Cytomegalovirus infection in Gambian infants leads to profound CD8 T-cell differentiation

Cytomegalovirus (CMV) infection is endemic in Gambian infants, with 62% infected by 3 months and 85% by 12 months of age. We studied the CD8 T-cell responses of infants to CMV following primary infection. CMV-specific CD8 T cells, identified with tetramers, showed a fully differentiated phenotype (CD28(-) CD62L(-) CD95(+) perforin(+) granzyme A(+) Bcl-2(low)). Strikingly, the overall CD8 T-cell population developed a similar phenotype following CMV infection, which persisted for at least 12 months. In contrast, primary infection was accompanied by up-regulation of markers of activation (CD45R0 and HLA-D) on both CMV-specific cells and the overall CD8 T-cell population and division (Ki-67) of specific cells, but neither pattern persisted. At 12 months of age, the CD8 T-cell population of CMV-infected infants was more differentiated than that of uninfected infants. Although the subpopulation of CMV-specific cells remained constant, the CMV peptide-specific gamma interferon response was lower in younger infants and increased with age. As the CD8 T-cell phenotype induced by CMV is indicative of immune dysfunction in the elderly, the existence of a similar phenotype in large numbers of Gambian infants raises the question of whether CMV induces a similarly deleterious effect.     

Herpesvirus-specific CD8 T cell immunity in old age: cytomegalovirus impairs the response to a coresident EBV infection

Aging in humans is associated with increased infections and the reduced proliferative capacity of T cells, part of the more global phenomenon termed immune senescence. The etiology of immune senescence is unknown but the accumulation of virus-specific memory T cells may be a contributory factor. We have examined CD8 T cell responses to two persistent herpesvirus infections, CMV and EBV, and to a recurrent virus infection, influenza, in different age cohorts of healthy donors using HLA-peptide tetramers and intracellular cytokine detection. Of these, CMV appears to be the most immunogenic, with the CD8 T cell response representing over 10% of the CD8 pool in many elderly donors. Interestingly, the effect of age upon EBV-specific responses depends upon donor CMV sero-status. In CMV seropositive donors, the magnitude of the EBV-specific immune response is stable with age, but in CMV seronegative donors, the response to EBV increases significantly with age. By contrast, the influenza-specific CD8 T cell immune response decreases with age, independent of CMV status. The functional activity of the herpesvirus-specific immune response decreases in elderly donors, although the characteristic phenotypes of CMV- and EBV-specific memory populations are retained. This demonstrates that aging is associated with a marked accumulation of CMV-specific CD8 T cells together with a decrease in immediate effector function. Moreover, infection with CMV can reduce prevailing levels of immunity to EBV, another persistent virus. These results suggest that carriage of CMV may be detrimental to the immunocompetent host by suppressing heterologous virus-specific immunity during aging.     

Functional Exhaustion of CD4+ T Lymphocytes during Primary Cytomegalovirus Infection

Human CMV establishes lifelong persistence after primary infection. Chronic CMV infection is associated with intermittent viral reactivation inducing high frequencies of CD4(+) T lymphocytes with potent antiviral and helper properties. Primary CMV infection is characterized by an intense viral replication lasting for several months. The impact of this prolonged exposure to high Ag loads on the functionality of CD4(+) T cells remains incompletely understood. In pregnant women with primary CMV infection, we observed that CMV-specific CD4(+) T lymphocytes had a decreased capacity to proliferate and to produce IL-2. A very large proportion of CMV-specific CD4(+) T cells had downregulated the expression of CD28, a costimulatory molecule centrally involved in the production of IL-2. Unexpectedly, both CD28(-) and CD28(+)CD4(+) T cells produced low levels of IL-2. This defective production of IL-2 was part of a larger downregulation of cytokine production. Indeed, CMV-specific CD4(+) T cells produced lower amounts of IFN-γ and TNF-α and showed lower functional avidity during primary as compared with chronic infection. Increased programmed death-1 expression was observed in CD28(+) CMV-specific CD4(+) T cells, and programmed death-1 inhibition increased proliferative responses. These results indicate that primary CMV infection is associated with the exhaustion of CMV-specific CD4(+) T cells displaying low functional avidity for viral Ags.     

