Cytomegalovirus-Specific T Cell Immunity Is Maintained in Immunosenescent Rhesus Macaques

Luka Čičin-Šain, Andrew W. Sylwester, Shoko I. Hagen, Don C. Siess, Noreen Currier, Alfred W. Legasse, Miranda B. Fischer, Caroline W. Koudelka, Michael K. Axthelm, Janko Nikolich-Žugich and Louis J. Picker
J Immunol August 15, 2011, 187 (4) 1722-1732; DOI: https://doi.org/10.4049/jimmunol.1100560

Abstract

Although CMV infection is largely benign in immunocompetent people, the specific T cell responses associated with control of this persistent virus are enormous and must be maintained for life. These responses may increase with advanced age and have been linked to an “immune risk profile” that is associated with poor immune responsiveness and increased mortality in aged individuals. Based on this association, it has been suggested that CMV-specific T cell responses might become dysfunctional with age and thereby contribute to the development of immune senescence by homeostatic disruption of other T cell populations, diminished control of CMV replication, and/or excess chronic inflammation. In this study, we use the rhesus macaque (RM) model of aging to ask whether the quantity and quality of CMV-specific T cell responses differ between healthy adult RMs and elderly RMs that manifest hallmarks of immune aging. We demonstrate that the size of the CD4+ and CD8+ CMV-specific T cell pools are similar in adult versus old RMs and show essentially identical phenotypic and functional characteristics, including a dominant effector memory phenotype, identical patterns of IFN-γ, TNF-α, and IL-2 production and cytotoxic degranulation, and comparable functional avidities of optimal epitope-specific CD8+ T cells. Most importantly, the response to and protection against an in vivo CMV challenge were identical in adult and aged RMs. These data indicate that CMV-specific T cell immunity is well maintained in old RMs and argue against a primary role for progressive dysfunction of these responses in the development of immune senescence.

Aging may be accompanied with a decline in immune function characterized by poor responses to vaccination and increased morbidity and mortality from infectious diseases (1–4). This functional decline is associated with complex, but characteristic, changes in both the innate and adaptive immune system, collectively referred to as “immune senescence” (5, 6). Among the most consistent and dramatic age-related changes are those that occur in T cell homeostasis and function, manifesting in blood as 1) decreased CD4/CD8 T cell ratios, 2) loss of naive cells and relative expansion of differentiated effector memory (EM) cells, 3) oligoclonality/clonal expansions, 4) poor proliferative responses, and 5) changes in cytokine secretion patterns (5, 7–12). These immunologic changes, which typically occur coordinately, have also been strongly correlated with evidence of persistent infection with the ubiquitous β-herpesvirus CMV, and together, these features constitute an immune risk profile (IRP) that in some studies has been predictive of increased mortality in aged individuals (7, 10–16).

These associations have led to the hypothesis that immune senescence may be infectious—a consequence of long-term exposure to, and immunologic control of, persistent infections, in particular, CMV (7, 13, 15, 17). CMV is among the most immunogenic of known viruses, eliciting stable frequencies of specific T cells in CMV+ adults that average 10% of both the CD4+ and CD8+ memory compartments in blood (18). Frequencies of CMV-specific T cells can be even higher in aged individuals (19–21), and given that CMV-specific T cell responses are characterized by 1) a dominant EM (CD28−, CD27−, CCR7−) phenotype, 2) functional characteristics commensurate with this phenotype (high effector cytokine production, relatively low IL-2 production, expression of cytotoxic apparatus, poor in vitro proliferation), and 3) highly hierarchical clonotypic repertoires (with the top clonotypes manifesting frequencies >1%), these responses clearly underlie many of the features of the IRP (7, 13, 15, 17, 22–28). Indeed, it has been postulated that an increasingly dysfunctional, proinflammatory, CMV-specific T cell response expands with advanced age, driving out other T cell populations and causing both the IRP and a significant component of age-associated immune deficiency (7, 13, 17, 29). However, overt CMV disease is rare in the elderly (30, 31), healthy aged individuals may also be CMV+ (32, 33), and other age-related mechanisms such as thymic involution and cessation of new T cell production clearly play a major role in naive cell deficiency (17, 34, 35) and, likely, the poor responsiveness of the elderly to new Ags. Thus, the large CMV-specific effector memory response may simply be better preserved than other T cell populations, persisting while other populations decline (36).

The issue of whether CMV infection plays a causal role in immune senescence and, if so, the understanding of the mechanism(s) by which this virus manifests these changes has critical implications for the clinical approach to elderly individuals. Obviously, if CMV infection and/or the CMV-specific T cell response are culpable in the development of immune senescence, therapy for immune senescence should be directed at preventing or treating CMV infection or interfering with the mechanism by which CMV and/or CMV-specific T cell responses cause the deleterious changes. Alternatively, if CMV and the CMV-specific T cell response are simply bystanders to the pathogenesis of functional immune senescence, therapy should be directed toward the non–CMV-related causal mechanisms. When manifest in middle-aged adults, the IRP does not portend a poor prognosis (37), suggesting a progressive process that develops in late middle age to advanced age. One possibility is that the CMV-specific T cell response deteriorates during this time frame, undergoing slow, but progressive, dysfunction, allowing more frequent and/or higher-magnitude episodes of viral replication. This antigenic stimulation might expand the CMV-specific response and elicit an ever-increasing level of dysfunction by exhausting any remaining nonsenescent T cell populations and/or stimulating production of dysfunctional progeny. Such a reinforcing cycle of viral replication and dysfunction might then cause generalized immune dysfunction by displacing naive and/or central memory T cell populations (13) and/or by creating a chronic inflammatory response that negatively affects the homeostasis of these other crucial T cell populations (38).

To address the question of whether CMV-specific T cell responses deteriorate with age and lose protective capacity, we turned to the rhesus macaque (RM) model. This model manifests the major features of immune senescence found in humans (35, 39–42), as well as a highly homologous rhesus cytomegalovirus (RhCMV) that manifests similar immunobiology as human cytomegalovirus (HCMV), including the same high-frequency EM-biased CD4+ and CD8+ T cell responses observed in CMV+ people (41, 43–46). Indeed, if chronic viral Ag exposure underlies deterioration of the CMV-specific T cell responses and ultimately the overall T cell compartment, the RM model might be expected to highlight this pathogenic mechanism as all colony-raised RMs become CMV+ in the first year of life, and RhCMV, unlike HCMV [which is shed intermittently in the chronic phase of infection (47–49)], is continually shed in the urine and saliva of the vast majority, if not all, animals (44, 46, 50). In this study, we compare in detail the CMV-specific CD4+ and CD8+ T cell responses in adult and aged RMs and demonstrate that both the ex vivo and in vivo function of these responses are unchanged with advanced age, even when other features of immune senescence are present. These results contradict the occurrence of a general deterioration of CMV-specific EM T cell function with advanced age and argue against the hypothesis that such age-associated deterioration is a requirement for development of immune senescence.

Materials and Methods

Animals

Colony-bred male and female RMs of Indian origin were maintained according to the federal, state, and local guidelines. All experiments were approved by the Institutional Animal Care and Use Committee at the Oregon National Primate Research Center. RMs with tumors, amyloidosis, or signs of significant clinical disease at the time of initial assignment were excluded from the study. The overall cohort consisted of 21 young adult (average = 7.8 y, range 7–10 y; 12 males, 9 females) and 36 old (average = 22.8 y, range 19–26 y, 15 males, 21 females) RMs, which naturally acquired RhCMV in their first year of life. The in vivo challenge assay was performed on a subset of this overall cohort consisting of 11 young adult (average = 8.5 y, range 7–10 y; 6 males, 5 females) and 9 old (average = 23.2 y, range 20–27 y; 4 males, 5 females) RMs. Pooled data from multiple independent experiments is shown throughout. The number of RMs used in any particular analysis was a variable subset of the overall cohort, dependent on the availability of RMs at the time of analysis or (where indicated) on defined inclusion criteria applied to the entire RM cohort. Bronchoalveolar lavage lymphocyte (BAL) collection and sampling was performed as previously described (41). In the RhCMV superinfection experiments, RMs were challenged by s.c. injection of 107 PFU RhCMV strain 68.1, as previously described (44).

Ags and Abs

RhCMV-specific CD4+ T cell responses were identified by the response to RhCMV (Cercopithecine herpesvirus 8) Ag preparations containing lysates of RhCMV (strain 68.1; ATCC VR-677)-infected monolayers of telomerized RM fibroblasts (44) combined with cell-free viral proteins concentrated from the culture medium of these infected cells (2:1 ratio of lysate to supernatant concentrate). Proteins were concentrated from infected cell supernatants by precipitation with 90% saturated ammonium sulfate, centrifugation (10,000 × g, 30 min, 4°C), resuspension in PBS, and finally, extensive dialysis against PBS (10,000 Da molecular mass exclusion). RhCMV-specific CD8+ T cell responses were identified by the response to consecutive, overlapping 15-mer peptide pools (11-aa overlap) spanning the entire sequences of immediate-early (IE)-1 and IE-2 RhCMV genes (Intavis, Reutlingen, Germany). The final concentration of each peptide in the pool was 2 μg/test. The mAbs L200 (CD4; AmCyan, PerCP–Cy5.5), SP34-2 (CD3; Pacific Blue, Alexa 700), SK-1 (CD8α; PerCP–Cy5.5, PE, unconjugated), CD28.2 (CD28; PE-TexasRed, PerCP–Cy5.5, unconjugated), DX2 (CD95; PE–Cy7, allophycocyanin, PE), 15053 (CCR7; FITC), B56 (Ki-67; FITC, PE), FN50 (CD69; PE, PE-TexasRed), 9F10 (CD49d; unconjugated), MAB11 (TNF-α; FITC, PE–Cy7), H3A3 (CD107a; FITC), H4B4 (CD107b; FITC), B27 (IFN-γ; allophycocyanin), and MQ1-17H12 (IL-2; PE) were obtained from BD Biosciences. mAb FN18 (CD3) was produced in-house and conjugated to Pacific blue or Alexa 700 using a conjugation kit from Life Technologies.

