Sex Differences in Adipose Tissue CD8+ T Cells and Regulatory T Cells in Middle-Aged Mice

Abstract

The prevalence of cardiovascular disease has increased among middle-aged women in the United States, yet has declined in middle-aged men. In experimental stroke, middle-aged females have larger strokes and greater inflammation than age-matched males or younger females. The mechanism underlying this shift from an "ischemia-protected" to an "ischemia-sensitive" phenotype in aging females is unknown. One potential factor is an age-related increase in systemic factors that induce inflammation. Increased abdominal fat deposition is seen in women during middle age. Adipose tissue plays a key role in obesity-induced systemic inflammation, including increased pro-inflammatory cytokines. We hypothesized that age and sex differences in adipose immune cells promote an augmented pro-inflammatory milieu in middle-aged females driven by a balance shift between pro-inflammatory and anti-inflammatory T cells. Abdominal adipose tissue immune cells from young (3-4 months) and middle-aged (15-16 months) male and female C57BL/6J mice were analyzed by flow cytometry. Plasma triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels were determined with colorimetric assays. Middle-aged mice had higher adipose tissue mass compared to young mice. Lipid profiling showed no sex differences in TG and LDL, but middle-aged females had lower HDL (0.84 ± 0.07 μg/μl) than middle-aged males (1.35 ± 0.06 μg/μl). Flow cytometry data demonstrated an age-associated increase in adipose tissue CD8⁺ T cells that was augmented by female sex, with middle-aged females having a higher percentage of CD8⁺ cells (34.4 ± 3.2% of CD3⁺ T cells) than middle-aged males (24.4 ± 2.2%). This increase in CD8⁺ T-cell proportion was adipose tissue-specific, as this change was not observed in blood. Middle-aged females had higher numbers of activated (CD69⁺) CD8⁺ T cells than males. In addition, female CD8⁺ T cells produced higher levels of IFN-γ, TNF-α, and granzyme B ex vivo, and females had higher adipose levels of IFN-γ, RANTES and MIP-1β than middle-aged males. In parallel, females had lower levels of regulatory T cells (Tregs), an anti-inflammatory T-cell subtype, compared to age-matched males. In conclusion, middle-aged females have a detrimental combination of elevated pro-inflammatory T cells and decreased anti-inflammatory Tregs in adipose tissue, which may promote a pro-inflammatory milieu and contribute to increased cardiovascular disease burden in aging females.

Keywords: CD8+ T cells; adipose tissue; aging; inflammation; regulatory T cells; sex differences.

Figure 1

Age-induced increase in adipose tissue mass and negatively shifted lipid profile in middle-aged females. (A) Perigonadal (epididymal in males and parametrial in females) adipose tissue mass normalized to body weight in young (3–4 months) and middle-aged (15–16 months) mice. Two-way ANOVA effect of age: p < 0.001, n = 9. (B) Plasma high-density lipoprotein (HDL) cholesterol and low-density lipoprotein cholesterol (LDL) in middle-aged mice. ***p < 0.001 unpaired t-test, n = 10. (C) Plasma triglycerides in middle-aged male and female mice. p = 0.32 unpaired t-test, n = 9–10.

Figure 2

Age-associated increases in adipose lymphoid cells and CD8⁺ T cells are pronounced in females. Flow cytometry quantification of lymphoid (CD45⁺CD11b⁻) and myeloid cell (CD45⁺CD11b⁺) percentages of (A) adipose tissue and (B) blood-derived immune cells. Two-way ANOVA lymphoid cells: effect of age p < 0.001, effect of sex p < 0.01; myeloid cells: effect of age p < 0.001, *p < 0.05, ***p < 0.001 Sidak’s multiple comparison’s test, n = 5. (C) Representative contour plots of CD4⁺ and CD8⁺ T-cell populations, with outliers displayed, in adipose tissue of young and middle-aged male and female mice. (D) Quantification of CD8⁺ T-cell percentages (CD3⁺CD8⁺) and (E) CD4⁺ T cells (CD3⁺CD4⁺) in adipose tissue of young and middle-aged mice using flow cytometry. Two-way ANOVA effect of age: p < 0.001, sex: p < 0.01. *p < 0.05 Sidak’s multiple comparison’s test, n = 8–9. (F) Representative histograms of CD8⁺CD69⁺ T cells in adipose tissue from young and middle-aged male and female mice. (G) Absolute number of activated CD8⁺ T cells (CD8⁺CD69⁺) normalized to bead counts and adipose tissue weights. Two-way ANOVA effect of age: p < 0.05, sex: p < 0.05, ***p < 0.001 Sidak’s multiple comparison’s test, n = 5.

Figure 3

Sex differences in adipose CD8⁺ T-cell cytokine production ex vivo and total adipose cytokine levels. (A) Representative contour plots of IFN-γ⁺CD8⁺ T cells after ex vivo stimulation with phorbol 12-myristate 13-acetate and ionomycin for 4 h at 37°C 5% CO₂. Quantification of (B) IFN-γ⁺CD8⁺ T cells, (C) IFN-γ⁺CD4⁺ T cells, (D) TNF-α⁺CD8⁺ T cells, and (E) Granzyme B (GzmB)⁺CD8⁺ T cells normalized to no stimulation conditions within each sex. Two-way ANOVA, *p < 0.05, ***p < 0.001 Sidak’s multiple comparison’s test, n = 5. Total adipose levels of (F) IFN-γ, (G) RANTES, and (H) MIP-1β in middle-aged mice by multiplex cytokine measurement. p ≤ 0.05 unpaired t-test, *p < 0.05 unpaired t-test with Welch’s correction, n = 8–10.

Figure 4

Males have higher levels of adipose regulatory T cells (Tregs) compared to age-matched females. Gating strategy (A), representative contour plots (B), and quantification (C) of CD4⁺CD25⁺FoxP3⁺ Tregs in adipose tissue from young and middle-aged male and female mice. Two-way ANOVA effect of sex: p < 0.001, **p < 0.01, ***p < 0.001 Sidak’s multiple comparison’s test, n = 9.

Figure 5

Age-associated increase in adipose CD8⁺ T cells and imbalanced immune response in middle-aged females. Middle-aged females had a greater increase in adipose effector CD8⁺ T cells than males, without a compensatory increase in anti-inflammatory regulatory T cells (Tregs). Higher levels of the CD8⁺ T-cell chemokine RANTES, MIP-1β, and the pro-inflammatory cytokines IFN-γ, TNF-α, and granzyme B (GzmB) were also observed in middle-aged females. We hypothesize that this imbalance may promote a pro-inflammatory milieu and contribute to increased cardiovascular disease burden in aging females.