Aging and human CD4(+) regulatory T cells

Abstract

Alterations in immunity that occur with aging likely contribute to the development of infection, malignancy and inflammatory diseases. Naturally occurring CD4(+) regulatory T cells (Treg) expressing high levels of CD25 and forkhead box P3 (FOXP3) are essential for regulating immune responses. Here we investigated the effect of aging on the number, phenotypes and function of CD4(+) Treg in humans. The frequency and phenotypic characteristics of CD4(+), FOXP3(+) T cells as well as their capacity to suppress inflammatory cytokine production and proliferation of CD4(+), CD25(-) T cells (target cells) were comparable in young (age <or=40) and elderly (age >or=65) individuals. However, when CD4(+), FOXP3(+) Treg and CD4(+), CD25(-) T cells were co-cultured at a ratio of 1:1, the production of anti-inflammatory cytokine IL-10 from CD4(+), CD25(-) T cells was more potently suppressed in the elderly than in the young. This finding was not due to changes in CTLA-4 expression or apoptosis of CD4(+), FOXP3(+) Treg and CD4(+), CD25(-) T cells. Taken together, our observations suggest that aging may affect the capacity of CD4(+), FOXP3(+) T cells in regulating IL-10 production from target CD4(+) T cells in humans although their other cellular characteristics remain unchanged.

Fig. 1

Correlation of CD25, FOXP3 and IL-7Rα expression by CD4⁺ T cells in young and elderly humans. Purified peripheral blood mononuclear cells (PBMCs) from young (n = 10) and elderly (n = 10) individuals were stained with Abs to CD4 and CD25, followed by permeabilization and staining with Abs to FOXP3 or isotype Abs. Some cells were additionally stained with Abs to IL-7Rα or isotype Abs. (A) Representative dot plots showing CD4⁺ T cell subsets expressing CD25 at different levels. (B) Representative histograms demonstrating the relationship of CD25 and FOXP3 expression by CD4⁺ T cells. (C) The frequency of CD4⁺ T cell subsets expressing CD25 differentially in young and elderly individuals. Error bars indicate standard error of the mean (SEM). (D) Representative histograms showing the expression of IL-7Rα on CD4⁺,FOXP3⁺ and FOXP3⁻ T cells. P values were obtained by the Mann-Whitney U test.

Fig. 2

Comparing the frequency of CD4⁺,FOXP3⁺ T cells and their naïve (CD45RA⁺) and memory (CD45RA⁻) phenotypes between young and elderly humans. PBMCs from young (n = 15) and elderly (n = 15) individuals were stained with Abs to CD4, CD45RA and IL-2/15Rβ, followed by permeabilization and staining with Abs to FOXP3 or isotype Abs. (A) Representative dot plots showing identification of FOXP3⁺ T cells in CD4⁺ T cells. Numbers in the plots indicate the frequency of FOXP3⁺ or isotype stained cells. (B) The frequency of FOXP3⁺ T cells in CD4⁺ T cells from young and elderly individuals. (C) Representative dot plots showing CD4⁺,FOXP3⁺ T cells with and without expressing CD45RA. Numbers in the plots indicate the frequency of CD45RA⁺ cells. (D) The frequency of CD4⁺,CD45RA⁻,FOXP3⁺ T cells and CD4⁺,CD45RA⁺,FOXP3⁺ T cells. (E) Representative histograms showing the expression of IL-2/-15Rβ on CD4⁺,FOXP3⁺ and FOXP3⁻ T cells. (F) MFI of IL-15Rβ expression on CD4⁺,FOXP3 in young and elderly individuals. P values were obtained by the Mann-Whitney U test.

