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CD4(+) CD56(+) natural killer T-like cells secreting interferon- are associated withincident coronary events.

Björkbacka, Harry; Berg, Katarina; Manjer, Jonas; Engelbertsen, Daniel; Wigren, Maria;Ljungcrantz, Irena; Andersson, Linda; Hedblad, Bo; Nordin Fredrikson, Gunilla; Nilsson, JanPublished in:Journal of Internal Medicine

DOI:10.1111/joim.12392

2016

Link to publication

Citation for published version (APA):Björkbacka, H., Berg, K., Manjer, J., Engelbertsen, D., Wigren, M., Ljungcrantz, I., Andersson, L., Hedblad, B.,Nordin Fredrikson, G., & Nilsson, J. (2016). CD4(+) CD56(+) natural killer T-like cells secreting interferon-γ areassociated with incident coronary events. Journal of Internal Medicine, 279(1), 78-88.https://doi.org/10.1111/joim.12392

Total number of authors:10

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CD4+CD56+ NKT-like cells secreting interferon-γ are associated with

incident coronary events

Harry Björkbacka*, Katarina E. Berg*, Jonas Manjer, Daniel Engelbertsen,

Maria Wigren, Irena Ljungcrantz, Linda Andersson, Bo Hedblad, Gunilla

Nordin Fredrikson, and Jan Nilsson

*Contributed equally

Department of Clinical Sciences Malmö, Lund University, Sweden

Running title: NKT-like cells and incident MI

Correspondence to:

Jan Nilsson

CRC 91:12, Jan Waldenströms gata 35, Skåne University Hospital, S-205 02

Malmö, Sweden

Phone: +46 40 39 12 30, Fax: +46 40 39 12 12, Email: Jan.Nilsson@med.lu.se

2

Abstract

Background: CD3+CD56+ NKT-like cells are a subset of T cells characterized

by expression of NK receptors and potent antitumor activity. They have also

been implicated in autoimmune disease and shown to be elevated in patients

with coronary disease.

Objectives: To investigate if high levels of CD3+CD56+ NKT-like cells are

associated with an increased incidence of cardiovascular disease and a lower

incidence of cancer.

Methods: A prospective study including 700 subjects participating in the

baseline investigation of the Malmö Diet and Cancer Study between 1991 and

1994. Leukocytes obtained at the baseline investigation and stored at -140°C

were thawed and CD3+CD56+ cells analyzed by flow cytometry. The incidence

of cancer and coronary events during a mean follow-up of 15 years were

obtained through national registers.

Results: Subjects in the lowest tertile of IFN-γ expressing CD4+CD56+ cells

were found to have an increased risk for incidence of coronary events (log-rank

test: P <0.05). This association remained significant when controlling for age,

sex, smoking, BMI, hypertension, diabetes, the Th1/Th2 and the Th1/Treg cell

ratio in a Cox proportional hazards regression model (hazard ratio 1.98, 95%

C.I. 1.24-3.16), but not when LDL/HDL ratio was included in the model. There

were no associations between CD3+CD56+ NKT-like cells and incident cancer.

Conclusions: The present study could not confirm the hypothesis that low levels

of CD3+CD56+ NKT-like cells are associated with a higher incidence cancer and

a lower incidence of CVD. Contrarily, we found that low levels of IFN-γ

expressing CD3+CD4+CD56+ NKT-like cells was associated with an increased

incidence of coronary events and that this association may be dependent on

lipoproteins.

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Key words: NKT-like cells, cardiovascular disease, cancer

4

Introduction

CD3+CD56+ T cells belong to a family of T cells that express NK markers but

in contrast to the classical invariant NKT cells they are not CD1d-alphaGalCer

restricted and are therefore referred to as NKT-like cells [1]. NKT-like cells

have a more diverse T cell repertoire than invariant NKT cells and are found

mainly in the spleen and in the bone-marrow [2]. They are rare early in life but

increase with age and constitute about 20% of all CD8+ T cells and about 5% of

all CD4+ T cells by the age of 80 years [3]. CD3+CD56+ T cells expand in

response to virus infection [4] and release cytokines that stimulate the

recruitment of innate immune cells [5]. CD3+CD56+ T cells have been shown

to possess antitumor activity [6] and have been postulated to play a role in

tumor surveillance [7]. High levels of CD3+CD56+ T cells are associated with

improved survival in chronic lymphatic leukemia, lung and colon cancer [8, 9].