Cytomegalovirus seropositivity drives the CD8 T cell repertoire toward greater clonality in healthy elderly individuals

The deterioration in immune function with aging is thought to make a major contribution to the increased morbidity and mortality from infectious disease in old age. One aspect of immune senescence is the reduction in CD8 T cell repertoire as due to the accumulation of oligoclonal, memory T cells and a reduction in the naive T cell pool. CD8 T cell clonal expansions accumulate with age, but their antigenic specificity remains unknown. In this study, we show that in elderly individuals seropositivity for human CMV leads to the development of oligoclonal populations of CMV-specific CTL that can constitute up to one-quarter of the total CD8 T cell population. Furthermore, CMV-specific CTL have a highly polarized membrane phenotype that is typical of effector memory cells (CD28(-), CD57(+), CCR7(-)). TCR analyses show that CMV-specific CTL have highly restricted clonality with greater restriction in the larger expansions. Clonal analysis of the total CD8 T cell repertoire was compared between CMV-seropositive and CMV-seronegative donors. Thirty-three percent more clonal expansions were observed in CMV-seropositive donors in comparison with seronegative individuals. These data implicate CMV as a major factor in driving oligoclonal expansions in old age. Such a dramatic accumulation of virus-specific effector CTL might impair the ability to respond to heterologous infection and may underlie the negative influence of CMV seropositivity on survival in the very elderly.     

Persistent and selective deficiency of CD4+ T cell immunity to cytomegalovirus in immunocompetent young children

Healthy young children who acquire CMV have prolonged viral shedding into the urine and saliva, but whether this is attributable to limitations in viral-specific immune responses has not been explored. In this study, we found that otherwise immunocompetent young children after recent primary CMV infection accumulated markedly fewer CMV-specific CD4(+) T cells that produced IFN-gamma than did adults. These differences in CD4(+) T cell function persisted for more than 1 year after viral acquisition, and did not apply to CMV-specific IFN-gamma production by CD8(+) T cells. The IFN-gamma-producing CD4(+) T cells of children or adults that were reactive with CMV Ags were mainly the CCR7(low) cell subset of memory (CD45R0(high)CD45RA(low)) cells. The decreased IFN-gamma response to CMV in children was selective, because their CCR7(low) memory CD4(+) T cells and those of adults produced similar levels of this cytokine after stimulation with staphylococcal enterotoxin B superantigen. CD4(+) T cells from children also had reduced CMV-specific IL-2 and CD154 (CD40 ligand) expression, suggesting an early blockade in the differentiation of viral-specific CD4(+) T cells. Following CMV acquisition, children, but not adults, persistently shed virus in urine, and this was observable for at least 29 mo postinfection. Thus, CD4(+) T cell-mediated immunity to CMV in humans is generated in an age-dependent manner, and may have a substantial role in controlling renal viral replication and urinary shedding.     

Quantification of T-cell dynamics during latent cytomegalovirus infection in humans

Cytomegalovirus (CMV) infection has a major impact on the T-cell pool, which is thought to be associated with ageing of the immune system. The effect on the T-cell pool has been interpreted as an effect of CMV on non-CMV specific T-cells. However, it remains unclear whether the effect of CMV could simply be explained by the presence of large, immunodominant, CMV-specific memory CD8⁺ T-cell populations. These have been suggested to establish through gradual accumulation of long-lived cells. However, little is known about their maintenance. We investigated the effect of CMV infection on T-cell dynamics in healthy older adults, and aimed to unravel the mechanisms of maintenance of large numbers of CMV-specific CD8⁺ T-cells. We studied the expression of senescence, proliferation, and apoptosis markers and quantified the in vivo dynamics of CMV-specific and other memory T-cell populations using in vivo deuterium labelling. Increased expression of late-stage differentiation markers by CD8⁺ T-cells of CMV+ versus CMV- individuals was not solely explained by the presence of large, immunodominant CMV-specific CD8⁺ T-cell populations. The lifespans of circulating CMV-specific CD8⁺ T-cells did not differ significantly from those of bulk memory CD8⁺ T-cells, and the lifespans of bulk memory CD8⁺ T-cells did not differ significantly between CMV- and CMV+ individuals. Memory CD4⁺ T-cells of CMV+ individuals showed increased expression of late-stage differentiation markers and decreased Ki-67 expression. Overall, the expression of senescence markers on T-cell populations correlated positively with their expected in vivo lifespan. Together, this work suggests that i) large, immunodominant CMV-specific CD8⁺ T-cell populations do not explain the phenotypical differences between CMV+ and CMV- individuals, ii) CMV infection hardly affects the dynamics of the T-cell pool, and iii) large numbers of CMV-specific CD8⁺ T-cells are not due to longer lifespans of these cells.     

