Tc17 CD8+ T cells accumulate in murine atherosclerotic lesions, but do not contribute to early atherosclerosis development.

AIMS: CD8+ T cells can differentiate into subpopulations that are characterized by a specific cytokine profile, such as the Tc17 population that produces interleukin-17. The role of this CD8+ T-cell subset in atherosclerosis remains elusive. In this study, we therefore investigated the contribution of Tc17 cells to the development of atherosclerosis. METHODS AND
RESULTS: Flow cytometry analysis of atherosclerotic lesions from apolipoprotein E-deficient mice revealed a pronounced increase in RORγt+CD8+ T cells compared to the spleen, indicating a lesion-specific increase in Tc17 cells. To study whether and how the Tc17 subset affects atherosclerosis, we performed an adoptive transfer of Tc17 cells or undifferentiated Tc0 cells into CD8-/- low-density lipoprotein receptor-deficient mice fed a Western-type diet. Using flow cytometry, we showed that Tc17 cells retained a high level of interleukin-17A production in vivo. Moreover, Tc17 cells produced lower levels of interferon-γ than their Tc0 counterparts. Analysis of the aortic root revealed that the transfer of Tc17 cells did not increase atherosclerotic lesion size, in contrast to Tc0-treated mice.
CONCLUSION: These findings demonstrate a lesion-localized increase in Tc17 cells in an atherosclerotic mouse model. Tc17 cells appeared to be non-atherogenic, in contrast to their Tc0 counterpart.

1. Introduction

Atherosclerosis, the most frequent underlying pathology of cardiovascular disease, is characterized by both the buildup of cholesterol as well as chronic inflammation within the wall of large- and medium-sized arteries. T cells are observed in both early and advanced atherosclerotic lesions and have been shown to contribute to lesion initiation and progression. Different subsets of CD4+ helper-T cells have been extensively described and studied in the context of atherosclerosis. A pro-atherogenic function is ascribed to the interferon-γ (IFN-γ)-producing T helper 1 (Th1) subset,, whereas the interleukin (IL)-10-producing regulatory T cells (Tregs) are atheroprotective., The role of the Th2 subset, characterized by the production of IL-4 and IL-5, is more controversial. Whereas the signature Th2 cytokines IL-4, IL-5, and IL-33 are reported to inhibit atherosclerosis development,,, reduced Th2 responses and IL-4 deficiency were also reported to decrease lesion formation, suggesting a pro-atherogenic role for Th2 cells as well. Finally, the IL-17-producing Th17 subset is known to drive autoimmunity and atherogenesis via activation of the endothelium, increasing pro-inflammatory cytokine production, and contributing to macrophage recruitment. In contrast, loss of suppressor of cytokine signalling (SOCS) 3 in T cells, resulting in increased IL-17 and IL-10 production, reduces atherosclerotic lesion development. This effect is mediated via the induction of an anti-inflammatory macrophage phenotype and a reduction in vascular inflammation. Interestingly, treatment with recombinant IL-17 resulted in reduced expression of vascular cell adhesion molecule-1 (VCAM-1) as well as reduced T-cell infiltration in the lesions, suggesting the aforementioned atheroprotective effects of SOCS3 knockout are at least in part mediated via IL-17.

In a similar vein to their CD4+ T-cell counterparts, CD8+ T cells can be categorized into subsets based on their cytokine production. Upon activation of CD8+ T cells, cytokines released by antigen-presenting cells (APCs) can influence the differentiation of the CD8+ T cells into different subsets. The cytokines IL-2 and IL-12 drive CD8+ T cells towards a Tc1 phenotype through the induction of the transcription factor T-box-containing protein expressed in T cells (T-bet)., Tc1 cells are known for their cytotoxic function and expression of effector molecules, such as granzymes, perforin, IFN-γ, and TNF-α., These cells confer protection against intracellular infections, as well as cancer. Alternatively, the release of IL-4 by the APCs polarizes CD8+ T cells towards a Tc2 phenotype. These cells express the transcription factor GATA3 and are characterized by the production of the cytokines IL-4, IL-5, and IL-13., This cell type is known to propagate allergic reactions and contribute to autoimmune disorders, such as arthritis. Finally, exposure to the cytokines IL-6, IL-21, and TGF-β drives CD8+ T cells to differentiate towards a Tc17 phenotype, by inducing the expression of the transcription factors RAR-related orphan nuclear receptor γt (RORγt) and IFN regulatory factor 4., Tc17 cells are characterized by their production of IL-17 and have been shown to play a pro-inflammatory role in several autoimmune disorders, such as multiple sclerosis, diabetes, and arthritis.,

The roles of CD8+ T-cell subsets in atherosclerosis remain largely unexplored, although some studies are suggesting that these cells may be involved. Tc1 cells have been implicated in atherogenesis, as IFN-γ-producing CD8+ T cells potentiated atherosclerosis development in apolipoprotein E-deficient (apoE−/−) mice. Additionally, IFN-γ produced by CD8+ T cells was shown to contribute to monopoiesis during early lesion development in low-density lipoprotein receptor-deficient (LDLr−/−) mice. Moreover, ApoE−/− mice deficient in E3-ligase CBL-B showed an increase in INF-γ and granzyme B-producing CD8+ T cells, resulting in enhanced macrophage killing and atherosclerosis. Finally, an increase in IL-17-producing CD8+ T cells in the circulation of humans has been associated with a higher incidence of myocardial infarction, hinting at a role for Tc17 cells in cardiovascular disease. However, direct evidence showing a causal relation between Tc17 cells and atherosclerosis is lacking.

