Impaired thymic export and apoptosis contribute to regulatory T-cell defects in patients with chronic heart failure

Abstract

Objective: Animal studies suggest that regulatory T (T(reg)) cells play a beneficial role in ventricular remodeling and our previous data have demonstrated defects of T(reg) cells in patients with chronic heart failure (CHF). However, the mechanisms behind T(reg-)cell defects remained unknown. We here sought to elucidate the mechanism of T(reg-)cell defects in CHF patients.

Methods and results: We performed flow cytometry analysis and demonstrated reduced numbers of peripheral blood CD4(+)CD25(+)FOXP3(+)CD45RO(-)CD45RA(+) naïve T(reg) (nT(reg)) cells and CD4(+)CD25(+)FOXP3(+)CD45RO(+)CD45RA(-) memory T(reg) (mT(reg)) cells in CHF patients as compared with non-CHF controls. Moreover, the nT(reg)/mT(reg) ratio (p<0.01), CD4(+)CD25(+)FOXP3(+)CD45RO(-) CD45RA(+)CD31(+) recent thymic emigrant T(reg) cell (RTE-T(reg)) frequency (p<0.01), and T-cell receptor excision circle levels in T(reg) cells (p<0.01) were lower in CHF patients than in non-CHF controls. Combined annexin-V and 7-AAD staining showed that peripheral T(reg) cells from CHF patients exhibited increased spontaneous apoptosis and were more prone to interleukin (IL)-2 deprivation- and CD95 ligand-mediated apoptosis than those from non-CHF individuals. Furthermore, analyses by both flow cytometry and real-time polymerase chain reaction showed that T(reg)-cell frequency in the mediastinal lymph nodes or Foxp3 expression in hearts of CHF patients was no higher than that of the non-CHF controls.

Conclusion: Our data suggested that the T(reg)-cell defects of CHF patients were likely caused by decreased thymic output of nascent T(reg) cells and increased susceptibility to apoptosis in the periphery.

Figure 1. Frequencies of the regulatory T (T_reg)-cell subset in CHF patients and non-CHF controls.

PBMCs of CHF patients (n = 52, 32 NIHF and 20 IHF) and non-CHF controls (n = 43) were included, and a 6-color flow cytometric analysis using mAbs specific for CD4, CD25, CD45RA, CD45RO, CD31 and Foxp3 was performed. a. Representative FACS images from a non-CHF control. Dot plots show CD45RA and CD45RO expression on gated CD4⁺ T cells. Naïve and memory CD4⁺ T cells were defined as CD45RA⁺CD45RO⁻ (R1) and CD45RA⁻CD45RO⁺ (R2), respectively. b. A small subpopulation of memory T_reg (mT_reg) (upper panel) and naïve T_reg (nT_reg) (lower panel) cells expressing both CD25 and Foxp3 was detectable. c. Histograms show the expression of CD31 on nT_reg cells. Recent thymic export-T_reg (RTE-T_reg) cells were identified as CD4⁺CD25⁺Foxp3⁺CD45RA⁺CD45RO⁻CD31⁺ cells. d. Frequencies of total T_reg, nT_reg, and mT_reg cells in different patient populations were determined as percentages of total CD4⁺ T cells. e. The ratio of nT_reg to mT_reg cells in different subject populations. f. RTE-T_reg cell frequency in different subject populations was presented as a percentage of total T_reg cells. *p<0.05 vs. non-CHF controls.

Figure 2,. Analysis of intracellular T-cell receptor excision circle (TREC) levels in purified T_reg cells from CHF patients and non-CHF controls.

a. CD4⁺CD25⁺CD127^low T_reg cells from CHF patients (n = 25, 14 NIHF and 11 IHF) and non-CHF controls (n = 15) were isolated by magnetic selection (left), and the TREC levels were determined by RT-PCR (right; *p<0.01) and compared to non-CHF controls. b. RTE-T_reg frequencies were plotted against TREC levels in purified T_reg cells from CHF patients and non-CHF controls (r = 0.75, p<0.001).

Figure 3. Correlation between CD4⁺CD25⁺ Foxp3⁺ T_reg cells and CD4⁺CD25⁺CD127^lowT_reg cells.

Frequencies of CD4⁺CD25⁺Foxp3⁺ T_reg cells were plotted against CD4⁺CD25⁺CD127^lowT_reg cells in 47 CHF patients and 38 non-CHF controls (r = 0.91, p<0.001).

Figure 4. Spontaneous apoptosis of T_reg cells from CHF patients and non-CHF controls.

PBMCs of 47 CHF patients and 38 non-CHF controls were stained with anti-CD4, anti-CD25, anti-CD127, annexin-V and 7-AAD and analyzed by flow cytometry. a. Representative FACS analyses from one non-CHF control and one CHF patient are shown. A small subpopulation of CD25⁺CD127^low/− cells were gated and identified as T_reg cells (left panels). The staining of annexin-V and 7-AAD was further analyzed on gated T_reg cells (middle), and apoptosis levels of the T_reg cells are calculated as percentage of annexin-V⁺7-AAD⁻ cells among 7-AAD⁻ cells (right; *p<0.01 vs. non-CHF controls). b. CD4⁺CD25⁺CD127^low/− T_reg cells from CHF patients (n = 25, 14 NIHF and 11 IHF) and non-CHF controls (n = 15) were isolated by magnetic selection (left), and the expression of both the anti-apoptotic gene Bcl-2 (top panel) and the pro-apoptotic gene Bak (bottom panel) was measured. *p<0.05 vs. non-CHF controls.

Figure 5. IL-2 deprivation and FasL-mediated T_reg-cell apoptosis.

PBMCs were stained with anti-CD4, anti-CD25, anti-CD127, and apoptosis was induced as described in Methods. a. IL-2 deprivation-mediated T_reg-cell apoptosis between CHF patients (n = 47) and non-CHF controls (n = 38).b. CD95 expression on gated T_reg cells from CHF patients (n = 47) and non-CHF controls (n = 38). c. CD95L induced a dose-dependent apoptosis of T_reg cells from CHF patients after incubation with CD95L for 12 h (upper panel; data are means from three separate experiments). Apoptosis of T_reg cells from CHF patients in the presence of 100 ng/ml CD95L was plotted against time (lower panel; data are means from three separate experiments). d. FasL-induced apoptosis of T_reg cells from CHF patients and non-CHF controls (100 ng/ml FasL for 12 hrs). e. ELISA determination of plasma soluble FasL levels in 47 CHF patients and 38 non-CHF controls. *p<0.05 vs. non-CHF controls.

Figure 6. T_reg cells in mediastinal lymph nodes and hearts.

a. Representative FACS dot plots showed the presence of CD4⁺CD25⁺Foxp3⁺ T_reg cells in the mediastinal lymph nodes. b. Percentages of CD4⁺CD25⁺Foxp3⁺ T_reg cells in the mediastinal lymph nodes were determined in six CHF patients (three with idiopathic cardiomyopathy and three with ischemic cardiomyopathy) and three controls without cardiomyopathy. c. Comparison of Foxp3 expression in the mediastinal lymph nodes and hearts of CHF and non-CHF controls. *p<0.05 vs. non-CHF controls.