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Keywords:

  • Angiogenesis;
  • CD1 molecules;
  • Cell migration;
  • Inflammation;
  • Invariant NKT cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Atherosclerosis, a chronic inflammatory lipid storage disease of large arteries, is complicated by cardiovascular events usually precipitated by plaque rupture or erosion. Inflammation participates in lesion progression and plaque rupture. Identification of leukocyte populations involved in plaque destabilization is important for effective prevention of cardiovascular events. This study investigates CD1d-expressing cells and invariant NKT cells (iNKT) in human arterial tissue, their correlation with disease severity and symptoms, and potential mechanisms for their involvement in plaque formation and/or destabilization. CD1d-expressing cells were present in advanced plaques in patients who suffered from cardiovascular events in the past and were most abundant in plaques with ectopic neovascularization. Confocal microscopy detected iNKT cells in plaques, and plaque-derived iNKT cell lines promptly produced proinflammatory cytokines when stimulated by CD1d-expressing APC-presenting α-galactosylceramide lipid antigen. Furthermore, iNKT cells were diminished in the circulating blood of patients with symptomatic atherosclerosis. Activated iNKT cell-derived culture supernatants showed angiogenic activity in a human microvascular endothelial cell line HMEC-1-spheroid model of in vitro angiogenesis and strongly activated human microvascular endothelial cell line HMEC-1 migration. This functional activity was ascribed to IL-8 released by iNKT cells upon lipid recognition. These findings introduce iNKT cells as novel cellular candidates promoting plaque neovascularization and destabilization in human atherosclerosis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Atherosclerosis is complicated by cardiovascular (CV) events, which usually occur when plaques rupture or erode. Vulnerable plaques prone to rupture are characterized by inflammation, plaque hemorrhage and abnormal apoptosis 1, 2, three processes that are spatially and temporally interconnected. Both innate and acquired immune responses can modulate atherosclerotic plaque development 3. Macrophages and T lymphocytes infiltrating the arterial wall during atherosclerosis 2, 4 produce proinflammatory cytokines, chemokines, metalloproteinases and mesenchymal growth factors that are all potentially involved in plaque growth and rupture but might also contribute to plaque remodeling and stabilization. A histopathological quantitative analysis has suggested that macrophages in the arterial wall seem to be protective in the early, but deleterious in the late stages of disease 5. T-cell populations with different functional capacities have been identified within atherosclerotic lesions 4 and contribute to the pathogenic complexity of the inflammatory process 6. In addition to inflammation, other mechanisms such as lipid retention 7, neovascularization 1, 5, 8 and tissue remodeling 9, 10 support plaque growth. How different leukocyte populations contribute to or are affected by these additional mechanisms remains elusive. T cells recognizing protein or lipid antigens within plaques are likely involved. Invariant NKT (iNKT) cells, which express a semi-invariant TCR made by Vα24 and Vβ11 chains, have attracted attention as lipid-responsive cells 11. These cells recognize lipid antigens presented by CD1d, a member of the CD1 family of antigen-presenting molecules 12. α-Galactosylceramide (αGalCer), a glycolipid antigen and potent activator of iNKT cells, accelerates atherosclerotic lesion formation in the ApoE−/− mouse model 13–15. CD1d-deficient and TCR Vα14-deficient mice, both lacking iNKT cells, are protected in this model of atherosclerosis 15–17. Moreover, in this model, adoptive transfer of iNKT cells markedly increases plaque burden 18. On the contrary, in the LDL receptor−/− mouse model, an atheroprotective role for iNKT cells has been described 19. Taken together, these animal studies provide strong evidence that iNKT cells are involved in atherosclerotic plaque development and progression.

No detailed investigations on iNKT cells in human atherosclerosis have yet been performed. Although CD1d protein is expressed in human atherosclerotic lesions 20, it remains unknown whether CD1d expression correlates with lesion severity or disease activity. This study examines CD1d-expressing cells and iNKT cells in human atherosclerotic lesions, their correlation with disease severity and activity and potential mechanisms for their involvement in plaque formation, progression and/or destabilization.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

CD1d+ cells in human atherosclerotic lesions are a sign of arterial vulnerability

