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Biochemical and Biophysical Research Communications 443 (2014) 864–870
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CD8+CD25+ T cells reduce atherosclerosis in apoE( / ) mice Jianchang Zhou 1, Paul C. Dimayuga 1, Xiaoning Zhao, Juliana Yano, Wai Man Lio, Portia Trinidad, Tomoyuki Honjo, Bojan Cercek, Prediman K. Shah, Kuang-Yuh Chyu ⇑ Oppenheimer Atherosclerosis Research Center, Division of Cardiology, Cedars-Sinai Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, United States
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Article history: Received 9 December 2013 Available online 14 December 2013 Keywords: Atherosclerosis CD8+CD25+ T cells Immune modulation Adoptive transfer
a b s t r a c t Background: It is increasingly evident that CD8+ T cells are involved in atherosclerosis but the specific subtypes have yet to be defined. CD8+CD25+ T cells exert suppressive effects on immune signaling and modulate experimental autoimmune disorders but their role in atherosclerosis remains to be determined. The phenotype and functional role of CD8+CD25+ T cells in experimental atherosclerosis were investigated in this study. Methods and results: CD8+CD25+ T cells were observed in atherosclerotic plaques of apoE( / ) mice fed hypercholesterolemic diet. Characterization by flow cytometric analysis and functional evaluation using a CFSE-based proliferation assays revealed a suppressive phenotype and function of splenic CD8+CD25+ T cells from apoE( / ) mice. Depletion of CD8+CD25+ from total CD8+ T cells rendered higher cytolytic activity of the remaining CD8+CD25 T cells. Adoptive transfer of CD8+CD25+ T cells into apoE( / ) mice suppressed the proliferation of splenic CD4+ T cells and significantly reduced atherosclerosis in recipient mice. Conclusions: Our study has identified an athero-protective role for CD8+CD25+ T cells in experimental atherosclerosis. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction The presence of CD8+ T cells in atherosclerotic lesions is widely demonstrated but studies investigating their role in atherogenesis have yielded contradictory results. It was reported that mice genetically deficient in CD8+ T cells did not have altered atherosclerosis development [1,2]. More recently, apoE( / ) mice severely depleted of CD8+ T cells using depleting antibodies showed significantly reduced atherosclerosis suggesting a pro-atherogenic role [3]. On the other hand, Fyfe et al. demonstrated that an atherogenic diet induced a three-fold increase in atherosclerotic lesion size in MHC I knockout mice which are CD8+ T cell deficient [4]. These discrepant findings may be attributed to different experimental designs. However, it is also possible that CD8+ T cells have subtype specificity in their functional role as suggested by our recent report wherein reduced neointima formation after vascular injury was rendered by CD8+CD28hi T cells but not by CD8+CD28+ T cells [5], and by the reported association between CD8+ T cell subsets and cardiovascular disease [6]. It is notable that CD8+ T cells have historically been referred to as T suppressor cells [7].
Evidence suggests that specific CD8+ T cell phenotypes may be involved in modulating atherosclerosis. It was reported that hypercholesterolemia activated CD8+CD28+ T cells that preceded CD4+ T cell activation in mice [8], and that residing CD8+ T cells in human atherosclerotic plaques have an activated phenotype with increased CD25 expression [9]. However, it remains unclear whether the observed phenotypes contribute to disease progression or if it operates to down-modulate pro-atherogenic immune signaling. For example, CD8+CD25+ T cells functioning as suppressors are involved in the modulation of autoimmune disorders with similar function to CD4+ Tregs [7,10–15]. The role of this phenotype in atherogenesis is highlighted by our previous report showing increased CD8+CD25+ T cells early after immunization of apoE( / ) mice with an apoB-100 derived peptide vaccine [16]. In this study, we characterized the phenotype and function of CD8+CD25+ T cells from apoE( / ) mice and defined their role in experimental atherosclerosis using adoptive cell transfer strategy. 2. Materials and methods 2.1. Animals
⇑ Corresponding author. Address: Division of Cardiology, Room 5539, CedarsSinai Heart Institute, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, United States. E-mail address: [email protected] (K.-Y. Chyu). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2013.12.057
Male apoE( / ) mice on a C57BL/6 background were purchased from Jackson Laboratories (Bar Harbor, Me), housed in a pathogenfree animal and kept on a 12-h day/night cycle with unrestricted access to water and regular mouse chow (5015, PMI Nutrition
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International) unless mentioned otherwise. The Institutional Animal Care and Use Committee of Cedars-Sinai Medical Center approved the experimental protocols. A group of 7 week-old apoE( / ) mice were fed regular mouse chow or an atherogenic diet (TD 88137, Harlan-Teklad) for 6 weeks. Mice were euthanized and the peripheral blood mononuclear cells were collected, stained and subjected to flow cytometric analysis. CD8+ T cells in atherosclerotic plaques were visualized by immunohistochemical staining of cryo-sections of aortic sinus of atherogenic diet-fed apoE( / ) mice euthanized at 25 weeks of age using standard immuno-staining protocol with a monoclonal anti-mouse CD8b antibody (BD Biosciences) as primary antibody. Negative control involved immunostaining with omission of primary antibody. Detection of CD8+CD25+ T cells in atherosclerotic plaques was assessed by enzyme digestion of plaques of apoE( / ) mice fed the atherogenic diet and euthanized at 29 weeks of age. Plaques were enzymatically digested (0.25 mg/ ml collagenase, 0.125 mg/ml elastase, 60 U/ml hyaluronidase in DMEM/F12 medium at 37 °C for 45 min) and subjected to flow cytometry. Flow cytometric analysis was performed by staining cells with FITC-conjugated CD8b and PE-conjugated CD25 antibodies (eBioscience) and analyzed on an LSR II apparatus (BD Biosciences). 2.2. Phenotypic characterization of CD8+CD25+ T cells Splenocytes pooled from two to three 13 week old male apoE( / ) mice fed regular mouse chow were characterized by flow cytometric analysis using standard protocols with antibodies from eBioscience unless otherwise indicated: anti-CD8b, anti-CD25, anti-CD28, anti-FoxP3, anti-IL-10, anti-TGF-b (R&D Systems), anti-IL-12, anti-IFN-c, anti-CCR7, anti-GITR (Biolegend), and anti-CTLA-4 (Biolegend). Cell permeabilized for staining intracellular molecules was performed using standard procedure. Gating and cut off were based on isotype controls. Data files were analyzed using Summit V4.3 software (DAKO). 2.3. Functional characterization of CD8+CD25+ T cells Pooled splenocytes from 13-week old male apoE( / ) mice fed regular mouse chow were purified by sorting with ARIA II Cell Sorter (BD Biosciences) for the following cell populations: CD8+CD25+, CD8+CD25 and CD4+CD25 T cells. Purified CD4+CD25 T cells (responder cells) were labeled with CFSE (Invitrogen, 2.5 lM at 37 °C for 10 min) and then cultured (1 105 cells in 100 ll culture medium) in the presence of CD3/CD28 Dynabeads (Invitrogen) at the cell-to-bead ratio of 1:2 with CD8+CD25+ or CD8+CD25 T cells (treatment groups) at a ratio of 1:1. The control was CD4+CD25 T cells without CD8+ T cells. Four days after co-culture, cells were stained for CD4 and analyzed by LSR II analyzer. The results are expressed as the CFSE mean fluorescent intensity (MFI). Cytolytic activity was assayed using bone marrow-derived dendritic cells (BMDCs) as target cells [17]. In brief, bone marrow cells from femurs and tibiae of male apoE( / ) mice were cultured in complete RPMI-1640 containing 10 ng/ml GM-CSF (R&D Systems) and 10 ng/ml IL-4 (Invitrogen). Immature DCs were harvested on day 8 and subcultured into new culture plates with 2 105 DCs in 1.5 ml medium. The negatively isolated CD8+ T cells or CD8+ T cells depleted of CD8+CD25+ cells were then co-culture with DCs at a CD8:DC ratio of 3:1 for 4 h, cells were then collected and processed for flow cytometric determination of CD11c and 7-AAD (eBioscience) by LSR II analyzer. CD11c positive cells stained with 7-AAD were identified as lysed target cells using target cell death without CD8+ T cells as baseline [17].
