The cytomegalovirus-specific CD4+ T-cell response expands with age and markedly alters the CD4+ T-cell repertoire

Immune function in the elderly is associated with a number of phenotypic and functional abnormalities, and this phenomenon of immune senescence is associated with increased susceptibility to infection. The immune response to pathogens frequently declines with age, but the CD8(+) T-cell response to cytomegalovirus (CMV) is unusual, as it demonstrates a significant expansion over time. Here we have documented the CD4(+) T-cell immune response to CMV in healthy donors of different ages. The magnitude of the CMV-specific CD4(+) T-cell immune response increases from a mean of 2.2% of the CD4(+) T-cell pool in donors below 50 years of age to 4.7% in donors aged over 65 years. In addition, CMV-specific CD4(+) T cells in elderly donors demonstrate decreased production of interleukin-2 and less dependence on costimulation. CMV seropositivity is associated with marked changes in the phenotype of the overall CD4(+) T-cell repertoire in healthy aged donors, including an increase in CD57(+) expression and a decrease in CD28 and CD27 expression, a phenotypic profile characteristic of immune senescence. This memory inflation of CMV-specific CD4(+) T cells contributes to evidence that CMV infection may be damaging to immune function in elderly individuals.     

Autocrine Production of β-Chemokines Protects CMV-Specific CD4+ T Cells from HIV Infection

Induction of a functional subset of HIV-specific CD4⁺ T cells that is resistant to HIV infection could enhance immune protection and decrease the rate of HIV disease progression. CMV-specific CD4⁺ T cells, which are less frequently infected than HIV-specific CD4⁺ T cells, are a model for such an effect. To determine the mechanism of this protection, we compared the functional response of HIV gag-specific and CMV pp65-specific CD4⁺ T cells in individuals co-infected with CMV and HIV. We found that CMV-specific CD4⁺ T cells rapidly up-regulated production of MIP-1α and MIP-1β mRNA, resulting in a rapid increase in production of MIP-1α and MIP-1β after cognate antigen stimulation. Production of β-chemokines was associated with maturational phenotype and was rarely seen in HIV-specific CD4⁺ T cells. To test whether production of β-chemokines by CD4⁺ T cells lowers their susceptibility to HIV infection, we measured cell-associated Gag DNA to assess the in vivo infection history of CMV-specific CD4⁺ T cells. We found that CMV-specific CD4⁺ T cells which produced MIP-1β contained 10 times less Gag DNA than did those which failed to produce MIP-1β. These data suggest that CD4⁺ T cells which produce MIP-1α and MIP-1β bind these chemokines in an autocrine fashion which decreases the risk of in vivo HIV infection.     

CMV-Independent Lysis of Glioblastoma by Ex Vivo Expanded/Activated Vδ1+ γδ T Cells

Vδ2^neg γδ T cells, of which Vδ1+ γδ T cells are by far the largest subset, are important effectors against CMV infection. Malignant gliomas often contain CMV genetic material and proteins, and evidence exists that CMV infection may be associated with initiation and/or progression of glioblastoma multiforme (GBM). We sought to determine if Vδ1+ γδ T cells were cytotoxic to GBM and the extent to which their cytotoxicity was CMV dependent. We examined the cytotoxic effect of ex vivo expanded/activated Vδ1+ γδ T cells from healthy CMV seropositive and CMV seronegative donors on unmanipulated and CMV-infected established GBM cell lines and cell lines developed from short- term culture of primary tumors. Expanded/activated Vδ1+ T cells killed CMV-negative U251, U87, and U373 GBM cell lines and two primary tumor explants regardless of the serologic status of the donor. Experimental CMV infection did not increase Vδ1+ T cell - mediated cytotoxicity and in some cases the cell lines were more resistant to lysis when infected with CMV. Flow cytometry analysis of CMV-infected cell lines revealed down-regulation of the NKG2D ligands ULBP-2, and ULBP-3 as well as MICA/B in CMV-infected cells. These studies show that ex vivo expanded/activated Vδ1+ γδ T cells readily recognize and kill established GBM cell lines and primary tumor-derived GBM cells regardless of whether CMV infection is present, however, CMV may enhance the resistance GBM cell lines to innate recognition possibly contributing to the poor immunogenicity of GBM.     