Cell preparation and stimulation

Citrated venous blood was centrifuged on a Ficoll-Hypaque density gradient (Sigma-Aldrich) to separate PBMCs. A small aliquot (100 μl) of whole blood was used at each bleed to define absolute lymphocyte counts in an Ac*T 5diff hematologic analyzer (Beckman-Coulter). PBMCs and BALs were washed in HBSS and resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated FCS (HyClone), 2 mM l-glutamine (Sigma-Aldrich), 1 mM Na-pyruvate (Sigma-Aldrich), and 50 μM 2-mercaptoethanol (Sigma-Aldrich) (R10). To evaluate RhCMV-specific CD4+ T cell responses, 106 cells in 1 ml R10 were placed in 17 mm × 100 mm polystyrene tubes precoated with goat Fab fragments (2.5 μg/ml) against mouse IgG (H+L), and then coated with costimulatory CD28 and CD49d Abs (2.5 μg/ml of each). Precoating and coating were done on stimulation day, in PBS, at 37°C for 1 h, with PBS washes between procedures and after coating. Six microliters of RhCMV lysate was added for 6 h total, in the presence of brefeldin A (10 μg/ml) for the last 5 h. Stimulation with 15-mer peptide pools was performed in the same volume format, but in polypropylene tubes. Costimulatory Abs were added directly in the tube (at 0.5 μg/ml each). To evaluate dynamic responses upon in vivo challenge, these assays were adapted to a 96-well plate format, where 5 × 105 cells in 200 μl R10 were stimulated with lysate or 15-mer peptide pools for 9 h (last 8 h in presence of brefeldin A), and costimulation as above. In all cases, a negative control tube that contained media and costimulatory Abs was set up in parallel.

Immunofluorescence staining and flow cytometric analysis

Flow cytometric analysis was performed on an LSR-II apparatus (BD Biosciences) with Pacific blue, AmCyan, FITC, PE, PE-TexasRed, PerCP–Cy5.5, PE–Cy7, allophycocyanin, Alexa 700, and allophycocyanin–Cy7 as the available fluorescent channels. Analysis was done with FlowJo software (Tree Star, Ashland, OR). In all cases, gating on the lymphocyte population was followed by the separation of the CD3+ T cell subset and progressive gating on CD4+ and CD8+ T cell subsets. Naive cells were defined by CD28 versus CD95 expression patterns, as previously described (41). Among memory T cells, quadrant analysis of the CD28 and CCR7 parameters defined the central, transitional effector, and effector and memory subsets, as described (51). For the detection of Ag-responding subsets, we gated the CD4+ or CD8+ subsets on intracellular expression of CD69 and any of the IFN-γ, TNF-α, or IL-2 cytokines (46). Boolean gates of any of these combinations were generated, and in select experiments, cells responding by production of any of the cytokines were phenotyped with respect to CD28 and CCR7 expression, as described (46). For the detection of degranulation responses to Ag stimulation, CD107a and CD107b Abs were added to the cells during the stimulation assay, so as to detect externalization of cytotoxic granule membrane proteins upon granule fusion with the cell membrane (25).

Functional avidity assessment

Immunodominant peptides indicated in Table I were used at graded concentrations from 100 μg/ml to 1 pg/ml to stimulate PBMCs for 6 h (last 5 h in presence of brefeldin A). IFN-γ, TNF-α, or CD107a/b responses were measured as detailed earlier and normalized to maximum response to generate dose-response curves and calculate peptide concentrations corresponding with 50% of maximum response.

RhCMV quantification using real-time PCR

RhCMV DNA in blood or BAL samples was quantified by quantitative PCR amplification of a segment of the RhCMV IE-2 gene, as previously described (44). Nucleic acid was purified from samples using a Magnapure Compact instrument (Roche Diagnostics) according to the manufacturer’s instructions. Total nucleic acid in the samples was quantified by absorbance at 260 nm using a Nanodrop ND-1000 spectrophotometer. Duplicate 10-μl samples of purified nucleic acid were analyzed, and the resulting copies per sample RhCMV IE-2 gene were divided by the micrograms of total nucleic acid in the sample.

TCRB spectratyping

TCRB transcripts were analyzed for CDR3 region length polymorphism by PCR spectratyping, as previously described (40). We monitored spectratypes in PBMCs over 3 y, with four time points roughly 1 y apart. All RMs were analyzed at least twice. TCRB spectratype profiles were independently evaluated for morphology by three independent researchers; profiles were defined as “Gaussian” or “clonal” when at least two assessments categorized a Vβ as such.

Statistical analysis

Statistical analysis was performed with methods indicated in the text and using SAS software version 9.2 (SAS Institute) or Prism 5 software (GraphPad). Assessments were considered non-significant if the p value was >0.05.

Results

Loss of naive cells and T cell diversity in old monkeys

The overall goal of this project was to characterize changes in the quantity and/or quality of RhCMV-specific T cell responses associated with old age. To accomplish this goal, we assembled a cohort of randomly selected adult (7–10 y of age) and old (19–26 y of age) RMs (roughly corresponding with 20–30 and 60–80 y of age, respectively, in the human life span). Our use of adult rather than juvenile RMs as the baseline population allowed us to characterize changes associated with aging and not with the maturation of the immune system. We first assessed the impact of aging on the size and composition of the overall T cell compartment to provide context for changes in the RhCMV-specific T cell populations. As shown in Fig. 1A, the absolute counts of total CD4+ and CD8+ T cells were comparable among the adult and old RM cohorts, but the relative composition of these subsets was significantly different. The proportion of naive cells within both the CD4+ and CD8+ T cell lineages, defined as CD28intCD95lo (41), was significantly lower in old monkeys (Fig. 1B), consistent with previous results (40, 41). Memory cells were subclassified by the expression of CD28 and CCR7 into central memory (CM) subsets (CD28+CCR7+), transitional effector memory (TransEM) cells (CD28+CCR7−), and EM cells (CD28−CCR7−) (51). With regard to these subsets, the old RMs manifested a significant increase in relative size of the EM population in both lineages, as well as a modest CD4+ T cell-specific increase in the proportion of CM cells (Fig. 1B). Notably, the proportion of TransEM cells, which include the highest fraction of proliferating cells and is a general reflection of ongoing EM cell production (51), was not different between the adult and aged RMs.

FIGURE 1.
FIGURE 1.

Comparison of CD4+ and CD8+ T cell subset distribution and TCRB-defined clonality in adult versus old RMs. A, Absolute lymphocyte counts were multiplied by the fraction of CD3+/CD4+ and CD3+/CD8+ small lymphocytes determined by flow cytometry to determine absolute CD4+ and CD8+ T cell counts in blood. Symbols show values obtained in individual adult or old monkeys; horizontal bars denote medians. The p value was obtained by a two-tailed Mann–Whitney U test. B, PBMCs were further analyzed for CD28, CCR7, and CD95 expression to determine frequencies of naive, CM, TransEM, and EM subsets within the overall CD4+ and CD8+ T cell populations (see text). Histograms show mean frequencies of the indicated subsets in the adult and old RM cohorts; error bars show SEM. Indicated p values were obtained by mixed-model ANOVA followed by step-down correction. C, PBMC RNA was PCR analyzed with 24 primers specific for each TCRVB family followed by PAGE and densitometry to identify Vβ families showing a Gaussian distribution of PCR product densities over their length. Symbols indicate frequencies of TCRVB chains with Gaussian PCR products in individual monkeys over 3 y. Horizontal lines are medians; p value was obtained by a two-tailed Mann–Whitney U test. D, The same analysis as in C was performed to identify TCRVB families carrying a single PCR band over at least two consecutive years. Average frequencies of their occurrence in the adult and old monkey cohort are indicated. Error bars show SEM; p value was obtained by a two-tailed Mann–Whitney U test.

A key characteristic of an aging immune system is a general loss of T cell repertoire diversity and the appearance of prominent clonal T cell expansions, reflecting both the decline of the highly diverse naive population and the expansion or relative preservation of dominant memory clonotypes (9, 15, 52). To establish whether our adult and aged RM cohorts differed with respect to repertoire diversity and frequency of clonal expansions, we monitored CDR3 region length polymorphism of TCRB transcripts by PCR spectratyping (53). Cell populations exhibiting high TCRB diversity exhibit a bell-shaped (Gaussian) distribution of CDR3 region lengths, whereas populations with narrow repertoire show distorted profiles and in extreme cases of repertoire reduction can show a single PCR product band. We analyzed all 24 TCRBV chains in these adult and old RMs over 3 consecutive years and quantified their relative percentage of profiles with Gaussian CDR3 size distributions and with highly skewed (single peak) distributions. As TCRBV populations with single peaks can represent reversible expansions of T cells that recently encountered an Ag (e.g., cells in the TransEM subset) or stable age-associated (potentially pathogenic) T cell clonal expansions (TCE; e.g., cells in the EM subset) (52, 54), we determined the frequencies of TCRBV families exhibiting the same TCE profile in at least two consecutive time points spaced at least a year apart. Notably, these analyses revealed that old RMs manifested significantly lower frequencies of Gaussian TCRB profiles (Fig. 1C) and a significantly higher frequency of TCRB profiles showing a stable, single peak (Fig. 1D). Overall, 58.8% of old RMs had at least one stable TCE, versus only 23.8% of young adult RMs, which was a significant difference (p = 0.014; two-tailed Fisher’s exact test). Taken together, these data argue for an age-associated skewing of the TCRB repertoire in the RMs of our old cohort.