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2.4. Adoptive transfer experiment T cells used for adoptive transfer were harvested from male apoE( / ) mice fed regular mouse chow at 13 weeks of age. CD8+ T cells were negatively isolated from pooled splenocytes using Dynal Mouse CD8 Negative Isolation Kit (Invitrogen) according to the manufacturer’s protocols. Purity of the isolated CD8+ T cells was P90%. The selected CD8+ T cells were then stained with PE anti-mouse CD25 antibody (eBioscience), and CD25+ T cells were collected by sorting with a MoFlo cell sorter (BD Biosciences), similar to a previously reported antibody based cell sorting method [18]. Pilot experiments using total CD8+ T cells at doses of 1 105 and 1 106 showed that the lower dose recipients had a trend for reduced atherosclerosis. We therefore used the 1 105 dose to investigate the effect of CD8+CD25+ subtype on atherosclerosis given their suppressor phenotype as previously reported [7,10–12]. The isolated CD8+CD25+ T cells or CD8+CD25 T cells were then adoptively transferred (1 105 cells/mouse) to naïve male apoE( / ) recipient mice at 7 weeks of age via tail vein injection. Homing experiments were performed using adoptively transferred CFSE-labeled CD8+ T cells [19,20] and mice were euthanized 3 days later. Aortic tissues were enzymatically digested (0.25 mg/ml collagenase, 0.125 mg/ml elastase, 60 U/ml hyaluronidase in DMEM/ F12 medium at 37 °C for 45 min) and subjected to flow cytometry. Size gating indicated that 0.49% of collected cells were lymphocytes, 0.2% of which were CFSE+ (Supplemental Fig. 1). Mice injected with PBS served as control. Recipient mice were fed regular mouse chow until 13 weeks of age when chow was switched to high cholesterol diet (TD 88137, Harlan-Teklad) until euthanasia at 25 weeks of age. Serum was collected to determine total and free serum cholesterol levels with colorimetric assays (Wako Diagnostics). Aortas were cleaned and stained en face with oil-red-O to assess the extent of atherosclerosis with computer-assisted histomorphometry with the samples blinded to the assessor. Hearts were embedded in OCT for sectioning of the aortic sinuses to analyze plaque size, lipid content by oil red-o staining, and T cell and macrophage content by CD3 and MOMA-2 antibody staining, respectively. 2.5. Ex-vivo cell proliferation Splenocytes from recipient mice were collected and labeled with CFSE. Cells were stimulated with CD3/CD28 Dynabeads (Invitrogen) at the cell-to-bead ratio of 1:1 for 4 days. Cells were then collected, stained with CD4 and CD8 antibodies as indicated and subjected to flow cytometry. The results are expressed as the CFSE mean fluorescent intensity (MFI). 2.6. Statistics Data are presented as mean ± SD. Number of animals in each group is listed in text or figure legend. Data were analyzed by ANOVA followed by Newman-Keuls multiple group comparison, or by t-test when appropriate. P < 0.05 was considered as statistically significant. 3. Results 3.1. Effect of atherogenic diet on peripheral blood CD8+CD25+ T cells Six weeks of high cholesterol diet significantly reduced CD8+CD25+ T cells in peripheral blood of apoE( / ) mice (11.2 ± 2.9% vs. 3.8 ± 1.8%, P = 0.0011), suggesting a role in
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Fig. 1. CD8+ T cells in atherosclerosis. (A) Feeding of apoE( / ) mice with an atherogenic diet (HC) for 6 weeks resulted in significantly reduced peripheral blood CD8+CD25+ T cells compared to mice fed normal chow (NC). CD8b+ gated peripheral blood cells were plotted against CD25 using isotype control as reference. Results are expressed as percent of CD8+ T cells. N = 5 each; ⁄P < 0.01. (B) Immuno-staining of aortic sinus of 25 week-old apoE( / ) mice showed presence of CD8b+ T cells (reddish-brown stain, arrows in C) in the atherosclerotic plaques (B and C; representative of 3 separate mice, 2 sections per mouse; D; negative control). Box in B marks area magnified as shown in C. Bar in B = 100 microns; bar in C = 10 microns. (E) Flow cytometric analysis of enzymatically digested plaques from aortic sinuses of 29 week-old mice showed presence of CD8+CD25+ T cells in atherosclerotic plaque-derived cells. Cells were gated first on CD8 and plotted on CD8 vs. CD25 (representative of 3 separate mice) as performed for the peripheral blood cells.
