Diverse specificities, phenotypes, and antiviral activities of cytomegalovirus-specific CD8+ T cells.

UNLABELLED: 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 HCMV-specific 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-γ). Finally, we also tested their functional antiviral capacity using a novel viral dissemination assay. We identified substantial CD8(+) T cell responses by IFN-γ 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. IMPORTANCE: Human 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.

Human cytomegalovirus latency-associated proteins elicit immune-suppressive IL-10 producing CD4⁺ T cells.

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 CDCD4⁺ 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.

How understanding immunology contributes to managing CMV disease in immunosuppressed patients: now and in future.

Several decades of research on human cytomegalovirus (HCMV) and the principal mammalian cytomegaloviruses which to varying degrees act as models of HCMV infection, particularly murine, guinea pig and rhesus CMV, have led to the recognition of the CMVs as interesting models of persistent infection with a large and complex DNA virus, which have been highly informative of the immunology and molecular pathogenesis of the virus-host relationship in the normal host. However, it is appropriate to ask how this relative wealth of knowledge has influenced the understanding and management of clinical disease due to HCMV. This article considers the immunology of cytomegalovirus in the normal human host, and the interrelated issue of the sites of HCMV latency and mechanisms of reactivation in the myeloid cell lineage, and in related in vitro model systems. The way in which this site of latency conditions the immune response, and emerging information on the special features of the adaptive immune response to HCMV during latency are also considered. Examples of HCMV disease associated with acquired immunosuppression, principally in the context of transplantation, but also as a consequence of HIV/AIDS and immune reconstitution inflammatory syndrome, are then discussed, with a particular emphasis on how understanding the immunology of persistent infection may contribute to managing CMV disease now and in future.

Human cytomegalovirus latency alters the cellular secretome, inducing cluster of differentiation (CD)4+ T-cell migration and suppression of effector function.

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.

Intracellular sequestration of the NKG2D ligand ULBP3 by human cytomegalovirus.

Human CMV (HCMV) encodes multiple genes that control NK cell activation and cytotoxicity. Some of these HCMV-encoded gene products modulate NK cell activity as ligands expressed at the cell surface that engage inhibitory NK cell receptors, whereas others prevent the infected cell from upregulating ligands that bind to activating NK cell receptors. A major activating NKR is the homodimeric NKG2D receptor, which has eight distinct natural ligands in humans. It was shown that HCMV is able to prevent the surface expression of five of these ligands (MIC A/B and ULBP1, 2, and 6). In this article, we show that the HCMV gene product UL142 can prevent cell surface expression of ULBP3 during infection. We further show that UL142 interacts with ULBP3 and mediates its intracellular retention in a compartment that colocalizes with markers of the cis-Golgi complex. In doing so, UL142 prevents ULBP3 trafficking to the surface and protects transfected cells from NK-mediated cytotoxicity. This is the first description of a viral gene able to mediate downregulation of ULBP3.

In vitro generation of human cytotoxic lymphocytes by virus. Viral glycoproteins induce nonspecific cell-mediated cytotoxicity without release of interferon.

Purified hemagglutinin and fusion glycoproteins of measles virus either in soluble form or inserted in artifical membranes bind to human peripheral blood lymphocytes and induce cell-mediated cytotoxicity (CMC) in a dose-response fashion. Both autologous and heterologous noninfected target cells are lysed in vitro. The expression of CMC is not inhibited by anti-measles virus antibody added to lymphocytes previously exposed to viral glycoproteins. THe killer lymphocytes are Fc receptor positive, both erythrocyte-rosetting and non-erythrocyte-rosetting, as assessed by both positive and negative selection experiments. The induction of nonspecific CMC by viral glycoproteins either in the soluble state or inserted into artificial membranes could be segregated from the CMC associated with whole virions. First, on kinetics studies, purified viral glycoproteins induced CMC more rapidly than did whole virus. Second, viral glycoprotein-produced response occurred in the absence of detectable release of interferon into the culture medium, whereas CMC activity due to whole virions was associated with interferon release. The fact that purified measles virus glycoproteins integrated into artificial membrane bilayers were as efficient as their soluble counterparts in inducing CMC suggests that the hydrophobic portion of the glycoproteins was not involved in the induction and expression of the lytic activity. Purified glycoproteins from lymphocytic choriomeningitis virus behave similarly, although this virus is unrelated to measles virus. It is inferred that interferon-independent CMC induced by viral glycoproteins might account for some of the biological reactions occurring early in the control of a viral infection.