Sustained high frequencies of specific CD4 T cells restricted to a single persistent virus

Replication of cytomegalovirus (CMV) is largely controlled by the cellular arm of the immune response. In this study the CMV-specific CD4 T-cell response was characterized in a cohort of apparently healthy individuals. In 11% of all individuals, extremely high frequencies, between 10 and 40%, were found. High-level frequencies of CMV-specific CD4 T cells persisted over several months and were not the result of an acute infection. Specific T cells were oligoclonal and were phenotypically and functionally characterized as mature effector cells, with both cytokine-secreting and proliferative potential. These high-level frequencies do not seem to compromise the immune response towards heterologous infections, and no signs of immunopathology were observed. Whereas a large temporary expansion of virus-specific T cells is well known to occur during acute infection, we now show that extremely high frequencies of virus-specific T cells may continuously exist in chronic CMV infection without overtly compromising the remaining protective immunity.     

Direct relationship between suppression of virus-specific immunity and emergence of cytomegalovirus disease in simian AIDS

Although opportunistic infections like cytomegalovirus (CMV) are common sequelae of end-stage AIDS, the immune events leading to CMV reactivation in human immunodeficiency virus (HIV)-infected individuals are not well defined. The role of cellular and humoral CMV-specific immune responses in immune control of latent CMV infection was evaluated prospectively in a cohort of 11 simian immunodeficiency virus (SIV)-infected CMV-seropositive rhesus macaques, 6 of whom had histologic evidence of CMV disease at death. Macaques with CMV disease differed from macaques without CMV disease in having significantly higher levels of plasma SIV RNA and CMV DNA and significantly lower titers of anti-CMV binding antibodies (Abs) at the time of death. A significant decline in anti-CMV Abs and CMV-specific CD4(+) and CD8(+) T lymphocytes over time was observed in the macaques with CMV disease, but not in the macaques without CMV disease. Reduction in CMV-specific CD8(+) T lymphocytes and anti-CMV neutralizing Abs was significantly correlated with a decline in CMV-specific CD4(+) T lymphocytes. Although declines in CMV-specific T lymphocytes alone were sufficient for reactivation of low-level CMV viremia, high-level viremia (>1,000 copies of CMV DNA per ml of plasma) was observed when anti-CMV neutralizing and binding Abs had also declined. Thus, the occurrence of CMV reactivation-associated disease in AIDS is associated with suppression of both cellular and humoral CMV-specific immune responses. The underlying mechanism may be a dysfunction of memory B and CD8(+) T lymphocytes associated with SIV-induced impairment of CMV-specific CD4(+) T-cell help.     

Clonotypic structure of the human CD4+ memory T cell response to cytomegalovirus

High steady-state frequencies of CMV-specific CD4(+) memory T cells are maintained in CMV-exposed subjects, and these cells are thought to play a key role in the immunologic control of this permanent infection. However, the essential components of this response are poorly defined. Here, we report the use of a step-wise application of flow cytometric and molecular techniques to determine the number and size of the TCR Vbeta-defined clonotypes within freshly obtained CMV-specific CD4(+) memory T cell populations of four healthy, CMV-exposed human subjects. This analysis revealed a stable clonotypic hierarchy in which 1-3 dominant clonotypes are maintained in concert with more numerous subdominant and minor clonotypes. These dominant clonotypes accounted for 10-50% of the overall CMV response, and comprised from 0.3 to 4.0% of peripheral blood CD4(+) T cells. Two subjects displayed immunodominant responses to single epitopes within the CMV matrix phosphoprotein pp65; these single epitope responses were mediated by a single dominant clonotype in one subject, and by multiple subdominant and minor clonotypes in the other. Thus, the CMV-specific CD4(+) T cell memory repertoire in normal subjects is characterized by striking clonotypic dominance and the potential for epitope focusing, suggesting that primary responsibility for immunosurveillance against CMV reactivation rests with a handful of clones recognizing a limited array of CMV determinants. These data have important implications for the understanding of mechanisms by which a genetically stable chronic viral pathogen such as CMV is controlled, and offer possible insight into the failure of such control for a genetically flexible pathogen like HIV-1.     