Here, we systematically investigated the presence of different CD8+ T-cell subsets in a murine model of atherosclerosis and observed an increase in the number of Tc17 cells within the lesions. We show that undifferentiated CD8+ T cells switch to a Tc1 phenotype when transferred into LDLR−/− mice on a Western-type diet (WTD). CD8+ T cells that are polarized towards Tc17 cells, however, produced lower levels of IFN-γ upon adoptive transfer and showed to be non-atherogenic. Tc0 cells, on the other hand, produced high levels of IFN-γ and enhanced atherosclerotic lesion formation.

2. Methods

2.1 Mice

C57Bl/6, CD8a−/−, LDLr−/−, and apoE−/− mice were purchased from Jackson Laboratory (Bar Harbor, Maine, USA) and bred in-house. CD8a−/− mice were crossed with LDLr−/− mice to obtain CD8a−/−LDLr−/− mice in-house, after which genotypes were verified by PCR. The mice were kept under standard laboratory conditions and food and water were provided ad libitum. For the development of advanced atherosclerotic lesions in apoE−/− mice, mice were kept on a chow diet for 35–49 weeks before analysis of CD8+ T-cell phenotypes in the lesion. Upon sacrifice, mice were subcutaneously anaesthetized with a lethal dose of ketamine (40mg/mL), sedazine (8mg/mL), and atropine (0.1mg/mL). All animal work were performed in compliance with the Dutch government guidelines and the Directive 2010/63/EU of the European Parliament. Experiments were approved by the Ethics Committee for Animal Experiments of Leiden University.

2.2 Cell preparation and flow cytometry

Mice were sacrificed as described above and blood, spleens, and aortas were harvested after in situ perfusion with phosphate-buffered saline (PBS, pH 7.4, Lonza). White blood cells were obtained by lysing blood samples two times for 2 min with lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA; pH 7.3). Single-cell suspensions of spleens were obtained by using a 70 μm cell strainer (Greiner Bio-One). Splenocytes were lysed for 1 min with lysis buffer to obtain white blood cells. Aortas were cleaned of perivascular fat, cut into small pieces, and digested by incubation with a digestion mix (collagenase I 450 U/mL, collagenase XI 250 U/mL, DNAse 120 U/mL, and hyaluronidase 120 U/mL; all Sigma–Aldrich) for 30 min at 37°C while shaking, and subsequently strained over a 70 μm strainer. Cells were stained with the appropriate antibodies (Supplementary material online, ). For intracellular staining, cells were fixed and permeabilized by using an intracellular staining kit (eBioscience) according to the manufacturer’s protocol. To detect cytokine production, cells were stimulated for 3.5 h with phorbol 12-myristate 13-acetate (PMA, 50 ng/mL, Sigma–Aldrich) and ionomycin (500 ng/mL, Sigma–Aldrich) in the presence of brefeldin A (ThermoScientific) in complete RPMI 1640 medium containing 25 mM HEPES (Lonza) supplemented with 5% foetal bovine serum (Greiner), 60 μM β-mercaptoethanol (Sigma), 100 U/mL mix of penicillin/streptomycin (Lonza), 1% non-essential amino acids (NEAA; Gibco), 1% sodium pyruvate (Sigma), and 2% l-glutamine (Lonza) at 37°C and 5% CO2. Flow cytometry analyses were performed on a Beckman Coulter Cytoflex S and FlowJo software (Treestar).