atherogenesis (Fig. 1A). This also indicated that CD8+ T cell phenotype changes occur early, in agreement with a previous report [8]. 3.2. CD8+CD25+ T cells in atherosclerotic plaques Although phenotype changes were observed early in the disease process, plaques were not yet present in the aortic sinus to a degree that could be analyzed. Therefore, mice were fed an atherogenic diet for 19 weeks for plaque development in the aortic sinus. Immunostaining for CD8b showed presence of CD8+ T cells in the atherosclerotic plaques of 25 week-old apoE( / ) mice fed an atherogenic diet (Fig. 1B–D). To further characterize the CD8+ T cells in the plaques, we used enzymatic digestion to collect cells for flow cytometry. To assure that the plaques were of sufficient size for tissue digestion, mice were fed the atherogenic diet for 23 weeks. Flow cytometric analysis indicated that 1.4 ± 0.1% of aortic sinus plaque-derived cells were CD8+ T cells (Fig. 1E), of which 79.9 ± 3.3% were CD25+ (Fig. 1E). Given the small amount of CD8+CD25+ T cells in the plaques, it is technically not feasible to isolate them in sufficient amount to characterize their phenotype or adoptively transfer them to the recipients to assess functions. This limitation led us to use splenic CD8+CD25+ T cells as an alternative for phenotypic and functional characterization in the subsequent experiments. 3.3. CD8+CD25+ T cells posses T-suppressor phenotype Surface staining indicated that more than 90% of the CD8+CD25+ T cells were CD28 positive compared to 50% of CD8+CD25 T cells
(Fig. 2A and B). There was also a significantly higher percentage of the CD8+CD25+ T cells that expressed GITR, CCR7, and surface TGF-b (Fig. 2B) when compared to CD8+CD25 T cells. Intra-cellular staining showed that CD8+CD25+ T cells had higher expression of CTLA-4, TGF-b, and IL-10 as well as increased percentage of FoxP3+ cells compared to the CD8+CD25 subset (Fig. 3A and B). These are consistent with the suppressor phenotype of CD8+CD25+ T cells previously described in mice and humans [10–15]. The significantly higher percentage of FoxP3+ cells in the CD8+CD25+ T cells population is also consistent with a previous report [21]. Further analysis of the intracellular-staining assay results showed low percentage and a lack of difference in the TGF-b+IL-10+FoxP3+ cells in the CD8+CD25+ compared to CD8+CD25 T cells (0.0 ± 0.0% and 1.4 ± 1.5%, respectively). On the other hand, a significant difference was found in the TGF-b+IL-10+FoxP3 population in CD8+CD25+ compared to CD8+CD25 T cell population (28.7 ± 7.8% vs. 2.6 ± 2.5%, respectively, P < 0.01). Interestingly, TGF-b IL-10 FoxP3+cells were also significantly higher in the CD8+CD25+ compared to CD8+CD25 T cells (16.1 ± 4.1% vs. 3.8 ± 1.2%, respectively, P < 0.01). 3.4. CD8+CD25+ T cells possess suppressor function in vitro Given that there are no universal surface markers for the suppressor phenotype of CD8+CD25+ T cells, functional assays to establish suppressor property of such T cells were further performed. First, the capacity to inhibit the proliferative response of CD4+CD25 T cells (responder cells) to anti-CD3 and anti-CD28
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Fig. 2. Characterization of surface markers of splenic CD8+CD25+ T cells. (A) Representative scatter plot of live splenic CD8+ cells gated into CD25+ or CD25 cells using isotype as reference (top panel, left and right, respectively) and analyzed for cell surface marker CD28 (bottom left and middle panels, respectively), GITR, CCR7, and TGF-b. Isotype for surface marker staining reference shown in bottom right panel. (B) Cell surface markers CD28, GITR, CCR7, and TGF-b are expressed as percentage of CD8+ cells gated on either CD25+ or CD25 cells. ⁄P < 0.05 vs. CD25+ cells. Spleens from 2 to 3 mice were pooled in 3 separate replicates.
stimulation was investigated. We confirmed that at the number ratio of 1:1 to responder cells, CD8+CD25+ T cells significantly inhibited the proliferative response of CD4+CD25 cells to antiCD3 and anti CD28 stimulation when compared to CD8+CD25 T cells (Fig. 3C). We further studied the suppressor capacity of CD8+CD25+ T cells on the cytolytic activity of CD8+ T cells. We depleted CD8+CD25+ T cells from total CD8+ T cells and compared the cytolytic activity using bone marrow derived dendritic cells (BMDCs) as target cells. CD25-depleted CD8+ T cells cells had a higher cytolytic activity against BMDCs when compared to total CD8+ T cells (Fig. 3D). This observation is consistent with the notion that CD8+CD25+ T cells possess suppressor property and depletion of CD8+CD25+ T cells from total CD8+ T cells rendered the remaining CD8+CD25 T cells with a higher cytolytic activity. Thus we have demonstrated the suppressor function of CD8+CD25+ T cells in vitro. Next we transferred CD8+ T cell subsets into apoE( / ) mice to test their effect on atherogenesis in vivo.
lating cholesterol levels (962 ± 366 mg/dL, 1101 ± 451 mg/dL, and 1205 ± 267 mg/dL, respectively); or body weight (35.4 ± 6.4 g, 32.7 ± 5.2 g and 39.2 ± 6.6 g, respectively). There was a trend for reduced aortic sinus plaque size in the CD8+CD25+ T cell recipient group (0.30 ± 0.09 mm2; N = 12) compared to the PBS and CD8+CD25 T cell recipient groups (0.34 ± 0.07 mm2; N = 11, and 0.40 ± 0.11 mm2; N = 8, respectively; p = 0.09 ANOVA), but did not reach statistical significance. No differences were noted in aortic sinus plaque lipid content (11.0 ± 2.5%, 10.3 ± 2.7%, and 9.9 ± 3.6%, respectively), and CD3+ T cell presence (110.1 ± 47.1 cells/mm2, 119.2 ± 46.2 cells/mm2, and 108.5 ± 44.5 cells/mm2, respectively) among the groups. However, both recipient groups had significantly reduced macrophage content in the plaques compared to PBS (CD8+CD25+: 1.4 ± 0.9%; CD8+CD25 : 0.8 ± 0.7%; PBS: 2.5 ± 1.3%; P < 0.05 by ANOVA).
3.5. Adoptive transfer of CD8+CD25+ T cells reduced atherosclerosis in recipient mice
Since T cell activation and expansion contribute to atherosclerosis lesion growth and disease aggravation, we assessed the effect of adoptive CD8+CD25+ T cell transfer on splenic T cell proliferation in recipient mice at the 25 week euthanasia time time point. There was significant reduction in the proliferation of CD4+ T cells from CD8+CD25+ T cell recipient mice compared to CD8+CD25 T cell recipient mice (Fig. 4B). CD8+ T cell proliferation in both recipient groups was not affected (Fig. 4B, right panel).
Adoptive transfer of 1 105 CD8+CD25+ T cells significantly reduced aortic atherosclerosis compared to injection of PBS or CD8+CD25 T cells as measured by aortic en-face ORO staining in recipient mice (5.4 ± 1.3%, 7.4 ± 2.0% and 7.1 ± 0.9% respectively, P = 0.007 by ANOVA; Fig. 4A), with no significant difference in circu-
3.6. Adoptive transfer of CD8+CD25+ T cells inhibited T cell proliferation in recipient mice
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Fig. 3. Characterization of intracellular markers and functional assay of splenic CD8+CD25+ T cells. (A) Representative scatter plot of CD25+ or CD25 gated CD8+ spleen cells as in Fig. 2. Intracellular staining was performed and the expression measured in mean fluorescence intensity (MFI; left panel, CTLA-4 expression as representative). Results were plotted on bar graphs and expression levels for CTLA-4, TGF-b, and IL-10 (right panel) or FoxP3 (B) compared between the groups. ⁄P < 0.05 vs. CD25+ cells. (C) Proliferative response of CD4+CD25 T cells to CD3/CD28 antibody stimulation expressed as decay of CFSE MFI signal. CD4+CD25 T cell proliferation was reduced when cocultured with CD8+CD25+ T cells compared to CD8+CD25 T cells (⁄P < 0.05). (D) Lytic activity against CD11c+ BMDCs was compared between total CD8+ T cells and CD8+CD25 T cells after CD25+ cell depletion. ⁄P < 0.05 vs. total CD8+ cells. Spleens from 2 to 3 mice were pooled in 3 separate replicates.