We quantified intimal macrophages and CD1d+ cells in arterial tissue obtained from asymptomatic (ASA) patients who never experienced CV events previously (n=21) and patients with symptomatic (SA) atherosclerosis who developed CV events in the past (n=15) using human arterial tissue microarrays (Fig. 1). Definition of CV events is described in the Materials and methods section. This approach permits correlation of histomorphological findings with disease activity and lesion severity. We analyzed a total of 108 arterial sectors obtained systematically from three different vascular beds (carotid, renal and iliac artery) of 36 patients (clinical characteristics summarized in Table 1). Plaque type according to the American Heart Association (AHA) classification 21, and numbers of CD1d-expressing cells, CD68+ macrophages and vWF-positive microvessels per-intima area were determined in serial histopathological sections. In a per-sector analysis, both CD68+ macrophages and CD1d+ cells were found more commonly in advanced lesions than at early plaque stages (Fig. 1A; Supporting Information Fig. 1 for CD1d+ staining controls). In a per-patient analysis, i.e. when the three observations in the iliac, renal and carotid artery for each patient were averaged, the density (number of cells per mm2) of CD68+ or CD1d+ cells did not differ between ASA and SA patients (Fig. 1B). On the contrary, when signs of ectopic neovascularization were also considered as a variable, SA patients had on average the highest numbers of CD1d+ cells (p<0.05) (Fig. 1B). It is remarkable that CD1d+ cells were virtually absent from lesions without signs of ectopic neovascularization (early lesions) and low in ASA patients. For the tissue microarray analysis, arterial rings were harvested on average of 24 h after death. We tested whether the number of detectable CD1d+ cells would fade with time after death but found no such correlation (data not shown).

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Figure 1. APC in atherosclerotic lesions. The number of CD68+ macrophages and CD1d+ cells per-intima area was determined with arterial tissue microarrays 5. For each arterial sector, the intima area was morphometrically measured and the CD1d+ and CD68+ cells in the intima counted (expressed as cells/mm2). (A) Macrophage and CD1d+ cell counts in arterial sectors affected by atherosclerotic lesions of increasing severity according to the AHA classification. (B) Quantitative analysis of macrophage and CD1d+ cell counts in arterial sectors according to the disease activity (i.e. whether patients suffered CV events during their lifetime or not and said to have ASA or SA atherosclerosis). Neovessels were detected as vWF-positive microvessels in the arterial intima 5 and plaques were scored positive (filled boxes) or negative (open boxes) with respect to this anatomical sign. Data in (A) and (B) are presented as box plots with median, interquartile range and 5–95 percentiles. n.s., not significant *p<0.05, **p<0.01, ***p<0.001, Mann–Whitney U-test. (§) compares all ASA with all SA.

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Table 1. Clinical characteristics of the 36 patients
 No. of CV events (n=21)CV events (n=15)p-Value
  • a)

    a) Mean±SD.

  • b)

    b) Myocardial infarction, angina pectoris with myocardial ischemia and revascularization.

  • c)

    c) Cerebrovascular ischemic stroke, transient ischemic attack and revascularization.

  • d)

    d) SA peripheral arterial occlusive disease, SA aortic aneurysm and revascularization.

  • e)

    e) Infection at death was defined by the presence of two or more of the following criteria: body temperature >38°C, C-reactive protein >50 mg/L, neutrophils (band forms) >10%, positive blood cultures.

CV risk factors
Diabetes mellitus – no. (%)1 (5)7 (47)0.004
Body mass index (kg/m2)a)23±626±50.06
Hypercholesterolemia – no. (%)2 (10)4 (27)0.17
Arterial hypertension – no. (%)4 (19)6 (40)0.17
Smoking – no. (%)3 (14)4 (27)0.35
Male sex – no. (%)13 (48)11 (50)0.90
Age (years)a)74±1479±90.12
History of CV disease
Coronary heart diseaseb) – no. (%)0 (0)15 (100) 
Cerebrovascular diseasec) – no. (%)0 (0)5 (33) 
Arterial occlusive diseased) – no. (%)0 (0)6 (40) 
Autopsy (hours after death)a)24±1224±130.94
Infection at death – no. (%)e)9 (43)8 (53)0.53

iNKT cells are found in atherosclerotic lesions

The presence of CD1d-expressing cells in advanced, unstable atherosclerotic lesions prompted a search for iNKT cells. Due to the predicted scarcity of these cells, different approaches were applied to investigate their presence in atherosclerotic plaques. Lesional arterial intima from five SA patients was examined by confocal microscopy. We demonstrated the presence of CD3+/Vα24+ and CD3+/ Vβ11+ cells, which represented up to 3% of total infiltrating CD3+ T cells in all lesions analyzed (Fig. 2A and Table 2). These findings suggest but do not prove the presence of iNKT cells. We therefore prepared cell suspensions from thrombendarterectomy specimens and performed costaining with anti-Vα24 and anti-Vβ11 mAb ex vivo (Fig. 2B). The identification of Vα24/Vβ11 double-positive cells with fluorescent microscopy provided evidence that iNKT cells are present in the diseased arterial wall.