Human cytomegalovirus-specific CD4+-T-cell cytokine response induces fractalkine in endothelial cells

Cytomegalovirus (CMV) infection has been linked to inflammation-related disease processes in the human host, including vascular diseases and chronic transplant rejection. The mechanisms through which CMV affects the pathogenesis of these diseases are for the most part unknown. To study the contributing role of the host immune response to CMV in these chronic inflammatory processes, we examined endothelial cell interactions with peripheral blood mononuclear cells (PBMC). Endothelial cultures were monitored for levels of fractalkine induction as a marker for initiating the host inflammatory response. Our results demonstrate that in the presence of CMV antigen PBMC from normal healthy CMV-seropositive donors produce soluble factors that induce fractalkine in endothelial cells. This was not observed in parallel assays with PBMC from seronegative donors. Examination of subset populations within the PBMC further revealed that CMV antigen-stimulated CD4(+) T cells were the source of the factors, gamma interferon and tumor necrosis factor alpha, driving fractalkine induction. Direct contact between CD4(+) cells and the endothelial monolayers is required for this fractalkine induction, where the endothelial cells appear to provide antigen presentation functions. These findings indicate that CMV may represent one member of a class of persistent pathogens where the antigen-specific T-cell response can result in the induction of fractalkine, leading to chronic inflammation and endothelial cell injury.     

Long-term cytomegalovirus infection leads to significant changes in the composition of the CD8+ T-cell repertoire, which may be the basis for an imbalance in the cytokine production profile in elderly persons

In spite of the present belief that latent cytomegalovirus (CMV) infection drives CD8+ T-cell differentiation and induces premature immune senescence, no systematic studies have so far been performed to compare phenotypical and functional changes in the CD8+ T-cell repertoire in CMV-infected and noninfected persons of different age groups. In the present study, number, cytokine production, and growth potential of naive (CD45RA+ CD28+), memory (CD45RA- CD28+), and effector (CD45RA+ CD28- or CD45RA- CD28-) CD8+ T cells were analyzed in young, middle-aged, and elderly clinically healthy persons with a positive or negative CMV antibody serology. Numbers and functional properties of CMVpp65(495-503)-specific CD8+ T cells were also studied. We demonstrate that aging as well as CMV infection lead to a decrease in the size of the naive CD8+ T-cell pool but to an increase in the number of CD8+ effector T cells, which produce gamma interferon but lack substantial growth potential. The size of the CD8+ memory T-cell population, which grows well and produces interleukin-2 (IL-2) and IL-4, also increases with aging, but this increase is missing in CMV carriers. Life-long latent CMV infection seems thus to diminish the size of the naive and the early memory T-cell pool and to drive a Th1 polarization within the immune system. This can lead to a reduced diversity of CD8 responses and to chronic inflammatory processes which may be the basis of severe health problems in elderly persons.     

Emergence of a CD4+CD28- granzyme B+, cytomegalovirus-specific T cell subset after recovery of primary cytomegalovirus infection

Cytotoxic CD4(+)CD28(-) T cells form a rare subset in human peripheral blood. The presence of CD4(+)CD28(-) cells has been associated with chronic viral infections, but how these particular cells are generated is unknown. In this study, we show that in primary CMV infections, CD4(+)CD28(-) T cells emerge just after cessation of the viral load, indicating that infection with CMV triggers the formation of CD4(+)CD28(-) T cells. In line with this, we found these cells only in CMV-infected persons. CD4(+)CD28(-) cells had an Ag-primed phenotype and expressed the cytolytic molecules granzyme B and perforin. Importantly, CD4(+)CD28(-) cells were to a large extent CMV-specific because proliferation was only induced by CMV-Ag, but not by recall Ags such as purified protein derivative or tetanus toxoid. CD4(+)CD28(-) cells only produced IFN-gamma after stimulation with CMV-Ag, whereas CD4(+)CD28(+) cells also produced IFN-gamma in response to varicella-zoster virus and purified protein derivative. Thus, CD4(+)CD28(-) T cells emerge as a consequence of CMV infection.     