Moreover, clinical trials using ex vivo expanded CD3+CD56+ T cells (so called

cytokine-induced killer cells) have demonstrated encouraging results both in

hematological malignancies and solid tumors [10]. However, increased levels of

CD3+CD56+ T cells have been also reported in several autoimmune diseases

including rheumatoid arthritis, sarcoidosis and Behcet’s uveitis [11-13] and the

expression of CD56 on T cells has been shown to correlate with a loss of CD28,

a hallmark of emerging immune-senescence [14] Bergström et al recently [15]

reported increased levels of CD3+CD56+ T cells in patients with coronary

artery disease and demonstrated that these cells were characterized by down-

regulation of CD28 and an increased expression of interferon (IFN)-γ. Since the

atherosclerotic disease processes has been shown to be modulated by adaptive

immune responses [16] it is an interesting possibility that this finding could

reflect a diminished ability to control autoimmune responses against oxidized

LDL and other self-antigens in atherosclerotic plaques. Against this background

it could be hypothesized that high levels of CD3+CD56+ NKT-like cells would

5

be associated with a lower risk of cancer as well as with a higher incidence of

coronary events (CE). To test this hypothesis we used mononuclear cells from

the Malmö Diet and Cancer (MDC) study biobank to study the association of

CD3+CD56+ T cells with incident cancer and CE during 15 years of follow-up.

Material and methods

Study population

The study cohort, consisting of 700 participants aged 63–68 years from the

MDC cardiovascular cohort has been previously described [17]. Briefly,

participants were followed from baseline examination in 1991-1994 until first

CVD or cancer event, emigration from Sweden, death or 31 December 2008,

whichever came first. A CVD event was defined as a fatal or nonfatal

myocardial infarction [i.e. International Classification of Diseases, 9th Revision

(ICD-9) code 410)], fatal or non-fatal stroke (ICD-9 codes 430, 431, 434 and

436) or death attributable to underlying coronary heart disease (ICD-9 codes

410–414). A total of 150 incident CVD events (84 coronary events and 66

strokes) were identified during the follow-up period. Classification of cancer

was based on ICD-9 codes 140-239. A total of 170 incident cancer events were

identified during the follow-up period. Hypertension was defined as blood

pressure ≥140/90 mm Hg or use of blood pressure-lowering medication,

smoking as current smoking. Blood pressure, body mass index (BMI), smoking

status, hs-CRP, and cholesterol and lipid levels were determined as previously

described [18, 19]. One subject was excluded because of incomplete clinical

data. The study was approved by the Regional Ethical Review Board in Lund

and was conducted in accordance with the Declaration of Helsinki. All

participants gave written consent.

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B-mode ultrasound

Analysis of common and bulb carotid intima–media thickness (IMT) was

performed using an Acuson 128 CT system with a 7-MHz transducer as

described previously [18].

Isolation of mononuclear leukocytes

Blood (15 mL) was collected in heparin tubes and layered on top of 15 mL

Lymphoprep before centrifugation at 1350 g for 12 min at room temperature.

The interface layer was carefully harvested and the cells were then washed twice

with 0.9% NaCl (the first centrifugation was at 600 g and the second at 300 g,

both for 10 min at room temperature). The cells were re-suspended in 1.7 mL

autologous serum and 1.6 mL cold RPMI 1640 medium with 20% DMSO was

added. The cells were frozen slowly by placing them in a Styrofoam box at -

80°C overnight. Frozen mononuclear cells were stored at -140°C.

Flow cytometry

Cells were then thawed to room temperature within 2 min; subsequently 4 mL

phosphate-buffered saline (PBS) containing 1% human serum (HS) at 37°C was

added over 1–2 min and a further 4 mL was added over approximately 30 s.