2.3 In vitro culture of Tc0 and Tc17 cells

Spleens, mesenteric lymph nodes, and iliac lymph nodes were isolated from C57Bl/6 mice after cervical dislocation. CD8+ T cells were isolated by using a negative selection magnetic CD8+ T-cell isolation kit (Milteny Biotec) according to the manufacturer’s protocol. About 0.3 × 106 cells were plated per well in a 96-well plate in a total volume of 200 μL complete RPMI (as stated above). In order to obtain undifferentiated Tc0 cells, the medium was supplemented with 20 U/mL IL-2 (Peprotech), 0.5 ng/mL IL-7 (Peprotech), 0.5 µg/mL soluble anti-CD3 (ThermoScientific), 0.5 µg/mL soluble anti-CD28 (ThermoScientific), and 10 µg/mL anti-IFN-γ (BioXcell). For Tc17 differentiation, the medium was supplemented with 20 U/mL IL-2 (Peprotech), 0.5 ng/mL IL-7 (Peprotech), 20 ng/mL IL-6 (Peprotech), 5 ng/mL TGF-β (BioLegend), 20 ng/mL IL-1β (Peprotech), 20 ng/mL IL-23 (R&D systems), 0.5 µg/mL soluble anti-CD3 (ThermoScientific), 0.5 µg/mL soluble anti-CD28 (ThermoScientific), 10 μg/mL anti-IL-4 (BioXcell), and 10 µg/mL anti-IFN-γ (BioXcell). The cells were incubated for 2 days at 37°C and 5% CO2, after which the medium was refreshed with the same cytokine stimulations, but without anti-CD3 and anti-CD28. The cells were incubated for one more day before analysis by flow cytometry or adoptive transfer.

2.4 RNA isolation and qPCR

RNA was isolated from in vitro-cultured Tc0 and Tc17 cells by phenol/chloroform extraction. cDNA was synthesized using Maxima H minus reverse transcriptase reagents (ThermoFisher, Bleiswijk, the Netherlands) according to the manufacturer’s protocol. After creating cDNA, the SensiMix SYBR low-ROX kit (GC Biotech, Waddinxveen, the Netherlands) was used to perform the quantitative PCR (qPCR). The primers used for the qPCR are listed in Supplementary material online, . The PCR was run under the following conditions: initial denaturation 10 min at 95°C, followed by denaturation at 95°C for 15 s and annealing/extension at 64°C for 40 s for 40 cycles. The transcription levels were normalized to the expression of β-actin.

2.5 Adoptive transfer

Blood samples of 100 μL were drawn via the tail vein in EDTA-containing tubes (Sarstedt) from 18 CD8−/−LDLr−/− mice between 8 and 14 weeks of age. Total cholesterol levels were assessed by using an enzymatic colorimetric assay (Roche Diagnostics). The mice were randomized into two groups based on age, weight, and plasma cholesterol levels. From the start of the experiment, mice were fed a WTD containing 0.25% cholesterol and 15% cocoa butter (Special Diet Services, Witham, Essex, UK) for 6 weeks. Every week, mice received intravenous injections of matched numbers of between 8.8 × 105 and 2.3 × 106 Tc0 or Tc17 cells, depending on the amount obtained during isolation (on an average 1.7 × 106 per injection). During the experiment, transfer efficiency was monitored by drawing blood after two and four injections of CD8+ T cells, 5 days after the mice received the last injection. The mice were sacrificed using an overdose of anaesthesia (15 mg ketamine, 75 μg atropine, and 3.75 mg xylazine), 1 week after the sixth injection as described above, and organs were isolated as described under cell preparation and flow cytometry.

2.6 Histological analysis

All hearts were embedded in optimal cutting temperature compound (Sakura) and horizontally sectioned towards the aortic axis and the aortic arch. Upon reaching of the aortic root, defined by the trivalve leaflets, 10 μm sections were collected. Lesion size analysis was performed on cryosections of the aortic root lesion stained with Oil-red O and haematoxylin (Sigma–Aldrich). Sirius red staining (Sigma–Aldrich) was performed on corresponding sections to determine collagen content, and Masson’s Trichrome staining (Sigma–Aldrich) to determine the necrotic area. Plaque macrophages were stained immunohistochemically by using a rat anti-mouse Monocytes/Macrophages antibody (MOMA, 1:1000, AbD Serotec) as a primary antibody, biotinylated rabbit anti-rat IgG (1:100; Vector) as a secondary antibody, and Vectastain ABC horseradish peroxidase in combination with ImmPACT Nova Red for visualization (Vector). Plaques were stained for VCAM-1 by using purified rat anti-mouse CD106 (1:100, BD Biosciences) as a primary antibody, biotinylated rabbit anti-rat IgG (1:200, Vector) as a secondary antibody, and Vectastain ABC horseradish peroxidase in combination with Immpact Nova Red for visualization (Vector). The average plaque size (in μm2) was calculated from five sequential sections. For all other analyses, three subsequent sections displaying the highest plaque content per mouse were analysed. All microscopic analyses were performed on a Leica DM-RE microscope using Leica QWin software and were blinded for independent analysis. The relative amount of collagen, macrophages, and necrosis in the atherosclerotic lesions was quantified by dividing the area stained positive for collagen, MOMA or that displaying necrosis by the total lesion surface area, and calculated as a percentage.

2.7 Statistical analysis

The data are presented as individual dot plots with bars denoting the mean, and the number of animals in each group is stated in the text. Data were tested for normal distribution by using a Shapiro–Wilk normality test and analysed by using a two-tailed Student’s t-test, Mann–Whitney test, one-way or two-way ANOVA, as appropriate. Statistical analysis was performed using Prism (GraphPad). Probability values of P < 0.05 were considered significant.

