Infectious disease remains a major cause of morbidity and mortality in all populations, and the elderly are at particular risk of clinical disease (8
). This has been attributed to a number of findings, of which a functional impairment of the immune system termed immune senescence is believed to be a major cause. Immune senescence is characterized by features such as reduction in the number of naive T cells and an accumulation of memory cells (1
). The combination of features such as an inverted CD4/CD8 ratio and reduced proliferative response to mitogen has been term the immune risk phenotype and has been directly associated with impaired survival in cohort studies of healthy elderly donors (20
). Primary CMV infection is usually silent, but the virus is not cleared from the host and enters a state of viral latency. The majority of individuals over the age of 65 years are CMV seropositive, although CMV seropositivity is generally not considered to be of clinical significance in the immunocompetent host.
However, the potential association between CMV infection and immune senescence is coming under increasing scrutiny. CMV seropositivity is associated with the development of the immune risk phenotype (21
), and an impaired immune response to influenza vaccination is observed in CMV-seropositive donors (25
). In contrast to the immune response to most infections, the CD8 T-cell immune response to CMV increases markedly with age, such that over 40% of the CD8 T-cell pool may comprise CMV-specific T cells in some donors (15
). It is possible that the development of such large memory T-cell expansions may be associated with an impairment of the immune response to heterologous agents. Age-related CD8 T-cell clonal expansions in mice are associated with an impaired immune response to viral infection, due to constriction of the CD8 T-cell repertoire (17
). However, the potential importance of the CMV-specific CD4 T-cell immune response in relation to immune function during aging has received less attention.
Here we show that in CMV-seropositive individuals there is an accumulation of CMV-specific CD4 T cells in association with aging. This supports recent data showing that the percentage of CMV-specific CD4 T cells increases with age, whereas other virus-specific CD4 T-cell responses do not (9
). The cytokine flow cytometry assay revealed that the CMV-specific immune response to viral lysate constitutes nearly 6% of the CD4 T-cell repertoire in the elderly, a frequency that is considerably greater than for other pathogen-specific immune responses. Nevertheless, indirect evidence suggests that this frequency is likely to be a significant underestimate of the total CMV-specific CD4+
T-cell response. Although lysate of CMV-infected cells is widely used as an antigenic source for the identification of CMV-specific CD4 T cells, the full complement of viral proteins may not be evenly presented by antigen-presenting cells due to competition for HLA class II binding. There may also be CMV-specific CD4 T cells that go undetected due to their inability to produce cytokines during in vitro analysis, and this has been shown repeatedly in the detection of CMV-specific CD8 T cells using major histocompatibility complex class I peptide tetramers (15
CMV-specific cytotoxic T lymphocytes in elderly donors demonstrated lower levels of IL-2 production, which is characteristic of CD4 T cells with a low activation threshold (12
). Production of IL-2 may be necessary in states of high antigen load or inflammation in situations requiring expansion of effector T cells. CMV-specific CD4 T cells are characterized by a CD28−
effector memory phenotype, and an IFN-γ+
cytokine profile is typical of this subset (23
). Murine data have shown that a memory CD4 T-cell subset with predominant expression of IL-2 is preferentially located within lymphoid tissue, and examination of the frequency and cytokine production of CMV-specific CD4 T cells within secondary lymphoid tissue will be of interest (22
The functional properties of the CMV-specific CD4 T-cell response show significant differences between young and elderly donors. The CMV-specific CD4+
T-cell response exhibits heterogeneity in the activation threshold based on the requirement for costimulation in the form of antibodies to CD28 or CD49d or activation of antigen-presenting cells by agents such as TNF-α (28
). Such differences in threshold can be found within a single clonotype and appear to be independent of T-cell receptor stimulation and consistent with an intrinsic activation threshold that is downstream of the T-cell receptor (6
). The young donors in our study exhibited this phenomenon, and the addition of costimulatory antibodies increased the magnitude of the detected CMV-specific immune response by a factor of 1.1- to 3.7-fold. In contrast, costimulation did not increase detection of the CMV-specific CD4 T-cell response in elderly donors, indicating that cells have a lower threshold level for activation.