Gradual dilution of the DMSO in the frozen cells avoided cell damage by

osmotic shock, and pre-warming the dilution medium helped to actively

compensate for changes in osmotic pressure. Cells were centrifuged and

suspended in RPMI 1640 supplemented with 10% HS (Invitrogen, Stockholm,

Sweden), 1% sodium pyruvate, 1% HEPES, 1% penicillin/streptomycin, 1% L-

glutamine and 0.1% β-mercaptoethanol (Gibco, Stockholm, Sweden) at a

concentration of 2x106 cells/mL. First, 4x105 cells/well were incubated with

PMA (10 ng), ionomycin (0.2 µg) and brefeldin A (1 µg, all from Sigma) for 4

hours at 37°C. Cells were washed and incubated with CD56-biotin, CD3-

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PE/Cy7, CD4-PB and CD8-AF700 for 30 min at 4°C. The samples were then

rinsed and incubated with strepavidin-PerCP for 20 min at 4°C. Cells were

washed and incubated with Fix/Perm solution and permeabilization buffer and

subsequently incubated with IFN-γ-PE (all antibodies were from Biolegend) for

30 min at 4°C. Samples were acquired on an ADP-Cyan flow cytometer

(Beckman Coulter) and analysis was performed using FlowJo 7.5.5 (Treestar

Inc.). Species-specific compensation beads (BD Biosciences, Stockholm,

Sweden) coupled to the respective antibodies was used to compensate

fluorescent signals and non-stained cells, and FMO (fluorescence-minus-one)

control samples were used to set the negative population. CD14++CD16-

monocytes, IFN-γ expressing Th1 cells, IL-4 expressing Th2 cells and

CD4+FoxP3+ regulatory T cells were analyzed as previously reported [17, 20,

21]. Cells used for analysis of CD14++CD16- monocytes were not incubated

with PMA/ionomycin and brefeldin A. Mononuclear leukocytes used in control

experiments to study the effect of freezing and thawing were obtained from

healthy volunteers. Plasma cytokine were determined by multiplexed

immunoassay (MesoScale Discovery).

Statistical analysis

Measures of skewness and kurtosis were used to test for normal distribution of

CD3+CD56+ T cell populations and clinical parameters. Differences between

means of normally distributed continuous variables were assessed with

independent sample t tests and differences in proportions between subjects with

and without CVD events were assessed using the chi-squared test. Differences

between means of non-normally distributed continuous variables were assessed

using the non-parametric Mann–Whitney test. Spearman’s rank correlation

coefficients were used to examine relationships among continuous variables.

The relation between CD3+CD56+ NKT-like cell subsets (in tertiles) and

incidence of first CVD events (coronary events or stroke) and cancer during

8

follow-up was assessed by Kaplan Meier survival curves and quantified by Log

rank test. A Cox proportional hazards regression model was used to assess the

hazard ratio (HR, and 95% confidence interval) of first CVD events in relation

to tertiles of CD3+CD56+ T cell subsets. The model included age, sex; diabetes,

blood pressure, lipid levels, smoking status and use of anti-diabetic or blood

pressure-lowering medication. The proportionality of the hazards assumption

was confirmed by visual inspection of log–negative log survival curves.

Results

Baseline clinical characteristics of the study cohort are shown in Table 1.

Coronary cases were more often male, had diabetes and received blood pressure-

lowering medication. They were also characterized by higher fasting blood

glucose, LDL/HDL ratio and systolic blood pressure (table 1). Stroke cases were

more often male, treated with anti-diabetic and blood pressure-lowering

medication, and had higher fasting blood glucose levels and diastolic blood

pressure. A total of 170 subjects developed cancer during follow-up. There were

no significant differences in baseline clinical characteristics between subjects

with or without incident cancer (data not shown). Eighteen subjects suffered

from both an incident coronary event and cancer.

CD3+CD56+ cell subsets were analyzed in thawed samples of mononuclear

leukocytes that had been frozen in autologous serum/DMSO at the MDC study

baseline investigation in 1991–1994 and stored at -140˚C. Flow cytometric

analysis using the viability marker 7AAD in thawed CD45+ leukocytes

demonstrated that 95% of the cells were alive.

9

Analysis of CD3+CD56+ cell subsets

The following populations were analyzed; CD3+CD56+, CD3+CD4+CD56+ and

CD3+CD8+CD56+ (see figure 1A for gating). Control experiments demonstrated

that freezing and thawing did not influence the size of the CD3+CD56+ T cell

population (figure 1B). However, it did result in a minor relative decrease of the

CD3+CD8+ population suggesting that these cells may be more sensitive to

freezing and thawing. The expression of IFN-γ in activated CD3+CD56+ T cell

populations were assessed as the fraction of cells demonstrating positive staining

as well as by the mean fluorescence intensity (MFI). The fraction of

CD3+CD56+ T cells of all lymphocytes was 5.4±3.8%. The fraction of CD4+

and CD8+ in the total population of CD3+CD56+ T cells were 25.6±12.4% and

40.2±16.7% respectively, while the fractions of IFN-γ expressing

CD3+CD4+CD56+ and CD3+CD8+CD56+ in the study cohort were 18.9±11.9%

and 34.9±18.5% , respectively (expressed as % of all CD3+CD56+ T cells).