3. Results

3.1 Increased expression of RORγt by CD8+ T cells derived from advanced murine atherosclerotic lesions

We investigated the presence of the different CD8+ T-cell subsets in the atherosclerotic lesions of apoE−/− mice with advanced atherosclerosis using flow cytometry. We focused on the difference in phenotype between CD8+ T cells derived from the aortic lesions and their counterparts in the spleen, as these cells can locally affect the lesion development and composition. We observed a significant decrease in the percentage of CD8+ T cells that produce IFN-γ within the lesions compared to their counterparts in the spleen (11.7% vs. 39.3%, Figure , representative FACS plots in Supplementary material online, ). This is in line with our previous research, showing a reduced number of cytokine-producing CD8+ T cells in the lesions of these mice, probably due to the immunosuppressive effects of increased CD39-expression on these cells. We were unable to detect any IL-4 secretion by aortic CD8+ T cells, whereas we did detect low levels of this cytokine in the splenic CD8+ T cells (Figure ). Conversely, we observed no production of IL-5 by splenic CD8+ T cells, whereas there was a low expression of this cytokine in their aortic counterparts (Figure ). Finally, we observed only very low expression levels of IL-17A in the CD8+ T cells derived from both sites, with no significant differences between the different sites (Figure ) and no production of IL-10 was detected (Figure ). Due to the low cytokines levels, we looked into the expression of the key transcription factors associated with the different Tc subsets in the lesions of these mice: T-bet, GATA3, RORγt, and FOXP3 for Tc1, Tc2, Tc17, and regulatory CD8+ T cells (TcReg), respectively. Interestingly, we observed a significant 45-fold increase in the percentage of CD8+ T cells that are positive for RORγt (Figure ), as well as a 19-fold increase in the percentage of GATA3-expressing CD8+ T cells (Figure ) and an almost two-fold increase in FOXP3 positive CD8+ T cells in the aorta compared to the spleen (Figure ), whereas the percentage of T-bet-expressing CD8+ T cells showed a three-fold decrease (Figure ). Of note, we only observed IFN-γ production by the T-bet positive CD8+ T cells in the aorta, but not by the RORγt or GATA3-expressing CD8+ T cells (Supplementary material online, ), confirming functionally distinct lineages. As there was such a pronounced increase in the RORγt-expressing lesional CD8+ T cells, we set out to further explore the role of Tc17 cells in atherosclerosis.

Murine atherosclerotic lesions display an increased expression of the Tc17-associated transcription factor RORγt as well as the Tc2-associated transcription factor GATA3 within the CD8+ T-cell compartment compared to the spleen. Flow cytometric analysis of IFN-γ+ (A), IL-4+ (B), IL-5+ (C), IL-17A+ (D), IL-10+(E), T-bet+ (F), GATA3+ (G), RORγt+ (H), and FOXP3 (I) CD8+ T cells in the aortas and spleens of apoE−/− mice stimulated for 3.5h with PMA and ionomycin. Cells were pre-gated on live, Thy1.2+CD8+ T cells. Representative FACS plots are available in Supplementary material online, . Individual data points and mean±SEM of n=5 (A, B, F), n=4 (C, D, G, H), or n = 4 vs. 3 (E, I) apoE−/− mice of 35–49 weeks old, data are representative of n = 3 independent experiments. Significance was determined by using an unpaired t-test (A, C) or a Mann–Whitney test (B, D, E, F, G, H, I). *P < 0.05, **P < 0.01, ***P < 0.001.

3.2 In vitro characterization of Tc0 and Tc17 cells

To evaluate the role of Tc17 cells in atherosclerotic lesion development, we decided to perform an adoptive transfer of Tc17 cells into LDLr−/− CD8−/− mice. First, we cultured undifferentiated CD8 T cells (Tc0) and Tc17 cells in vitro, based on previously published protocols.,, CD8+ T cells were isolated from wild-type mice and activated by using anti-CD3 and anti-CD28 antibodies. Tc0 were cultured for 3 days in medium supplemented with IL-2, IL-7, and anti-IFN-γ. Tc17 cells were differentiated for 3 days in medium supplemented with IL-2, IL-7, IL-6, IL-1β, TGF-β, IL-23, anti-IL-4, and anti-IFN-γ. Flow cytometry analysis revealed that our approach led to a robust Tc17 phenotype, with a 19-fold increase in the percentage of cells positive for IL-17A in the Tc17 cells compared to the Tc0 cells (24.8% vs. 1.3%, Figure , representative FACS plots shown in Supplementary material online, ), associated with a five-fold increase in RORγt-expressing cells (13.2% vs. 2.7%, Figure ), and a reduction in T-bet-expressing cells (7.3% vs. 25.1%, Figure ). Moreover, both Tc0 and Tc17 produced low amounts of IFN-γ (4.5% and 4.9%, respectively, Figure ), indicating that these cells do not display a Tc1 phenotype. We observed a low production of IL-5 by both subsets, although the Tc0 subset produced three-fold more IL-5 compared to the Tc17 subset (2.4% vs. 0.8%, Figure ). The percentage of GATA3+ cells was low in both groups and did not differ between the two subsets (Figure ), indicating the cultured cells do not display a Tc2 phenotype. Finally, we confirmed the Tc17 phenotype on transcriptional level and show increased expression of RORyt and CCR6, another established Th17 marker (Supplementary material online, ).