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Figure 2. iNKT cells in atherosclerotic lesions. (A) Lesional T cells in situ. Cryosections of intima of arterial rings from five patients with advanced atherosclerotic lesions were examined by confocal microscopy for the presence of T cells coexpressing CD3 and TCR Vβ11 (upper panels) or CD3 and TCR Vα24 chains (lower panels). Quantitative evaluation of cells positive for CD3/TCR Vα24 or CD3/TCR Vβ11 is summarized in Table 2. (B) Confocal analysis of iNKT cells freshly isolated from atherosclerotic plaques. Collagenase-released cells from fresh arterial tissue biopsies were collected by cytospin, costained with anti-TCR Vα24 and anti-TCR Vβ11 and analyzed by confocal microscopy. The data are representative of four independent experiments. All scale bars represent 2 μm.

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Table 2. Lesional T cells in situ: confocal estimation of numbers of cells positive for CD3 and TCR Vα24 or TCR Vβ11a)
PatientAHA plaque typeTCR Vα24+/total CD3+cells (%)TCR Vβ11+/total CD3+cells (%)
  • a)

    a) Values in parentheses express the numbers of Vα24+/CD3+ or Vβ11+/CD3+ cells as a percentage of total CD3+ cells and are given for each individual patient' biopsy and as the mean±SD for all five biopsies examined.

1IV5/163 (3.1)8/143 (5.6)
2IV5/274 (1.8)3/255 (1.2)
3V5/180 (2.8)6/192 (3.1)
4V2/152 (1.3)2/121 (1.6)
5VI8/153 (5.2)7/185 (3.8)
  (2.8±1.5)(3.1±1.8)

Next, we performed dual fluorescence confocal microscopy of lesional tissue from eight SA patients using anti-CD1d and anti-TCR Vα24-Jα18 (6B11) mAb, which recognizes the iNKT-specific invariant TCR Vα chain 22, 23. Representative micrographs unequivocally demonstrating the presence of iNKT cells in atherosclerotic lesions are shown in Fig. 3. In some instances, there was evidence of colocalization of the iNKT TCR with CD1d and even iNKT TCR and CD1d polarization toward each other. These findings could indicate an ongoing activation of iNKT cells within the atherosclerotic tissue.

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Figure 3. Identification of iNKT cells in atherosclerotic lesions. Panels from left to right show the staining of iNKT cells with anti-TCR Vα24-Jα18, of CD1d+ cells, iNKT cells (in red) merged to CD1d+ cells (in green), and of nuclei (Hoechst). Scale bars: 10 μm. Stainings were performed on eight patient tissue specimens and two representative stainings are shown. UPN, unique patient number. Boxed regions/arrows indicate colocalization of the iNKT TCR with CD1d (UPN 259) and iNKT TCR and CD1d polarization towards each other (UPN 189).

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To confirm and formally prove that iNKT cells reside in atherosclerotic lesions, we isolated and expanded iNKT cells from thrombendarterectomy specimens obtained from SA patients and performed phenotypic and functional studies. We stimulated plaque-derived T cells with αGalCer and CD1d-expressing cells to facilitate the selective expansion of iNKT cells and succeeded in establishing six bulk T-cell lines. Flow cytometry analysis using five-color staining showed that 60–90% of CD3+ cells were coexpressing TCR Vα24 and Vβ11 chains (Fig. 4A). In all lines, Vα24+Vβ11+ cells were also stained with αGalCer-loaded soluble human CD1d dimers, thus confirming the lipid specificity and CD1d restriction of their TCR. Five of the six iNKT cell isolates were CD4+ (representative shown in Fig. 4A) and all six were CD8. Plaque-derived iNKT cells stimulated with αGalCer produced large amounts of IL-4, TNF-α, IFN-γ and GM-CSF (Fig. 4B). We compared the six plaque-derived iNKT cell lines and 66 blood-derived iNKT cell clones with respect to their responsiveness to αGalCer. The ED50 was calculated after measurement of IFN-γ (Fig. 4C), TNF-α, IL-4 and GM-CSF (data not shown) release. For all cytokines, plaque-derived iNKT cells exhibited ED50 values at least tenfold lower than peripheral blood-derived iNKT cells. Taken together, these results prove that the iNKT cells present within atherosclerotic lesions have phenotypic and functional features of bona fide iNKT cells 24 and react to αGalCer with unusual high efficiency.