Accordingly, 73.8% of the CD3+CD4+CD56+ and 86.8% of CD3+CD8+CD56+ T

cells expressed IFN-γ when activated. CD3+CD56+ T cells have been referred to

as NKT-like cells because they as classical invariant NKT cells express NK cell

receptors [1]. To determine the relation between CD3+CD56+ T cells and

classical NKT cells in the present study we used α-galactosylceramide-loaded

CD1d tetramers. Only a minor fraction of the CD3+CD56+ T cells (1.3±0.2%)

demonstrated positive tetramer staining and most of the α-galactosylceramide

tetramer-positive cells were CD56 negative (figure 1C). CD56+, but not CD56-,

cells expressed IFN-γ.

Associations of CD3+CD56+ T cells with other leukocyte populations and

plasma cytokines

10

Generally CD3+CD56+ T cell populations and their expression of IFN-γ were

strongly associated with the expression of IFN-γ (Th1 cells) and IL-4 (Th2 cells)

in conventional (CD56-) T cells and more weakly with the presence of classical

pro-inflammatory CD14++ CD16- monocytes (table 2). There were also

significant inverse correlations between the CD3+CD56+ T cell populations and

the level of circulating Tregs (table 2). Notably, most associations were stronger

for IFN-γ expressing CD3+CD56 T cells than for the corresponding total

CD3+CD56+ T cell populations. A high number of CD3+CD56+ T cells in

plasma were associated with increased levels of IL-8 but lower levels of IL-13

in plasma (table 3).

CD3+CD56+ T cells and cancer

There were no significant differences in baseline CD3+CD56+ T total cell

number or fraction of CD3+CD56+ NKT-like cells between those with incident

cancer and those that remained free of cancer during follow-up (table 4).

Plotting CD3+CD56+ NKT-like cell tertiles into Kaplan-Meier curves also

revealed no association with the incident cancer (figure 2 A and B and data not

shown). When subjects with incident cancer were studied separately we found

we found stronger associations  between plasma IL-2 levels and both total

CD4+CD56+ and IFN-γ expressing CD4+CD56+ NKT-like cells (-0.17, p<0.05

for both), but otherwise there were no major differences in these associations

between those with and without incident cancer (data not shown).

CD3+CD56+ T cells and cardiovascular disease

There was no significant difference in CD3+CD56+ cell numbers or percent of

the different NKT-like cell subsets between those with or without an incident

cardiovascular event (table 4). However, Kaplan–Meier curves of event-free

survival showed an increased incidence of coronary events in the group with the

11

lowest tertiles of CD4+CD56+IFN-γ+ T cells as compared to the two highest

tertiles (figure 2; log-rank test: P <0.05). This association remained significant

when controlling for age, sex, smoking, BMI, hypertension, diabetes, the

Th1/Th2 and the Th1/Treg cell ratio in a Cox proportional hazards regression

model (hazard ratio 1.98, 95% C.I. 1.24-3.16), but not when LDL/HDL ratio

was included in the model (hazard ratio: 1.61; 95% C.I. 0.92-2.82). There was

no significant association between other CD3+CD56+ T cell tertiles and

cardiovascular events. Moreover, there were no significant associations between

CD3+CD56+ T cell populations (in total numbers or as percent) and intima-

media thickness in the common carotid artery or the carotid bulb (data not

shown). When subjects with incident coronary events were studied separately

we found we found stronger associations  between IL-5 and both CD4+CD56+

and CD8+CD56+ NKT-like cells (-0.23, p<0.05 and r=0.22, p<0.05,

respectively), but otherwise there were no major differences between those with

and without coronary events (data not shown).

Association of CD3+CD56+ T cells with cardiovascular risk factors

Correlations between CD3+CD56+ T cells and cardiovascular risk factor were

generally weak but the general trend was that CD3+CD56+ T cells correlated

positively with BMI and triglycerides and inversely with age and HDL (table 5).

