A chronic, T cell mediated inflammatory process determines the growth and development of atherosclerotic lesions [1]–[3]. Although the majority of the inflammatory cells in atherosclerotic lesions are macrophages, up to 20 percent of the cells are T lymphocytes [2], [4]. Moreover, activation of T cells is considered to play an important role in the process of atherosclerotic plaque destabilization which may initiate plaque disruption and the onset of acute coronary syndromes[5]–[7]

In general, the effector functions of activated T cells are under tight control of a special subset of T cells, the regulatory T cells (Treg). Treg play a central role in inducing and maintaining immunologic tolerance and the termination of immune responses. Deficiency or dysfunction of these cells lead to autoimmunity or aggravated pathogen-induced inflammation[8], [9]. An important population of Treg is formed by the naturally occurring Treg, phenotypically characterized by a high constitutive expression of CD25, the alpha chain of the interleukin-2 receptor, and also referred to as CD4⁺CD25⁺ regulatory T cells[10]. In tissues these cells are best identified by their expression of transcription factor FOXP3 (Forkhead Box Protein P3), a member of the forkhead winged helix protein family of transcription factors[11].

Since atherosclerotic plaque inflammation also relates to autoimmunity, likely induced by oxidized LDL epitopes [12], a role for Treg in atherosclerotic inflammatory process could be anticipated. Indeed, experimental studies have shown that adoptive transfer of Treg prevent the development of plaques in mouse models of atherosclerosis[13]–[15]. Furthermore, it was recently shown that the numbers of Treg are decreased in the peripheral blood of patients with acute coronary syndromes[16]. However, the in situ presence of Treg in human atherosclerotic plaques has not yet been investigated.

In the present study we systematically evaluated the distribution of regulatory T cells in all developmental stages of human plaques (type 1 to VI according to the classification of the American Heart Association, AHA) with the use of monoclonal antibodies against regulatory T cell markers FOXP3 and GITR. Cellular specificity of FOXP3 and GITR for T cells was investigated with immunodoublestains visualized with spectral imaging [17]. Moreover, using the AHA classification of lesions, we compared specifically the relative frequency of Treg in early, stable, and high risk (vulnerable and thrombosed) plaques. In order to get insight in the numbers of Treg that participated in atherosclerosis, we compared these data with the Treg counts in normal human skin and in the inflammatory infiltrates encountered in various types of T cell mediated inflammatory skin diseases.

Regulatory T cells play a central role in inducing and maintaining immunologic tolerance and the termination of immune responses, and deficiency or dysfunction of these cells lead to autoimmunity or aggravated pathogen-induced inflammation[8], [9]. Also in atherosclerotic disease the presence and function of regulatory T cells may have important implications, because of their potential suppressive effects on the in situ inflammatory response. In the present study we investigated the presence of naturally occurring regulatory T cells in normal and atherosclerotic vessels by studying the presence of FOXP3 and GITR positive T cells. In normal intima T cells are virtually absent. T cells and Treg are present in atherosclerotic plaques, but the frequency of the latter is low (0.5–5%). Despite these relatively low numbers, related to the total number of T cells present in the plaque, we found that high risk plaques (AHA IV, Va and VI) contain significantly more FOXP3 positive T cells compared to early lesions (AHA 1, II and III). It is known that the onset of acute coronary syndromes is associated with changes in the inflammatory response in these lesions, including a shift in phenotype of intraplaque T cells: the frequency of activated T lymphocytes is significantly increased in unstable lesions [5], [21]. Furthermore, it is has been shown that the onset of acute coronary syndromes is associated with the antigen driven, oligoclonal proliferation of certain T cell subsets[6]. It appears that as a result of such an increase in T cell mediated inflammatory activity, also the frequency of Treg increases in these unstable lesions.

At present, most studies on Treg in relation to (auto) immunity have been performed on peripheral blood Treg. Peripheral blood Treg are relatively easily detectable by their high constitutive expression of CD25 (CD4⁺CD25^high subset). In humans, decreased frequency of peripheral blood CD4⁺CD25^high Treg have been linked to different types of autoimmune diseases like rheumatoid arthritis, type-1 diabetes, multiple sclerosis and systemic lupus erythematosus [22]–[25]. Recently it was shown that the frequency of Treg is also decreased in the peripheral blood of patients with acute coronary syndromes[16], [26]. Our study is the first describing (low numbers) of Treg also inside human plaques.

In the present study we used FOXP3 and GITR to analyze the presence of Treg in situ. GITR is directly involved in maintaining immunological tolerance by GITR-GITRligand interactions [27], [28]. However, recently activated T lymphocytes may also express GITR, and therefore the expression of GITR by T lymphocytes should be taken with caution. Still, in the present study we found that, apart from the adventitia in advanced atherosclerotic plaques, the distribution of GITR and FOXP3 were comparable, and the results obtained with GITR were in line with the observations obtained with FOXP3. Transcription factor FOXP3 is now generally considered as the most reliable antigen to identify naturally occurring Treg in situ [17], [29], [30]. Only recently reliable monoclonal antibodies specific for FOXP3 became available[31], and therefore there are not many data available yet on the distribution or frequency of Treg in solid tissues. In the present study we observed that the mean frequency of FOXP3 positive Treg in atherosclerotic intima, but also in atherosclerotic adventitia was in the range of the 0.5–5% of the total number of CD3 positive T cells. By contrast, in a previous study [17], performed under identical methodological conditions, we found that the frequency of FOXP3 positive T cells in normal skin and a panel of inflammatory skin diseases was in the range of 25–30%, which is significantly higher compared to the situation in atherosclerotic intima and adventitia. It thus appears that, at least compared to normal and various T cell mediated inflammatory skin diseases, the frequency of Treg in atherosclerotic tissue is very low. This raises two important questions: first, what the reason for this low frequency of Treg in atherosclerotic tissue, and the second, does this have biological and/or clinical implications.

Recently, it was shown that oxidized lipoproteins inhibited FOXP3 expression and Treg function of mouse effector cells in vitro[15]. Thus, it could be that oxidized lipids, present already in the intima in the earliest stages of atherosclerosis, locally inhibit FOXP3 expression and Treg function in vivo. Indeed, we found that the frequency of Treg is not only relatively low in advanced, but also in early lesions. Similarly, the low frequency of FOXP3⁺ Treg in the adventitia could be the result of plaque derived lipoproteins, transported via Vasa vasorum microvessels to the adventitia. On the other hand, it is also possible that other mechanisms are responsible for the low numbers of Treg, like a decreased homing of Treg into vascular tissue. Still, it also important to note the frequency of Treg may be highly variable between different solid tissues, and so far we only compared (inflammatory) skin and atherosclerotic vessels.

The second important question that should be addressed concerns the biological and clinical implications of this low frequency of Treg in atherosclerotic tissue. In this respect it is first important to emphasize again that both experimental, as well as studies in humans have clearly shown that T cells and T cell activation are very important in development of atherosclerotic lesions, and the onset of acute cardiovascular syndromes [32]. Furthermore, it has been shown that increasing the numbers of Treg in atherosclerosis prone (ApoE-/-) mice by means of adoptive transfer leads to smaller atherosclerotic lesions[13]–[15], implicating that Treg do appear capable modifying plaque volume in vivo. These observations, taken together with the findings on the in situ expression of Treg in human plaques of the present study make it tempting to speculate that such low umber of Treg do have implications could be explain the smoldering chronic inflammatory process in the longstanding course of atherosclerosis.