4. Discussion In this study, we show that splenic CD8+CD25+ T cells in apoE( / ) mice have suppressor phenotypic markers and function. Adoptive transfer of CD8+CD25+ T cells reduced atherosclerotic lesions and reduced CD4+ T cell proliferation. The role of CD8+ T cells in atherosclerosis remains unclear. A recent report using global depletion of CD8+ T cells in apoE( / ) mice supports a pro-atherogenic role for these cells [3]. But T cells are not homogenous in phenotype, consistent with the complexity of the immune function in atherosclerosis. Given that there are different phenotypes, we first assessed which of these responded to the inflammatory challenge of an atherogenic diet. Our recent report of an increase of CD8+CD25+ T cells early after immunization of apoE( / ) mice with an apoB-100 derived peptide vaccine [16] led us to speculate that CD8+CD25+ T cells may be important in modulating atherosclerosis. Our results show that the CD8+CD25+ T cell subtype appears to be negatively impacted by the atherogenic diet. Their possible role was supported by their presence in advanced atherosclerotic plaques. However, characterization of the phenotype within the plaques was severely restricted by the scant number of these cells. Thus, we used splenic CD8+CD25+ T cells instead to investigate their role in atherosclerosis. We first characterized the phenotype of splenic CD8+CD25+ T cells. The high percentage of CD8+CD25+ T cells co-expressing CD28, TGF-b, GITR and CCR7, with increased intracellular expression levels of FoxP3, CTLA-4, TGF-b, and IL-10 compared to CD8+CD25 T cells in our study demonstrated a similar phenotype
to previously described CD8+CD25+ suppressor T cell populations [7,10–15]. Interestingly, based on our intracellular staining assay, it appears that the CD8+CD25+. T cells have two subpopulations, one that is TGF-b+IL-10+FoxP3 and the other that is TGF-b IL-10 FoxP3+. This indicated that other than CD25, phenotyping the cells using typical markers would not be definitive; the functional profile would be more relevant. Our study revealed a suppressive function similar to that reported for CD8+CD25+ suppressor cells and CD4+CD25+ Treg cells, suggesting this subset of CD8+ cells may function as suppressor cells regulating immune activation [10,12,15]. The observation that removal of CD8+CD25+ T cells from total CD8+ T cells rendered the remaining CD8+CD25 T cells more cytolytic against target cells further supported the observation that CD8+CD25+ T cells function as suppressors. The suppressor function of CD8+CD25+ T cells in atherosclerosis was verified by adoptive cell transfer experiments. Atherosclerosis was significantly reduced by adoptive transfer of CD8+CD25+ T cells into apoE( / ) mice, while transfer of the same number of CD8+CD25 T cells did not have a significant effect. There was a trend for reduced plaque size in the aortic sinus as well but did not reach statistical significance, suggesting differential magnitude of effect on different sites. Disease progression is known to occur at a higher rate in the aortic sinus [22]. It is possible that that the CD8+CD25+. T cell function is limited to slower progressing plaques. The effect of reducing aortic atherosclerosis by a single injection of 1 105 CD8+CD25+ T cells (30% decrease) to 7-week old mice is comparable to that by multiple transfer of a total of 9 105
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Fig. 4. Effect of adoptive transfer of CD8+CD25+ T cells on atherosclerosis. (A) Representative photographs of en-face oil red-O staining of aortas from the experimental groups (left panel); Bar = 5 mm. Histomorphometric analysis of plaque area as a percentage of total aortic area indicating significantly reduced atherosclerosis in CD8+CD25+ T cell recipient mice (right panel). PBS group, N = 11; CD8+CD25+ T cell group, N = 12; CD8+CD25 T cell group, N = 10. ⁄P < 0.05 vs. PBS and CD8+CD25 . (B) CD4+ T cell gate from spleens of recipient mice at 25 weeks of age (left panel). Representative CFSE histogram of CD4+ T cell gated splenocytes from recipient mice cultured for 4 days with CD3/ CD28 antibody stimulation (second panel), and plotted on a bar graph (third panel). Similar gating strategy was used to assess CD8+ T cell proliferation (right panel). N = 3–7 per group. ⁄P < 0.05 vs. CD8+CD25 T cell recipient mice.
CD4+CD25+ T cells into mice starting at 3 months of age [18]. The observation that proliferation of ex-vivo cultured CD4+ T cells from CD8+CD25+ T cell recipient mice was suppressed suggests that the transferred CD8+CD25+ T cells possessed suppressor function regulating other immune cells involved in atherosclerotic disease progression. It is unclear why there was preferential inhibition of proliferation in the CD4+ T cell population. It is possible that the kinetics of the effect of the CD8+CD25+ T cell transfer differs between CD4+ and CD8 T+ cells. CD8+ T cells have phenotypic and functional subtypes. This is likely to be modulated by the disease context, as well as the stage of a particular disease. We have recently identified other CD8+ T cell phenotypes involved in neointima formation [5,23] and in the protective effects of a vaccine against atherosclerosis [16]. Our results are in agreement with a recent report showing that CD8+ T cells are significantly involved in the atherosclerosis disease process [3]. Recent reports have shown that CD8+ T cell presence in human atherosclerotic plaques increase with time and plaque complexity [24], with increased activation markers as well as cytolytic function [25]. The importance of defining CD8+ T cell subsets in atherosclerosis was recently highlighted in a report describing associations between CD8+ T cells and carotid atherosclerotic disease [6]. In conclusion, adoptive transfer of CD8+CD25+ T cells significantly blunted the development of atherosclerosis in apoE( / ) mice via the possible mechanism of suppressing T cell activation and proliferation in the recipient mice. Our findings present evidence of a novel suppressive function for CD8+CD25+ T cells in murine atherosclerosis.