Discussion

CD3+CD56+ T cells belong to a family of T cells that express NK markers but

are distinct from classical invariant NKT cells by not being CD1d- restricted and

are therefore referred to as NKT-like cells [1]. The present study tested the

hypothesis that the level of circulating CD3+CD56+ NKT-like cells is associated

with the risk for development of CVD and cancer. Since CD3+CD56+ T cells

12

have potent anti-tumor properties [6] that low frequencies of CD3+CD56+ T

cells is associated with a more rapid progression of chronic lymphatic leukemia,

lung and colon cancer [8, 9] and that ex vivo generated CD3+CD56+ T cells are

used in clinical trials for treatment of cancer [10] it could be anticipated that in a

general population high levels of these cells would be associated with a lower

risk of cancer. However, we found no evidence for an association between

circulating levels of CD3+CD56+ NKT-like cells and the incidence of cancer in

the present study. The reason for the lack of association between cancer and

CD3+CD56+ NKT-like cells in present study remains to be fully elucidated.

However, it should be kept in mind that previous studies have investigated the

relation between these cells and the clinical outcome of prevalent cancer rather

the association with risk for cancer development. It is thus possible that

CD3+CD56+ NKT-like cells are important for targeting existing cancer cells than

in protecting against the development of new cancers.

With respect to the risk for CVD we found that subjects with low levels of IFN-γ

expressing CD3+CD4+CD56+ NKT-like cells had an increased incidence of

coronary events, whereas no association were found with the incidence of

stroke. The association between the fraction of IFN-γ expressing

CD3+CD4+CD56+ NKT-like cells and incidence of coronary events was

independent of age, sex, smoking, BMI, hypertension and diabetes. However,

the association lost statistical significance when also adjusting for HDL or the

LDL/HDL ratio. Interestingly, the fraction of IFN-γ expressing

CD3+CD4+CD56+ T cells correlated significantly with HDL but not with LDL.

Atherosclerosis is a chronic inflammatory arterial disease responsible for the

development of most cases of myocardial infarction and stroke [22]. The

arterial inflammation in atherosclerosis is primarily driven by accumulation and

oxidation of low density lipoprotein (LDL)-derived lipids. Evidence from

13

studies performed in animal models of atherosclerosis suggest that this

inflammation can be modulated by adaptive immunity and that both protective

and pathogenic immune responses are activated in response to arterial

accumulation of oxidized LDL [23-26]. Immune responses mediated by CD4+

Th1, CD8+ T cells and NKT cells have been found to aggravate the disease

process while regulatory T cells (Treg) are protective. The role of CD4+ Th2

and B cells is more complex and both protective and pathogenic subtypes have

been described. Against this background there has been an increasing interest in

exploring possible clinical associations between immune cells and risk for

development of cardiovascular disease, but this has so far only been investigated

in a limited number of prospective studies. It has been reported that low levels

of CD4+ Th2 and Tregs as well as high levels of CD8+ T cells are associated

with an increased risk for development of acute coronary events [20, 21, 27],

while low levels of CD4+ Th2 and CD40+ B cells are associated with increased

risk of stroke [28]. Autoimmune responses against epitopes in oxidized LDL are

believed to aggravate atherosclerosis [24]. Since CD3+CD56+ NKT-like cells are

increased in several autoimmune diseases [11-14] and as well as in patients with

coronary disease [15] there is a possibility that they could be involved in the

regulation of tolerance in atherosclerotic plaques. The correlation between

CD3+CD56+ T cells and HDL suggest some type of interaction with lipoprotein

lipids. Invariant NKT cells are a subset of T cells that share characteristics with

both conventional T cells and natural killer cells and that are responsive to lipid

antigens presented on the MHC class I-like molecule CD1 [29, 30].

CD3+CD56+ T cells have been referred to as NKT-like cells because both

express the NK cell marker CD56 [1, 31]. We used α-galactosylceramide

tetramers to identify CD1d-restricted NKT cells and found that most of these

were not CD56 positive. Indeed, only about one percent of the CD3+CD56+ T

cells were CD1d-restricted. However, it cannot be excluded that CD3+CD56+

NKT-like cells also respond to lipids presented on other CD1 molecules. The

14

association between low levels of IFN-γ expressing CD3+CD4+CD56+ T cells

and increased risk of coronary events could argue for a protective role of these

cells in cardiovascular disease. Most experimental studies have shown that

inhibition of presentation of lipid antigens through the CD1d–NKT cell

pathways results in a decreased development of atherosclerosis in

hypercholesterolemic mice [32-35]. Again, this would argue against

responsiveness to lipid antigens as the explanation to the association between

CD3+CD56+ NKT-like cells and coronary events.