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Figure 4. iNKT cells from atherosclerotic plaque tissue. (A) A representative (total, six) bulk T-cell line isolated from plaques and expanded after stimulation with αGalCer. T cells were stained with anti-CD3, anti-CD4, anti-TCR Vα24 and anti-TCR Vβ11 and with αGalCer-loaded CD1d dimers. The FACS gating strategy is shown in Supporting Information Fig. 7. Left panel shows density plot after gating on CD3+ cells. The right panel shows density plot after gating on CD3+Vα24+Vβ11+ cells. (B) Cytokine release from one representative plaque-derived iNKT cell line after in vitro stimulation with αGalCer. Empty circles show cytokine release in the presence of the maximum dose of αGalCer and absence of CD1d-expressing APC. Results are expressed as mean±SD of triplicate determinations. One of the three representative experiments is shown. Similar results were obtained with the other five cell lines in at least two experiments. (C) Potency of αGalCer on 66 iNKT cell clones established from PBMC (open symbols) or with six iNKT cell lines isolated from plaque tissue (closed symbols). ED50 here defines the αGalCer dose inducing half-maximal IFN-γ release. Each point represents the ED50 value of one titration experiment, and for each group the median and interquartile range is given. ***p>0.001, unpaired Student's t-test.

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Circulating iNKT cell numbers are reduced in patients with SA atherosclerosis

Next, we investigated iNKT cells in the blood from three groups of donors, namely SA patients, age-matched control patients free of CV events in the past (i.e. ASA) and young healthy individuals. iNKT cells were detected in PBMC with four-color immunofluorescence analysis using anti-CD3ε, anti-TCR Vα24, anti-TCR Vβ11 mAb and αGalCer-loaded CD1d dimers. We detected a significant (p≤0.001) reduction of circulating iNKT cells in SA patients compared with either ASA patients or young healthy individuals (Fig. 5). A reduction was also observed in the ASA patients as compared with the young healthy individuals (p<0.01), possibly reflecting an age-related effect on this lymphocyte subset 25. These findings raise interesting issues regarding the fate of iNKT cells in peripheral blood of SA atherosclerotic patients: are they reduced because of lack of proliferative responsiveness to stimulatory lipids, increased apoptosis or increased extravasation into tissues?

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Figure 5. Circulating iNKT cells are reduced in atherosclerosis patients. Distribution of iNKT cells in PBMC from healthy young donors (N) and from age-matched patients with ASA atherosclerosis or patients with SA atherosclerosis. iNKT cells were detected by FACS with αGalCer-loaded CD1d dimers (A and B) or with anti-TCR Vα24 and anti-TCR Vβ11 (C). The FACS gating strategy is shown in Supporting Information Fig. 7. In order to have a statistically quantifiable number of iNKT cells, acquisition of at least 5 million CD3+ cells was performed. Data are reported as percentage after gating on CD3+ cells (A) or as percentage of total PBMC (B and C). Box plots with median, interquartile range and 5–95 percentiles are presented. *p<0.05, **p<0.01, ***p<0.001, Mann–Whitney U-test.

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Characterization of proatherosclerotic activity of iNKT cells

The presence of CD1d+ cells and iNKT cells within advanced atherosclerotic lesions, particularly in patients with SA disease, led us to investigate whether this T-lymphocyte population has a role in key processes of plaque formation and destabilization. Following αGalCer stimulation, plaque-derived iNKT cells release proinflammatory and potential angiogenic modulators (Supporting Information Fig. 2). Both plaque- and blood-derived iNKT cells secreted the same type of cytokines (data not shown).

Since neovascularized arterial sectors had the highest numbers of CD1d+ cells, subsequent investigations focused on the effects of iNKT activation on angiogenic behavior of human microvascular endothelial cell line HMEC-1 (EC). We examined angiogenic potential of conditioned medium (CM) derived from iNKT cell cultures stimulated with (CM+) or without (CM−) αGalCer using the EC-spheroid model of in vitro microvascular sprout formation as a global functional test for angiogenesis. Visualization of spheroids indicated that CM+ induced greater sprout outgrowth than CM− (Fig. 6A). Morphometric analysis showed a significant increase in both the number (Fig. 6B) and the length (Fig. 6C) of sprouts. CM collected from cultures containing αGalCer but lacking either CD1d+-APC or iNKT cells, or both, failed to enhance sprout outgrowth (Supporting Information Fig. 3). Taken together, these data confirm that antigen-stimulated iNKT cells can promote angiogenesis in vitro.

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Figure 6. Antigen activation of iNKT cells increases sprout outgrowth from EC spheroids. Conditioned media derived from iNKT cell cultures stimulated without (open bars) or with αGalCer (filled bars) were examined using the EC-spheroid model of in vitro angiogenesis. (A) Representative images of spheroids 24 h after exposure to conditioned media. Spheroids were morphometrically analyzed for total sprout number (B) and total sprout length (C). Bars undermarked “no APC” indicate the response to medium from iNKT cells cultured alone. Data are mean±SD from six experiments, each performed in triplicate. *p<0.05, **p<0.01, Student's t-test. A second iNKT cell clone elicited similar proangiogenic effects (Supporting Information Fig. 3).

