Low numbers of FOXP3 positive regulatory T cells are present in all developmental stages of human atherosclerotic lesions

Abstract

Background: T cell mediated inflammation contributes to atherogenesis and the onset of acute cardiovascular disease. Effector T cell functions are under a tight control of a specialized T cell subset, regulatory T cells (Treg). At present, nothing is known about the in situ presence of Treg in human atherosclerotic tissue. In the present study we investigated the frequency of naturally occurring Treg cells in all developmental stages of human atherosclerotic lesions including complicated thrombosed plaques.

Methodology: Normal arteries, early lesions (American Heart Association classification types I, II, and III), fibrosclerotic plaques (types Vb and Vc) and 'high risk' plaques (types IV, Va and VI) were obtained at surgery and autopsy. Serial sections were immunostained for markers specific for regulatory T cells (FOXP3 and GITR) and the frequency of these cells was expressed as a percentage of the total numbers of CD3+ T cells. Results were compared with Treg counts in biopsies of normal and inflammatory skin lesions (psoriasis, spongiotic dermatitis and lichen planus).

Principle findings: In normal vessel fragments T cells were virtually absent. Treg were present in the intima during all stages of plaque development (0.5-5%). Also in the adventitia of atherosclerotic vessels Treg were encountered, in similar low amounts. High risk lesions contained significantly increased numbers of Treg compared to early lesions (mean: 3.9 and 1.2%, respectively). The frequency of FOXP3+ cells in high risk lesions was also higher compared to stable lesions (1.7%), but this difference was not significant. The mean numbers of intimal FOXP3 positive cells in atherosclerotic lesions (2.4%) was much lower than those in normal (24.3%) or inflammatory skin lesions (28%).

Conclusion: Low frequencies of Treg in all developmental stages of human plaque formation could explain the smoldering chronic inflammatory process that takes place throughout the longstanding course of atherosclerosis.

Figure 1. Histology of normal human artery.

Hematoxylin&eosin overview (A, bar = 200 µm) and detailed CD3 (B, bar = 50 µm) staining of a normal human (mammary) artery. CD3 positive T cells are rare in normal human intima.

Figure 2. Histology of different types of atherosclerotic arteries.

Hematoxylin&Eosin overview (A, E, I, bar = 200 µm) and detailed CD3 (B, F, J, bar = 50 µm), FOXP3(C, G, K) and GITR(D, H, L) stains of three different types of atherosclerotic lesions: AHA class II (A–D), AHA class Va (E–H) and AHA class VI (I–L). CD3 positive T cells are frequently present in atherosclerotic tissue, but Treg are scarce.

Figure 3. Double staining images after spectral imaging.

A: FOXP3 (green) and CD3 (red) in atherosclerotic intima. Nuclei are depicted in blue. Inset shows double stained cell. Note nuclear staining of transcription factor FOXP3 and cytoplasmatic CD3 positivity; B: GITR (green) and CD3 (red) in atherosclerotic intima. Inset shows one double and one single stained cell; C: GITR (green) and CD3 (red) in atherosclerotic adventitia. GITR positive cells do not co-express CD3. D: GITR (green) and CD20 (red). No double stained cells. E: GITR (green) and CD138 (red). Inset shows double stained cells. F: GITR (green) and CD68 (red). No double stained cells.

Figure 4. Frequency of FOXP3 and GITR positive T cells in atherosclerotic tissue.

Values are expressed as a percentage (±SD) of the total number of T lymphocytes. Plaques were categorized according to the AHA classification (A and B, respectively) or categorized as early, fibrosclerotic and vulnerable lesions (C and D, respectively). *: P<0.05

Figure 5. Treg in atherosclerotic tissue compared to normal skin and inflammatory dermatoses.

Frequency of FOXP3 (A) and GITR (B) positive T cells in atherosclerotic and dermal specimens. Values are expressed as percentage (±SD) of the total number of T lymphocytes. The frequency of Treg in atherosclerotic intima and adventitia are significantly lower compared to both normal skin as well as a panel of inflammatory dermatoses (psoriasis, spongiotic dermatitis and lichen planus). **: P<0.005