+CCR7+CD27+CD28+) at baseline (>88 years) correlated with significantly better survival, while there was a tendency for the reciprocal accumulation of late-differentiated effector memory cells (CD45RA−CCR7−CD27−CD28−) also to associate with better survival. These findings suggest that better retention of memory cells specific for previously encountered antigens may provide a survival advantage in this particular population. Given the prevalence of Cytomegalovirus (CMV) and its reported association with immunosenescence, we tested whether memory for this potential pathogen was relevant to survival. We found that individuals mounting an exclusively pro-inflammatory ex vivo response (TNF, IFN-γ, IL-17) to the major CMV target molecules pp65 and IE1 had a significant survival advantage over those also having anti-inflammatory responses (IL-10). These findings suggest that higher levels of naïve T cells may not necessarily be associated with a survival advantage and imply that the nature of immunosurveillance against CMV may be crucial for remaining longevity, at least in the very elderly.Electronic supplementary materialThe online version of this article (doi:10.1007/s11357-012-9425-7) contains supplementary material, which is available to authorized users.

Phenotypic and functional immune attributes predicting survival in very elderly people (47.7)

Abstract The low level of naïve T-cells commonly observed in the elderly is thought to be causally associated with mortality. However this has not been tested in longitudinal studies. Here, we present correlations between peripheral T-cell subsets and 8-year survival in individuals from the population-based prospective Leiden 85-plus Study. Counter-intuitively, a lower frequency of naïve (CD45RA+CCR7+CD27+CD28+) CD8+ T-cells at baseline (&amp;gt;88 years) correlated with significantly better survival, while there was a tendency for the reciprocal accumulation of late-differentiated effector memory cells (CD45RA-CCR7-CD27-CD28-) also to associate with better survival, suggesting that better retention of memory cells specific for previously encountered antigens provided a survival advantage in this particular population. Given the prevalence of Cytomegalovirus (CMV) and its reported association with immunosenescence, we tested whether memory for this potential pathogen was relevant to survival. We found that individuals mounting an exclusively pro-inflammatory ex-vivo response (TNF, IFN-γ, IL-17) to the major CMV target molecules pp65 and IE1 had a significant survival advantage over those also having anti-inflammatory responses (IL-10). These findings suggest that higher levels of naïve T cells are not necessarily associated with a survival advantage and imply that the nature of immunosurveillance against CMV may be crucial for remaining longevity, at least in the very elderly.

Generation of Polyclonal CMV-specific T Cells for the Adoptive Immunotherapy of Glioblastoma

Glioblastoma (GBM) is the most common primary brain cancer in adults and is virtually incurable. Recent studies have shown that cytomegalovirus (CMV) is present in majority of GBMs. To evaluate whether the CMV antigens pp65 and IE1, which are expressed in GBMs, could be targeted by CMV-specific T cells, we measured the frequency of T cells targeting pp65 and IE1 in the peripheral blood of a cohort of 11 sequentially diagnosed CMV-seropositive GBM patients, and evaluated whether it was feasible to expand autologous CMV-specific T cells for future clinical studies. All 11 CMV-seropositive GBM patients had T cells specific for pp65 and IE1 in their peripheral blood assessed by IFNγ enzyme-linked immunospot assay. However, the precursor frequency of pp65-specific T cells was decreased in comparison with healthy donors (P=0.001). We successfully reactivated and expanded CMV-specific T cells from 6 out of 6 GBM patients using antigen-presenting cells transduced with an adenoviral vector encoding pp65 and IE1. CMV-specific T-cell lines contained CD4 as well as CD8 T cells, recognized pp65 and IE1 targets and killed CMV-infected autologous GBM cells. Infusion of such CMV-specific T-cell lines may extend the benefits of T-cell therapy to patients with CMV GBMs.