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Soluble factors released by iNKT cells promote EC migration

Angiogenesis is a complex process and both proliferation and migration of EC contribute to this phenomenon 8, 26. To identify which of these activities is modulated in response to iNKT cell activation, we compared the effects of CM on proliferation and migration of EC in monolayer cultures. CM+ derived from different iNKT cells did not activate EC proliferation (Supporting Information Fig. 4) but did induce cell migration. Two methods were used to evaluate migration. In the first, confluent EC monolayers were scrape-wounded and migration into the wound was recorded over a 12 h period by time-lapse videomicroscopy. This wound-healing assay showed more rapid migration for EC cultured in the presence of CM+ (Fig. 7A and B, and Supporting Information Fig. 5). Representative videos showing EC motility in the presence of CM− (Supporting Information Fig5 video1-CM-.avi) and CM+ (Supporting Information Fig5 video2-CM+.avi) are given in the Supporting Information. The second assay quantified transmigration of EC in a Boyden-chamber and also demonstrated enhanced migration of EC toward CM+ (Fig. 7C). These data suggest a chemokine-like effect on EC angiogenic behavior.

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Figure 7. Antigen activation of iNKT cells promotes EC migration. Confluent monolayers of EC were scrape-wounded and the subsequent rate of wound closure monitored over a time period of 12 h by time lapse videomicroscopy. Acquired images were processed and analyzed using CellR software. (A) Representative images illustrating EC migration in the presence of CM+ or CM−. White lines indicate the location of the wound front and arrows indicate migration path length. (B) Quantitative analysis of the rate of EC migration from the initial wound front into the wound area (path length versus time). The data are representative of at least 30 experiments, each one performed in duplicate and values are given as averaged path length measurements±SD from triple fixed observation fields/well. CM+ from different iNKT clones similarly enhanced motility (Supporting Information Fig. 5). (C) EC transmigration toward CM+ and CM− in Boyden chamber chemotaxis assay was quantified after a 6- h incubation. Data are reported as mean±SD from three experiments each performed in duplicate. ***p<0.001, Student's t-test. The CM used in the illustrated experiments were obtained using HeLa cells as APC. In other experiments, C1R cells were used as APC with comparable results (data not shown).

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IL-8 is produced by iNKT cells and induces EC migration

IL-8, a pleiotropic chemokine with known angiogenic activity in vitro and in vivo27, 28, was among the numerous factors released by activated iNKT cells (Supporting Information Fig. 2). iNKT cells isolated from plaques (Fig. 8A) and peripheral blood (Supporting Information Fig. 6) showed strong intracellular staining for IL-8 when stimulated with αGalCer, proving that they readily produce this chemokine. To determine the contribution of iNKT cell-released IL-8 to the angiogenic potential of CM+, wound-healing assays were conducted in the presence of anti-IL-8 blocking Ab or using CM which had been immunodepleted of IL-8 prior to assay. Both treatments completely abrogated the enhanced EC migration (Fig. 8B). Basal EC migration was not affected by inclusion of anti-IL-8 Ab or IL-8-depletion, excluding nonspecific inhibitory effects of the Ab. Therefore, the enhanced migration response of EC to CM+ is dependent on IL-8 released by activated iNKT cells.

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Figure 8. Antigen-activated iNKT cells produce IL-8 which promotes EC migration. (A) Intracellular IL-8 analyzed by FACS in plaque-derived lines either resting (APC alone) or activated with APC+αGalCer or with PMA+ionophore. Cells were stained intracellularly with anti-IL-8 and anti-CD3 mAb. Percentages of CD3+ cells producing IL-8 are indicated. The FACS gating strategy is shown in Supporting Information Fig. 7. (B) Effects of inclusion of anti-IL-8 blocking mAb (upper panels) and of IL-8 immunodepletion (lower panels) of CM on EC migration examined by wound assay. Upper graphs: assays were performed in the absence (open circles) and presence of neutralizing anti-IL-8 mAb or isotype control IgG (closed or open triangles, respectively). Lower graphs: assays were performed with untreated CM (open circles), or CM subjected to immunodepletion protocols using neutralizing anti-IL-8 mAb or isotype control IgG (closed or open squares, respectively). Data shown are the average path length measurements±SD from triple fixed observation fields/well of duplicate samples. Similar results were obtained in three experiments using CM from different iNKT cell clones. The CM used in the illustrated experiments were obtained using HeLa cells as APC. In other experiments, C1R cells were used as APC with comparable results (data not shown).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Our study investigated iNKT cells in human atherosclerosis. We found that cells expressing CD1d are present in advanced atherosclerotic plaques and lesions from patients with active, SA disease. In patients with SA atherosclerosis, vascularized plaques had the highest number of CD1d+ cells. We identified the presence of iNKT cells in atherosclerotic lesions and characterized their function after isolation from plaques. iNKT cells from plaques show a high reactivity to the αGalCer antigen and may promote neovascularization in an IL-8-dependent manner. Our study suggests that iNKT cells contribute to the predisposition of atherosclerotic plaques to rupture.