Acknowledgments This study was supported by the Heart Foundation, the Spielberg Cardiovascular Research Fund and the Skirball Foundation. The funding sources had no involvement in the design, conduct, and reporting of the paper. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbrc.2013.12.057. References [1] R. Elhage, P. Gourdy, L. Brouchet, J. Jawien, M.J. Fouque, C. Fievet, X. Huc, Y. Barreira, J.C. Couloumiers, J.F. Arnal, F. Bayard, Deleting TCR alpha beta+ or CD4+ T lymphocytes leads to opposite effects on site-specific atherosclerosis in female apolipoprotein E-deficient mice, Am. J. Pathol. 165 (2004) 2013–2018. [2] D. Kolbus, I. Ljungcrantz, I. Soderberg, R. Alm, H. Bjorkbacka, J. Nilsson, G.N. Fredrikson, TAP1-deficiency does not alter atherosclerosis development in Apoe / mice, PLoS One 7 (2012) e33932. [3] T. Kyaw, A. Winship, C. Tay, P. Kanellakis, H. Hosseini, A. Cao, P. Li, P. Tipping, A. Bobik, B.H. Toh, Cytotoxic and proinflammatory CD8+ T lymphocytes promote development of vulnerable atherosclerotic plaques in apoE-deficient mice, Circulation 127 (2013) 1028–1039. [4] A.I. Fyfe, J.H. Qiao, A.J. Lusis, Immune-deficient mice develop typical atherosclerotic fatty streaks when fed an atherogenic diet, J. Clin. Invest. 94 (1994) 2516–2520. [5] P.C. Dimayuga, K.Y. Chyu, W.M. Lio, X. Zhao, J. Yano, J. Zhou, T. Honjo, P.K. Shah, B. Cercek, Reduced neointima formation after arterial injury in CD4 / mice is mediated by CD8+CD28hi T cells, J. Am. Heart Assoc. 2 (3) (2013) e000155.
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CD8+CD25+ T cells reduce atherosclerosis in apoE( / ) mice Jianchang Zhou 1, Paul C. Dimayuga 1, Xiaoning Zhao, Juliana Yano, Wai Man Lio, Portia Trinidad, Tomoyuki Honjo, Bojan Cercek, Prediman K. Shah, Kuang-Yuh Chyu ⇑ Oppenheimer Atherosclerosis Research Center, Division of Cardiology, Cedars-Sinai Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, United States
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Article history: Received 9 December 2013 Available online 14 December 2013 Keywords: Atherosclerosis CD8+CD25+ T cells Immune modulation Adoptive transfer
a b s t r a c t Background: It is increasingly evident that CD8+ T cells are involved in atherosclerosis but the specific subtypes have yet to be defined. CD8+CD25+ T cells exert suppressive effects on immune signaling and modulate experimental autoimmune disorders but their role in atherosclerosis remains to be determined. The phenotype and functional role of CD8+CD25+ T cells in experimental atherosclerosis were investigated in this study. Methods and results: CD8+CD25+ T cells were observed in atherosclerotic plaques of apoE( / ) mice fed hypercholesterolemic diet. Characterization by flow cytometric analysis and functional evaluation using a CFSE-based proliferation assays revealed a suppressive phenotype and function of splenic CD8+CD25+ T cells from apoE( / ) mice. Depletion of CD8+CD25+ from total CD8+ T cells rendered higher cytolytic activity of the remaining CD8+CD25 T cells. Adoptive transfer of CD8+CD25+ T cells into apoE( / ) mice suppressed the proliferation of splenic CD4+ T cells and significantly reduced atherosclerosis in recipient mice. Conclusions: Our study has identified an athero-protective role for CD8+CD25+ T cells in experimental atherosclerosis. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction The presence of CD8+ T cells in atherosclerotic lesions is widely demonstrated but studies investigating their role in atherogenesis have yielded contradictory results. It was reported that mice genetically deficient in CD8+ T cells did not have altered atherosclerosis development [1,2]. More recently, apoE( / ) mice severely depleted of CD8+ T cells using depleting antibodies showed significantly reduced atherosclerosis suggesting a pro-atherogenic role [3]. On the other hand, Fyfe et al. demonstrated that an atherogenic diet induced a three-fold increase in atherosclerotic lesion size in MHC I knockout mice which are CD8+ T cell deficient [4]. These discrepant findings may be attributed to different experimental designs. However, it is also possible that CD8+ T cells have subtype specificity in their functional role as suggested by our recent report wherein reduced neointima formation after vascular injury was rendered by CD8+CD28hi T cells but not by CD8+CD28+ T cells [5], and by the reported association between CD8+ T cell subsets and cardiovascular disease [6]. It is notable that CD8+ T cells have historically been referred to as T suppressor cells [7].
Evidence suggests that specific CD8+ T cell phenotypes may be involved in modulating atherosclerosis. It was reported that hypercholesterolemia activated CD8+CD28+ T cells that preceded CD4+ T cell activation in mice [8], and that residing CD8+ T cells in human atherosclerotic plaques have an activated phenotype with increased CD25 expression [9]. However, it remains unclear whether the observed phenotypes contribute to disease progression or if it operates to down-modulate pro-atherogenic immune signaling. For example, CD8+CD25+ T cells functioning as suppressors are involved in the modulation of autoimmune disorders with similar function to CD4+ Tregs [7,10–15]. The role of this phenotype in atherogenesis is highlighted by our previous report showing increased CD8+CD25+ T cells early after immunization of apoE( / ) mice with an apoB-100 derived peptide vaccine [16]. In this study, we characterized the phenotype and function of CD8+CD25+ T cells from apoE( / ) mice and defined their role in experimental atherosclerosis using adoptive cell transfer strategy. 2. Materials and methods 2.1. Animals
⇑ Corresponding author. Address: Division of Cardiology, Room 5539, CedarsSinai Heart Institute, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, United States. E-mail address: [email protected] (K.-Y. Chyu). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2013.12.057
Male apoE( / ) mice on a C57BL/6 background were purchased from Jackson Laboratories (Bar Harbor, Me), housed in a pathogenfree animal and kept on a 12-h day/night cycle with unrestricted access to water and regular mouse chow (5015, PMI Nutrition