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.     

Maturation and Mip-1β Production of Cytomegalovirus-Specific T Cell Responses in Tanzanian Children,Adolescents and Adults: Impact by HIV and Mycobacterium tuberculosis Co-Infections

It is well accepted that aging and HIV infection are associated with quantitative and functional changes of CMV-specific T cell responses. We studied here the expression of Mip-1β and the T cell maturation marker CD27 within CMVpp65-specific CD4⁺ and CD8⁺ T cells in relation to age, HIV and active Tuberculosis (TB) co-infection in a cohort of Tanzanian volunteers (≤16 years of age, n = 108 and ≥18 years, n = 79). Independent of HIV co-infection, IFNγ⁺ CMVpp65-specific CD4⁺ T cell frequencies increased with age. In adults, HIV co-infection further increased the frequencies of these cells. A high capacity for Mip-1β production together with a CD27^low phenotype was characteristic for these cells in children and adults. Interestingly, in addition to HIV co-infection active TB disease was linked to further down regulation of CD27 and increased capacity of Mip-1β production in CMVpp65-specific CD4⁺ T cells. These phenotypic and functional changes of CMVpp65-specific CD4 T cells observed during HIV infection and active TB could be associated with increased CMV reactivation rates.     

Decreased frequency of cytomegalovirus (CMV)-specific CD4+ T lymphocytes in simian immunodeficiency virus-infected rhesus macaques: inverse relationship with CMV viremia

The frequency of cytomegalovirus (CMV)-specific CD4+ T lymphocytes was determined in CMV-seropositive rhesus macaques with or without simian immunodeficiency virus (SIV) infection by using the sensitive assays of intracellular cytokine staining and gamma interferon ELISPOT. Both techniques yielded 3- to 1,000-fold-higher frequencies of CMV-specific CD4+ T lymphocytes than traditional proliferative limiting dilution assays. The median frequency of CMV-specific CD4+ T lymphocytes in 23 CMV-seropositive SIV-negative macaques was 0.63% (range, 0.16 to 5.8%). The majority of CMV-specific CD4+ T lymphocytes were CD95(pos) and CD27(lo) but expressed variable levels of CD45RA. A significant reduction (P < 0.05) in the frequency of CMV-specific CD4+ T lymphocytes was observed in pathogenic SIV-infected macaques but not in macaques infected with live attenuated strains of SIV. CMV-specific CD4+ T lymphocytes were not detected in six of nine pathogenic SIV-infected rhesus macaques. CMV DNA was detected in the plasma of four of six of these macaques but in no animal with detectable CMV-specific CD4+ T lymphocytes. In pathogenic SIV-infected macaques, loss of CMV-specific CD4+ T lymphocytes was not predicted by the severity of CD4+ T lymphocytopenia. Neither was it predicted by the pre-SIV infection frequencies of CD45RA(neg) or CCR5(pos) CMV-specific CD4+ T lymphocytes. However, the magnitude of activation, as evidenced by the intensity of CD40L expression on CMV-specific CD4+ T lymphocytes pre-SIV infection, was three- to sevenfold greater in the two macaques that subsequently lost these cells after SIV infection than in the two macaques that retained CMV-specific CD4+ T lymphocytes post-SIV infection. Future longitudinal studies with these techniques will facilitate the study of CMV pathogenesis in AIDS.     

Cytomegalovirus-Specific T Cells Persist at Very High Levels during Long-Term Antiretroviral Treatment of HIV Disease

Background

In healthy, HIV seronegative, CMV seropositive adults, a large proportion of T cells are CMV-specific. High-level CMV-specific T cell responses are associated with accelerated immunologic aging (“immunosenesence”) in the elderly population. The impact of untreated and treated HIV infection on the frequency of these cells remains undefined.