In order to perform quantitative immunohistochemical analysis of inflammatory cells in atherosclerotic plaques, we took advantage of the arterial tissue microarray technique which permitted us to compare serial sections of 108 arterial sectors from 36 patients. Our approach, recently reproduced 29, facilitated evaluation of associations between the presence of CD68+ macrophages and CD1d+ cells and disease activity, lesion severity (i.e. plaque stage) and plaque neovascularization. CD68+ macrophages were found in all samples analyzed, even in those without lesions, as reported 5, and were slightly increased in very advanced plaques. On the contrary, CD1d+ cells were virtually absent from the normal arterial intima or in early plaque stages, whereas they were increased in advanced lesions particularly in the presence of neovessels. Expression of CD1d in human atherosclerotic plaques has been reported in two studies 20, 30. However, sample numbers were small and no correlations were made with clinical stage, disease activity or histological hallmarks, leaving open the question of whether CD1d expression correlates with lesion grade. In our study, a substantial number of CD1d+ cells were observed in advanced lesions (AHA type >IV) and particularly in lesions with signs of neovascularization, thus demonstrating a close correlation with advanced disease. The preferential localization of CD1d+ cells in areas with neovascularization could be explained by their efficient recruitment into vascularized plaques 31 and/or by their capacity to promote plaque neovascularization. Our data are in accordance with the concept that CD1d may present lipid antigens locally to specific T cells, including iNKT cells, which in turn may release angiogenic factors and contribute to neovascularization.

In the diseased arterial wall, we found T cells expressing Vα24 or Vβ11 TCR chains, which are used by iNKT cells. Flow cytometric analysis of freshly isolated iNKT cells was not possible due to small biopsy size and the minute number of resident iNKT cells. This technical limitation prevented exact quantification of iNKT cells and analysis of expressed activation markers. Therefore, the activation status of iNKT cells within lesions remains unknown. However, we could identify iNKT cells in lesional tissue by several methodological approaches, namely (i) detection of TCR Vα24-Jα18 with the 6B11 mAb; (ii) detection of Vα24 and Vβ11 coexpressing T cells freshly isolated from lesions and (iii) expansion and functional characterization of iNKT cell lines from plaques. We observed an intraplaque infiltration of iNKT cells and a significant reduction of iNKT cells in circulating blood in SA patients. This relative accumulation in plaques could be caused by homing and retention following local activation and/or proliferation upon antigen recognition.

The presence of iNKT cells in lesions has been inferred in mouse atherosclerosis models by molecular investigations and not cellular isolation. In pioneering studies on ApoE-deficient mice under high cholesterol diet, the presence of iNKT cells was suggested by RT-PCR 13, 14, 32. However, to date, iNKT cells have neither been isolated from plaques nor functionally characterized. In one study, CD3+CD161+ cells were histologically detected in carotid specimens and appeared with a frequency of 0.3–2% among plaque-infiltrating T cells 30. CD161 is expressed by a variety of T lymphocytes and therefore is not a specific marker for iNKT cells. In a second study, all CD3+ cells expanded from aortic aneurysms expressed the CD161 marker 33, suggesting an abnormal proliferation of this cell type in vitro. Since the expression of semi-invariant TCR Vα24/Vβ11 was not investigated, the presence of iNKT cells was not confirmed. We isolated plaque-infiltrating iNKT cells, which were Vα24+Vβ11+. They also bound αGalCer-loaded CD1d dimers, providing clear evidence that they are classical iNKT cells, and were efficiently activated by αGalCer-loaded CD1d-expressing APC to release IL-4, IFN-γ, GM-CSF and TNF-α. Thus, in atherosclerotic plaques, there is accumulation of cells expressing phenotypic and functional features of bona fide iNKT cells.