Lysis of measles virus-infected cells by the purified cytolytic alternative complement pathway and antibody.

The dependence of antibody-and-complement-mediated lysis of virus-infected cells on the alternative pathway was examined utilizing the isolated cytolytic alternative pathway--a system consisting of the six purified proteins of the alternative pathway of activation (C3, factors B and D, beta 1H, C3b inactivator and properdin), and the five proteins of the membrane attack pathway (C5--9) of complement. HeLa cells acutely infected with measles virus were lysed by anti-viral IgG and the isolated cytolytic alternative pathway with an efficiency comparable to whole human serum. IgG and its F(ab')2 fragment were equally effective in inducing lysis by the isolated cytolytic alternative pathway, binding of approximately equal to 5 X 10(7) molecules per cell being required for 50% lysis; in contrast, no lysis occurred when equivalen or greater amounts of Fab' were bound to the virus-infected cell. Properdin was required for lysis. No lysis occurred if properdin was deleted from the isolated cytolytic alternative pathway, and lysis was diminished by 80% in properdin-depleted serum. Uptake of [125I]C3b from the isolated alternative pathway onto measles virus-infected cells occurred in the absence of properdin, but was accelerated in the presence of properdin. The 11 proteins of the isolated cytolytic alternative pathway are thus sufficient for lysis of measles virus-infected cells bearing anti-viral IgG or F(ab')2 without any other serum protein.

Human cytomegalovirus-specific cytotoxic T cells. Relative frequency of stage-specific CTL recognizing the 72-kD immediate early protein and glycoprotein B expressed by recombinant vaccinia viruses.

CTL are held to be an important host defense mechanism in persistent herpes-virus infections. We have therefore studied the nature and specificity of human cytomegalovirus (HCMV)-specific CTL in normal persistently infected individuals. This was achieved by using vaccinia recombinants encoding viral genes expressed at different stages of the virus replicative cycle, a structural glycoprotein gB (vac.gB) and the major 72-kD immediate early nonstructural protein (vac.IE) of HCMV, combined with limiting dilution analysis of the CTL response. In two subjects, 43 and 58% of HCMV CTL precursors (CTLp) lysed vac.IE-infected cells, in contrast to less than 6% lysing gB-infected cells. HCMV-specific CTL could also be generated by secondary in vitro stimulation with vac.gB- but not vac.IE-infected autologous fibroblasts. The high frequency of 72-kD IE protein-specific CTL suggests that this is at least a major recognition element for the HCMV-specific CTL response in asymptomatic persistently infected individuals, and CTL with this specificity may be important in maintaining the normal virus/host equilibrium.

Metabolism of the fifth component of complement, and its relation to metabolism of the third component, in patients with complement activation.

The metabolism of the fifth component of complement (C5), and its relatonship to metabolism of the third component of complement (C3), has been studied in normal subjects and patients by simultaneous administration of radioiodine labeled C5 and C3. In seven normal subjects the fractional catabolic rate of C5 ranged from 1.5 to 2.1% of the plasma pool/h and extravascular/intravascular distribution ratio from 0.22 to 0.78, these values being similar to those obtained for C3, and synthesis rate from 71 to 134 mug/kg per h, In patients with complement activation the increase in fractional catabolic rate of C5 was nearly always less than that of C3. The data also showed that there was increased extravascular distribution of C3 and C5 in most patients and considerable extravascular catabolism of both proteins in some. However, there were differences in metabolic parameters between patients with different types of complement activation. In patients with systemic lupus erythematosus, fractional catabolism and extravascular distribution of C3 and C5 were both increased, and there was marked extravascular catabolism of both proteins. There was increased fractional catabolism and extravascular distribution of C3 in patients with mesangiocapillary nephritis and (or) partial lipodystrophy, and fractional catabolism of C5 was also increased in three of six studies although distribution of C5 was always within the normal range; however, in two patients with nephritic factor in their serum fractional catabolism of C5 was normal despite markedly increased C3 turnover, suggesting that in patients with alternative pathway activation by nephritic factor little or no C5 convertase is generated.

Human cytomegalovirus-specific immunity following haemopoietic stem cell transplantation.