Methodology/Principal Findings

We measured the proportion of CD4+ and CD8+ T cells responding to CMV pp65 and IE proteins was measured using flow cytometry in 685 unique HIV seronegative and seropositive individuals. The proportion of CMV-specific CD8+ T cells was consistently higher in the HIV-seropositive subjects compared to the HIV-seronegative subjects. This HIV effect was observed even in patients who lacked measurable immunodeficiency. Among the HIV-seropositive subjects, CMV-specific CD8+ T cell responses were proportionately lower during recent infection, higher during chronic untreated infection and higher still during long-term antiretroviral treated infection. The CD8+ T cell response to just two CMV proteins (pp65 and IE) was approximately 6% during long-term therapy, which was over twice that seen in HIV-seronegative persons. CMV-specific CD4+ T cell responses followed the same trends, but the magnitude of the effect was smaller.

Conclusions/Significance

Long-term successfully treated HIV infected patients have remarkably high levels of CMV-specific effector cells. These levels are similar to that observed in the elderly, but occur at much younger ages. Future studies should focus on defining the potential role of the CMV-specific inflammatory response in non-AIDS morbidity and mortality, including immunosenescence.     

T Lymphocytes from Chagasic Patients Are Activated but Lack Proliferative Capacity and Down-Regulate CD28 and CD3ζ

Background

Chronic persistent infections have been associated with T lymphocytes functional impairment. The aim of this study was to compare the activation status, the proliferative potential and the expression of CD28 and CD3ζ chain on T lymphocytes between chronic chagasic patients and uninfected controls.

Methodology/Principal Findings

Forty-two chronic chagasic patients, 28 healthy individuals and 32 non-chagasic cardiomyopathy donors were included. Peripheral blood was marked for CD3, CD4, CD8, HLA-DR, CD28, CD38 and intracellular CD3ζ. Peripheral blood mononuclear cells were stained with carboxyfluorescein diacetate succinimidylester and incubated with T. cruzi lysate or phytohemagglutinin for five days. Cells from 3 healthy controls were incubated with T. cruzi trypomastigotes separated with transwells; and the expression of CD3ζ chain and proliferation index was determined. Heart-infiltrating cells from two chronic chagasic patients were tested for the aforementioned cellular markers. Chagasic patients displayed higher frequencies of CD4+/HLA-DR+/CD38+ (8.1%±6.1) and CD8+/HLA-DR+/CD38+ (19.8±8.9) T cells in comparison with healthy (1.6±1.0; 10.6±8.0) and non-chagasic cardiomyopathy donors (2.9±2.9; 5.8±6.8). Furthermore, the percentage of CD4+ activated T cells was higher in chagasic patients with cardiac involvement. CD8+ T cells proliferation index in chagasic donors (1.7±0.3) was lower when compared with healthy (2.3±0.3) and non-chagasic cardiomyopathy individuals (3.1±1.1). The frequencies of CD4+/CD28+ and CD8+/CD28+ T cells, as well as the CD3ζ^bright/CD3ζ^dim% ratios in CD4+ and CD8+ were lower in chagasic patients when compared with both control groups. The CD3ζ^bright/CD3ζ^dim% ratio and proliferative indexes for CD4+ and CD8+ T lymphocytes decreased gradually in those cells cultivated with parasites and displayed lower values than those incubated with medium alone. Finally, heart-infiltrating T cells from two T. cruzi infected patients also expressed activation markers and down-regulate CD28 and CD3ζ.

Conclusions

CD8+ T lymphocytes from chagasic donors displayed reduced proliferative capacity, which might be associated with CD3ζ down-regulation and diminished CD28 expression on CD4 T cells.     