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International) unless mentioned otherwise. The Institutional Animal Care and Use Committee of Cedars-Sinai Medical Center approved the experimental protocols. A group of 7 week-old apoE( / ) mice were fed regular mouse chow or an atherogenic diet (TD 88137, Harlan-Teklad) for 6 weeks. Mice were euthanized and the peripheral blood mononuclear cells were collected, stained and subjected to flow cytometric analysis. CD8+ T cells in atherosclerotic plaques were visualized by immunohistochemical staining of cryo-sections of aortic sinus of atherogenic diet-fed apoE( / ) mice euthanized at 25 weeks of age using standard immuno-staining protocol with a monoclonal anti-mouse CD8b antibody (BD Biosciences) as primary antibody. Negative control involved immunostaining with omission of primary antibody. Detection of CD8+CD25+ T cells in atherosclerotic plaques was assessed by enzyme digestion of plaques of apoE( / ) mice fed the atherogenic diet and euthanized at 29 weeks of age. Plaques were enzymatically digested (0.25 mg/ ml collagenase, 0.125 mg/ml elastase, 60 U/ml hyaluronidase in DMEM/F12 medium at 37 °C for 45 min) and subjected to flow cytometry. Flow cytometric analysis was performed by staining cells with FITC-conjugated CD8b and PE-conjugated CD25 antibodies (eBioscience) and analyzed on an LSR II apparatus (BD Biosciences). 2.2. Phenotypic characterization of CD8+CD25+ T cells Splenocytes pooled from two to three 13 week old male apoE( / ) mice fed regular mouse chow were characterized by flow cytometric analysis using standard protocols with antibodies from eBioscience unless otherwise indicated: anti-CD8b, anti-CD25, anti-CD28, anti-FoxP3, anti-IL-10, anti-TGF-b (R&D Systems), anti-IL-12, anti-IFN-c, anti-CCR7, anti-GITR (Biolegend), and anti-CTLA-4 (Biolegend). Cell permeabilized for staining intracellular molecules was performed using standard procedure. Gating and cut off were based on isotype controls. Data files were analyzed using Summit V4.3 software (DAKO). 2.3. Functional characterization of CD8+CD25+ T cells Pooled splenocytes from 13-week old male apoE( / ) mice fed regular mouse chow were purified by sorting with ARIA II Cell Sorter (BD Biosciences) for the following cell populations: CD8+CD25+, CD8+CD25 and CD4+CD25 T cells. Purified CD4+CD25 T cells (responder cells) were labeled with CFSE (Invitrogen, 2.5 lM at 37 °C for 10 min) and then cultured (1 105 cells in 100 ll culture medium) in the presence of CD3/CD28 Dynabeads (Invitrogen) at the cell-to-bead ratio of 1:2 with CD8+CD25+ or CD8+CD25 T cells (treatment groups) at a ratio of 1:1. The control was CD4+CD25 T cells without CD8+ T cells. Four days after co-culture, cells were stained for CD4 and analyzed by LSR II analyzer. The results are expressed as the CFSE mean fluorescent intensity (MFI). Cytolytic activity was assayed using bone marrow-derived dendritic cells (BMDCs) as target cells [17]. In brief, bone marrow cells from femurs and tibiae of male apoE( / ) mice were cultured in complete RPMI-1640 containing 10 ng/ml GM-CSF (R&D Systems) and 10 ng/ml IL-4 (Invitrogen). Immature DCs were harvested on day 8 and subcultured into new culture plates with 2 105 DCs in 1.5 ml medium. The negatively isolated CD8+ T cells or CD8+ T cells depleted of CD8+CD25+ cells were then co-culture with DCs at a CD8:DC ratio of 3:1 for 4 h, cells were then collected and processed for flow cytometric determination of CD11c and 7-AAD (eBioscience) by LSR II analyzer. CD11c positive cells stained with 7-AAD were identified as lysed target cells using target cell death without CD8+ T cells as baseline [17].
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2.4. Adoptive transfer experiment T cells used for adoptive transfer were harvested from male apoE( / ) mice fed regular mouse chow at 13 weeks of age. CD8+ T cells were negatively isolated from pooled splenocytes using Dynal Mouse CD8 Negative Isolation Kit (Invitrogen) according to the manufacturer’s protocols. Purity of the isolated CD8+ T cells was P90%. The selected CD8+ T cells were then stained with PE anti-mouse CD25 antibody (eBioscience), and CD25+ T cells were collected by sorting with a MoFlo cell sorter (BD Biosciences), similar to a previously reported antibody based cell sorting method [18]. Pilot experiments using total CD8+ T cells at doses of 1 105 and 1 106 showed that the lower dose recipients had a trend for reduced atherosclerosis. We therefore used the 1 105 dose to investigate the effect of CD8+CD25+ subtype on atherosclerosis given their suppressor phenotype as previously reported [7,10–12]. The isolated CD8+CD25+ T cells or CD8+CD25 T cells were then adoptively transferred (1 105 cells/mouse) to naïve male apoE( / ) recipient mice at 7 weeks of age via tail vein injection. Homing experiments were performed using adoptively transferred CFSE-labeled CD8+ T cells [19,20] and mice were euthanized 3 days later. Aortic tissues were enzymatically digested (0.25 mg/ml collagenase, 0.125 mg/ml elastase, 60 U/ml hyaluronidase in DMEM/ F12 medium at 37 °C for 45 min) and subjected to flow cytometry. Size gating indicated that 0.49% of collected cells were lymphocytes, 0.2% of which were CFSE+ (Supplemental Fig. 1). Mice injected with PBS served as control. Recipient mice were fed regular mouse chow until 13 weeks of age when chow was switched to high cholesterol diet (TD 88137, Harlan-Teklad) until euthanasia at 25 weeks of age. Serum was collected to determine total and free serum cholesterol levels with colorimetric assays (Wako Diagnostics). Aortas were cleaned and stained en face with oil-red-O to assess the extent of atherosclerosis with computer-assisted histomorphometry with the samples blinded to the assessor. Hearts were embedded in OCT for sectioning of the aortic sinuses to analyze plaque size, lipid content by oil red-o staining, and T cell and macrophage content by CD3 and MOMA-2 antibody staining, respectively. 2.5. Ex-vivo cell proliferation Splenocytes from recipient mice were collected and labeled with CFSE. Cells were stimulated with CD3/CD28 Dynabeads (Invitrogen) at the cell-to-bead ratio of 1:1 for 4 days. Cells were then collected, stained with CD4 and CD8 antibodies as indicated and subjected to flow cytometry. The results are expressed as the CFSE mean fluorescent intensity (MFI). 2.6. Statistics Data are presented as mean ± SD. Number of animals in each group is listed in text or figure legend. Data were analyzed by ANOVA followed by Newman-Keuls multiple group comparison, or by t-test when appropriate. P < 0.05 was considered as statistically significant. 3. Results 3.1. Effect of atherogenic diet on peripheral blood CD8+CD25+ T cells Six weeks of high cholesterol diet significantly reduced CD8+CD25+ T cells in peripheral blood of apoE( / ) mice (11.2 ± 2.9% vs. 3.8 ± 1.8%, P = 0.0011), suggesting a role in
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Fig. 1. CD8+ T cells in atherosclerosis. (A) Feeding of apoE( / ) mice with an atherogenic diet (HC) for 6 weeks resulted in significantly reduced peripheral blood CD8+CD25+ T cells compared to mice fed normal chow (NC). CD8b+ gated peripheral blood cells were plotted against CD25 using isotype control as reference. Results are expressed as percent of CD8+ T cells. N = 5 each; ⁄P < 0.01. (B) Immuno-staining of aortic sinus of 25 week-old apoE( / ) mice showed presence of CD8b+ T cells (reddish-brown stain, arrows in C) in the atherosclerotic plaques (B and C; representative of 3 separate mice, 2 sections per mouse; D; negative control). Box in B marks area magnified as shown in C. Bar in B = 100 microns; bar in C = 10 microns. (E) Flow cytometric analysis of enzymatically digested plaques from aortic sinuses of 29 week-old mice showed presence of CD8+CD25+ T cells in atherosclerotic plaque-derived cells. Cells were gated first on CD8 and plotted on CD8 vs. CD25 (representative of 3 separate mice) as performed for the peripheral blood cells.