The herpesvirus Human Cytomegalovirus (HCMV) is an important opportunistic infection in recipients of allogeneic haemopoietic stem cell transplants, in whom HCMV-specific CD8+ and CD4+ T-cell responses are impaired. The nature of the HCMV-specific T-cell response in healthy virus carriers has been characterised in detail. High frequencies of circulating CD8+ T-cells that recognise defined viral peptides are maintained for years, and include individual CD8+ clones that have undergone extensive clonal expansion and phenotypic diversification in vivo. Following stem cell transplantation, the kinetics of HCMV-specific CD8+ T-cell reconstitution in the recipient are related to the presence or absence of antigen-experienced CD8+ T-cells transferred via the allograft, and to the presence of the virus in the recipient. We discuss recent progress in our understanding of HCMV-specific immunity in healthy virus carriers and in recipients after alloSCT.

Automation of limiting dilution cytotoxicity assays.

Limiting dilution analysis is a valuable technique for the quantitation and clonal analysis of immunocompetent cells. However, manual processing of the large number of samples necessary for satisfactory statistical analysis is laborious, and consequently results in inaccuracies reflected as increased standard errors. We describe the application of an automated robotic liquid handling tool to process samples in limiting dilution cytotoxicity assays. Our studies have shown that automated liquid handling is more accurate than manual methods, and that errors are limited. This results in savings of both time and resources. Furthermore, the process may be adapted for the safe, remote handling of sterile cell cultures, and human pathogens.

Unusual megaloblastic anaemia wiht multinucleate erythroblasts: two cases with septicaemia and acute renal failure.

The case histories and blood pictures of two patients who had cardiac lesions, septicaemia, and renal failure and terminally developed a leucoerythroblastic anaemia with megloblastic features associated with multinucleate erythroblasts, are described. Though folate deficiency may have made a minor contribution to the blood abnormalities, it is considered that some other disturbance in erythropoiesis was responsible for the bizarre blood and bone marrow changes in these patients. Similar cases reported in the earlier literature are reviewed.

Pathogenesis of human cytomegalovirus disease and the kidney.

Uncertainty still surrounds many aspects of the pathogenesis of human cytomegalovirus (HCMV) infection. During asymptomatic infection HCMV is probably present in a small fraction of peripheral blood mononuclear cells (most likely monocytes); in active infection HCMV is detectable in a wider range of cells. In vitro experiments suggest that cellular control of transcription of HCMV immediate early (IE) gene expression is one key determinant of HCMV reactivation. The cytotoxic T cell response to IE and other virus gene products is probably important in subsequently limiting dissemination of HCMV infection. The basis for any association between active HCMV disease and allograft rejection is unclear, although homologies between cellular and viral proteins suggest possible mechanisms. Although HCMV may be associated with tubulointerstitial disease, there is no unequivocally established role for HCMV in the pathogenesis of human glomerular disease.

Clinical aspects and management of cytomegalovirus infection.

The previous article discussed recent advances in understanding the biology of HCMV infection. Here we discuss current approaches to the clinical management of HCMV disease, and how understanding the biology of the virus may affect these.

Latency and reactivation of human cytomegalovirus.

The sequence analysis of herpesviruses suggests they have been evolving with their individual vertebrate hosts for millions of years, and their divergence parallels that of the hosts they infect. Given this time they have been learning to live with their individual hosts, it is not surprising that they have become extremely well adapted to doing so without causing much in the way of obvious disease. A key feature of their strategy for persisting in the host is the ability of all herpesviruses to establish latent infection-a state in which no, or only a very limited set of, viral genes are expressed in cells in which viral DNA persists. The alpha herpesviruses (herpes simplex and varicella zoster virus) establish latency in neuronal cells in sensory ganglia: these are long lived non-dividing cells and the alpha herpesviruses persist in these with expression of only the latency associated transcripts-although the function of these RNA transcripts remains incompletely understood. The principal gamma herpesvirus of humans, Epstein Barr virus (EBV), is latent mainly in B lymphocytes: EBV persistence in B cells is associated with expression of a limited set of viral genes encoding functions necessary for the maintenance of the episomal viral DNA as B cells divide.The mechanism by which the principal beta herpesvirus of humans-human cytomegalovirus (HCMV) persists, is also incompletely understood and the subject of this review. Understanding how HCMV persists has clinical relevance in that its transmission to seronegative recipients might be more easily prevented, and the mechanisms by which it produces disease in the neonate and immunocompromised hosts more easily understood, if we knew more about the cells in which the virus is latent and the way in which it reactivates.

Analysis of cytotoxic T-cell responses to human cytomegalovirus infection.

Inferential evidence from studies of immunosuppressed humans indicates the control of disease due to human cytomegalovirus (HCMV) in the persistently infected host (virus carrier) depends on the cellular immune response: cytotoxic T lymphocytes (CTL) appear to be a particularly important component of this response (1,2).