Decreased Proportion of Cytomegalovirus Specific CD8 T-Cells but No Signs of General Immunosenescence in Alzheimer’s Disease

Cytomegalovirus (CMV) has been suggested as a contributing force behind the impaired immune responsiveness in the elderly, with decreased numbers of naïve T-cells and an increased proportion of effector T-cells. Immunological impairment is also implicated as a part of the pathogenesis in Alzheimer’s disease (AD). The aim of this study was to investigate whether AD patients present with a different CMV-specific CD8 immune profile compared to non-demented controls. Blood samples from 50 AD patients and 50 age-matched controls were analysed for HLA-type, CMV serostatus and systemic inflammatory biomarkers. Using multi-colour flow cytometry, lymphocytes from peripheral blood mononuclear cells were analysed for CMV-specific CD8 immunity with MHC-I tetramers A01, A02, A24, B07, B08 and B35 and further classified using CD27, CD28, CD45RA and CCR7 antibodies. Among CMV seropositive subjects, patients with AD had significantly lower proportions of CMV-specific CD8 T-cells compared to controls, 1.16 % vs. 4.13 % (p=0.0057). Regardless of dementia status, CMV seropositive subjects presented with a lower proportion of naïve CD8 cells and a higher proportion of effector CD8 cells compared to seronegative subjects. Interestingly, patients with AD showed a decreased proportion of CMV-specific CD8 cells but no difference in general CD8 differentiation.     

γδ T Cells Confer Protection against Murine Cytomegalovirus (MCMV)

Cytomegalovirus (CMV) is a leading infectious cause of morbidity in immune-compromised patients. γδ T cells have been involved in the response to CMV but their role in protection has not been firmly established and their dependency on other lymphocytes has not been addressed. Using C57BL/6 αβ and/or γδ T cell-deficient mice, we here show that γδ T cells are as competent as αβ T cells to protect mice from CMV-induced death. γδ T cell-mediated protection involved control of viral load and prevented organ damage. γδ T cell recovery by bone marrow transplant or adoptive transfer experiments rescued CD3ε^−/− mice from CMV-induced death confirming the protective antiviral role of γδ T cells. As observed in humans, different γδ T cell subsets were induced upon CMV challenge, which differentiated into effector memory cells. This response was observed in the liver and lungs and implicated both CD27⁺ and CD27⁻ γδ T cells. NK cells were the largely preponderant producers of IFNγ and cytotoxic granules throughout the infection, suggesting that the protective role of γδ T cells did not principally rely on either of these two functions. Finally, γδ T cells were strikingly sufficient to fully protect Rag^−/−γc^−/− mice from death, demonstrating that they can act in the absence of B and NK cells. Altogether our results uncover an autonomous protective antiviral function of γδ T cells, and open new perspectives for the characterization of a non classical mode of action which should foster the design of new γδ T cell based therapies, especially useful in αβ T cell compromised patients.     

Control of Murine Cytomegalovirus Infection by γδ T Cells

Infections with cytomegalovirus (CMV) can cause severe disease in immunosuppressed patients and infected newborns. Innate as well as cellular and humoral adaptive immune effector functions contribute to the control of CMV in immunocompetent individuals. None of the innate or adaptive immune functions are essential for virus control, however. Expansion of γδ T cells has been observed during human CMV (HCMV) infection in the fetus and in transplant patients with HCMV reactivation but the protective function of γδ T cells under these conditions remains unclear. Here we show for murine CMV (MCMV) infections that mice that lack CD8 and CD4 αβ-T cells as well as B lymphocytes can control a MCMV infection that is lethal in RAG-1^-/- mice lacking any T- and B-cells. γδ T cells, isolated from infected mice can kill MCMV infected target cells in vitro and, importantly, provide long-term protection in infected RAG-1^-/- mice after adoptive transfer. γδ T cells in MCMV infected hosts undergo a prominent and long-lasting phenotypic change most compatible with the view that the majority of the γδ T cell population persists in an effector/memory state even after resolution of the acute phase of the infection. A clonotypically focused Vγ1 and Vγ2 repertoire was observed at later stages of the infection in the organs where MCMV persists. These findings add γδ T cells as yet another protective component to the anti-CMV immune response. Our data provide clear evidence that γδ T cells can provide an effective control mechanism of acute CMV infections, particularly when conventional adaptive immune mechanisms are insufficient or absent, like in transplant patient or in the developing immune system in utero. The findings have implications in the stem cell transplant setting, as antigen recognition by γδ T cells is not MHC-restricted and dual reactivity against CMV and tumors has been described.     