In separate studies, we have confirmed functional differences in T cell responses in our adult versus young RM cohorts, showing that the old cohort exhibited significantly weaker Ab and CD8+ T cell responses to an immunization with a live modified vaccinia strain Ankara (35). Thus, the old RM cohort studied in this article exhibited significant changes in the composition and function of the T cell compartment, consistent with immune senescence, and reminiscent of the IRP defined in aged human subjects.

RhCMV-specific CD4+ T cell responses in adult versus old monkeys

RhCMV-specific CD4+ T cell responses were quantified in both PBMCs and BALs, the latter representing an accessible mucosal effector site (41), by in vitro stimulation with whole viral Ag preparations derived from RhCMV-infected fibroblasts and were assessed for CD69 upregulation and IFN-γ, TNF-α, and IL-2 production by CD4+ memory T cells. The median frequency of T cells within the CD4+ memory compartment in blood (PBMC) specifically responding to RhCMV Ag with a cytokine response (any of the three cytokines analyzed, in any combination) was significantly higher in the adult RMs than that in the old RMs (Fig. 2A, left), but there was no difference in these frequencies in the BAL compartment [Fig. 2A, right; note that BAL T cells are essentially 100% memory in phenotype (41)]. To compare the profile of cytokine responses in adult versus old RMs, we analyzed the frequency of RhCMV-responding cells exhibiting any combination of the three cytokines and defined the contribution of each cytokine profile to the total response. Aging-related changes in these cytokine synthesis profiles were not observed in either PBMC or BAL responses (Fig. 2B). In summary, our data indicate that aging is associated with a modest decline in the frequency of CMV-specific CD4+ T cells in the blood memory compartment, but not in the BAL memory compartment, and no qualitative changes in cytokine profiles were observed in either site.

FIGURE 2.
FIGURE 2.

Comparison of RhCMV-specific CD4+ T cell responses in adult versus old RMs. A, PBMCs and BALs were assessed for CD4+ T cell responses to RhCMV Ag preparations with cytokine flow cytometry as described in Materials and Methods. The frequency of CD4+ T cells expressing CD69 and any combination of IFN-γ, TNF-α, and IL-2 expression after incubation with and without RhCMV Ag preparation was determined, with the difference between these frequencies reported as the RhCMV-specific CD4+ T cell response frequency. These values were then normalized to the memory population size in each RM. Symbols show individual monkeys; horizontal lines denote medians. The p value was obtained by a two-tailed Mann–Whitney U test. B, To determine if the pattern of IFN-γ, TNF-α, and IL-2 production by RhCMV Ag-stimulated CD4+ T cells differed in adult versus old RMs, the contribution of each possible combination of these cytokines to the overall response was determined by Boolean gating and statistically analyzed by a mixed-effects regression model analysis. Histograms indicate average frequencies of each of the indicated patterns in adult versus old responder cells; error bars are SEM.

RhCMV-specific CD8+ T cell responses in adult versus old monkeys

We next characterized RhCMV-specific responses in CD8+ T cells in PBMCs and BALs. As Ag preparations composed of lysates and/or supernatants from RhCMV-infected cells are suboptimal Ags for the induction of MHC class I-restricted (CD8+) T cell responses, we used a pool of overlapping, consecutive 15-mer peptides spanning two immunodominant RhCMV genes (IE-1 and IE-2) to evaluate RhCMV-specific CD8+ T cell responses in our cohorts. The rationale for selection of IE-1 and IE-2 for evaluation of RhCMV-specific CD8+ T cells was twofold. First, pan-proteome CD8+ T cell response analysis of HCMV-specific responses in HCMV+ humans has demonstrated that IE-1 and IE-2 are among the most frequently recognized HCMV proteins and manifest the highest response frequencies of all HCMV proteins (18). In addition, IE-1–specific CD8+ responses have been closely correlated with clinical outcome after transplantation (55, 56). Second, pilot studies in RM demonstrated that IE-1 and IE-2 were targeted by CD8+ T cells in the vast majority (albeit not all) of monkeys with robust response frequencies (far greater in both frequency and response size than responses to two RhCMV pp65 homologs; data not shown).

As shown in Fig. 3A and 3B, neither the distribution of overall response frequencies nor the cytokine profiles of RhCMV(IE)-specific CD8+ T cell responses in PBMCs or BALs were significantly different between the adult and old RM cohorts. Moreover, the frequency of cells undergoing Ag-triggered externalization of cytotoxic granules after IE peptide exposure was not significantly different between adult and old RMs in both PBMCs and BALs. Four old animals showed low (<0.2%) responses to the IE-1 plus IE-2 peptide pools in PBMCs and in BALs by all measures. As not all RhCMV+ RMs recognize epitopes within the IE proteins (data not shown), it is probable that these RMs were not deficient in RhCMV responsiveness but rather simply fell into this nonrecognition group. In any case, the exclusion of these four RMs from the analysis only decreased the difference between adult and old cohorts (data not shown), and therefore this consideration did not alter the conclusion that CD8+ responses to CMV Ag did not significantly change with age by these criteria.

FIGURE 3.
FIGURE 3.

Comparison of RhCMV(IE)-specific CD8+ T cell responses in adult versus old RMs. A, PBMCs and BALs were assessed for CD8+ T cell responses to mixes of consecutive, overlapping (11-aa overlap) 15-mer peptides composing the RhCMV IE-1 and IE-2 proteins with cytokine flow cytometry as described in Materials and Methods. The frequency of CD8+ T cells expressing CD69 and any combination of IFN-γ, TNF-α, and IL-2 expression after incubation with and without RhCMV IE peptides was determined, with the difference between these frequencies reported as the RhCMV-specific CD8+ T cell response frequency. These values were then normalized to the memory population size in each RM. Symbols show individual monkeys; horizontal lines denote medians. The p value was obtained by a two-tailed Mann–Whitney U test. B, To determine if the pattern of IFN-γ, TNF-α, and IL-2 production by RhCMV (IE) peptide-stimulated CD8+ T cells differed in adult versus old RMs, the contribution of each possible combination of these cytokines to the overall response was determined by Boolean gating and statistically analyzed by a mixed-effects regression model analysis. Histograms indicate average frequencies of each of the indicated patterns in adult versus old responder cells; error bars are SEM. Significance was assessed by mixed-effects regression model analysis. C, The frequency of CD8+ T cells manifesting externalization of cytotoxic granules in response to RhCMV (IE) peptide versus control stimulation in PBMCs (left) and BALs (right) was determined by CD107a and CD107b staining. These values were then normalized to the memory population size in each RM. Symbols show individual monkeys; horizontal lines denote medians. The p value was obtained by two-tailed Mann–Whitney U test.

Absolute CD4+ and CD8+ T cell response in old monkeys

To compare further the size of RhCMV-specific CD4+ and CD8+ T cell populations, we determined the absolute count of these cells in the blood in the adult and old RMs using the same intracellular cytokine assays described in Figs. 2 and 3. Importantly, we found no significant difference in the absolute counts of RhCMV-specific CD4+ or CD8+ T cells between adult and old monkeys (Fig. 4). Note that while the frequency of CD4+ T cell responders in the CD4+ memory T cell pool was somewhat decreased in old RMs (Fig. 2A), the increased number of total circulating memory cells in the old cohort compensated for this reduction (Fig. 4).

FIGURE 4.
FIGURE 4.

Comparison of absolute counts of RhCMV-specific CD4+ and CD8+ T cells in adult versus old RMs. PBMCs were stimulated to elicit cytokine responses in CD4+ or CD8+ T cells as shown in Figs. 2 and 3, respectively. Blood lymphocytes were counted in parallel, and this value was multiplied by the fraction of CD4+ or CD8+ RhCMV-specific lymphocytes to define the absolute count of RhCMV-specific CD4+ and CD8+ T cells. Symbols show individual monkeys; horizontal lines denote medians. The p value was obtained by a two-tailed Mann–Whitney U test.

Phenotypes of RhCMV-specific T cells in adult and old monkeys

Both HCMV-specific and RhCMV-specific T cells in blood manifest a predominantly EM phenotype (23, 24, 41, 46). To determine if aging accentuates this phenotypic bias, PBMCs were stimulated with RhCMV Ag (whole Ag preparations and IE peptides, as in Figs. 2A and 3A), and cytokine-responding cells were categorized by CD28 and CCR7 surface expression into CM, TransEM, or EM subsets of memory cells, as described in Fig. 1. In parallel, PBMCs were stimulated by the superantigen Staphylococcus enterotoxin B (SEB) and similarly phenotyped to demonstrate the distribution of a generic functional response among the CM, TransEM, or EM subsets. As shown in Fig. 5A, RhCMV-specific CD4+ T cells manifested a similar EM bias in both adult and old RMs, in contrast to the predominant CM phenotype of SEB-responsive cells. As shown in Fig. 1, the overall CD8+ memory compartment is much more EM-biased than the CD4+ memory compartment, and this overall bias increases with age. In keeping with this, the SEB-responsive CD8+ T cell subset was more EM-biased in old RMs than adult RMs (Fig. 5B). Importantly, however, the RhCMV(IE)-specific CD8+ T cell responses were even more EM-biased than the overall or SEB-responsive population, and this marked bias was similar in the adult and old RM cohorts. In conclusion, RhCMV-specific T cells are highly EM-biased in both adult and old RMs, and aging did not increase the extent of this bias.

FIGURE 5.
FIGURE 5.