Importantly, the iNKT cell lines isolated from plaques all showed an extremely low threshold of activation when stimulated with αGalCer. The same low threshold was found only in a minor fraction of iNKT cell clones isolated from peripheral blood. These findings might suggest that due to antigen recognition and expansion in plaques, there is a preferential accumulation of iNKT cells expressing TCR with high responsiveness to αGalCer in vitro. Whether high reactivity to αGalCer reflects high reactivity to lipids accumulating within plaques is unknown since endogenous self-lipid antigens stimulating iNKT cells remain poorly characterized. An additional and nonmutually exclusive possibility is that plaque-derived iNKT cells lack NK inhibitory receptors and therefore are activated by very low doses of antigen. Intriguingly, in a model of atherosclerosis in ApoE-deficient mice, iNKT cells with low expression of the inhibitory Ly49 receptors showed proatherogenic activity which was more pronounced than that of Ly49-positive iNKT cells 34. The high reactivity of plaque-derived human iNKT cells deserves further investigations, since it remains unclear whether the entire plaque infiltrating iNKT cell population shows this unusual behavior.

How iNKT cell activation exerts proatherogenic effects remains an open issue. One potential mechanism relates to inflammation, which in human atherosclerosis is characteristically progressive. Since human iNKT cells isolated from plaques do release proinflammatory cytokines, their chronic in situ activation by lipid antigens might lead to lesion progression. This hypothesis is in line with many studies conducted in mice. Injection of αGalCer increases size and number of plaques in a mouse atherosclerosis model 15. This experimental iNKT cell activation elicits massive release of Th1 and Th2 cytokines and elevation in plasma levels of IL-6 and monocyte chemoattractant protein 1, which have been proposed to enhance local inflammation 11, 15. In humans, atherosclerosis is a slowly progressive disease and there is as yet no evidence of massive inflammation in the arterial wall in early lesions. Instead a chronic inflammatory reaction may apply, probably together with other disease-promoting mechanisms.

A second pathogenic mechanism concerns neovascularization. The significant association of CD1d+ cells with neovascularization in plaques suggests that iNKT cells may be involved in angiogenic processes. Our findings revealed that iNKT cell activation by antigen has proangiogenic effects as shown by enhanced microvascular sprout formation in an in vitro assay of angiogenesis. This effect was associated with EC migration as demonstrated by enhanced EC motility in both wound-healing and transmigration Boyden chamber assays.

Among the multiple cytokines that were produced by activated iNKT cells, IL-8 was the most promising candidate to further investigate. IL-8 was detected previously in the supernatant from lipid-stimulated blood-derived iNKT cells 35. We found that plaque-derived iNKT cells produce IL-8 as shown by intracellular staining. Further, the enhanced EC migration was dependent on release of IL-8 from iNKT cells since the migration response was abrogated by IL-8-blockade or IL-8 immunodepletion. The participation of IL-8 in atherosclerotic lesion progression is suggested by several studies 36. IL-8 has been detected in atheromatous tissue 28, 37, 38 and can be induced in monocytes by oxidized LDL and cholesterol 37, 39. Functionally, IL-8 contributes to intimal macrophage accumulation 40, to endothelial adhesiveness for monocytes 41, has mitogenic and chemoattractant effects on smooth muscle cells 42 and may also facilitate plaque recruitment of CD8+ effector T cells with high cytotoxic potential 43. IL-8 has been proposed as an important mediator of angiogenesis in CV lesions contributing to plaque growth 28. It is tempting to speculate that iNKT cells, when chronically activated by lipid antigens in the arterial wall, exert both promigratory and proinflammatory functions which become important for plaque neovascularization and destabilization. These functions might be shared with resident monocytes and other T cells recognizing specific antigens in plaques.

In conclusion, our studies have revealed CD1d+ cells in advanced, vascularized atherosclerotic lesions from patients with active disease. We have identified the presence of iNKT cells within plaques, isolated plaque iNKT cells and demonstrated their high sensitivity to antigen stimulation and their proinflammatory and proangiogenic potential in vitro. By these mechanisms, iNKT cells might participate in plaque growth and destabilization. Gathering evidence suggests that atherosclerosis is an autoimmune disease treatable with immunotherapeutic approaches 44. Our observations invoke iNKT cells and CD1d-expressing cells as additional potential candidate targets for immunopreventative interventions.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Patients and arterial tissues

All investigations with human subjects and tissues were approved by the regional ethical review board and performed in accordance with institutional guidelines. The arterial tissue microarrays have been described previously 5. In brief, 0.5 cm long arterial ring segments were obtained systematically during autopsy from 36 deceased patients who were treated for a broad variety of medical conditions at the Department of General Medicine of an academic medical center (Cantonal Hospital Bruderholz). The arterial rings were removed always at the same anatomical site regardless of the local lesion severity: 2 cm before the bifurcation for the left common carotid, 2 cm after branching from the aorta for the left renal and 2 cm after the aortic bifurcation for the left iliac artery. The deceased patients entering this study were not selected but were by intention prospectively included in order to circumvent any relevant selection bias. Clinical characteristics are summarized in Table 1. Fifteen of these patients were known to have SA, active atherosclerosis and to have suffered from CV events, defined as myocardial infarction, angina pectoris with signs of myocardial ischemia, cerebrovascular ischemic stroke, transient ischemic attack, peripheral arterial occlusive disease, SA aortic aneurysm or any arterial revascularization procedure to treat atherosclerosis 5.