atherogenesis (Fig. 1A). This also indicated that CD8+ T cell phenotype changes occur early, in agreement with a previous report [8]. 3.2. CD8+CD25+ T cells in atherosclerotic plaques Although phenotype changes were observed early in the disease process, plaques were not yet present in the aortic sinus to a degree that could be analyzed. Therefore, mice were fed an atherogenic diet for 19 weeks for plaque development in the aortic sinus. Immunostaining for CD8b showed presence of CD8+ T cells in the atherosclerotic plaques of 25 week-old apoE( / ) mice fed an atherogenic diet (Fig. 1B–D). To further characterize the CD8+ T cells in the plaques, we used enzymatic digestion to collect cells for flow cytometry. To assure that the plaques were of sufficient size for tissue digestion, mice were fed the atherogenic diet for 23 weeks. Flow cytometric analysis indicated that 1.4 ± 0.1% of aortic sinus plaque-derived cells were CD8+ T cells (Fig. 1E), of which 79.9 ± 3.3% were CD25+ (Fig. 1E). Given the small amount of CD8+CD25+ T cells in the plaques, it is technically not feasible to isolate them in sufficient amount to characterize their phenotype or adoptively transfer them to the recipients to assess functions. This limitation led us to use splenic CD8+CD25+ T cells as an alternative for phenotypic and functional characterization in the subsequent experiments. 3.3. CD8+CD25+ T cells posses T-suppressor phenotype Surface staining indicated that more than 90% of the CD8+CD25+ T cells were CD28 positive compared to 50% of CD8+CD25 T cells
(Fig. 2A and B). There was also a significantly higher percentage of the CD8+CD25+ T cells that expressed GITR, CCR7, and surface TGF-b (Fig. 2B) when compared to CD8+CD25 T cells. Intra-cellular staining showed that CD8+CD25+ T cells had higher expression of CTLA-4, TGF-b, and IL-10 as well as increased percentage of FoxP3+ cells compared to the CD8+CD25 subset (Fig. 3A and B). These are consistent with the suppressor phenotype of CD8+CD25+ T cells previously described in mice and humans [10–15]. The significantly higher percentage of FoxP3+ cells in the CD8+CD25+ T cells population is also consistent with a previous report [21]. Further analysis of the intracellular-staining assay results showed low percentage and a lack of difference in the TGF-b+IL-10+FoxP3+ cells in the CD8+CD25+ compared to CD8+CD25 T cells (0.0 ± 0.0% and 1.4 ± 1.5%, respectively). On the other hand, a significant difference was found in the TGF-b+IL-10+FoxP3 population in CD8+CD25+ compared to CD8+CD25 T cell population (28.7 ± 7.8% vs. 2.6 ± 2.5%, respectively, P < 0.01). Interestingly, TGF-b IL-10 FoxP3+cells were also significantly higher in the CD8+CD25+ compared to CD8+CD25 T cells (16.1 ± 4.1% vs. 3.8 ± 1.2%, respectively, P < 0.01). 3.4. CD8+CD25+ T cells possess suppressor function in vitro Given that there are no universal surface markers for the suppressor phenotype of CD8+CD25+ T cells, functional assays to establish suppressor property of such T cells were further performed. First, the capacity to inhibit the proliferative response of CD4+CD25 T cells (responder cells) to anti-CD3 and anti-CD28
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Fig. 2. Characterization of surface markers of splenic CD8+CD25+ T cells. (A) Representative scatter plot of live splenic CD8+ cells gated into CD25+ or CD25 cells using isotype as reference (top panel, left and right, respectively) and analyzed for cell surface marker CD28 (bottom left and middle panels, respectively), GITR, CCR7, and TGF-b. Isotype for surface marker staining reference shown in bottom right panel. (B) Cell surface markers CD28, GITR, CCR7, and TGF-b are expressed as percentage of CD8+ cells gated on either CD25+ or CD25 cells. ⁄P < 0.05 vs. CD25+ cells. Spleens from 2 to 3 mice were pooled in 3 separate replicates.
stimulation was investigated. We confirmed that at the number ratio of 1:1 to responder cells, CD8+CD25+ T cells significantly inhibited the proliferative response of CD4+CD25 cells to antiCD3 and anti CD28 stimulation when compared to CD8+CD25 T cells (Fig. 3C). We further studied the suppressor capacity of CD8+CD25+ T cells on the cytolytic activity of CD8+ T cells. We depleted CD8+CD25+ T cells from total CD8+ T cells and compared the cytolytic activity using bone marrow derived dendritic cells (BMDCs) as target cells. CD25-depleted CD8+ T cells cells had a higher cytolytic activity against BMDCs when compared to total CD8+ T cells (Fig. 3D). This observation is consistent with the notion that CD8+CD25+ T cells possess suppressor property and depletion of CD8+CD25+ T cells from total CD8+ T cells rendered the remaining CD8+CD25 T cells with a higher cytolytic activity. Thus we have demonstrated the suppressor function of CD8+CD25+ T cells in vitro. Next we transferred CD8+ T cell subsets into apoE( / ) mice to test their effect on atherogenesis in vivo.
lating cholesterol levels (962 ± 366 mg/dL, 1101 ± 451 mg/dL, and 1205 ± 267 mg/dL, respectively); or body weight (35.4 ± 6.4 g, 32.7 ± 5.2 g and 39.2 ± 6.6 g, respectively). There was a trend for reduced aortic sinus plaque size in the CD8+CD25+ T cell recipient group (0.30 ± 0.09 mm2; N = 12) compared to the PBS and CD8+CD25 T cell recipient groups (0.34 ± 0.07 mm2; N = 11, and 0.40 ± 0.11 mm2; N = 8, respectively; p = 0.09 ANOVA), but did not reach statistical significance. No differences were noted in aortic sinus plaque lipid content (11.0 ± 2.5%, 10.3 ± 2.7%, and 9.9 ± 3.6%, respectively), and CD3+ T cell presence (110.1 ± 47.1 cells/mm2, 119.2 ± 46.2 cells/mm2, and 108.5 ± 44.5 cells/mm2, respectively) among the groups. However, both recipient groups had significantly reduced macrophage content in the plaques compared to PBS (CD8+CD25+: 1.4 ± 0.9%; CD8+CD25 : 0.8 ± 0.7%; PBS: 2.5 ± 1.3%; P < 0.05 by ANOVA).
3.5. Adoptive transfer of CD8+CD25+ T cells reduced atherosclerosis in recipient mice
Since T cell activation and expansion contribute to atherosclerosis lesion growth and disease aggravation, we assessed the effect of adoptive CD8+CD25+ T cell transfer on splenic T cell proliferation in recipient mice at the 25 week euthanasia time time point. There was significant reduction in the proliferation of CD4+ T cells from CD8+CD25+ T cell recipient mice compared to CD8+CD25 T cell recipient mice (Fig. 4B). CD8+ T cell proliferation in both recipient groups was not affected (Fig. 4B, right panel).