Polyfunctional T Cells Accumulate in Large Human Cytomegalovirus-Specific T Cell Responses

Large cytomegalovirus (CMV)-specific CD8 T-cell responses are observed in both young and, somewhat more often, old people. Frequent CMV reactivation is thought to exhaust these cells and render them dysfunctional so that larger numbers of them are needed to control CMV. Expansions of CMV-specific CD4 T cells are also seen but are less well studied. In this study, we examined the T-cell response to the dominant CMV pp65 and IE-1 antigens in healthy CMV-infected people across a wide age range (20 to 84 years) by using multicolor flow cytometry. CMV-specific T cells were characterized by the activation markers CD40 ligand (CD40L), interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) and the memory markers CD27 and CD45RA. The proportions of effector memory T cells increased in large responses, as did the proportions of polyfunctional CD8 (IFN-γ(+) IL-2(+/-) TNF-α(+)) and CD4 (CD40L(+/-) IFN-γ(+) IL-2(+) TNF-α(+)) T-cell subsets, while the proportion of naïve T cells decreased. The bigger the CD4 or CD8 T-cell response to pp65, the larger was the proportion of T cells with an advanced memory phenotype in the entire (including non-CMV-specific) T-cell compartment. In addition, the number of activation markers per cell correlated with the degree of T-cell receptor downregulation, suggesting increased antigen sensitivity in polyfunctional cells. In summary, our findings show that polyfunctional CMV-specific T cells were not superseded by dysfunctional cells, even in very large responses. At the same time, however, the memory subset composition of the entire T-cell compartment correlated with the size of the T-cell response to CMV pp65, confirming a strong effect of CMV infection on the immune systems of some, but not all, infected people.

H2B homology region of major immediate–early protein 1 is essential for murine cytomegalovirus to disrupt nuclear domain 10, but is not important for viral replication in cell culture

Cytomegalovirus (CMV) major immediate-early protein 1 (IE1) has multiple functions and is important for efficient viral infection. As does its counterpart in human CMV, murine CMV (MCMV) IE1 also functions as a disruptor of mouse-cell nuclear domain 10 (ND10), where many different gene-regulation proteins congregate. It still remains unclear how MCMV IE1 disperses ND10 and whether this dispersion could have any effect on viral replication. MCMV IE1 is 595 aa long and has multiple functional domains that have not yet been fully analysed. In this study, we dissected the IE1 molecule by truncation and/or deletion and found that the H2B homology domain (amino acid sequence NDIFERI) is required for the dispersion of ND10 by IE1. Furthermore, we made additional deletions and point mutations and found that the minimal truncation in the H2B homology domain required for IE1 to lose the ability to disperse ND10 is just 3 aa (IFE). Surprisingly, the mutated IE1 still interacted with PML and co-localized with ND10 but failed to disperse ND10. This suggests that binding to ND10 key protein is essential to, but not sufficient for, the dispersal of ND10, and that some other unknown mechanism must be involved in this biological procedure. Finally, we generated MCMV with IFE-deleted IE1 (MCMVdlIFE) and its revertant (MCMVIFERQ). Although MCMVdlIFE lost the ability to disperse ND10, plaque assays and viral gene production assays showed that the deletion of IFE did not increase viral replication in cell culture. We conclude that the dispersion of ND10 appears not to be important for MCMV replication in a mouse-cell culture.

The synergistic effects of cytomegalovirus IE2 and myocardin on cardiomyocyte hypertrophy

Human cytomegalovirus immediate early proteins (CMV IEs) are involved in transcriptional activities of both host and virus gene expression. This study shows that the transcriptional activity of myocardin in regulating cardiomyocyte hypertrophy is enhanced by co-expressing CMV IE2. Forced expression of IE2 increases the augmented cell size of neonatal rat cardiac myocytes induced by myocardin, as well as the mRNA and protein levels of hypertrophic genes, whereas deletion of CArG boxes in the atrial natriuretic factor (ANF) promoter attenuates the effect of CMV IE2 with myocardin. In conclusion, CMV IE2 synergistically stimulates myocardin transactivity in the hypertrophic marker gene ANF in a CArG box-dependent manner. Our study indicates that the association of CMV IEs with myocardin-induced transcription may be involved in myocardin-mediated cardiac hypertrophy.