Co-ordinated isolation of CD8+ and CD4+ T cells recognizing a broad repertoire of cytomegalovirus pp65 and IE1 epitopes for highly specific adoptive immunotherapy

Background aimsAdoptive transfer of cytomegalovirus (CMV)-specific memory T cells can be used for treatment of CMV reactivation after allogeneic stem cell transplantation. As co-ordinated CD8⁺ and CD4⁺ T cells specific for a broad repertoire of CMV epitopes may be most effective for adoptive immunotherapy, the aim of this study was to isolate these cells from peripheral blood of CMV seropositive donors, irrespective of their HLA type.MethodsActivation of CMV-specific CD8⁺ and CD4⁺ T cells was compared after stimulation of donor peripheral blood with minimal epitope peptides, pools of overlapping 15-mer peptides or full-length protein. Furthermore, the kinetics of interferon (IFN)-γ production after stimulation was analyzed to determine the optimal time-point for IFN-γ-based isolation of CMV-specific T cells. The specificity, phenotype and functionality of generated T-cell lines were analyzed.ResultsCMV protein-spanning 15-mer peptide pools induced simultaneous activation of both CD8⁺ and CD4⁺ CMV-specific T cells, while full-length CMV protein only efficiently activated CD4⁺ CMV-specific T cells. Isolation of IFN-γ-secreting cells at the peak of the IFN-γ response after 4-h stimulation with CMV pp65 and IE1 peptide pools resulted in efficient enrichment of CMV-specific T cells. The T-cell lines contained high frequencies of CD8⁺ and CD4⁺ T cells recognizing multiple CMV pp65 and IE1 epitopes, and produced IFN-γ and tumor necrosis factor (TNF)-α upon specific restimulation.ConclusionsThis study provides a feasible strategy for the rapid generation of clinical-grade CD8⁺ and CD4⁺ T-cell lines with high specificity for multiple CMV pp65 and IE1 epitopes, which may be used for effective adoptive immunotherapy.     

CD4+ T-cell reconstitution reduces cytomegalovirus in the immunocompromised brain

Cytomegalovirus (CMV) infection is the most common opportunistic infection of the central nervous system in patients with human immunodeficiency virus or AIDS or on immunosuppressive drug therapy. Despite medical management, infection may be refractory to treatment and continues to cause significant morbidity and mortality. We investigated adoptive transfer as an approach to treat and prevent neurotropic CMV infection in an adult immunodeficient mouse model. SCID mice were challenged with intracranial murine CMV (MCMV) and reconstituted with MCMV- or vesicular stomatitis virus (VSV)-sensitized splenocytes, T cells, or T-cell subsets. T cells labeled with vital dye or that constitutively generated green fluorescent protein (GFP) were identified in the brain as early as 3 days following peripheral transfer. Regardless of specificity, activated T cells localized to regions of the brain containing CMV, however, only those specific for CMV were effective at clearing virus. Reconstitution with unsorted MCMV-immune splenocytes, enriched T-cell fractions, or CD4(+) cells significantly reduced virus levels in the brain within 7 days and also prevented clinical disease, in significant contrast with mice given VSV-immune unsorted splenocytes, MCMV-immune CD8(+) T cells, and SCID control mice. Results suggest CMV-immune T cells (particularly CD4(+)) rapidly cross the blood-brain barrier, congregate at sites of specific CMV infection, and functionally eliminate acute CMV within the brain. In addition, when CMV-immune splenocytes were administered prior to a peripheral CMV challenge, CMV entry into the immunocompromised brain was prevented. Systemic adoptive transfer may be a rapid and effective approach to preventing CMV entrance into the brain and for reducing neurotropic infection.     

T cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is associated with impaired proliferative, cytokine, and cytotoxic effector functions of HCV-specific CD8(+) T cells that probably contribute significantly to viral persistence. Here, we investigated the potential role of T cells with a CD4(+)CD25(+) regulatory phenotype in suppressing virus-specific CD8(+) T-cell proliferation during chronic HCV infection. In vitro depletion studies and coculture experiments revealed that peptide specific proliferation as well as gamma interferon production of HCV-specific CD8(+) T cells were inhibited by CD4(+)CD25(+) T cells. This inhibition was dose dependent, required direct cell-cell contact, and was independent of interleukin-10 and transforming growth factor beta. Interestingly, the T-cell-mediated suppression in chronically HCV-infected patients was not restricted to HCV-specific CD8(+) T cells but also to influenza virus-specific CD8(+) T cells. Importantly, CD4(+)CD25(+) T cells from persons recovered from HCV infection and from healthy blood donors exhibited significantly less suppressor activity. Thus, the inhibition of virus-specific CD8(+) T-cell proliferation was enhanced in chronically HCV-infected patients. This was associated with a higher frequency of circulating CD4(+)CD25(+) cells observed in this patient group. Taken together, our results suggest that chronic HCV infection leads to the expansion of CD4(+)CD25(+) T cells that are able to suppress CD8(+) T-cell responses to different viral antigens. Our results further suggest that CD4(+)CD25(+) T cells may contribute to viral persistence in chronically HCV-infected patients and may be a target for immunotherapy of chronic hepatitis C.     

Preferential infection shortens the life span of human immunodeficiency virus-specific CD4+ T cells in vivo

CD4(+) T-cell help is essential for effective immune responses to viruses. In human immunodeficiency virus (HIV) infection, CD4(+) T cells specific for HIV are infected by the virus at higher frequencies than other memory CD4(+) T cells. Here, we demonstrate that HIV-specific CD4(+) T cells are barely detectable in most infected individuals and that the corresponding CD4(+) T cells exhibit an immature phenotype compared to both cytomegalovirus (CMV)-specific CD4(+) T cells and other memory CD4(+) T cells. However, in two individuals, we observed a rare and diametrically opposed pattern in which HIV-specific CD4(+) T-cell populations of large magnitude exhibited a terminally differentiated immunophenotype; these cells were not preferentially infected in vivo. Clonotypic analysis revealed that the HIV-specific CD4(+) T cells from these individuals were cross-reactive with CMV. Thus, preferential infection can be circumvented in the presence of cross-reactive CD4(+) T cells driven to maturity by coinfecting viral antigens, and this physical proximity rather than activation status per se is an important determinant of preferential infection based on antigen specificity. These data demonstrate that preferential infection reduces the life span of HIV-specific CD4(+) T cells in vivo and thereby compromises the generation of effective immune responses to the virus itself; further, this central feature in the pathophysiology of HIV infection can be influenced by the cross-reactivity of responding CD4(+) T cells.     

Distinct mechanisms of CD4+ and CD8+ T-cell activation and bystander apoptosis induced by human immunodeficiency virus type 1 virions

Apoptosis of uninfected bystander T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) infection. HIV-1 envelope/receptor interactions and immune activation have been implicated as contributors to bystander apoptosis. To better understand the relationship between T-cell activation and bystander apoptosis during HIV-1 pathogenesis, we investigated the effects of the highly cytopathic CXCR4-tropic HIV-1 variant ELI6 on primary CD4(+) and CD8(+) T cells. Infection of primary T-cell cultures with ELI6 induced CD4(+) T-cell depletion by direct cell lysis and bystander apoptosis. Exposure of primary CD4(+) and CD8(+) T cells to nonreplicating ELI6 virions induced bystander apoptosis through a Fas-independent mechanism. Bystander apoptosis of CD4(+) T cells required direct contact with virions and Env/CXCR4 binding. In contrast, the apoptosis of CD8(+) T cells was triggered by a soluble factor(s) secreted by CD4(+) T cells. HIV-1 virions activated CD4(+) and CD8(+) T cells to express CD25 and HLA-DR and preferentially induced apoptosis in CD25(+)HLA-DR(+) T cells in a CXCR4-dependent manner. Maximal levels of binding, activation, and apoptosis were induced by virions that incorporated MHC class II and B7-2 into the viral membrane. These results suggest that nonreplicating HIV-1 virions contribute to chronic immune activation and T-cell depletion during HIV-1 pathogenesis by activating CD4(+) and CD8(+) T cells, which then proceed to die via apoptosis. This mechanism may represent a viral immune evasion strategy to increase viral replication by activating target cells while killing immune effector cells that are not productively infected.