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

Abstract

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

FIG. 1.

The frequency of CMV-specific CD4 T cells is increased in elderly donors. Whole blood from CMV-seropositive donors was incubated with CMV viral lysate at 37°C for 6 h or with mock lysate as a control. The blood was then lysed, fixed, permeabilized, and stained with monoclonal antibodies. The frequency of the cells responsive to CMV was determined by the fraction of CD4 T cells which upregulated CD69 and expressed detectable amounts of intracellular TNF-α, IFN-γ, and IL-2. A. Representative flow cytometric profiles from a single elderly donor for the isotype control (i) and IFN-γ (ii) and IL-2 (iii) cytokine responses to CMV lysate. B. The frequency of CMV-specific CD4 T cells in individual donors is expressed as a percentage of the total CD4 T-cell pool and shown as a single data point. The mean value within the group is indicated as a horizontal line. (i) The mean percentage of CMV-specific CD4 T cell producing IFN-γ was 2% in young donors (range, 0.4 to 5.78%) and 4.3% in elderly donors (0.3 to 32%) (P = 0.03). (ii) The mean percentage of CMV-specific CD4 T cells producing TNF-α was 2.2% in young donors (range, 0.51 to 4.7%) and 4.7% in elderly donors (0.3 to 16.7%) (P = 0.08). (iii) The mean percentage of CMV-specific CD4 T cells producing IL-2 was 1.1% in young donors (range, 0.1 to 4.8%) and 1.3% in elderly donors (0.26 to 4.9%) (P = 0.9).

FIG. 2.

The ratio of IL-2/IFN-γ production by CMV-specific CD4 T cells is decreased in elderly donors. Whole blood stimulation and cytokine flow cytometry were used to determine IFN-γ and IL-2 production in response to CMV viral lysate. PBMC were stimulated with CMV lysate, and the percentage of cells which produced IL-2 or IFN-γ was determined and expressed as a ratio (IL-2/IFN-γ). Data from each donor are expressed as a single data point, with the median indicated by a horizontal line. The median value was 0.63 in the young donors and 0.37 in elderly donors (P = 0.003).

FIG. 3.

Influence of addition of costimulatory antibodies on detection of CMV-specific CD4 T cells in young or elderly donors. PBMC were stimulated with CMV lysate in the presence or absence of costimulatory antibodies to CD28 or CD49d. A. Magnitude of the CMV-specific CD4 T-cell response determined by CFC in the presence or absence of costimulatory antibodies in 11 young (i) and 11 elderly (ii) donors. B. Mean ratio of CMV-specific CD4 T cells detected in the presence or absence of costimulatory antibodies in young or elderly donors. The mean ratio was 2.2 (range, 1.07 to 3.90) in young donors. In elderly donors the mean ratio was 1, with a range of 0.40 to 1.48 (P = 0.0002).

FIG. 4.

Phenotypic analyses of CMV-specific CD4 T-cell responses in young and elderly asymptomatic virus carriers. PBMC from donors were stimulated with CMV lysate and then surface stained with monoclonal antibodies specific for each of the phenotypic markers shown, followed by cytoplasmic IFN-γ staining. Representative data are shown for two young (A and B) and two elderly (C and D) donors. (E) Summarized data showing CD45RA, CD45RO, CD27, and CD28 expression in all young and elderly donors tested are also shown. The y axis represents the percent of CMV-specific CD4 T cells expressing each marker.

FIG. 5.

Expression of differentiation and activation markers on peripheral blood CD4⁺ T cells of healthy CMV-seropositive and -seronegative donors. PBMC from CMV-seronegative (n = 10) and CMV-seropositive (n = 10) donors aged >65 years were stained with FITC, PE, and PC5-conjugated MAb. CD4⁺ CD3⁺ lymphocytes were gated and analyzed for the expression of a range of phenotypic markers on the third color. The mean percentages of CD4 T cells expressing individual phenotypic markers in CMV-seronegative or CMV-seropositive donors were as follows: CD45RA (53 and 45%, respectively; P = 0.0.07); CD45R0 (43 and 53%; P = 0.012) CD27 (94 and 77%; P = 0.00016); CD28 (99 and 86%; P = 0.00014); CD57 (0.92 and 11%; P = 0.0006); CD38 (44 and 38%; P = 0.3); HLA-DR (8.3 and 13%; P = 0.1); CCR7 (86 and 69%; P = 0.0015).