Tc0 and Tc17 cells demonstrate phenotypical differences in cytokine production and transcription factor expression. Flow cytometric analysis of IL-17A+ (A), RORγt+ (B), IFN-γ+ (C), T-bet+ (D), IL-5+ (E), and GATA3+ (F). CD8+ T cells isolated from C57Bl/6 mice and polarized for 3 days towards Tc0 or Tc17 cells. Cells were stimulated for 3.5h with PMA and ionomycin and pre-gated on live, Thy1.2+CD8+ T cells. Representative FACS plots are available in Supplementary material online, . Individual data points and mean±SEM of n=3, Representative of three independent experiments. Significance was determined by using an unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

3.3 Adoptively transferred CD8+ T cells migrate to the atherosclerotic lesion and affect the local CD4+ T-cell population

To determine the effect of Tc17 cells on the development of atherosclerosis, in vitro cultured Tc0 or Tc17 cells were adoptively transferred into LDLr−/− mice that were also deficient in CD8 (CD8−/−LDLr−/− mice) and therefore had no endogenous CD8+ T-cell population (Supplementary material online, , overview of experimental setup: Supplementary material online, ). Besides the absence of CD8+ T cells in these mice, the composition of blood leucocytes in CD8−/−LDLr−/− mice was comparable to that of LDLr−/− mice (Supplementary material online, ), with only minor changes in monocyte activation (Supplementary material online, ). Deletion of CD8a may also result in the ablation of CD8+ dendritic cells (DCs). Although, CD8+ DCs have a dominant role in antigen cross-presentation, DCs from CD8−/−LDLr−/− were able to induce CD8+ T-cell expansion to a similar extend as DCs from LDLr−/− mice, after exposure to ovalbumin in vivo and in vitro (Supplementary material online, and D). This suggests cross-presentation in CD8−/−LDLr−/− mice is not impaired. In line with reports in CD8-deficient apoE−/− mice,, atherosclerotic lesion development was not affected by ablation of CD8+ T cells, as atherosclerotic lesions were comparable in size and composition after feeding LDLr−/− mice and CD8−/−LDLr−/− mice (age 8–15 weeks) a WTD for 6 weeks (Supplementary material online, ).

The adoptive transfer with in vitro expanded Tc0 (control group) or Tc17 (treatment group) cells resulted in an increase in the CD8+ T-cell population in the blood of both treatment groups over time (Figure ). At 4 weeks post the first adoptive transfer, the circulating CD8+ T cells in the Tc17-treated group showed a substantially (four-fold) increased production of IL-17A compared to those in the Tc0-treated group (6.4% vs. 1.7%, Figure ), suggesting a stable Tc17 phenotype. Interestingly, we observed plasticity in the Tc0 subset, as these cells showed an increase in IFN-γ production from 4.5% at baseline to 53.6% 4 weeks after the first transfer. The Tc17 cells also showed an increased IFN-γ production from 4.9% to 44.9% (Figures and). Therefore, we analysed the relative amount of splenic IFN-γ+CD8+ T cells at sacrifice. Again, we observed that in the Tc0-treated group a larger fraction of the cells produce IFN-γ (75.9%) compared to those in the Tc17-treated group (30.5%, Figure ), although in both groups, the percentage of IFN-γ+ cells was notably higher than directly after in vitro differentiation (Figure ). In line with our expectations, the Tc17-treated group still displayed more IL-17A+ cells as compared to the Tc0 group (7.6% vs. 2.2%, Figure ), albeit less compared to the in vitro cytokine production levels at the moment of injection (Figure ). Moreover, at sacrifice, 86.1% of CD8+ T cells in the Tc0 group expressed T-bet, whereas in the Tc17 group this comprised 46.1% of the total CD8+ population (Figure ), suggesting the majority of the injected Tc0 cells had converted to a Tc1 phenotype. We observed a non-significant 1.2-fold increase in RORγt expression in the Tc17-treated group compared to the controls (Figure ). Of note, the adoptively transferred CD8+ T cells were able to proliferate in vivo, as we observed 30.2% and 20.0% Ki-67 expression in the Tc0 and Tc17 groups, respectively (Figure ). No differences were observed in the percentage of FoxP3+CD8+ T cells between the different groups (Supplementary material online, ).