Many CMV-specific CD4 T cells lose expression of CD28, and anti-CD28 antibody is therefore unlikely to act as a costimulatory stimulus to this population. However, CD28−
CMV-specific CD4 T cells are also seen in considerable numbers in young donors (27
). There was no correlation between the magnitude of the CMV-specific response and response to costimulation, suggesting that maintenance, rather than expansion, of CD4+
T-cell clones is associated with an alteration in signaling threshold.
The factors that determine the threshold of an antigen-specific response are uncertain, but repeated episodes of antigenic stimulation may be one factor. CMV latency is associated with episodes of subclinical viremia (19
), and it is likely that CMV carriage for many decades exposes the donor to intermittent viral reactivation with consequent repeated activation of T-cell immunity. The physiological significance of this change in activation threshold is uncertain, but T cells with lower thresholds for activation are more likely to be able to control viral reactivation in the setting of minimal inflammation and low antigen load. They may thus be well adapted to control low levels of CMV reactivation with minimal bystander damage and exhibit a form of pathogen control based on a high cytotoxic T-lymphocyte-low-antigen-load model. It has also been shown by others that CMV-specific CD4 T cells are driven to replicative exhaustion in elderly CMV-seropositive hosts (9
), although incidents of virus-associated disease in such patients do not occur. Whether this becomes problematic at a later stage for such individuals remains unknown.
The phenotype of CMV-specific CD4+
T cells indicated that cells had an effector memory profile with almost complete loss of CCR7 and CD62L. Most cells expressed the memory marker CD45RO, but a significant minority had a revertant phenotype with high-level expression of CD45RA although, in contrast to CD8+
, this population did not increase with age. The proportion of cells which had lost expression of CD27 and CD28 showed an increase with age, in agreement with a previous report (9
). The accumulation of effector memory CMV-specific CD4+
T cells was reflected in a marked alteration in the global CD4+
T-cell repertoire in elderly donors. CD27−
CD4 T-cell populations were increased from 6% and 1% of the CD4 T-cell pool, respectively, in CMV-seronegative donors to over 23% and 13% in the CMV-seropositive cohort (4
). It is likely that most of the CD4+
CD4 T-cell population has specificity for CMV, as documented in a recent report (27
). CD57 expression is associated with a highly differentiated cytotoxic phenotype (18
) and increases markedly from less than 1% of the CD4 T-cell repertoire to over 10% in CMV-seropositive donors. These data suggest that CMV-specific CD4 T cells may represent up to 10% of the T-cell pool in healthy elderly donors, although it is also possible that CD27 and CD28 loss is induced on T cells which are not specific for CMV peptides due to cytokine-mediated effects (9
). An accumulation of CD4 T cells expressing the activation marker HLA-DR or CD45RO, the CD45 isoform expressed on antigen-experienced T cells, is also observed in association with CMV seropositivity. Such an expansion of memory T cells may impair the number of naïve T cells within the CD4 T-cell repertoire, and this may be reflected in the 17% decrease in the percentage of CD4+
T cells in CMV-seropositive donors.
CMV-specific CD8 T cells are markedly expanded in elderly donors, and it may be possible that the CMV-specific CD4 T cells are providing help for CD8 T-cell expansion.
No correlation was observed between the magnitude of CMV-specific CD8 and CD4 immune responses in individual donors in our study (data not shown). However, an association cannot be formally ruled out, because the CD8 data were derived using a panel of tetramers that covers common but, importantly, not all HLA class I types (15
CMV seropositivity is associated with the accumulation of large numbers of virus-specific CD4 T cells and a profound alteration of the global T-cell repertoire. These features may impair the ability of the immune response to respond to heterologous antigen, and this was assessed by analysis of the response of CD4 T cells to stimulation in vitro with superantigen. Among CMV-seropositive donors, a nonsignificant reduction was observed in the percentage of T cells that produced IL-2 in response to superantigen, and while further studies are required this may reflect a reduction in the naïve and central memory CD4 T cells in CMV-seropositive donors.
CMV infection in immunocompetent donors has generally been regarded as being of negligible consequence for the health of the host, but accumulating data indicate that this may be a naïve assumption. Efforts to control the spread of CMV infection or to limit levels of CMV viremia within an infected host may serve to protect the immune system from the accumulation of potentially crippling levels of cellular immune responses.