Adoptive transfer of 1 105 CD8+CD25+ T cells significantly reduced aortic atherosclerosis compared to injection of PBS or CD8+CD25 T cells as measured by aortic en-face ORO staining in recipient mice (5.4 ± 1.3%, 7.4 ± 2.0% and 7.1 ± 0.9% respectively, P = 0.007 by ANOVA; Fig. 4A), with no significant difference in circu-
3.6. Adoptive transfer of CD8+CD25+ T cells inhibited T cell proliferation in recipient mice
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Fig. 3. Characterization of intracellular markers and functional assay of splenic CD8+CD25+ T cells. (A) Representative scatter plot of CD25+ or CD25 gated CD8+ spleen cells as in Fig. 2. Intracellular staining was performed and the expression measured in mean fluorescence intensity (MFI; left panel, CTLA-4 expression as representative). Results were plotted on bar graphs and expression levels for CTLA-4, TGF-b, and IL-10 (right panel) or FoxP3 (B) compared between the groups. ⁄P < 0.05 vs. CD25+ cells. (C) Proliferative response of CD4+CD25 T cells to CD3/CD28 antibody stimulation expressed as decay of CFSE MFI signal. CD4+CD25 T cell proliferation was reduced when cocultured with CD8+CD25+ T cells compared to CD8+CD25 T cells (⁄P < 0.05). (D) Lytic activity against CD11c+ BMDCs was compared between total CD8+ T cells and CD8+CD25 T cells after CD25+ cell depletion. ⁄P < 0.05 vs. total CD8+ cells. Spleens from 2 to 3 mice were pooled in 3 separate replicates.
4. Discussion In this study, we show that splenic CD8+CD25+ T cells in apoE( / ) mice have suppressor phenotypic markers and function. Adoptive transfer of CD8+CD25+ T cells reduced atherosclerotic lesions and reduced CD4+ T cell proliferation. The role of CD8+ T cells in atherosclerosis remains unclear. A recent report using global depletion of CD8+ T cells in apoE( / ) mice supports a pro-atherogenic role for these cells [3]. But T cells are not homogenous in phenotype, consistent with the complexity of the immune function in atherosclerosis. Given that there are different phenotypes, we first assessed which of these responded to the inflammatory challenge of an atherogenic diet. Our recent report of an increase of CD8+CD25+ T cells early after immunization of apoE( / ) mice with an apoB-100 derived peptide vaccine [16] led us to speculate that CD8+CD25+ T cells may be important in modulating atherosclerosis. Our results show that the CD8+CD25+ T cell subtype appears to be negatively impacted by the atherogenic diet. Their possible role was supported by their presence in advanced atherosclerotic plaques. However, characterization of the phenotype within the plaques was severely restricted by the scant number of these cells. Thus, we used splenic CD8+CD25+ T cells instead to investigate their role in atherosclerosis. We first characterized the phenotype of splenic CD8+CD25+ T cells. The high percentage of CD8+CD25+ T cells co-expressing CD28, TGF-b, GITR and CCR7, with increased intracellular expression levels of FoxP3, CTLA-4, TGF-b, and IL-10 compared to CD8+CD25 T cells in our study demonstrated a similar phenotype
to previously described CD8+CD25+ suppressor T cell populations [7,10–15]. Interestingly, based on our intracellular staining assay, it appears that the CD8+CD25+. T cells have two subpopulations, one that is TGF-b+IL-10+FoxP3 and the other that is TGF-b IL-10 FoxP3+. This indicated that other than CD25, phenotyping the cells using typical markers would not be definitive; the functional profile would be more relevant. Our study revealed a suppressive function similar to that reported for CD8+CD25+ suppressor cells and CD4+CD25+ Treg cells, suggesting this subset of CD8+ cells may function as suppressor cells regulating immune activation [10,12,15]. The observation that removal of CD8+CD25+ T cells from total CD8+ T cells rendered the remaining CD8+CD25 T cells more cytolytic against target cells further supported the observation that CD8+CD25+ T cells function as suppressors. The suppressor function of CD8+CD25+ T cells in atherosclerosis was verified by adoptive cell transfer experiments. Atherosclerosis was significantly reduced by adoptive transfer of CD8+CD25+ T cells into apoE( / ) mice, while transfer of the same number of CD8+CD25 T cells did not have a significant effect. There was a trend for reduced plaque size in the aortic sinus as well but did not reach statistical significance, suggesting differential magnitude of effect on different sites. Disease progression is known to occur at a higher rate in the aortic sinus [22]. It is possible that that the CD8+CD25+. T cell function is limited to slower progressing plaques. The effect of reducing aortic atherosclerosis by a single injection of 1 105 CD8+CD25+ T cells (30% decrease) to 7-week old mice is comparable to that by multiple transfer of a total of 9 105
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Fig. 4. Effect of adoptive transfer of CD8+CD25+ T cells on atherosclerosis. (A) Representative photographs of en-face oil red-O staining of aortas from the experimental groups (left panel); Bar = 5 mm. Histomorphometric analysis of plaque area as a percentage of total aortic area indicating significantly reduced atherosclerosis in CD8+CD25+ T cell recipient mice (right panel). PBS group, N = 11; CD8+CD25+ T cell group, N = 12; CD8+CD25 T cell group, N = 10. ⁄P < 0.05 vs. PBS and CD8+CD25 . (B) CD4+ T cell gate from spleens of recipient mice at 25 weeks of age (left panel). Representative CFSE histogram of CD4+ T cell gated splenocytes from recipient mice cultured for 4 days with CD3/ CD28 antibody stimulation (second panel), and plotted on a bar graph (third panel). Similar gating strategy was used to assess CD8+ T cell proliferation (right panel). N = 3–7 per group. ⁄P < 0.05 vs. CD8+CD25 T cell recipient mice.
CD4+CD25+ T cells into mice starting at 3 months of age [18]. The observation that proliferation of ex-vivo cultured CD4+ T cells from CD8+CD25+ T cell recipient mice was suppressed suggests that the transferred CD8+CD25+ T cells possessed suppressor function regulating other immune cells involved in atherosclerotic disease progression. It is unclear why there was preferential inhibition of proliferation in the CD4+ T cell population. It is possible that the kinetics of the effect of the CD8+CD25+ T cell transfer differs between CD4+ and CD8 T+ cells. CD8+ T cells have phenotypic and functional subtypes. This is likely to be modulated by the disease context, as well as the stage of a particular disease. We have recently identified other CD8+ T cell phenotypes involved in neointima formation [5,23] and in the protective effects of a vaccine against atherosclerosis [16]. Our results are in agreement with a recent report showing that CD8+ T cells are significantly involved in the atherosclerosis disease process [3]. Recent reports have shown that CD8+ T cell presence in human atherosclerotic plaques increase with time and plaque complexity [24], with increased activation markers as well as cytolytic function [25]. The importance of defining CD8+ T cell subsets in atherosclerosis was recently highlighted in a report describing associations between CD8+ T cells and carotid atherosclerotic disease [6]. In conclusion, adoptive transfer of CD8+CD25+ T cells significantly blunted the development of atherosclerosis in apoE( / ) mice via the possible mechanism of suppressing T cell activation and proliferation in the recipient mice. Our findings present evidence of a novel suppressive function for CD8+CD25+ T cells in murine atherosclerosis.
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