CD8+ T-cell populations increase over time upon adoptive transfer in both treatment groups. (A) Analysis of percentages of CD8+ T cells in the blood of the CD8−/−LDLr−/− mice after two and four injections of Tc0 or Tc17 cells and at sacrifice. Cells were pre-gated on live, Thy1.2+ cells, mean±SEM. (B) IL-17A and (C) IFN-γ production by the CD8+ T cells in the blood after four injections, as analysed by flow cytometry. Cells were stimulated for 3.5h with PMA and ionomycin and pre-gated on live, Thy1.2+CD8+ T cells. Individual data points and mean±SEM, n = 9 mice per group, mice that did not show detectable numbers of CD8+ T cells were not included in the analysis of panels (B) and (C). Significance was determined by using a two-way ANOVA with Bonferroni’s multiple comparisons (A) or by using an unpaired t-test (B and C). **P < 0.01.

Adoptive transfer of Tc17 cells in CD8−/−LDLr−/− mice skews the CD4+ T cells towards a less inflammatory phenotype in the aortic microenvironment. Flow cytometric analysis of IFN-γ+ (A), IL-17A+ (B) T-bet+ (D) RORγt+ (E), and Ki-67+ (G) CD8+ T cells in the spleens of the CD8−/−LDLr−/− mice that received the adoptive transfer of Tc0 or Tc17 cells at the time of sacrifice. Representative FACS plots are shown in (C, F, I), cytokine-positive populations were gated based on unstimulated controls. Cells were pre-gated on live, Thy1.2+CD8+ T cells. (H) percentages of CD8+ T cells in the aortas of the CD8−/−LDLr−/− mice at the time of sacrifice, analysed by flow cytometry. Cells were pre-gated on live, Thy1.2+ cells. Flow cytometry analysis of IFN-γ+ (J) and T-bet+ (K) CD4+ T cells in the aortas of the CD8−/−LDLr−/− mice at the time of sacrifice. Representative FACS plots are shown in (L), cytokine-positive populations were gated based on unstimulated controls. Cells were pre-gated on live, Thy1.2+CD4+ T cells. Cells were stimulated for 3.5h with PMA and ionomycin. Individual data points and mean±SEM, n = 9 mice per group. Significance was determined by using a Mann–Whitney test (A, B, D, G) or by using an unpaired t-test (C, E, F, H, I). *P < 0.05, **P < 0.01, ***P < 0.001.

Besides the spleen and blood, we were able to detect CD8+ T cells in the aortic lesions of both Tc0- and Tc17-recipient mice at the time of sacrifice (Figure ), illustrating that the adoptively transferred CD8+ T cells migrate into the plaques. However, as the total number of lymphocytes within murine aortas is low, these numbers did not allow us to distinguish the different CD8+ T-cell subsets. Interestingly, we did observe changes in the CD4+ T-cell compartment in the lesion, showing a significant increase in IFN-γ production (Figure ,  P < 0.05) and T-bet expression (Figure ,  P < 0.05) in the Tc0-treated group, suggesting a skewing towards an inflammatory Th1 phenotype. This Th1 skewing effect was minimal and not statistically different after Tc17 transfer (Figure ).

3.4 Adoptive transfer of Tc17 cells does not accelerate atherosclerotic lesion development in CD8-deficient atherosclerotic mice

We next assessed how the adoptive transfer of CD8+ T cells affects atherosclerosis development. The weight of the mice was unaffected, whereas cholesterol levels in serum were significantly increased by CD8+ T-cell transfer of both Tc0 as well as Tc17 cells (Supplementary material online, and E). Plaque size was assessed in the aortic root lesions of the hearts. Interestingly, neutral lipid staining of the lesions revealed that the average lesion size of Tc17-transferred CD8 T cells was equal to CD8−/−LDLr−/− mice that did not receive a transfer (Figure ). However, Tc0 mice that received the adoptively transferred Tc0 cells showed a 57.6% increase in lesion size, compared to the Tc17-treated group (Figure ). The increase in lesion size in the Tc0-treated group is partially due to an elevation in total macrophage accumulation, as MOMA positive are increased 2.8-fold, although this did not reach statistical significance. The relative plaque composition appeared to be similar in the Tc17- and Tc0-treated groups, as no change in the percentage of MOMA positive, necrotic area, or collagen positive area was observed (Figure ). Analysis of the VCAM-1+ area in the caps of the lesion revealed a 1.5-fold increase in the Tc0-treated group, although this did not reach significance (P = 0.21, Supplementary material online, and B).