Comparison of the phenotypically defined differentiation state of RhCMV-specific T cell responses in adult versus old RMs. PBMCs were stimulated with SEB (200 ng/ml), whole RhCMV Ag preparations, RhCMV(IE) peptide mixes, or no Ag, as described in Figs. 2 and 3. Cells were analyzed for the expression of CD3, CD4, CD8, CD69, IFN-γ, TNF-α, and IL-2, as described in Figs. 2 and 3, and in addition, the surface markers CD28 and CCR7, with the CD28 versus CCR7 phenotype (CM, TransEM, and EM designations as in Fig. 1) determined for all responding cells (CD69+ plus any combination of the three cytokines). To provide sufficient events for accurate phenotypic analysis, the data set was restricted to RMs with specific response frequencies of >0.4%. A, Analysis of CD4+ T cells. B, Analysis of CD8+ T cells. Horizontal lines show medians. Significance assessed by Kruskal–Wallis test, with Dunn’s postanalysis. Of note, parametric statistical analysis by ANOVA with Bonferroni postanalysis revealed an age-related difference between the CD8+ T cell populations responding to SEB, at a significance of p < 0.05, but not CD8+ T cell populations to IE, or any of the responding CD4+ T cell populations.

Function, phenotype, and triggering thresholds of CD8+ T cell responses to immunodominant CMV peptide epitopes in adult and old monkeys

The 15-mer peptides composing the IE-1/IE-2 peptide mixes are not optimal ligands for triggering CD8+ T cell responses (57), and the responses to these mixes likely include variable combinations of responses to different epitopes in each animal, factors that together might obscure differences in the function of RhCMV-specific CD8+ T cells in adult versus old RMs. To compare more precisely the functional responses in adult versus old RMs, we screened our cohorts for common CD8+ T cell responses to optimal IE-1 and IE-2 9-mer epitopes. Three epitopes with vigorous responses common to multiple RMs in both age cohorts were identified (Table I). CD8+ T cells responding to these immunodominant optimal epitopes displayed an even higher EM bias than the overall RhCMV(IE)-specific CD8+ T cell responses, but again, the adult and old RMs showed essentially identical phenotype profiles (Fig. 6A). In addition, the cytokine profile of the CD8+ T cells responding to these immunodominant optimal peptides were indistinguishable between the adult and old RM cohorts (Fig. 6B). As TCRs bind to peptide–MHC complexes with varying avidity, and high-avidity responses provide better protection against viral pathogens (58), we next investigated the functional avidity of CD8+ T cells to these immunodominant peptides by determining the dependence of the cytokine and degranulation responses on the dose of added peptide. The normalized dose-response curves of CD107–, IFN-γ–, or TNF-α–responding CD8+ T cells upon stimulation with graded peptide concentrations is shown in Fig. 6C. Notably, whereas dose dependence of the CD107 response to peptide VTTLGMALY showed a minor shift to the right in old monkeys, other functions triggered by this peptide and all functions triggered by a different peptide (SGVLPENVP) manifested statistically indistinguishable dose-response profiles. Taken together, these data argue against a general decline in CD8+ T cell responsiveness to immunodominant RhCMV epitopes with aging in RMs.

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Table I.

CD8+ T cell response to optimal IE peptides (% responding in the blood memory population)

FIGURE 6.
FIGURE 6.

Comparison of the phenotype, cytokine profile, and avidity of CD8+ T cells responding to immunodominant peptides in adult versus old RMs. A, CD8+ cells responding to the immunodominant 9-mer peptides shown in Table I were phenotyped by CD28 and CCR7 surface expression as in Fig. 5. Symbols show individual adult versus old RMs; horizontal lines are medians. Significance was assessed by Kruskal–Wallis test, with Dunn’s postanalysis. B, Cytokine profiles of CD8+ T cells responding to immunodominant 9-mer peptides shown in Table I were determined as in Fig. 3. Histograms indicate average values for adult versus old RMs; error bars are SEM. Significance was assessed by mixed-effects regression model analysis. C, PBMCs were stimulated with serial dilutions of the VTTLGMALY (top panels) or SGVLPENVP (bottom panels) peptides. The percentage of CD8+ T cells responding to these peptides with CD107 surface expression or intracellular production of IFN-γ or TNF-α was determined as in Fig. 3 and was normalized to the maximum response for each RM. Dose-response curves were defined by regression analysis in each RM, and adult and old RMs were compared by mixed-model analysis.

Response of adult and old RMs to CMV challenge

Although ex vivo functional assays of RhCMV-specific T cells revealed few differences in the quantity or quality of these responses in adult versus old RMs, these data do not rule out a defect in the ability of aged RhCMV-specific T cells to respond to in vivo challenge. To address directly the in vivo capabilities of RhCMV-specific T cell responses, we challenged a subset of the adult (n = 11) and old (n = 9) RM cohorts with infectious RhCMV and compared the ability of adult versus old RMs to respond to and control RhCMV superinfection (a superphysiologic challenge designed to reveal otherwise subclinical deficits in anti-RhCMV immunity). As a measure of such superinfection, levels of RhCMV DNA were monitored by quantitative PCR of both PBMCs and BALs. RhCMV DNA was not detectable in either cell population in any RM prior to challenge, and even after challenge, PBMCs remained negative in both adult and old RMs, consistent with previous studies of RhCMV superinfection of adult RMs (44). However, RhCMV DNA appeared in the BALs of both adult and old RMs after challenge, starting at day 14 and peaking at postinfection day 70, prior to a slow decline (Fig. 7). Importantly, the kinetics and levels of RhCMV replication in BALs were remarkably similar in adult and old RMs, strongly arguing that aging does not impair the ability of RhCMV-specific T cell responses to control RhCMV. This in vivo challenge also allowed assessment of the T cell proliferative response to RhCMV-Ag stimulation in vivo, as determined by the change in the frequency of RhCMV-specific T cells in blood and BALs, as well as the induction of the proliferation-associated marker Ki-67 on RhCMV-specific T cells postchallenge (41). In this regard, although the average frequency of RhCMV-specific CD4+ T cells in blood was slightly, but significantly, higher in adult RMs compared with that in old RMs, both the fold increase in these frequencies and the kinetics and level of Ki-67 induction on these cells were indistinguishable in adult versus old RMs (Fig. 8A–C). In keeping with their marked EM bias (59), frequencies of RhCMV(IE)-specific CD8+ T cells did not increase after challenge, and Ki-67 induction was modest; however, these profiles were also essentially identical in the adult versus old RM cohorts (Fig. 8D–F). Thus, the response to and control of RhCMV challenge was equivalent in the adult and old RM cohorts.

FIGURE 7.
FIGURE 7.

Comparison of RhCMV kinetics in adult and old monkeys after infectious RhCMV challenge. Adult (n = 11) or old (n = 9) RMs were challenged by s.c. injection of 107 PFU RhCMV. RhCMV genomes in PBMCs or BALs were quantified by quantitative PCR and standardized to genomic host DNA. Average ± SEM of RhCMV copy numbers (y-axis) at indicated postchallenge days (x-axis) are indicated. LD, limit of detection.

FIGURE 8.
FIGURE 8.

Comparison of the in vivo proliferation response of RhCMV-specific T cells in adult versus old RMs after infectious RhCMV challenge. The adult and old RMs challenged as shown in Fig. 7 were followed over time for frequencies of RhCMV-specific T cells in the circulating memory compartment by cytokine flow cytometry. Responding cells were defined by IFN-γ and/or TNF-α expression in CD69+ cells. A–C, CD4+ T cells responding to whole RhCMV Ag preparations. D–F, CD8+ T cells responding to RhCMV(IE) peptide mixes. Response frequencies are shown both as average ± SEM frequencies (A, D) and as average ± SEM fold change from the prechallenge baseline (average of day −7 and day 0 values) (B, E). Responding cells were simultaneously analyzed for expression of the proliferation marker Ki-67 (41) with results presented as average ± SEM for percent Ki-67+ of the RhCMV-specific T cells (C, F). Statistical analysis was by repeated-measures ANOVA.

Discussion

Primate CMVs have evolved a unique immunobiologic relationship with their hosts. In the absence of disease and in most cases overt infection, these viruses elicit and maintain remarkably high frequency T cell responses, considerably higher than those of other common viral infections and similar to those of overtly pathogenic chronic infections such as HIV (18, 60–64). Moreover, these responses manifest a distinctive highly EM-biased phenotype (22–25, 28, 59). Although the basis of this unique immunogenicity remains to be experimentally confirmed, HCMV is thought to, and RhCMV is known to, persist as a very low level continuous or frequently intermittent infection, rather than as a completely or predominantly quiescent, latent infection (44, 65, 66). This characteristic would provide chronic, low-level Ag exposure, which might drive the characteristic EM differentiation of CMV-specific T cells and maintain the very high CMV-specific T cell response frequencies over the lifetime of the host. Note that this chronic Ag exposure is not so high as to induce the T cell “functional exhaustion” that characterizes pathogenic chronic viral infection (67, 68), as CMV-specific T cells are highly functional (23, 25, 46, 60, 69). Indeed, the rapidity of CMV reactivation after the onset of potent immunosuppressive therapy, the potential seriousness of CMV disease in this setting, and the fact that the CMV is almost invariably controlled without overt disease in immunocompetent individuals, even into old age, attests to the in vivo function of these T cell responses (30, 31, 47, 65). Moreover, the fact that the highly EM-biased SIV-specific T cell responses elicited by RhCMV vectors encoding SIV Ags have shown promising efficacy in protection against pathogenic SIV challenge in RM suggests that “CMV-like” T cell responses can effectively function against heterologous pathogens (46). However, as indicated above, the association of CMV infection and the IRP has led to the hypothesis that lifelong control of CMV might have adverse consequences in old age, with the massive immune responses against CMV possibly causing or materially contributing to the immune senescence and the consequent increased morbidity and mortality of infectious diseases in the elderly (7, 11–13, 15, 17, 21). More recently, it has also been suggested that CMV control itself might be compromised in the elderly, leading to occult infection and associated chronic inflammation that might compromise function of diverse organs and physiologic processes (14, 70).