Expansion of human CMV-specific cytotoxic T lymphocytes to a clinical scale: a simple culture system using tetrameric HLA-peptide complexes

BACKGROUND: Recipients of allogeneic stem cell transplants (SCT) are at risk of human CMV infection during their immunocompromised period. The increasing number of reports of CMV isolates resistant to ganciclovir after transplantation has led us to attempt to develop alternative strategies for preventing or treating CMV infection. This study describes a system for generating sufficient numbers of CMV-specific cytotoxic T lymphocytes (CTL) for adoptive immunotherapy after SCT. METHODS: CMV-specific CTL were isolated from a single blood draw of a CMV-seropositive donor using PE-labeled HLA-A*0201/pp65(495-503) tetramers and anti-PE magnetic beads. A mixture of a tetramer-positive population and CD4(+) T lymphocytes was expanded to sufficient numbers for clinical application with IL-2 and immobilized anti-CD3 stimulation. RESULT: Starting from 50 mL of blood, we generated >10(7)/m(2) tetramer-positive CTL within 2 weeks. Flow cytometric analysis of expanded lymphocytes showed that purity of CMV peptide-specific CTL was >75%. Upon stimulation of HLA-A*0201-restricted CMV peptide, expanded CD8 T lymphocytes produced intracellular IFN-gamma. Purified CTL exhibited cytotoxic activity against CMV peptide-pulsed T2 cells and CMV-infected HLA-A*0201-positive fibroblasts, but not against HLA mismatched or uninfected target cells. Alloreactivity could be excluded in MLC. DISCUSSION: This simple, rapid culture system can be useful for adoptive immunotherapy after allogeneic SCT. We are now trying to adapt our laboratory scale study to a clinical scale study under good manufacturing practices (GMP) conditions.     

Isolation of Highly Suppressive CD25+FoxP3+ T Regulatory Cells from G-CSF-Mobilized Donors with Retention of Cytotoxic Anti-Viral CTLs: Application for Multi-Functional Immunotherapy Post Stem Cell Transplantation

Previous studies have demonstrated the effective control of cytomegalovirus (CMV) infections post haematopoietic stem cell transplant through the adoptive transfer of donor derived CMV-specific T cells (CMV-T). Strategies for manufacturing CMV immunotherapies has involved a second leukapheresis or blood draw from the donor, which in the unrelated donor setting is not always possible. We have investigated the feasibility of using an aliquot of the original G-CSF-mobilized graft as a starting material for manufacture of CMV-T and examined the activation marker CD25 as a targeted approach for identification and isolation following CMVpp65 peptide stimulation. CD25+ cells isolated from G-CSF-mobilized apheresis revealed a significant increase in the proportion of FoxP3 expression when compared with conventional non-mobilized CD25+ cells and showed a superior suppressive capacity in a T cell proliferation assay, demonstrating the emergence of a population of Tregs not present in non-mobilized apheresis collections. The expansion of CD25+ CMV-T in short-term culture resulted in a mixed population of CD4+ and CD8+ T cells with CMV-specificity that secreted cytotoxic effector molecules and lysed CMVpp65 peptide-loaded phytohaemagglutinin-stimulated blasts. Furthermore CD25 expanded cells retained their suppressive capacity but did not maintain FoxP3 expression or secrete IL-10. In summary our data indicates that CD25 enrichment post CMV stimulation in G-CSF-mobilized PBMCs results in the simultaneous generation of both a functional population of anti-viral T cells and Tregs thus illustrating a potential single therapeutic strategy for the treatment of both GvHD and CMV reactivation following allogeneic haematopoietic stem cell transplantation. The use of G-CSF-mobilized cells as a starting material for cell therapy manufacture represents a feasible approach to alleviating the many problems incurred with successive donations and procurement of cells from unrelated donors. This approach may therefore simplify the clinical application of adoptive immunotherapy and broaden the approach for manufacturing multi-functional T cells.     