In the present study we found significant associations between circulating

CD3+CD8+CD56+ NKT-like cells and plasma levels of the pro-inflammatory

cytokines, such as IFN-γ and TNF-α, as well as the chemokine IL-8. There was

also an inverse association between CD3+CD8+CD56+ T cells and the Th2-type

cytokine IL-13. IFN-γ expressing CD3+CD56+ NKT-like cells correlated

strongly with the presence of IFN-γ expressing Th1 cells and IL-4 expressing

Th2 cells suggesting that they occur as part of a more general immune response.

High levels of Th2 cells have previously been associated with a decreased risk

for coronary events while no such association was found for Th1 cells [21]. It

should also be noted that we observed a highly significant inverse correlation

between IFN-γ expressing CD3+CD56+ NKT-like cells and Tregs in the present

study. There is convincing evidence from experimental studies that Tregs have a

protective function in atherosclerosis [36, 37] and we have recently shown that

subjects with low levels of Tregs are at increased risk for coronary events [20].

Accordingly, it is unlikely that the association between high levels of IFN-γ

expressing CD3+CD56+ NKT-like cells and a lower incidence of coronary

events involves increased activation of Tregs. The observation that the

association between IFN-γ expressing CD3+CD56+ NKT-like cells and incident

coronary events remained significant when controlling for both the Th1/th2 as

15

well as the Th1/Treg ratio suggest that it is independent of interaction with other

T cell types.

There are some limitations of the present study that should be considered. The

studies were performed on cells that had been stored at -140 ˚C for several

years. As compared to initiating new prospective studies this has the obvious

advantage of allowing studies to be completed within a relatively short period of

time but it remains to be clarified how representative thawed cells are of the

original cell population. Although we did not observe any reduction of the

fraction of CD3+CD56+ NKT-like cells in response to freezing and thawing of

freshly isolated cells we did see a minor decrease in the fraction of CD3+CD8+ T

cells which may have influenced the results of our study. However, around 30%

of the CD3+CD56+ cells in the study cohort were CD4+ and CD8+ double

negative. It cannot be excluded that this reflects a loss of CD4+ and CD8+ with

extended freezing that we were unable to detect in our analyses of freshly

isolated cells. Moreover, though this study was relatively large in comparison

with many other clinical studies using characterization of leukocyte subtypes by

flow cytometry it still is small in terms of power to predict clinical outcomes.

Hence, it cannot be excluded that a larger study would have been able to detect

other association between CD3+CD56+ NKT-like cells and cancer.

In conclusion, the present study could not confirm the hypothesis that low levels

of CD3+CD56+ NKT-like cells are associated with a higher incidence cancer and

a lower incidence of CVD. Contrarily, we found that low levels of IFN-γ

expressing CD3+CD4+CD56+ NKT-like cells was associated with an increased

incidence of coronary events. The findings provide the first clinical support for a

role of CD3+CD4+CD56+ NKT-like cells in CVD.

16

Acknowledgements

We are grateful to Ragnar Alm for expert technical assistance. This work was

supported by grants from the Swedish Research Council; the Swedish Heart-

Lung foundation; Swedish Foundation for Strategic Research; the Albert

Påhlsson foundation and Skåne University Hospital foundation.