PBMC were obtained from a second cohort of 269 in-patients hospitalized for any reason and who, with written informed consent, participated in a cross-sectional observational study of atherosclerosis 45. Twenty-eight of this second cohort had previous CV events in more than one organ system; among these SA patients, the ten oldest individuals (median age: 78, range 76–83 years) were selected for the analysis of the number of circulating iNKT cells (Supporting Information Table 1). From this second cohort, 110 of the 269 patients never reported any CV events in the past; among them, the ten oldest subjects (median age: 79, range 78–83 years) were selected as an age-matched ASA group. The age of these patients matches that of SA and ASA patients from whom tissue microarrays were generated. As an additional control group, ten healthy young individuals free of any clinical evidence for atherosclerosis (median age 29, range 26–33 years) were included. PBMC from whole blood were isolated and processed as described previously 46. Multicolor FACS was used to characterize iNKT cells in total PBMC as described in Supporting Information.

Analysis of iNKT cells in arterial tissue

Fresh-frozen, OCT-embedded arterial rings obtained at autopsy from 13 SA patients with advanced grade atherosclerotic plaques (AHA grade IV, V or VI) were variously used for the identification of TCR Vα24+ or TCR Vβ11+ T cells (n=5) and of TCR Vα24-Jα18+ T cells (n=8) as detailed in Supporting Information.

Collagenase-assisted release of lymphocytes from fresh arterial tissue obtained from SA patients with advanced lesions undergoing thrombendarterectomy was performed as described previously 47 with some modifications (Supporting Information). After staining with anti-TCR Vα24-FITC and anti-TCR Vβ11-biotin/streptavidin-Cy5 cells were collected by cytospin and analyzed for TCR Vα24+Vβ11+ cells by confocal microscopy. In some experiments, the released lymphocytes were resuspended in complete RPMI-1640 medium (Supporting Information), split, seeded into individual wells of a 96-well plate and subjected to two rounds of restimulation with DC obtained as described previously 48 plus 100 ng/mL αGalCer (kind gift of Kirin Breweries) and addition of anti-MHC class I and anti-MHC class II mAb (W6/32 and L243, both from ATCC) to avoid activation of MHC-restricted alloreactive T cells. The expanded plaque tissue-derived cells were assessed for the presence of iNKT cells by multicolor FACS (Supporting Information and figure legends) and antigen-presentation assays.

In vitro study procedures

Materials and methods for these studies are fully detailed and referenced in Supporting Information. The following human cell lines and clones were used: MOLT-4 expressing negligible CD1d (ATCC CRL 1582), C1R-hCD1d and HeLa-hCD1d 49 as APC lines, human microvascular EC line HMEC-1 (EC), iNKT cell clones from PBMC of healthy donors and iNKT cell lines from plaques. To generate CM, iNKT cells were cultured with APC pulsed with αGalCer or vehicle, and supernatant was harvested after 48 h. Cytokines in CM were quantified by sandwich ELISA, or by Multiplex analysis (BioRad; Human17-Plex Panel 171-A11171) in selected experiments. Assays testing angiogenic effects of CM on EC included the EC-spheroid model of in vitro angiogenesis, proliferation, Boyden-chamber transmigration and videomicroscopy of wound closure.

Statistical analysis

Patient data were compared between groups using Mann–Whitney U-test. Data from in vitro experiments were compared using the unpaired Student's two-tailed t-test and are given as mean±SD. All analyses were performed using GraphPad Prism software (version 5.03). Differences were considered significant at p<0.05.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

This study was supported by EEC grant MOLSTROKE (Molecular basis of vascular events leading to thrombotic stroke) LSHM-CT-2004 Contract Number 005206 (to T. J. R., B. C. B. and G. D. L.), the Swiss National Science Foundation grants 3100A0-109918 (to G. D. L.), 3100-118096 (to B. C. B.) and 310000-118468/1 (to T. J. R.), Herzkreislauf Stiftung (to T. J. R., P. E.), Swiss Cardiology Foundation (to T. J. R., P. E.), and the Stiftung für Medizinische Forschung, Basel, Switzerland (to B. C. B.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors thank Ed Palmer for reading the manuscript and helpful discussions.

Conflict of interest: The authors declare no financial or commercial conflict of interest.

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  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

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