Adoptive transfer of Tc17 cells does not affect atherosclerotic lesion size, nor plaque composition, while a transfer of Tc0 cells accelerates lesion formation. (A) Quantification of lesion size in the aortic roots by Oil-red O staining and representative pictures of the trivalve area of CD8−/−LDLr−/−mice treated with Tc0 or Tc17 cells and the no-treatment group. (B) Quantification of relative monocyte/macrophage content in the aortic root lesions by MOMA staining and representative pictures of the lesions. (C) Quantification of relative necrotic core area and (D) relative collagen content in the aortic root lesions by Masson’s Trichrome staining and representative pictures of the lesions. Individual data points and mean±SEM, n=8 or 9 mice per group. Significance was determined by using an unpaired t-test. Outliers were removed after a Grubbs’ test. *P < 0.05, **P < 0.01, ***P < 0.001.

4. Discussion

CD8+ T cells play an important role in the adaptive immune response, responding to intracellular pathogens. Recently, CD8+ T-cell subsets such as Tc1, Tc2, and Tc17, which are characterized by their cytokine production resulting from different environmental cues, have been reported to also play a role in various autoimmune disorders. We report a large increase in Tc17 cells in the atherosclerotic lesion microenvironment specifically and show that adoptively transferred Tc17 cells do not contribute to the progression of atherosclerosis, while atherosclerotic lesion development was enhanced in Tc0-treated mice.

It is of particular interest to investigate the phenotype and function of CD8+ T cells within the lesion, as we have previously reported that CD8+ T cells can locally affect the lesion development and composition. However, it is difficult to determine the presence of the different CD8+ T-cell subsets within the lesional microenvironment based on their cytokine production, as the production of inflammatory cytokines produced by CD8+ T cells within the lesions is reduced. Indeed, here we report a reduced percentage of IFN-γ+ lesion-derived CD8+ T cells compared to their counterparts in the spleen and were hardly able to detect any IL-4, IL-5, or IL-17A production above background levels in the aorta of old apoE−/− mice. Therefore, we set out to measure the transcription factors associated with the Tc1, Tc2, and Tc17 subsets instead. There is a reduced percentage of CD8+ T cells expressing T-bet in aortic lesions compared to splenic CD8+ T cells, suggesting that the pro-inflammatory Tc1 subset is not enriched in the lesion environment. However, we observed a modest increase in the percentage of cells expressing GATA3 in the lesions, implying an increase in Tc2 cells compared to lymphoid tissues. Strikingly, the percentage of cells expressing RORγt was strongly increased within the lesion microenvironment, which indicates a relative enrichment of Tc17 cells at the site of disease. It has previously been reported that 3 months of high-fat diet feeding in apoE−/− mice results in increased IL-17A production by splenic T cells, although the cell type responsible for this increase was not specifically identified. We observed only very low percentages of IL-17+CD8+ T cells in the spleens of apoE−/− mice, which were kept on a chow diet. Nonetheless, this study suggests that the inflammatory stimuli associated with the development of atherosclerosis may drive Tc17 skewing. Moreover, another analysis of the entire T-cell compartment showed an increase in IL-17A production in the aorta compared to the spleens of apoE−/− mice fed a WTD for 15 weeks. As there are enhanced levels of the Tc17-polarizing cytokines IL-1β and IL-6 in the plaque,, the atherosclerotic environment in the lesion may indeed drive the local T cells to differentiate towards a Tc17 phenotype, or stimulate increased recruitment of these cells.

In vitro polarization of isolated CD8+ T cells from wild-type mice resulted in successful differentiation towards a Tc17 phenotype, as described previously using a similar differentiation protocol. Some basal levels of IFN-γ were produced by both the Tc0 and Tc17 cells, which is in agreement with available data., There was an increased expression of T-bet in the Tc0 subset compared to the Tc17 subset, which we hypothesize is due to the natural tendency of CD8+ T cells to differentiate towards an inflammatory effector phenotype upon the addition of IL-2, anti-CD3, and anti-CD28 antibodies. Indeed, we found that the addition of anti-IFN-γ to the Tc0 conditions induced a great reduction in the T-bet expression, strengthening this hypothesis. However, upon adoptive transfer, Tc0 cells up-regulated their expression of T-bet and IFN-γ production, indicating a switch towards the Tc1 phenotype. The Tc17 cells retained IL-17A production in vivo, though at lower levels than after in vitro differentiation. Finally, the RORγt expression was down-regulated in these cells. Indeed, previous work using antigen-specific Tc17 cells has shown that these cells can convert to IFN-γ-producing cells, although they retain some of their IL-17A production. Similar plasticity has been reported for CD4+ Th17 subsets., To date, the molecular mechanisms underlying these switches in phenotype remain unknown. However, the pro-inflammatory environment in the atherosclerotic mouse model may contribute to the increased production of IFN-γ, as hypercholesterolaemia results in increased inflammatory responses., As CD8+ T cells activated by using anti-CD3 and CD28 antibodies tend to differentiate towards an effector phenotype,, it is likely that absence of anti-IFN-γ, that was present in vitro, as well as the inflammatory signalling induced by the WTD-feeding in our mouse model, drives the switch towards a Tc1 phenotype. Indeed, the Tc0 cells showed a similar phenotype in vivo to that which we observed for the splenic CD8+ T cells in the atherosclerotic apoE−/− mice. Tc17 cells continued to produce IL-17A, but also gained the ability to produce IFN-γ. This may be explained by different transcriptional programs that are at work within this subset. IL-12, a cytokine known to be up-regulated in atherosclerotic mice, is able to induce repressive epigenetic modification of the SOCS3 promoter. As SOCS3 is an essential mediator of IL-17 production, increased IL-12 levels in the blood can stimulate the conversion of Tc17 cells towards a mixed Tc1/Tc17 phenotype, associated with an increased IFN-γ production. This is in agreement with our work, in which we observed maintenance of the Tc17 cytokine profile, but additional acquired characteristics of Tc1 cells. Previous work has demonstrated reciprocal plasticity between CD4+ Th17 and Treg cells, since Th17 polarized cells can differentiate into Tregs in vivo and vice versa. From this, it could be suggested that Tc17 cells can differentiate into regulatory CD8+ T cells via similar transcriptional mechanisms in our model. However, we observed no differences in FoxP3-expressing CD8+ T cells in our studies.

