Cytomegalovirus Infection- and Age-Dependent Changes in Human CD8+ T-Cell Cytokine Expression Patterns

T cells are strongly affected by immune aging, a phenomenon that leads to increased susceptibility to infections and decreased vaccination efficacy in elderly individuals. Cytomegalovirus (CMV) infection induces vigorous T-cell immune responses in humans and is thought to be a driving force of immune aging. In the present study we analyzed CMV-induced quantitative and qualitative differences in the cytokine-expressing T-cell repertoire from individuals of different age groups after in vitro stimulation. The CMV pp65 peptide pool and the superantigen Staphylococcus enterotoxin B (SEB) induced higher proportions of CD8⁺ effector T cells expressing gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and granulocyte-macrophage colony-stimulating factor in the oldest study group, while only SEB induced increased responses in the middle-aged study group. Notably, CMV-specific multiple cytokine expression patterns revealed higher proportions of IFN-γ- and TNF-α-coexpressing CD8⁺ T cells exclusively in the oldest study group. These qualitative differences were absent in SEB-induced CD8⁺ effector T cells, although quantitative differences were detected. We report age-dependent qualitative changes in CMV-specific CD8⁺ T-cell cytokine patterns which are biocandidate markers of immune exhaustion in elderly individuals.The term immune aging describes a conglomerate of features that affect immune functions and phenotype at late stages of human life (reviewed in reference 24). Clinically, immune aging is associated with higher susceptibility to diseases caused by “new” respiratory infections with bacteria (e.g., pneumococci) and viruses (e.g., influenza virus) (6, 7). The phenotype of immune aging includes several features and affects both innate and adaptive immunity. In particular, marked changes in T-cell immunity have been well documented (18, 20). These differences include changes on the single-cell level (e.g., T-cell receptor TCR]- and coreceptor-dependent signal transduction reviewed in reference 21]), as well as changes on the T-cell population level, which show (i) a decline in naïve T-cell numbers, (ii) a reduction in TCR repertoire diversity, and (iii) increases in memory and effector T-cell proportions (reviewed in reference 24). Longitudinal studies in human populations indicate that a subset of immune aging features can be used as biomarkers of mortality (25, 33). These studies identified cytomegalovirus (CMV) as a biomarker in the so-called immune risk profile (IRP).Human CMV infection is common in all parts of the world, with infection rates between 60% in developed countries and up to 100% in developing countries (11). The CMV infection rate steadily increases during an individual''s life time, with the highest prevalence in the very old (9). CMV infection is usually asymptomatic and leads to life-long persistence of the pathogen. Reactivation and development of clinical symptoms are rare and largely restricted to immunocompromised and immunosuppressed patients (10, 29). CMV infection rapidly induces immunodominant T-cell responses, mainly against two proteins, the immediate-early protein 1 (IE-1) and the matrix 65-kDa phosphoprotein (pp65) (14, 19, 34). This T-cell response is like a double-edged sword, because studies in humans and animal models suggest a protective role against CMV reactivation on the one hand (1, 12, 31) and, on the other hand, the T-cell response leads to vigorous and persistent clonal expansion of CD8⁺ T cells (reviewed in reference 22). These T-cell clones reach high numbers and thus dominate the peripheral blood T-cell repertoire (8, 28). In contrast to other chronic infection- or vaccine-induced T-cell responses, CMV-specific T-cell numbers are stable or even increase over time and lead to extremely high proportions of clonally expanded T cells in very old individuals (26). CMV-specific CD8⁺ and CD4⁺ T cells show some phenotypic peculiarities and continuously become dysfunctional or exhausted (5, 26). It has been suggested that CMV-induced changes in T-cell repertoire and function, although crucial for protection against CMV reactivation, challenge the maintenance of a balanced T-cell repertoire and in this way compromise host immunity against infections and efficacies of new vaccines in elderly individuals (reviewed in reference 24). Furthermore, it has been hypothesized that CMV infection itself is the driving force of immune aging (reviewed in reference 16).Quantitative assessment of T-cell responses (e.g., gamma interferon IFN-γ] and tumor necrosis factor alpha TNF-α] expression) is a common tool used to predict disease susceptibility and vaccination efficacy, but for some diseases (e.g., tuberculosis) these parameters are not sufficient for use as biomarkers of protection (13). Recent studies have strengthened the crucial role of qualitative differences in the T-cell response (i.e., T-cell-expressed cytokine patterns) for the prediction of disease susceptibility (3, 17, 36). Our own previous studies suggest a role of granulocyte-macrophage colony-stimulating factor (GM-CSF) as a T-cell biomarker in childhood tuberculosis (23).Here we analyzed proportions of cytokine-expressing T-cell subpopulations (i.e., for IFN-γ, TNF-α, GM-CSF, and interleukin-2 IL-2]) and determined the patterns of cytokine coexpression after in vitro stimulation with CMV pp65 and the superantigen staphylococcus enterotoxin B (SEB) in individuals from different age groups. In this way we characterized concomitantly age-dependent, CMV-specific and nonspecific, quantitative as well as qualitative differences in the T-cell response.