PD-1/PD-L1 expression on CD(4+) T cells and myeloid DCs correlates with the immune pathogenesis of atrial fibrillation

Abstract

Although immuno-inflammatory response contributes to pathogenesis of AF, molecular and cellular mechanism in this process remains poorly understood. Recently, increasing evidence suggests that Programmed death-1 (PD-1)/PD-1 ligand (PD-L) pathway may be a potential pathway participating in AF pathogenesis. In this study, we detected the PD-1 and PD-L1, 2 expression on peripheral blood function cells by flow cytometry in 91 atrial fibrillation (AF) patients and 35 healthy volunteers. The expression of PD-1 on CD(4+) T cells and PD-L1 on myeloid dendritic cells (mDCs) in AF patients is significantly down-regulated compared with healthy volunteers. In addition, the extent of PD-1/PD-L1 down-regulation is closely related with AF burden. More importantly, Allogeneic mixed leukocyte reactions (MLR) shows that the mDCs PD-L1 down-regulation is associated with increased T cell (CD(4+) and CD(8+)) proliferation, increased type 1 effector cytokines (IL-2 and IFN-γ) secretion, and decreased type 2 effector cytokine (IL-10) secretion. Then, PD-L1 up-regulation by the stimulation of IFN-α can significantly convert this representation. Collectively, our report suggest that T(CD(4+))/mDCs-associated PD-1/PD-L1 pathway plays a key role in AF immune regulation. PD-1/PD-L1 down-regulation in AF patients promotes T cells function and may contribute to AF pathogenesis.

Keywords: Programmed death-1; atrial fibrillation; myloid dendritic cell; programmed death-ligand 1.

Figure 1

Expression of PD-1 on T cells from AF patients. Fresh heparinized peripheral blood were stained with a mixture of mAbs and analysed by flow cytometry. (A and C) Representative dot plots of PD-1 staining for studied individuals. (B and D) Percentages of PD-1+ CD⁴⁺ T cells and PD-1+ CD⁸⁺ T cells are shown respectively. •, control group; ▪, paroxysmal AF patient; ▴, persistent AF patient. Horizontal bars, mean values. Values of P are shown. P-values were calculated by anova and LSD.

Figure 2

Expression of PD-L1 on mDCs, macrophages, CD⁴⁺ and CD⁸⁺ T cells from AF patients. Fresh heparinized peripheral blood were stained with a mixture of mAbs and analysed by flow cytometry. (A, C, E and G) Representative dot plots of PD-L1 staining for studied individuals. (B, D, F and H) Percentages of PD-L1+ mDCs+, PD-L1+ macrophages+, PD-L1+ CD⁴⁺ T cells and PD-L1+ CD⁸⁺ T cells are shown respectively. •, control group; ▪, paroxysmal AF patient; ▴, persistent AF patient. Horizontal bars, mean values. Values of P are shown. P-values were calculated by anova and LSD.

Figure 3

Expression of PD-L2 on mDCs, macrophages, CD⁴⁺ and CD⁸⁺ T cells from AF patients. Fresh heparinized peripheral blood were stained with a mixture of mAbs and analysed by flow cytometry. (A, C, E and G) Representative dot plots of PD-L2 staining for studied individuals. (B, D, F and H) Percentages of PD-L2+ mDCs+, PD-L2+ macrophages+, PD-L2+ CD⁴⁺ T cells and PD-L2+ CD⁸⁺ T cells are shown respectively. •, control group; ▪, paroxysmal AF patient; ▴, persistent AF patient. Horizontal bars, mean values. Values of P are shown. P-values were calculated by anova and LSD.

Figure 4

Expression of PD-1 on CD⁴⁺ T cells and PD-L1 on mDCs from group F and group O. The expression of PD-1 on CD⁴⁺ T cells and PD-L1 on mDCs were shown as a box with the 25th to 75th percentiles containing the median line and the lines showing the 1st to 99th percentiles. Values of P are shown. P-values were calculated by Mann–Whitney U-test.

Figure 5

Induction of PD-L1 expression on mDCs by IFN-α in vitro. The isolated mDCs were cultured with medium only or 10,000 U/ml IFN-α in 96-well plates for 72 hrs. PD-L1 was stained with a control Ab (filled histograms) or PD-L1 mAb (open histograms). Numbers in histograms represent mean fluorescence intensity of PD-L1 expression. Data shown are one of three similar experiments.

Figure 6

Effects of PD-1/PD-L1 pathway on T cell proliferation costimulated by mDCs. mDCs isolated from patients (n = 10) or healthy controls (n = 10) were cocultured at different ratios with CD⁴⁺ or CD⁸⁺ T cells from a normal individual (as the third party), with or without treatment of IFN-α. After 5 days at 37°C, proliferation of alloreactive T cells was assessed by pulsing the cultures with [³H] thymidine for the last 18 hrs. Results are expressed as mean cpm ± SD of triplicate wells. ○, healthy control; ▽, persistent AF patient; ▴, IFN-α-treated mDCs from patient groups; •, addition of anti-PD-L1 mAbs; and □, addition of isotype control IgG1.

Figure 7

Up-regulation of PD-L1 can restore cytokine production by responsive cells in MLR. Data are expressed in pg/ml. •, healthy control; ▪, AF patients; ▴, IFN-α-treated mDCs from AF group; ▾, addition of anti-PD-L1 mAbs; and ♦, addition of isotype control IgG1. Horizontal bars, median values. Values of P are shown. P-values were calculated by Mann–Whitney U-test.