Disclosures

None

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17 Berg KE, Ljungcrantz I, Andersson L, et al. Elevated CD14++CD16- Monocytes Predict Cardiovascular Events. Circulation Cardiovascular genetics 2012; 5: 122-31. 18 Hedblad B, Nilsson P, Janzon L, Berglund G. Relation between insulin resistance and carotid intima-media thickness and stenosis in non-diabetic subjects. Results from a cross-sectional study in Malmo, Sweden. Diabet Med 2000; 17: 299-307. 19 Persson M, Hedblad B, Nelson JJ, Berglund G. Elevated Lp-PLA2 levels add prognostic information to the metabolic syndrome on incidence of cardiovascular events among middle-aged nondiabetic subjects. Arterioscler Thromb Vasc Biol 2007; 27: 1411-6. 20 Wigren M, Bjorkbacka H, Andersson L, et al. Low Levels of Circulating CD4+FoxP3+ T Cells Are Associated With an Increased Risk for Development of Myocardial Infarction But Not for Stroke. Arterioscler Thromb Vasc Biol 2012; 32: 2000-4. 21 Engelbertsen D, Andersson L, Ljungcrantz I, Wigren M, Hedblad B, Nilsson J, Bjorkbacka H. T-helper 2 immunity is associated with reduced risk of myocardial infarction and stroke. Arterioscler Thromb Vasc Biol 2013; 33: 637-44. 22 Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 2005; 352: 1685-95. 23 Buono C, Binder CJ, Stavrakis G, Witztum JL, Glimcher LH, Lichtman AH. T-bet deficiency reduces atherosclerosis and alters plaque antigen-specific immune responses. Proc Natl Acad Sci U S A 2005; 102: 1596-601. 24 Hansson GK, Hermansson A. The immune system in atherosclerosis. Nature immunology 2011; 12: 204-12. 25 Libby P, Lichtman AH, Hansson GK. Immune effector mechanisms implicated in atherosclerosis: from mice to humans. Immunity 2013; 38: 1092-104. 26 Libby P. Inflammation in atherosclerosis. Nature 2002; 420: 868-74. 27 Kolbus D, Ljungcrantz I, Andersson L, Hedblad B, Fredrikson GN, Bjorkbacka H, Nilsson J. Association between CD8+ T-cell subsets and cardiovascular disease. J Intern Med 2013; 274: 41-51. 28 Mantani PT, Ljungcrantz I, Andersson L, et al. Circulating CD40+ and CD86+ B cell subsets demonstrate opposing associations with risk of stroke. Arterioscler Thromb Vasc Biol 2014; 34: 211-8. 29 Gapin L, Godfrey DI, Rossjohn J. Natural Killer T cell obsession with self-antigens. Current opinion in immunology 2013; 25: 168-73. 30 Brutkiewicz RR. CD1d ligands: the good, the bad, and the ugly. J Immunol 2006; 177: 769-75. 31 Berzins SP, Smyth MJ, Baxter AG. Presumed guilty: natural killer T cell defects and human disease. Nat Rev Immunol 2011; 11: 131-42. 32 Tupin E, Nicoletti A, Elhage R, Rudling M, Ljunggren HG, Hansson GK, Berne GP. CD1d-dependent Activation of NKT Cells Aggravates Atherosclerosis. J Exp Med 2004; 199: 417-22. 33 Major AS, Wilson MT, McCaleb JL, et al. Quantitative and qualitative differences in proatherogenic NKT cells in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 2004; 24: 2351-7. 34 Nakai Y, Iwabuchi K, Fujii S, et al. Natural killer T cells accelerate atherogenesis in mice. Blood 2004; 104: 2051-9. 35 VanderLaan PA, Reardon CA, Sagiv Y, et al. Characterization of the natural killer T-cell response in an adoptive transfer model of atherosclerosis. Am J Pathol 2007; 170: 1100-7. 36 Ait-Oufella H, Salomon BL, Potteaux S, et al. Natural regulatory T cells control the development of atherosclerosis in mice. Nat Med 2006; 12: 178-80.

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37 Mallat Z, Ait-Oufella H, Tedgui A. Regulatory T-cell immunity in atherosclerosis. Trends Cardiovasc Med 2007; 17: 113-8.

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Figure legends

Figure 1. (A) Flow cytometric gating of the NKT populations CD3+CD56+,

CD3+CD4+CD56+ and CD3+CD8+CD56+ from mononuclear cells (MNLs). The

expression of IFN-g in the different CD3+CD56+ cell populations were assessed

as the fraction of cells demonstrating positive staining as well as by the mean

fluorescence intensity (MFI). (B) Fraction of CD3+CD56+, CD4+ and CD8+ T

cells in healthy controls before and after freezing and thawing. (C) Fraction of

CD3+CD56+ and α-galactosylceramide-CD1d tetramer-positive T cells (out of

all CD3+ T cells) in unstimulated and PMA/ionomycin stimulated cells from

healthy controls. (D) Gating for α-galactosylceramide-CD1d tetramer-positive

cells.

Figure 2. Kaplan-Meier curves of event-free survival of a cancer, coronary event

or death. Tertiles of (A) CD4+CD56+ NKT-like cells, (B) CD8+CD56+ NKT-like

cells and (C) lowest versus the two highest tertiles of CD4+CD56+IFN-γ+ NKT-

like cells (expressed as % of all CD3+CD56+ cells for all) are shown.