In this study, we adapted immunological techniques which have been widely applied to the study of adults to a large pediatric population of over 1000 children. The aim of the study was to define the quantity and quality of antiviral responses in early infection and relate any changes in these to age. Long term longitudinal studies of CMV specific populations are difficult to perform in healthy pediatric populations; nevertheless, using similar large scale cross sectional approaches we have previously observed a clearly dynamic host-virus relationship in adult infection [9, 25].
The pattern of age-related accumulation observed in this cross-sectional analysis revealed strong responses in the first year of life, consistent with acquisition of infection during this period. The acquisition of cellular immunity can be regarded as a good marker for viral infection, at a time when serological responses might be confounded by the presence of maternally transmitted antibody.
In particular, we evaluated in detail the frequencies of specific CD8+ T cells during the first four years of life (Table 3, 4). The results showed that initial expansion occurred very early in both HLA-A2 and A24 individuals. These findings support the idea that the primary infection with HCMV commonly occurs during the first or second year of life as suggested in previous studies [2, 26–28]. Long-term repetitive antigen stimulation through continuous low level virus reactivation is considered to be responsible for the maintenance of very high levels of CMV specific "effector memory" CD8+ T cells thereafter [6–8]. Why such cells accumulate over time and particularly in the elderly is not known, nor indeed is their efficacy in containing viral reactivation or reinfection. In the pediatric and young adult populations studied here no apparent "inflation" was seen in our analysis, although typical expanded populations were seen in elderly controls. It is possible that "inflation" of responses only occurs after a certain period of host-virus interaction, as is the case in MCMV . Alternatively it may be missed due to the cross-sectional design of the study, Overall however, it does appear that for at least the first couple of decades, the host virus balance remains roughly in status quo.
In this study, responsiveness in both ICS and ELISPOT assays was significantly correlated with tetramer staining. High frequencies of HCMV specific CD8+ cells were observed in even in 0–2 year-old infants, and, although the number of infants examined was relatively small, these findings suggest that infants infected with HCMV have a functional CD8+ T cell response to HCMV similar to that in adults. Moreover, all of these infants but one, who were positive for HCMV tetramer under 1-year-old, were asymptomatic in this study. Those observations are consistent with a recent smaller study which showed that newborns with congenital HCMV infection were asymptomatic and had functional and mature HCMV-specific CD8+ T cells [18, 29]. A recent study reported that IFN-γ secretion produced by HCMV-tetramer positive T cells in old individuals was lower than in young individuals . However, the secretion of IFN-γ produced by tetramer reactive cells in elderly individuals was not compared with that in young individuals in this study.
Based on an analysis of CD28/CD27 expression, the stage of "maturation" or "differentiation" of HCMV-specific CD8+ T cells can be defined. CD28+ CD27+ T cells are considered to be naïve cells or early-differentiated T cells, progressing to CD28-CD27- T cells, thought to be fully differentiated T cells [10–14]. We had expected that the predominant phenotype of HCMV-specific CD8+ T cells in the youngest group might be CD28+CD27+. However, the CD28-CD27+, intermediate differentiated phenotype, was predominant (43.2% of CD8+ tetramer positive T cells). Subsets of CD28+CD27+ (naïve or early-differentiated phenotype) and CD28-CD27- (late-differentiated phenotype) represented 34.9% and 20.8% of CD8+ tetramer positive T cells, respectively. The same proportion of HCMV-specific T cells was observed in the 5–9 yr. and 10–14 yr. groups, and also in infants under one-year-old (data not shown). These findings suggest that "mature" CD8+ T cells developed consistently in young infants. In the elderly group, the proportion of CD28-CD27+ (intermediate-differentiated phenotype) T cells was decreased, as the proportion of CD28-CD27- (late-differentiated phenotype) increased substantially. This finding broadly supports the idea that the lineage differentiation pattern of HCMV specific CD8+ cells is CD28+CD27+ → CD28-CD27+→ CD28-CD27-, although it suggests that the movement of cells across this spectrum is not continuous over time. Interestingly, however, we also noted a small proportion of CD28+CD27- T-cells which rose to 22% of tetramer positive CD8+ T-cells in the elderly group.
Naïve CD8+ T cells express CD45RA, and this is uniformly expressed in cord blood. While antigen-experienced CD8+ T cells initially express CD45RO, re-expression of CD45RA may occur and this has been described as a state of late or terminal differentiation – although longitudinal studies of single cells have not been performed. According to this scheme, it was expected that a subset of CD45RA+CD45RO- tetramer+ cells would accumulate with age. In contrast, the CD45RA+CD45RO- subset was predominant in all of the age-groups, even in the youngest children and the proportion did not change through life. There are two possibilities to explain these observations. First, this state may be driven by continuous interaction with antigen. However, this hypothesis is not entirely consistent with the results of CD28/CD27 subsets of this study, in which CD28-CD27- "late" differentiated cells, were predominant in only the elderly. Alternatively, the levels of CD45 isoforms expression correlate poorly with the stages of antigen-driven T cell differentiation. This is supported by a previous study, which showed that CD45RA expression was not correlated to the differentiation phenotypes (28). Expression of other cell surface markers, such as CCR7, CD57 or CD85j might correlate more closely with the evolution of T cell responses over time [31–37].
In this study, HLA-A2+ subjects were selected by flow cytometry using the HLA-A2 mAb. However, the HLA-A2 molecular type of Japanese is classified into three subtypes. HLA-A*0201, A*0206, and A*0207 representing 10%, 10%, and 3%, of Japanese, respectively . Because the difference among HLA subtypes may in principle affect the antigen processing and the presentation of CTL epitopes in the context of HLA class I molecules, the magnitude of CTL response might depend on HLA subtype . However, the frequency of individuals staining positive for the HLA-A2 pp65 tetramer was higher than that in a previous study, in a Caucasian population expressing predominantly HLA-A*0201 . In addition, there was no difference in the percentage of tetramer/CD8 in the 20–29-year-old group between the previous study and this study. Those observations suggest that the difference among the three subtypes, did not substantially influence the results of tetramer staining. However, further studies are required to determine whether the difference among HLA-A2 subtypes can influence functional assays.
CMV sero-status of subjects was not evaluated in this study. We reported that 77% of HCMV-seropositive HLA-A2 subjects were positive for CMV tetramer . These findings suggest that the result of tetramer assay can be related to sero-status, because tetramer reactive T cells are memory T cells. However, further studies are required to assess whether the results of tetramer assays are consistent with the HCMV sero-status in different population.
In previous study , we showed "memory inflation" in an HLA-A2 positive population. However, the subject was a Caucasioan population. Therefore, there was a possibility that the frequency of HCMV specific tetramer might be different in a Japanese population. In order to confirm "memory inflation" in Japanese, HLA-A2 positive elderly subjects were tested. In this study, we could confirm that "memory inflation" was present in a Japanese population. However, HLA-A24 elderly subjects were not tested because HLA-A24 subjects were not examined in our previous study.
In conclusion, the maintenance of large populations of HCMV-specific CD8+ T cells was observed throughout childhood, with substantial responses to infection in early infancy. Overall major age-related changes in tetramer+ CD8+ T cells were not observed in the first two decades of life and tetramer+ CD8+ cells of young infants showed a mature phenotype and function similar to that in adults. These data provide novel insight into the relative maturity of the infant antiviral response, and the impact of CMV on the childhood immune system. Additionally, they demonstrate the technical feasibility of detailed cellular immunological studies in pediatric populations, undertaken at a large scale as may be applicable in other childhood infections and vaccine programs.