Perhaps the most likely mechanism that could link CMV infection or anti-CMV immunity to aging pathogenesis would be the development in old age of an intrinsic senescence of the CMV-specific T cell response itself. Given that CMV is often acquired in early childhood and that CMV-specific T cells are, as suggested above, almost certainly exposed to chronic antigenic stimulation, it is reasonable to speculate that after decades of “use,” including many years without a functional thymus to provide new naive cells to replenish and diversify these responses (71), CMV-specific T cells might develop critically short telomeres, accumulate mutations, and thus simply wear out (15, 19, 20, 29, 72). Hypothetically, this might lead to diminished control of CMV replication, increasing the frequency and/or magnitude of reactivation episodes, which in turn might result in direct pathogenicity by frequent or continuous occult infection(s) and chronic inflammation in critical tissues and/or indirect pathogenicity by driving expansion of an abnormal CMV-specific T cell response or by stimulating chronic immune activation, thereby displacing or dysregulating the homeostasis of other crucial T cell populations. Conceivably, these conditions might set up a reinforcing spiral of deterioration in which failing CMV-specific immunity leads to CMV replication, which further compromises both the CMV-specific response and, as collateral damage, the overall immune system, leading to further CMV replication (as well as other infectious complications), and further immune compromise.

This concept has been sustained by reports that either the CMV-specific T cell response itself and/or the CD28− EM population it dominates comprise the major proportion of PBLs in elderly individuals (particularly those with the highest short-term mortality), that these populations manifest replicative senescence and other functional “abnormalities,” that CMV detection in secretions is more common in the elderly, and that elderly individuals with the highest quartile of CMV-specific Ab levels have increased mortality (7, 13, 15–17, 19, 20, 29, 70, 72). There are, however, several crucial caveats in interpreting these observations. First, the domination of CMV-specific EM cells in the elderly has only been reported as a relative dominance in peripheral blood. The overall number of CMV-specific T cells in the body has not been enumerated in adult versus elderly individuals, and thus the dominance of CMV-specific T cells might result from loss of non-EM populations around a stable EM population that includes the vast majority of the CMV-specific T cells. CMV− seronegative individuals would lack the benchmark of the large CMV-specific population by which the relative loss of other populations in blood can be visualized. In tissue, CMV-specific T cells are present at high frequency in extralymphoid effector sites, but these cells constitute only a minor component of T cells in the lymph nodes supporting naive and CM T cell homeostasis [(73) and L. Picker, unpublished observations], arguing against a direct displacement of naive and CM T cells by CMV-specific T cells. The replicative senescence and other “abnormal” functions attributed to CMV-specific T cells (low IL-2, etc.) are in fact intrinsic characteristics of EM cell differentiation and typify CMV-specific T cell populations from individuals of all ages, including children, and even heterologous responses elicited by CMV vectors in the RM model (22, 46, 59). Moreover, the skewed clonotypic hierarchies of CMV-specific T cells are also characteristic of mature CMV-specific T cell responses regardless of age (22, 26, 44, 74). Finally, the association of CMV secretion or elevated levels of CMV Abs with sick or “soon to be sick” elderly individuals is not surprising given CMV replication can increase with stress (65, 75). It is therefore possible that CMV replication and increased levels of CMV-specific immunity in such end-of-life individuals is initiated by their other age-associated disease, rather than vice versa.

A crucial issue in the investigation of a possible causal role of CMV in immune senescence is the question of whether CMV-specific T cell populations do or do not become dysfunctional with age and therefore are less able to control the virus. In this study, we addressed this question in the RM model, which includes both a human-like immune senescence syndrome and a CMV virus, RhCMV, which is biologically highly homologous to HCMV and which allows for infectious challenge with RhCMV. We compared the frequency of RhCMV-specific CD4+ and CD8+ T cells in blood and lung airspace (a representative mucosal interface effector site), the absolute number and differentiation phenotype of these cells in blood, and both ex vivo and in vivo function of these cells in cohorts of adult and old RMs, the latter cohort showing laboratory and functional evidence of immune senescence. Overall, these studies failed to show biologically significant differences in adult versus old CMV-specific T cell responses, most importantly including any difference in the response to and control of infectious RhCMV challenge. In a previously reported study, we found that the clonotypic hierarchies of RhCMV-specific CD4+ T cell responses were similar in adult and old RMs (44). Taken together, these results are not consistent with a general deterioration of RhCMV-specific T cells with old age and therefore argue against a major contribution of such deterioration to immune senescence in this model. There are, however, several caveats to these analyses. First, we did observe a minor but statistically significant shift to the left in one functional response to one immunodominant peptide. Second, our analysis of RhCMV-specific CD8+ T cells was limited to those specific for IE-1 and IE-2. Although this specificity is undoubtedly an important component of the overall RhCMV-specific response (55, 56), many other CMV proteins are targeted by CD8+ T cells (18), and the IE-1– and IE-2–specific CD8+ T cell response may not be representative of CD8+ T cell responses to all RhCMV proteins. However, given the equivalence of adult and old RMs in the control of, and functional response to, a superphysiologic in vivo RhCMV challenge, we do not believe these caveats change our overall interpretation that cellular immunity to RhCMV is generally well preserved in old age. In this regard, it is notable that transcriptional analysis of highly purified CD8+/CD28− cells in young and elderly people revealed remarkable similarity, whereas analysis of CD8+/CD28+ populations revealed changes in the old donors (76), results that also suggest relative preservation of EM T cell integrity with age compared with naive and CM T cells.

These data do not, by any means, rule out the participation of CMV infection in the pathogenesis of immune senescence in humans, but they do help refine the possible mechanisms by which such participation might occur. For example, because healthy elderly individuals may harbor CMV infection and the association between the IRP and aging mortality is not uniform across all human populations (32, 33), it is possible that other genetic or environmental predispositions might be required for failure of CMV-specific immunity or other age-associated contribution of CMV to aging pathogenesis. RMs and human populations that lack such putative predispositions would lack CMV-mediated acceleration of immune senescence, but still would be susceptible to other age-related immune deterioration such as lack of thymopoiesis and loss of naive populations (17). In addition, CMV might participate in the pathogenesis of immune senescence by mechanisms that do not require general deterioration of the CMV-specific T cell response. In this regard, chronic, nonpathogenic viral infections have been shown to reset the innate immune system in animal models, in some instances providing augmented innate immune-mediated protection against heterologous pathogens (77). This innate immune resetting may have a negative effect on immune homeostasis when maintained over long periods of time and/or when associated with other pathology such as thymic involution (78). Finally, CMV may only participate in aging pathogenesis when associated with multisystem failure, providing an additional insult to a collapsing overall physiology (75). In this scenario, dysfunction of the CMV-specific T cell response might only occur in old individuals with significant ongoing disease and would not be observed in otherwise healthy elderly subjects. As RMs manifesting significant disease at time of assignment were excluded from the old RM cohort studied here, our study would not have identified such secondary failure of RhCMV-specific immunity.

The β-herpesviruses in general, and the CMV family of viruses specifically, emerged prior to mammalian radiation, and therefore, these viruses have coevolved with their mammalian hosts, leading to an evolutionarily “negotiated” balance between the host immune system and the virus. With this balance, the virus enjoys pervasiveness in a large host population that could not occur with unchecked pathogenicity. For the host, the prevention of pathogenicity within reproductively relevant members of the population eliminates the evolutionary cost of carrying the infection. The unique high frequency of EM-biased T cell responses elicited by primate CMVs are clearly an integral part of this balance in these species and should be considered normal and adaptive in children, adults, and most elderly. This, of course, does not preclude the involvement of CMV or these protective T cell responses in disease in the elderly, a common theme in aging (7, 40), but does suggest that the mechanisms responsible for any such involvement are likely to be complex. Our data demonstrate a remarkable preservation of CMV-specific T cell responses in a primate model, arguing against a simple model in which progressive deterioration or dysregulation of these responses releases CMV from immune control in old individuals and/or otherwise contributes to immune dysfunction. Further work in both the human system and non-human primate models will be required to confirm or rule out other potential mechanisms linking CMV infection or CMV-specific immunity to age-related disease and mortality and/or to define cofactors that might facilitate CMV-mediated pathogenesis in a subset of susceptible elderly individuals.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Nate Whizin for cytometry assistance with data collection and Tomi Mori for assistance with statistical analysis.

Footnotes

  • This work was supported by National Institutes of Health Awards P01 AG023664 and R01 AI082529 (to L.J.P. and J.N.-Ž.) and by the Oregon National Primate Research Center National Institutes of Health Core Grant P51 RR00163.

  • Abbreviations used in this article:

    BAL
    bronchoalveolar lavage lymphocyte
    CM
    central memory
    EM
    effector memory
    HCMV
    human cytomegalovirus
    IE
    immediate early
    IRP
    immune risk profile
    RhCMV
    rhesus cytomegalovirus
    RM
    rhesus macaque
    SEB
    Staphylococcus enterotoxin B
    TransEM
    transitional effector memory.

  • Received February 23, 2011.
  • Accepted June 6, 2011.