Of note, the Tc0 cells were more proliferative than their Tc17 counterparts. Possibly, the Tc0 cells resemble a more naïve phenotype as they are less fixed in their transcriptional program towards a certain phenotype. This enables them to proliferate more vigorously upon antigen recognition in vivo, compared to their more differentiated counterparts.

The injected CD8+ T cells were able to infiltrate the lesions in vivo, supporting the notion that local CD8+ T-cell interactions in the lesions may have contributed to the observed differences between the Tc17- and Tc0-treated groups. However, we were unable to establish differences between the different treatment groups in expression of transcription factors or the production of cytokines by these aortic CD8+ T cells, as the cell numbers were too low to draw any significant conclusions about subsets within the population. Interestingly, no change in plaque size or composition could be identified between the Tc17-treated group and non-treated CD8−/−LDLr−/− mice, whereas transfer of Tc0 cells significantly increased the size of atherosclerotic lesions. This strongly suggests Tc17 are not atherogenic and their enrichment in the plaques of CD8 competent mice does not drive plaque progression and instability. Previous reports have shown an atheroprotective effect of IL-17, as increased expression of IL-17 reduced lesion development and neutralization of IL-17 accelerated atherosclerosis. The proposed atheroprotective effect of IL-17 might contribute to the reduced pathogenicity of Tc17 cells. Alternatively, the reduced levels of IFN-γ produced by Tc17 may also account for this. IFN-γ is able to augment macrophage activation, which may, in turn, contribute to increased atherosclerosis development. IFN-γ−/−apoE−/− mice have been shown to display a large reduction in atherosclerosis compared to controls, associated with a decrease in lesion cellularity but an increase in lesional collagen content. In another study, administration of IFN-γ to apoE−/− mice resulted in a two-fold increase in lesion size, mediated by an increase in both T cells as well as APCs. CD8+ T-cell-derived IFN-γ has previously been shown to have limited impact on lesion size and stability, but this study was performed in lymphocyte-deficient apoE−/− mice, which may overlook the effect of CD8+ T-cell-derived IFN-γ on CD4+ T cells. We observed an increase in the total macrophage content of the lesions in the Tc0 group compared to the Tc17 and no transfer group, which could be mediated by the increased IFN-γ that is produced in these mice. In fact, we observed an increase in T-bet expressing Th1 CD4+ T cells in the lesion microenvironment of the Tc0-treated mice. IFN-γ is an important regulator of T-bet expression within CD4+ T cells, suggesting that the increase in Th1 cell population within the lesions of the Tc0-treated mice could be due to the increase in IFN-γ levels. In addition to the inflammatory effects of the adoptively transferred cells, this increase in the Th1 cell population within the lesions further promotes inflammation and atherogenesis, as these cells also display atherogenic functions. Thus, both the percentual increase in IL-17A as well as the percentual decrease in IFN-γ production by the Tc17 cells compared to the Tc0 cells may have contributed to the reduce pathogenic effects observed here.

5. Conclusion

In conclusion, we have shown an enrichment in Tc17 cells in the plaque microenvironment of atherosclerotic mice. The enrichment of Tc17 cells does not affect the progression of atherosclerosis, in contrast to their Tc0 counterpart. These findings demonstrate the presence of different Tc subsets within atherosclerotic lesions and warrant further research to the function of these subsets in the plaque microenvironment.

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Authors’ contributions

J.v.D., M.J.M.d.J., W.J., J.K., and B.S. designed the experiments. J.v.D., M.J.M.d.J., N.B., R.J.T.L., M.E.v.O., N.K., A.C.F., and I.B. performed the experiments and analysed data. J.v.D., M.J.M.d.J., and B.S. wrote the article with contributions from A.C.F., W.J., I.B., and J.K.

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