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.     

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.     

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.     

Large scale analysis of pediatric antiviral CD8+ T cell populations reveals sustained, functional and mature responses

Background

Cellular immunity plays a crucial role in cytomegalovirus (CMV) infection and substantial populations of CMV-specific T cells accumulate throughout life. However, although CMV infection occurs during childhood, relatively little is know about the typical quantity and quality of T cell responses in pediatric populations.

Methods

One thousand and thirty-six people (Male/Female = 594/442, Age: 0–19 yr.; 959 subjects, 20–29 yr.; 77 subjects) were examined for HLA typing. All of 1036 subjects were tested for HLA-A2 antigen. Of 1036 subjects, 887 were also tested for HLA-A23, 24 antigens. In addition, 50 elderly people (Male/Female = 11/39, Age: 60–92 yr.) were also tested for HLA-A2 antigen. We analyzed the CD8+ T cell responses to CMV, comparing these to responses in children and young. The frequencies, phenotype and function CD8+ T cells for two imunodominant epitopes from pp65 were measured.

Results

We observed consistently high frequency and phenotypically "mature" (CD27 low, CD28 low, CD45RA+) CMV-specific CD8+ T cell responses in children, including those studied in the first year of life. These CD8+ T cells retained functionality across all age groups, and showed evidence of memory "inflation" only in later adult life.

Conclusion

CMV consistently elicits a very strong CD8+ T cell response in infants and large pools of CMV specific CD8+ T cells are maintained throughout childhood. The presence of CMV may considerably mould the CD8+ T cell compartment over time, but the relative frequencies of CMV-specific cells do not show the evidence of a population-level increase during childhood and adulthood. This contrast with the marked expansion ("inflation") of such CD8+ T cells in older adults. This study indicates that large scale analysis of peptide specific T cell responses in infants is readily possible. The robust nature of the responses observed suggests vaccine strategies aimed at priming and boosting CD8+ T cells against major pathogens (including HIV, malaria and CMV itself) could be successful in this age-group.     

Differential CMV-specific CD8+ effector T cell responses in the lung allograft predominate over the blood during human primary infection

Acquisition of T cell responses during primary CMV infection in lung transplant recipients (LTRs) appear critical for host defense and allograft durability, with increased mortality in donor+/recipient- (D+R-) individuals. In 15 D+R- LTRs studied, acute primary CMV infection was characterized by viremia in the presence or absence of pneumonitis, with viral loads higher in the lung airways/allograft compared with the blood. A striking influx of CD8+ T cells into the lung airways/allograft was observed, with inversion of the CD4+:CD8+ T cell ratio. De novo CMV-specific CD8+ effector frequencies in response to pooled peptides of pp65 were strikingly higher in lung mononuclear cells compared with the PBMC and predominated over IE1-specific responses and CD4+ effector responses in both compartments. The frequencies of pp65-specific cytokine responses were significantly higher in lung mononuclear cells compared with PBMC and demonstrated marked contraction with long-term persistence of effector memory CD8+ T cells in the lung airways following primary infection. CMV-tetramer+CD8+ T cells from PBMC were CD45RA- during viremia and transitioned to CD45RA+ following resolution. In contrast, CMV-specific CD8+ effectors in the lung airways/allograft maintained a CD45RA- phenotype during transition from acute into chronic infection. Together, these data reveal differential CMV-specific CD8+ effector frequencies, immunodominance, and polyfunctional cytokine responses predominating in the lung airways/allograft compared with the blood during acute primary infection. Moreover, we show intercompartmental phenotypic differences in CMV-specific memory responses during the transition to chronic infection.