Inverse Associations between Obesity Indicators and Thymic T-Cell Production Levels in Aging Atomic-Bomb Survivors

<div><p>Reduction of the naive T-cell population represents a deteriorating state in the immune system that occurs with advancing age. In animal model studies, obesity compromises the T-cell immune system as a result of enhanced adipogenesis in primary lymphoid organs and systemic inflammation. In this study, to test the hypothesis that obesity may contribute to the aging of human T-cell immunity, a thousand atomic-bomb survivors were examined for obesity status and ability to produce naive T cells, i.e., T-cell receptor excision circle (TREC) numbers in CD4 and CD8 T cells. The number of TRECs showed a strong positive correlation with naive T cell numbers, and lower TREC numbers were associated with higher age. We found that the TREC number was inversely associated with levels of obesity indicators (BMI, hemoglobin A1c) and serum CRP levels. Development of type-2 diabetes and fatty liver was also associated with lower TREC numbers. This population study suggests that obesity with enhanced inflammation is involved in aging of the human T-cell immune system. Given the fact that obesity increases the risk of numerous age-related diseases, attenuated immune competence is a possible mechanistic link between obesity and disease development among the elderly.</p></div

Regression analyses of TRECs.

a<p>Dependent variable: CD4 TRECs.</p>b<p>Dependent variable: CD8 TRECs.</p

Regression analyses of TRECs using multiple obesity indicators.

a<p>Age, gender, radiation dose, alcohol consumption, smoking, and cancer were also adjusted.</p>b<p>A forward stepwise procedure was used for 7 obesity-related variables: BMI, past BMI, total cholesterol, CRP, diabetes, fatty liver, and hypertension. Four variables (past BMI, CRP, diabetes, and fatty liver) were consequently selected (significant level to select, p<0.2) to construct statistical models.</p

GEE multivariate analysis.

a<p>Dependent variable: the bivariate variable of standardized CD4 TRECs and CD8 TRECs.</p>b<p>Effect of TREC2 represents the difference of the expectations of CD4 TRECs and CD8 TRECs, or the subtraction of the CD4 TREC expectation from the CD8 TREC expectation.</p

Study subjects (N = 1073).

a<p>Median (5–95% percentiles).</p>b<p>Number (%).</p>c<p>TREC stands for T-cell receptor excision circle.</p

Repopulation of hematopoiesis by irradiated human HSPCs in secondary recipients.

<p>(A) Changes in chimerism of human hematopoietic cells in the PB of secondary recipients. At 2 weeks post-irradiation, CD34⁺ human HSPCs were purified from the primary recipient mice, and 2 x 10⁶ CD34⁺ HSPCs were transplanted into secondary NOG recipients. Chimerism of CD45⁺ human and mouse hematopoietic cells in the PB are shown in upper panels. The percentages of CD11b⁺ myeloid cells, CD19⁺ B cells, and CD3⁺ T cells in CD45⁺ human hematopoietic cells in the PB are shown in lower panels. PB data of NOG mice just before the irradiation (Pre, n = 27) are also indicated at week 0. Data are presented as mean ± S.D. (0 Gy, n = 5; 0.5 Gy, n = 5; 1.0 Gy, n = 5). (B) Total BM cell numbers and chimerism of CD45⁺ human and mouse hematopoietic cells at 12 weeks after secondary transplantation. (C) Absolute numbers of CD34⁺CD38^- HSCs, CD34⁺CD38⁺ HPCs, CMPs and LMPPs in the BM (bilateral femurs and tibiae) (0 Gy, n = 5; 0.5 Gy, n = 5; 1.0 Gy, n = 5). (D) Numbers of colony-forming cells included in 1,000 CD34⁺ human HSPCs recovered from the BM 12 weeks after secondary transplantation (0–1.0 Gy, n = 3 each). The data in Fig 3B-D are presented as scatter diagrams with median values (bars). <i>P</i>-values for trend were calculated by nonparametric Jonckheere-Terpstra test.</p

Impaired activation of B cell regulator genes in irradiated human HSPCs.