References

  1. ↵
    1. Ginaldi L.,
    2. M. F. Loreto,
    3. M. P. Corsi,
    4. M. Modesti,
    5. M. De Martinis
    . 2001. Immunosenescence and infectious diseases. Microbes Infect. 3: 851–857.
    OpenUrlCrossRefPubMed
    1. High K. P.
    2004. Infection as a cause of age-related morbidity and mortality. Ageing Res. Rev. 3: 1–14.
    OpenUrlCrossRefPubMed
    1. Hakim F. T.,
    2. R. E. Gress
    . 2007. Immunosenescence: deficits in adaptive immunity in the elderly. Tissue Antigens 70: 179–189.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Aspinall R.,
    2. G. Del Giudice,
    3. R. B. Effros,
    4. B. Grubeck-Loebenstein,
    5. S. Sambhara
    . 2007. Challenges for vaccination in the elderly. Immun. Ageing 4: 9.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Weiskopf D.,
    2. B. Weinberger,
    3. B. Grubeck-Loebenstein
    . 2009. The aging of the immune system. Transpl. Int. 22: 1041–1050.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Linton P. J.,
    2. K. Dorshkind
    . 2004. Age-related changes in lymphocyte development and function. Nat. Immunol. 5: 133–139.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Derhovanessian E.,
    2. A. Larbi,
    3. G. Pawelec
    . 2009. Biomarkers of human immunosenescence: impact of cytomegalovirus infection. Curr. Opin. Immunol. 21: 440–445.
    OpenUrlCrossRefPubMed
    1. Kovaiou R. D.,
    2. B. Grubeck-Loebenstein
    . 2006. Age-associated changes within CD4+ T cells. Immunol. Lett. 107: 8–14.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Posnett D. N.,
    2. R. Sinha,
    3. S. Kabak,
    4. C. Russo
    . 1994. Clonal populations of T cells in normal elderly humans: the T cell equivalent to “benign monoclonal gammapathy.” J. Exp. Med. 179: 609–618.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Wikby A.,
    2. P. Maxson,
    3. J. Olsson,
    4. B. Johansson,
    5. F. G. Ferguson
    . 1998. Changes in CD8 and CD4 lymphocyte subsets, T cell proliferation responses and non-survival in the very old: the Swedish longitudinal OCTO-immune study. Mech. Ageing Dev. 102: 187–198.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Olsson J.,
    2. A. Wikby,
    3. B. Johansson,
    4. S. Löfgren,
    5. B. O. Nilsson,
    6. F. G. Ferguson
    . 2000. Age-related change in peripheral blood T-lymphocyte subpopulations and cytomegalovirus infection in the very old: the Swedish longitudinal OCTO immune study. Mech. Ageing Dev. 121: 187–201.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Wikby A.,
    2. B. Johansson,
    3. J. Olsson,
    4. S. Löfgren,
    5. B. O. Nilsson,
    6. F. Ferguson
    . 2002. Expansions of peripheral blood CD8 T-lymphocyte subpopulations and an association with cytomegalovirus seropositivity in the elderly: the Swedish NONA immune study. Exp. Gerontol. 37: 445–453.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Pawelec G.,
    2. A. Akbar,
    3. C. Caruso,
    4. R. Effros,
    5. B. Grubeck-Loebenstein,
    6. A. Wikby
    . 2004. Is immunosenescence infectious? Trends Immunol. 25: 406–410.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Vescovini R.,
    2. C. Biasini,
    3. A. R. Telera,
    4. M. Basaglia,
    5. A. Stella,
    6. F. Magalini,
    7. L. Bucci,
    8. D. Monti,
    9. T. Lazzarotto,
    10. P. Dal Monte,
    11. et al
    . 2010. Intense antiextracellular adaptive immune response to human cytomegalovirus in very old subjects with impaired health and cognitive and functional status. J. Immunol. 184: 3242–3249.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Hadrup S. R.,
    2. J. Strindhall,
    3. T. Køllgaard,
    4. T. Seremet,
    5. B. Johansson,
    6. G. Pawelec,
    7. P. thor Straten,
    8. A. Wikby
    . 2006. Longitudinal studies of clonally expanded CD8 T cells reveal a repertoire shrinkage predicting mortality and an increased number of dysfunctional cytomegalovirus-specific T cells in the very elderly. J. Immunol. 176: 2645–2653.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Wang G. C.,
    2. W. H. Kao,
    3. P. Murakami,
    4. Q. L. Xue,
    5. R. B. Chiou,
    6. B. Detrick,
    7. J. F. McDyer,
    8. R. D. Semba,
    9. V. Casolaro,
    10. J. D. Walston,
    11. L. P. Fried
    . 2010. Cytomegalovirus infection and the risk of mortality and frailty in older women: a prospective observational cohort study. Am. J. Epidemiol. 171: 1144–1152.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Brunner S.,
    2. D. Herndler-Brandstetter,
    3. B. Weinberger,
    4. B. Grubeck-Loebenstein
    . 2011. Persistent viral infections and immune aging. Ageing Res. Rev. 10: 362–369.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Sylwester A. W.,
    2. B. L. Mitchell,
    3. J. B. Edgar,
    4. C. Taormina,
    5. C. Pelte,
    6. F. Ruchti,
    7. P. R. Sleath,
    8. K. H. Grabstein,
    9. N. A. Hosken,
    10. F. Kern,
    11. et al
    . 2005. Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects. J. Exp. Med. 202: 673–685.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Ouyang Q.,
    2. W. M. Wagner,
    3. A. Wikby,
    4. S. Walter,
    5. G. Aubert,
    6. A. I. Dodi,
    7. P. Travers,
    8. G. Pawelec
    . 2003. Large numbers of dysfunctional CD8+ T lymphocytes bearing receptors for a single dominant CMV epitope in the very old. J. Clin. Immunol. 23: 247–257.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Ouyang Q.,
    2. W. M. Wagner,
    3. W. Zheng,
    4. A. Wikby,
    5. E. J. Remarque,
    6. G. Pawelec
    . 2004. Dysfunctional CMV-specific CD8(+) T cells accumulate in the elderly. Exp. Gerontol. 39: 607–613.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Vescovini R.,
    2. C. Biasini,
    3. F. F. Fagnoni,
    4. A. R. Telera,
    5. L. Zanlari,
    6. M. Pedrazzoni,
    7. L. Bucci,
    8. D. Monti,
    9. M. C. Medici,
    10. C. Chezzi,
    11. et al
    . 2007. Massive load of functional effector CD4+ and CD8+ T cells against cytomegalovirus in very old subjects. J. Immunol. 179: 4283–4291.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Komatsu H.,
    2. A. Inui,
    3. T. Sogo,
    4. T. Fujisawa,
    5. H. Nagasaka,
    6. S. Nonoyama,
    7. S. Sierro,
    8. J. Northfield,
    9. M. Lucas,
    10. A. Vargas,
    11. P. Klenerman
    . 2006. Large scale analysis of pediatric antiviral CD8+ T cell populations reveals sustained, functional and mature responses. Immun. Ageing 3: 11.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Kern F.,
    2. E. Khatamzas,
    3. I. Surel,
    4. C. Frömmel,
    5. P. Reinke,
    6. S. L. Waldrop,
    7. L. J. Picker,
    8. H. D. Volk
    . 1999. Distribution of human CMV-specific memory T cells among the CD8pos. subsets defined by CD57, CD27, and CD45 isoforms. Eur. J. Immunol. 29: 2908–2915.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Appay V.,
    2. P. R. Dunbar,
    3. M. Callan,
    4. P. Klenerman,
    5. G. M. Gillespie,
    6. L. Papagno,
    7. G. S. Ogg,
    8. A. King,
    9. F. Lechner,
    10. C. A. Spina,
    11. et al
    . 2002. Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections. Nat. Med. 8: 379–385.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Casazza J. P.,
    2. M. R. Betts,
    3. D. A. Price,
    4. M. L. Precopio,
    5. L. E. Ruff,
    6. J. M. Brenchley,
    7. B. J. Hill,
    8. M. Roederer,
    9. D. C. Douek,
    10. R. A. Koup
    . 2006. Acquisition of direct antiviral effector functions by CMV-specific CD4+ T lymphocytes with cellular maturation. J. Exp. Med. 203: 2865–2877.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Bitmansour A. D.,
    2. S. L. Waldrop,
    3. C. J. Pitcher,
    4. E. Khatamzas,
    5. F. Kern,
    6. V. C. Maino,
    7. L. J. Picker
    . 2001. Clonotypic structure of the human CD4+ memory T cell response to cytomegalovirus. J. Immunol. 167: 1151–1163.
    OpenUrlAbstract/FREE Full Text
    1. Almanzar G.,
    2. S. Schwaiger,
    3. B. Jenewein,
    4. M. Keller,
    5. D. Herndler-Brandstetter,
    6. R. Würzner,
    7. D. Schönitzer,
    8. B. Grubeck-Loebenstein