Fig. 3

Comparing the capacity of CD4⁺,CD25^bright T cells in suppressing the proliferation and cytokine production of CD4⁺,CD25⁻ T cells between young and elderly humans. PBMCs from young and elderly individuals were stained with Abs to CD4 and CD25 and sorted into CD4⁺,CD25^bright (R1 in Fig. 1A, Treg) and CD4⁺,CD25⁻ (R4 in Fig. 1A, Target) T cells. (A) In some experiment, the latter cells were stained with CFSE and mixed with CD4⁺,CD25^bright T cells at various ratios as indicated in the figure and incubated for 7 days in the presence of anti-CD3 and -CD28 Abs. Proliferating target cells were identified based on CFSE staining using flow cytometry. Numbers on histograms indicate the frequency of proliferating target cells. Representative data from a healthy elderly individual. (B) Proliferation inhibitory index was calculated as described in methods. Circles and error bars indicate the mean and standard deviation (SD), respectively. (C–D) Sorted CD4⁺,CD25^bright and CD4⁺,CD25⁻ T cells were co-cultured for 7 days at various ratios in the presence of anti-CD3 and CD28 Abs. Cytokine levels in tissue culture supernatant were measured using a multiplex cytokine assay kit. (C) Inhibitory index for cytokine production was calculated as described in methods. (D) IL-10 levels (pg/ml) in tissue culture supernatant. Bars and error bars indicate the mean and SEM, respectively. P values were obtained by the Mann-Whitney U test.

Fig. 3

Comparing the capacity of CD4⁺,CD25^bright T cells in suppressing the proliferation and cytokine production of CD4⁺,CD25⁻ T cells between young and elderly humans. PBMCs from young and elderly individuals were stained with Abs to CD4 and CD25 and sorted into CD4⁺,CD25^bright (R1 in Fig. 1A, Treg) and CD4⁺,CD25⁻ (R4 in Fig. 1A, Target) T cells. (A) In some experiment, the latter cells were stained with CFSE and mixed with CD4⁺,CD25^bright T cells at various ratios as indicated in the figure and incubated for 7 days in the presence of anti-CD3 and -CD28 Abs. Proliferating target cells were identified based on CFSE staining using flow cytometry. Numbers on histograms indicate the frequency of proliferating target cells. Representative data from a healthy elderly individual. (B) Proliferation inhibitory index was calculated as described in methods. Circles and error bars indicate the mean and standard deviation (SD), respectively. (C–D) Sorted CD4⁺,CD25^bright and CD4⁺,CD25⁻ T cells were co-cultured for 7 days at various ratios in the presence of anti-CD3 and CD28 Abs. Cytokine levels in tissue culture supernatant were measured using a multiplex cytokine assay kit. (C) Inhibitory index for cytokine production was calculated as described in methods. (D) IL-10 levels (pg/ml) in tissue culture supernatant. Bars and error bars indicate the mean and SEM, respectively. P values were obtained by the Mann-Whitney U test.

Fig 4

Measuring CTLA-4 and Fas expression by CD4⁺,FOXP3⁺ T cells as well as apoptosis of CD4⁺,CD25^bright and CD25⁻ T cells in young and elderly humans. (A – D) PBMCs from young (n = 15) and elderly (n = 15) individuals were stained with Abs to CD4, followed by permeabilization and staining with Abs to FOXP3, CTLA-4, Fas or isotype Ab. Representative histograms showing the expression of CTLA-4 (A) and Fas (C) by CD4⁺,FOXP3⁻ and FOXP3⁺ T cells. MFI of CTLA-4 (B) and Fas (D) expression by CD4⁺,FOXP3⁺ T cells. (E) PBMCs from young (n = 8) and elderly (n = 9) individuals were stained with Abs to CD4 and CD25 and sorted into CD4⁺,CD25^bright (Treg) and CD4⁺,CD25⁻ T (Target) cells. The latter cells were stained with CFSE and incubated for 7 days with CD4⁺,CD25^bright T cells at 1:1 ratio in the presence of anti-CD3/-CD28 Abs. The frequency of live Treg and target cells was measured using annexin V staining after incubation. P values were obtained by the Mann-Whitney U test.

Fig. 5

Chemokine receptor expression on CD4⁺,FOXP3⁺ T cells in young and elderly humans. PBMCs from young (n = 15) and elderly (n = 15) individuals were stained with Abs to CD4, CCR7, CCR4, CCR5, CXCR3 or isotype Abs, followed by permeabilization and staining with Abs to FOXP3. MFI of CCR7, CCR4, CCR5 and CXCR3 expression on CD4⁺,FOXP3⁺ T cells was calculated. P values were obtained by the Mann-Whitney U test.

Fig. 6

A conceptual model showing the potential role for FOXP3⁺ Treg in interfering IL-10 production from CD4⁺,CD25⁻ T cells in the elderly that leads to increased morbidity and mortality of sepsis.