<p>(A) Quantitative RT-PCR analysis of <i>IL7R</i>, <i>EBF1</i> and <i>PAX5</i> in CD34^<b>+</b> human HSPCs from humanized mice at 2 weeks post-irradiation and at 12 weeks post-secondary transplantation (n = 3 each). (B) Quantitative RT-PCR analysis of <i>p16INKA</i>, <i>HOXA9</i> and <i>MEIS1</i> in CD34^<b>+</b> human HSPCs from humanized mice at 2 weeks post-irradiation and at 12 weeks post-secondary transplantation (n = 3 each). Relative expression levels of each gene mRNA were adjusted in reference to <i>GAPDH</i> mRNA expression levels. Data are shown as the mean e adjust <i>P</i> < 0.05, **<i>P</i> < 0.01 and ***<i>P</i> < 0.001. The t-test was used for comparison of mean values.</p

Early effects of irradiation on human HSPCs in humanized mice.

<p>(A) Changes in chimerism of human hematopoietic cells in the PB after irradiation. At 12 weeks post-irradiation, the mice were randomly grouped into three categories and irradiated with 0, 0.5 or 1.0 Gy for each group. At 2 weeks post-irradiation, mice were sacrificed to evaluate early effects of irradiation. White blood cell (WBC) counts in the PB (left panel) and chimerism of CD45⁺ human (middle panel) and mouse (right panel) hematopoietic cells in the PB are presented as scatter diagrams with median values (bars) (n = 9 each). PB data of NOG mice just before the irradiation (Pre) are presented as controls (n = 27). (B) The percentages of CD11b⁺ myeloid cells, CD19⁺ B cells, and CD3⁺ T cells in CD45⁺ human hematopoietic cells in the PB of NOG mice just before the irradiation (Pre, n = 27) and at 2 weeks after irradiation (0–1.0 Gy, n = 9 each). (C) Total BM cell numbers (left panel) and chimerism of CD45⁺ human (middle) and mouse (right) hematopoietic cells in the BM just before the irradiation (Pre, n = 6) and at 2 weeks after irradiation (0–1.0 Gy, n = 9 each). (D) Absolute numbers of CD34⁺CD38^- HSCs, CD34⁺CD38⁺ HPCs, CMPs and LMPPs in the BM (bilateral femurs and tibiae) just before the irradiation (Pre, n = 6) and 2 weeks after irradiation (0–1.0 Gy, n = 9 each). (E) Numbers of colony-forming cells included in 1,000 CD34⁺ human HSPCs recovered at 2 weeks after irradiation (0–1.0 Gy, n = 3 each). Bars in scatter diagrams indicate median values. <i>P</i>-values for trend were calculated by nonparametric Jonckheere-Terpstra test.</p

Long-term radiation effects on human HSPCs in primary recipient mice.

<p>Changes in chimerism of human hematopoietic cells in the PB of primary recipients at 12 weeks post-irradiation. Chimerism of CD45^<b>+</b> human and mouse hematopoietic cells in the PB are shown in upper panels. The percentages of CD11b^<b>+</b> myeloid cells, CD19^<b>+</b> B cells, and CD3^<b>+</b> T cells in CD45^<b>+</b> human hematopoietic cells in the PB are shown in lower panels. PB data of NOG mice just before the irradiation (Pre) are also indicated. Data are presented as scattering diagrams with medium values (bars) (Pre, n = 15; 0 Gy, n = 5; 0.5 Gy, n = 5; 1.0 Gy, n = 5). <i>P</i>-values for trend were calculated by nonparametric Jonckheere-Terpstra test. The t-test was used for comparison of mean values.</p

<i>In vitro</i> differentiation of B cells from human HSPCs irradiated in humanized mice.

<p>Frequency of B cell-producing CD34^<b>+</b> HSPCs irradiated in humanized mice. Human CD34^<b>+</b> HSPCs were recovered from the primary recipients at 2 weeks post-irradiation. Graded numbers of purified CD34^<b>+</b> HSPCs (50, 10^<b>2</b>, 5 x 10^<b>2</b>, 10^<b>3</b>, 5 x 10^<b>3</b>, 10^<b>4</b> cells) were co-cultured with TSt-4 stromal cells in the presence of SCF, IL-7 and Flt3L (10 ng /ml each) for 4 weeks. Generation of CD19^<b>+</b> B cells was determined by flow cytometric analysis. The frequency of B–cell producing HSPCs was calculated using L-Calc software. The % of wells with B cell production and frequency of B-cell producing CD34^<b>+</b> HSPCs are summarized in the table and each data is plotted in the bottom panel. *** <i>P</i> < <b>0.001.</b> The frequencies in non-irradiated and irradiated cells were compared by the <b>χ2</b>-test for a 2 x 2 contingency table.</p