    . 2005. 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. J. Virol. 79: 3675–3683.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Sacre K.,
    2. G. Carcelain,
    3. N. Cassoux,
    4. A. M. Fillet,
    5. D. Costagliola,
    6. D. Vittecoq,
    7. D. Salmon,
    8. Z. Amoura,
    9. C. Katlama,
    10. B. Autran
    . 2005. Repertoire, diversity, and differentiation of specific CD8 T cells are associated with immune protection against human cytomegalovirus disease. J. Exp. Med. 201: 1999–2010.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Fletcher J. M.,
    2. M. Vukmanovic-Stejic,
    3. P. J. Dunne,
    4. K. E. Birch,
    5. J. E. Cook,
    6. S. E. Jackson,
    7. M. Salmon,
    8. M. H. Rustin,
    9. A. N. Akbar
    . 2005. Cytomegalovirus-specific CD4+ T cells in healthy carriers are continuously driven to replicative exhaustion. J. Immunol. 175: 8218–8225.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Rafailidis P. I.,
    2. E. G. Mourtzoukou,
    3. I. C. Varbobitis,
    4. M. E. Falagas
    . 2008. Severe cytomegalovirus infection in apparently immunocompetent patients: a systematic review. Virol. J. 5: 47.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Morunglav M.,
    2. I. Theate,
    3. G. Bertin,
    4. P. Hantson
    . 2010. CMV enteritis causing massive intestinal hemorrhage in an elderly patient. Case Report Med. 2010: pii: 385795.
    OpenUrl
  32. ↵
    1. Colonna-Romano G.,
    2. A. N. Akbar,
    3. A. Aquino,
    4. M. Bulati,
    5. G. Candore,
    6. D. Lio,
    7. P. Ammatuna,
    8. J. M. Fletcher,
    9. C. Caruso,
    10. G. Pawelec
    . 2007. Impact of CMV and EBV seropositivity on CD8 T lymphocytes in an old population from West-Sicily. Exp. Gerontol. 42: 995–1002.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Derhovanessian E.,
    2. A. B. Maier,
    3. R. Beck,
    4. G. Jahn,
    5. K. Hähnel,
    6. P. E. Slagboom,
    7. A. J. de Craen,
    8. R. G. Westendorp,
    9. G. Pawelec
    . 2010. Hallmark features of immunosenescence are absent in familial longevity. J. Immunol. 185: 4618–4624.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Douek D. C.,
    2. R. D. McFarland,
    3. P. H. Keiser,
    4. E. A. Gage,
    5. J. M. Massey,
    6. B. F. Haynes,
    7. M. A. Polis,
    8. A. T. Haase,
    9. M. B. Feinberg,
    10. J. L. Sullivan,
    11. et al
    . 1998. Changes in thymic function with age and during the treatment of HIV infection. Nature 396: 690–695.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Cicin-Sain L.,
    2. S. Smyk-Pearson,
    3. N. Currier,
    4. L. Byrd,
    5. C. Koudelka,
    6. T. Robinson,
    7. G. Swarbrick,
    8. S. Tackitt,
    9. A. Legasse,
    10. M. Fischer,
    11. et al
    . 2010. Loss of naive T cells and repertoire constriction predict poor response to vaccination in old primates. J. Immunol. 184: 6739–6745.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Nikolich-Zugich J.,
    2. B. D. Rudd
    . 2010. Immune memory and aging: an infinite or finite resource? Curr. Opin. Immunol. 22: 535–540.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Wikby A.,
    2. I. A. Månsson,
    3. B. Johansson,
    4. J. Strindhall,
    5. S. E. Nilsson
    . 2008. The immune risk profile is associated with age and gender: findings from three Swedish population studies of individuals 20-100 years of age. Biogerontology 9: 299–308.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Bruunsgaard H.,
    2. M. Pedersen,
    3. B. K. Pedersen
    . 2001. Aging and proinflammatory cytokines. Curr. Opin. Hematol. 8: 131–136.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Janković V.,
    2. I. Messaoudi,
    3. J. Nikolich-Zugich
    . 2003. Phenotypic and functional T-cell aging in rhesus macaques (Macaca mulatta): differential behavior of CD4 and CD8 subsets. Blood 102: 3244–3251.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Cicin-Sain L.,
    2. I. Messaoudi,
    3. B. Park,
    4. N. Currier,
    5. S. Planer,
    6. M. Fischer,
    7. S. Tackitt,
    8. D. Nikolich-Zugich,
    9. A. Legasse,
    10. M. K. Axthelm,
    11. et al
    . 2007. Dramatic increase in naive T cell turnover is linked to loss of naive T cells from old primates. Proc. Natl. Acad. Sci. USA 104: 19960–19965.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Pitcher C. J.,
    2. S. I. Hagen,
    3. J. M. Walker,
    4. R. Lum,
    5. B. L. Mitchell,
    6. V. C. Maino,
    7. M. K. Axthelm,
    8. L. J. Picker
    . 2002. Development and homeostasis of T cell memory in rhesus macaque. J. Immunol. 168: 29–43.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Haberthur K.,
    2. F. Engelman,
    3. A. Barron,
    4. I. Messaoudi
    . 2010. Immune senescence in aged nonhuman primates. Exp. Gerontol. 45: 655–661.
    OpenUrlCrossRefPubMed
  43. ↵
    1. Powers C.,
    2. K. Früh
    . 2008. Rhesus CMV: an emerging animal model for human CMV. Med. Microbiol. Immunol. (Berl.) 197: 109–115.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Price D. A.,
    2. A. D. Bitmansour,
    3. J. B. Edgar,
    4. J. M. Walker,
    5. M. K. Axthelm,
    6. D. C. Douek,
    7. L. J. Picker
    . 2008. Induction and evolution of cytomegalovirus-specific CD4+ T cell clonotypes in rhesus macaques. J. Immunol. 180: 269–280.
    OpenUrlAbstract/FREE Full Text
    1. Kaur A.,
    2. M. D. Daniel,
    3. D. Hempel,
    4. D. Lee-Parritz,
    5. M. S. Hirsch,
    6. R. P. Johnson
    . 1996. Cytotoxic T-lymphocyte responses to cytomegalovirus in normal and simian immunodeficiency virus-infected rhesus macaques. J. Virol. 70: 7725–7733.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    1. Hansen S. G.,
    2. C. Vieville,
    3. N. Whizin,
    4. L. Coyne-Johnson,
    5. D. C. Siess,
    6. D. D. Drummond,
    7. A. W. Legasse,
    8. M. K. Axthelm,
    9. K. Oswald,
    10. C. M. Trubey,
    11. et al
    . 2009. Effector memory T cell responses are associated with protection of rhesus monkeys from mucosal simian immunodeficiency virus challenge. Nat. Med. 15: 293–299.
    OpenUrlCrossRefPubMed
  47. ↵
    1. Pass R. F.
    2001. Cytomegalovirus. In Fields Virology, 4th Ed. Knipe D. M., P. M. Howley, eds. Lippincott/The Williams and Wilkins Co., Philadelphia, PA, p. 2675–2705.
    1. Clarke L. M.,
    2. A. Duerr,
    3. J. Feldman,
    4. M. F. Sierra,
    5. B. J. Daidone,
    6. S. H. Landesman
    . 1996. Factors associated with cytomegalovirus infection among human immunodeficiency virus type 1-seronegative and -seropositive women from an urban minority community. J. Infect. Dis. 173: 77–82.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    1. Arora N.,
    2. Z. Novak,
    3. K. B. Fowler,
    4. S. B. Boppana,
    5. S. A. Ross
    . 2010. Cytomegalovirus viruria and DNAemia in healthy seropositive women. J. Infect. Dis. 202: 1800–1803.
    OpenUrlAbstract/FREE Full Text
  50. ↵
    1. Hansen S. G.,
    2. C. J. Powers,
    3. R. Richards,
    4. A. B. Ventura,
    5. J. C. Ford,
    6. D. Siess,
    7. M. K. Axthelm,
    8. J. A. Nelson,
    9. M. A. Jarvis,
    10. L. J. Picker,
    11. K. Früh
    . 2010. Evasion of CD8+ T cells is critical for superinfection by cytomegalovirus. Science 328: 102–106.
    OpenUrlAbstract/FREE Full Text
  51. ↵
    1. Picker L. J.,
    2. E. F. Reed-Inderbitzin,
    3. S. I. Hagen,
    4. J. B. Edgar,
    5. S. G. Hansen,
    6. A. Legasse,
    7. S. Planer,
    8. M. Piatak Jr..,
    9. J. D. Lifson,
    10. V. C. Maino,
    11. et al
    . 2006. IL-15 induces CD4 effector memory T cell production and tissue emigration in nonhuman primates. J. Clin. Invest. 116: 1514–1524.
    OpenUrlCrossRefPubMed
  52. ↵
    1. Clambey E. T.,
    2. J. W. Kappler,
    3. P. Marrack
    . 2007. CD8 T cell clonal expansions & aging: a heterogeneous phenomenon with a common outcome. Exp. Gerontol. 42: 407–411.
    OpenUrlCrossRefPubMed
  53. ↵
    1. Mosley R. L.,
    2. M. M. Koker,
    3. R. A. Miller
    . 1998. Idiosyncratic alterations of TCR size distributions affecting both CD4 and CD8 T cell subsets in aging mice. Cell. Immunol. 189: 10–18.
    OpenUrlCrossRefPubMed
  54. ↵
    1. Messaoudi I.,
    2. J. Lemaoult,
    3. J. A. Guevara-Patino,
