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

Abstract

In spite of the present belief that latent cytomegalovirus (CMV) infection drives CD8+ T-cell differentiation and induces premature immune senescence, no systematic studies have so far been performed to compare phenotypical and functional changes in the CD8+ T-cell repertoire in CMV-infected and noninfected persons of different age groups. In the present study, number, cytokine production, and growth potential of naive (CD45RA+ CD28+), memory (CD45RA- CD28+), and effector (CD45RA+ CD28- or CD45RA- CD28-) CD8+ T cells were analyzed in young, middle-aged, and elderly clinically healthy persons with a positive or negative CMV antibody serology. Numbers and functional properties of CMVpp65(495-503)-specific CD8+ T cells were also studied. We demonstrate that aging as well as CMV infection lead to a decrease in the size of the naive CD8+ T-cell pool but to an increase in the number of CD8+ effector T cells, which produce gamma interferon but lack substantial growth potential. The size of the CD8+ memory T-cell population, which grows well and produces interleukin-2 (IL-2) and IL-4, also increases with aging, but this increase is missing in CMV carriers. Life-long latent CMV infection seems thus to diminish the size of the naive and the early memory T-cell pool and to drive a Th1 polarization within the immune system. This can lead to a reduced diversity of CD8 responses and to chronic inflammatory processes which may be the basis of severe health problems in elderly persons.

FIG. 1.

Aging and CMV infection affect the differentiation of CD8⁺ T cells. Four CD8⁺ T-cell subpopulations were defined according to the expression of CD45RA and CD28 within gated CD8⁺⁺ T cells. (A) The distribution of these subpopulations is demonstrated by showing the example of two young, two middle-aged, and two elderly persons, one without and one with latent CMV infection in each age group. (B) Distribution of naïve (I), memory (II), effector CD45RA⁻ (III), and effector CD45RA⁺ (IV) CD8 T cells in the different cohorts. □, CMV antibody (Ab) negative; ▨, CMV Ab positive. Data are presented as the mean ± SEM, and statistical comparisons between the groups were performed. Significant differences are indicated as follows: *, CMV Ab^- versus CMV Ab⁺ persons within each age group; †, young versus elderly; #, middle-aged versus elderly within persons of one CMV Ab status.

FIG. 2.

Aging and CMV infection lead to an imbalance in the cytokine production profile of the total unpurified CD8⁺ T-cell population. The production of IL-2, IL-4, and IFN-γ in response to stimulation with PMA-ionomycin was studied. (A) Cytokine production profile of the total gated CD8⁺ T-cell population in CMV antibody (Ab)-negative and -positive persons of different age groups. Statistical comparisons between the groups were done, and the results are presented as means ± SEM. Significant differences are shown as follows: *, CMV Ab^- versus CMV Ab⁺ persons within each age group; †, young versus elderly; #, middle-aged versus elderly within persons of one CMV Ab status. (B) The example of two young, two middle-aged, and two elderly persons is shown, one without and one with latent CMV infection in each age group. CD8 cells producing either IL-2, IL-4, or IFN-γ were gated and analyzed for their expression of CD45RA and CD28.

FIG. 3.

(A) A CD25⁺CD8⁺ T-cell subpopulation of substantial size is found within the CD45RA⁻ CD28⁺ memory population in elderly persons without CMV infection. This population is decreased in size in elderly persons with latent CMV infection as well as in younger age groups. A representative example of CD25-expressing cells (black dots) superimposed on a double staining of CD45RA and CD28 in gated CD8⁺ cells (gray dots) in the different study groups is shown. (B) The frequency of CD25-expressing cells in CD8⁺ T cells is shown in young, middle-aged, and in elderly persons with (▨) and without (□) latent CMV infection. Bars represent means ± SEM. Significant differences are indicated as follows: *, CMV antibody-negative (Ab⁻) versus CMV Ab⁺ persons; †, young versus elderly; #, middle-aged versus elderly persons without latent CMV infection.

FIG. 4.

CMVpp65_495-503-specific CD8⁺ T cells have an effector phenotype and produce IFN-γ. (A) Representative example of CMVpp65_495-503 tetramer binding CD8⁺ T cells in a CMV antibody (Ab)-negative donor and in one young, one middle-aged, and one elderly person with a positive CMV Ab serology. (B) CD28 CD45RA double staining of CMVpp65_495-503 tetramer binding cells within the CD8⁺ gate. A typical example of one young, one middle-aged, and one elderly person is shown. Figures in the diagram represent the percentage of cells of each phenotype within the total CMVpp65_495-503 tetramer binding population, which was considered 100%. (C) Representative example showing the expression of CD25, CD62L, and perforin as well as the production of IL-2, IL-4, and IFN-γ in CMVpp65_495-503 tetramer binding cells within the CD8⁺ gate. Cytokines were analyzed following stimulation with PMA and ionomycin. The diagram represents the staining of cells from one elderly person with a positive CMV Ab serology. Similar results were obtained in all other HLA-A2-positive donors with a positive CMV Ab serology from the three age groups.

FIG. 5.

Following antigenic stimulation, CMVpp65_495-503-specific CD28⁺ CD8⁺ T cells have a more pronounced growth potential than their CD28⁻ counterparts. CD28⁺, CD28⁻ CD45RA⁻, and CD28⁻ CD45RA⁺ T cells were isolated, as described in Materials and Methods, and stimulated with CMVpp65_495-503 (NLVPMVATV) (2 μg/ml) in the presence of IL-2 (20 ng/ml) for 1 week. Stainings of unpurified cells before subset isolation are shown in the middle of the graph (box): CD28 and CD45RA expression levels in gated CD8 cells as well as in cells gated on the CMVpp65_495-503 tetramer binding population are shown. The percentages of cells within each subpopulation that were tetramer positive before in vitro culture are shown outside the box under the heading Pre. Tetramer binding cells in the three subpopulations following 1 week's culture in the presence of CMVpp65_495-503 peptide and IL-2 are also shown outside the box. The fold increase in the percentage of peptide-specific cells is indicated for each culture. AS, antigenic stimulation. The experiment was performed in cells from two young and two elderly CMV antibody-positive persons. The graph shows a characteristic example of one young and one elderly person.