    4. B. M. Metzner,
    5. J. Nikolich-Zugich
    . 2004. Age-related CD8 T cell clonal expansions constrict CD8 T cell repertoire and have the potential to impair immune defense. J. Exp. Med. 200: 1347–1358.
    OpenUrlAbstract/FREE Full Text
  55. ↵
    1. Nickel P.,
    2. G. Bold,
    3. F. Presber,
    4. D. Biti,
    5. N. Babel,
    6. S. Kreutzer,
    7. J. Pratschke,
    8. C. Schönemann,
    9. F. Kern,
    10. H. D. Volk,
    11. P. Reinke
    . 2009. High levels of CMV-IE-1-specific memory T cells are associated with less alloimmunity and improved renal allograft function. Transpl. Immunol. 20: 238–242.
    OpenUrlCrossRefPubMed
  56. ↵
    1. Bunde T.,
    2. A. Kirchner,
    3. B. Hoffmeister,
    4. D. Habedank,
    5. R. Hetzer,
    6. G. Cherepnev,
    7. S. Proesch,
    8. P. Reinke,
    9. H. D. Volk,
    10. H. Lehmkuhl,
    11. F. Kern
    . 2005. Protection from cytomegalovirus after transplantation is correlated with immediate early 1-specific CD8 T cells. J. Exp. Med. 201: 1031–1036.
    OpenUrlAbstract/FREE Full Text
  57. ↵
    1. Maecker H. T.,
    2. H. S. Dunn,
    3. M. A. Suni,
    4. E. Khatamzas,
    5. C. J. Pitcher,
    6. T. Bunde,
    7. N. Persaud,
    8. W. Trigona,
    9. T. M. Fu,
    10. E. Sinclair,
    11. et al
    . 2001. Use of overlapping peptide mixtures as antigens for cytokine flow cytometry. J. Immunol. Methods 255: 27–40.
    OpenUrlCrossRefPubMed
  58. ↵
    1. Messaoudi I.,
    2. J. A. Guevara Patiño,
    3. R. Dyall,
    4. J. LeMaoult,
    5. J. Nikolich-Zugich
    . 2002. Direct link between mhc polymorphism, T cell avidity, and diversity in immune defense. Science 298: 1797–1800.
    OpenUrlAbstract/FREE Full Text
  59. ↵
    1. Lanzavecchia A.,
    2. F. Sallusto
    . 2005. Understanding the generation and function of memory T cell subsets. Curr. Opin. Immunol. 17: 326–332.
    OpenUrlCrossRefPubMed
  60. ↵
    1. Waldrop S. L.,
    2. C. J. Pitcher,
    3. D. M. Peterson,
    4. V. C. Maino,
    5. L. J. Picker
    . 1997. Determination of antigen-specific memory/effector CD4+ T cell frequencies by flow cytometry: evidence for a novel, antigen-specific homeostatic mechanism in HIV-associated immunodeficiency. J. Clin. Invest. 99: 1739–1750.
    OpenUrlCrossRefPubMed
    1. Asanuma H.,
    2. M. Sharp,
    3. H. T. Maecker,
    4. V. C. Maino,
    5. A. M. Arvin
    . 2000. Frequencies of memory T cells specific for varicella-zoster virus, herpes simplex virus, and cytomegalovirus by intracellular detection of cytokine expression. J. Infect. Dis. 181: 859–866.
    OpenUrlAbstract/FREE Full Text
    1. Sester M.,
    2. U. Sester,
    3. S. Alarcon Salvador,
    4. G. Heine,
    5. S. Lipfert,
    6. M. Girndt,
    7. B. Gärtner,
    8. H. Köhler
    . 2002. Age-related decrease in adenovirus-specific T cell responses. J. Infect. Dis. 185: 1379–1387.
    OpenUrlAbstract/FREE Full Text
    1. Hammarlund E.,
    2. M. W. Lewis,
    3. S. G. Hansen,
    4. L. I. Strelow,
    5. J. A. Nelson,
    6. G. J. Sexton,
    7. J. M. Hanifin,
    8. M. K. Slifka
    . 2003. Duration of antiviral immunity after smallpox vaccination. Nat. Med. 9: 1131–1137.
    OpenUrlCrossRefPubMed
  64. ↵
    1. Betts M. R.,
    2. D. R. Ambrozak,
    3. D. C. Douek,
    4. S. Bonhoeffer,
    5. J. M. Brenchley,
    6. J. P. Casazza,
    7. R. A. Koup,
    8. L. J. Picker
    . 2001. Analysis of total human immunodeficiency virus (HIV)-specific CD4(+) and CD8(+) T-cell responses: relationship to viral load in untreated HIV infection. J. Virol. 75: 11983–11991.
    OpenUrlAbstract/FREE Full Text
  65. ↵
    1. Reinke P.,
    2. S. Prösch,
    3. F. Kern,
    4. H. D. Volk
    . 1999. Mechanisms of human cytomegalovirus (HCMV) (re)activation and its impact on organ transplant patients. Transpl. Infect. Dis. 1: 157–164.
    OpenUrlCrossRefPubMed
  66. ↵
    1. Sinclair J.,
    2. P. Sissons
    . 2006. Latency and reactivation of human cytomegalovirus. J. Gen. Virol. 87: 1763–1779.
    OpenUrlCrossRefPubMed
  67. ↵
    1. Barber D. L.,
    2. E. J. Wherry,
    3. D. Masopust,
    4. B. Zhu,
    5. J. P. Allison,
    6. A. H. Sharpe,
    7. G. J. Freeman,
    8. R. Ahmed
    . 2006. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature 439: 682–687.
    OpenUrlCrossRefPubMed
  68. ↵
    1. Day C. L.,
    2. D. E. Kaufmann,
    3. P. Kiepiela,
    4. J. A. Brown,
    5. E. S. Moodley,
    6. S. Reddy,
    7. E. W. Mackey,
    8. J. D. Miller,
    9. A. J. Leslie,
    10. C. DePierres,
    11. et al
    . 2006. PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature 443: 350–354.
    OpenUrlCrossRefPubMed
  69. ↵
    1. Makedonas G.,
    2. N. Hutnick,
    3. D. Haney,
    4. A. C. Amick,
    5. J. Gardner,
    6. G. Cosma,
    7. A. R. Hersperger,
    8. D. Dolfi,
    9. E. J. Wherry,
    10. G. Ferrari,
    11. M. R. Betts
    . 2010. Perforin and IL-2 upregulation define qualitative differences among highly functional virus-specific human CD8 T cells. PLoS Pathog. 6: e1000798.
    OpenUrlCrossRefPubMed
  70. ↵
    1. Stowe R. P.,
    2. E. V. Kozlova,
    3. D. L. Yetman,
    4. D. M. Walling,
    5. J. S. Goodwin,
    6. R. Glaser
    . 2007. Chronic herpesvirus reactivation occurs in aging. Exp. Gerontol. 42: 563–570.
    OpenUrlCrossRefPubMed
  71. ↵
    1. Vezys V.,
    2. D. Masopust,
    3. C. C. Kemball,
    4. D. L. Barber,
    5. L. A. O’Mara,
    6. C. P. Larsen,
    7. T. C. Pearson,
    8. R. Ahmed,
    9. A. E. Lukacher
    . 2006. Continuous recruitment of naive T cells contributes to heterogeneity of antiviral CD8 T cells during persistent infection. J. Exp. Med. 203: 2263–2269.
    OpenUrlAbstract/FREE Full Text
  72. ↵
    1. Monteiro J.,
    2. F. Batliwalla,
    3. H. Ostrer,
    4. P. K. Gregersen
    . 1996. Shortened telomeres in clonally expanded CD28-CD8+ T cells imply a replicative history that is distinct from their CD28+CD8+ counterparts. J. Immunol. 156: 3587–3590.
    OpenUrlAbstract
  73. ↵
    1. Ellefsen K.,
    2. A. Harari,
    3. P. Champagne,
    4. P. A. Bart,
    5. R. P. Sékaly,
    6. G. Pantaleo
    . 2002. Distribution and functional analysis of memory antiviral CD8 T cell responses in HIV-1 and cytomegalovirus infections. Eur. J. Immunol. 32: 3756–3764.
    OpenUrlCrossRefPubMed
  74. ↵
    1. Price D. A.,
    2. J. M. Brenchley,
    3. L. E. Ruff,
    4. M. R. Betts,
    5. B. J. Hill,
    6. M. Roederer,
    7. R. A. Koup,
    8. S. A. Migueles,
    9. E. Gostick,
    10. L. Wooldridge,
    11. et al
    . 2005. Avidity for antigen shapes clonal dominance in CD8+ T cell populations specific for persistent DNA viruses. J. Exp. Med. 202: 1349–1361.
    OpenUrlAbstract/FREE Full Text
  75. ↵
    1. Limaye A. P.,
    2. K. A. Kirby,
    3. G. D. Rubenfeld,
    4. W. M. Leisenring,
    5. E. M. Bulger,
    6. M. J. Neff,
    7. N. S. Gibran,
    8. M. L. Huang,
    9. T. K. Santo Hayes,
    10. L. Corey,
    11. M. Boeckh
    . 2008. Cytomegalovirus reactivation in critically ill immunocompetent patients. JAMA 300: 413–422.
    OpenUrlCrossRefPubMed
  76. ↵
    1. Lazuardi L.,
    2. D. Herndler-Brandstetter,
    3. S. Brunner,
    4. G. T. Laschober,
    5. G. Lepperdinger,
    6. B. Grubeck-Loebenstein
    . 2009. Microarray analysis reveals similarity between CD8+CD28- T cells from young and elderly persons, but not of CD8+CD28+ T cells. Biogerontology 10: 191–202.
    OpenUrlCrossRefPubMed
  77. ↵
    1. Barton E. S.,
    2. D. W. White,
    3. J. S. Cathelyn,
    4. K. A. Brett-McClellan,
    5. M. Engle,
    6. M. S. Diamond,
    7. V. L. Miller,
    8. H. W. Virgin IV.
    . 2007. Herpesvirus latency confers symbiotic protection from bacterial infection. Nature 447: 326–329.
    OpenUrlCrossRefPubMed
  78. ↵
    1. Sauce D.,
    2. M. Larsen,
    3. S. Fastenackels,
    4. A. Duperrier,
    5. M. Keller,
    6. B. Grubeck-Loebenstein,
    7. C. Ferrand,
    8. P. Debré,
    9. D. Sidi,
    10. V. Appay
    . 2009. Evidence of premature immune aging in patients thymectomized during early childhood. J. Clin. Invest. 119: 3070–3078.
    OpenUrlCrossRefPubMed
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