Increased Cholesterol Content in Gammadelta (γδ) T Lymphocytes Differentially Regulates Their Activation

Gammadelta (γδ) T lymphocytes respond quickly upon antigen encounter to produce a cytokine response. In this study, we sought to understand how functions of γδ T cells are differentially regulated compared to αβ T cells. We found that cholesterol, an integral component of the plasma membrane and a regulator of TCR signaling, is increased in γδ T cells compared to αβ T cells, and modulates their function. Higher levels of activation markers, and increased lipid raft content in γδ cells suggest that γδ T cells are more activated. Cholesterol depletion effectively decreased lipid raft formation and activation of γδ T cells, indicating that increased cholesterol content contributes to the hyper-activated phenotype of γδ T cells, possibly through enhanced clustering of TCR signals in lipid rafts. TCR stimulation assays and western blotting revealed that instead of a lower TCR threshold, enhanced TCR signaling through ERK1/2 activation is likely the cause for high cholesterol-induced rapid activation and proliferation in γδ T cells. Our data indicate that cholesterol metabolism is differentially regulated in γδ T cells. The high intracellular cholesterol content leads to enhanced TCR signaling and increases activation and proliferation of γδ T cells.


Introduction
Most T cells express the ab T cell receptor (TCR). However, a small subset of T cells expresses the c and d chains of the TCR. These cd T cells represent ,3-5% of total CD3 + T cells in human peripheral blood and recognize non-peptide antigens such as lipids and phosphorylated nucleotides, as well as antigens that do not require processing and presentation by MHC molecules [1,2]. Antigen-naive cd T cells can react quickly, within hours after pathogen infection, and thus serve an innate immunity-like role before ab T cells and other adaptive immune responses could take place [2,3]. A cd T cell response is key to numerous pathogenic processes, as these cells have been shown to facilitate adaptive immune responses through various mechanisms [4]. For instance, cd T cells promote the maturation of naïve dendritic cells during viral infection, possibly through the production of proinflammatory cytokines such as TNFa, IFNc, and IL-6 [5]. cd T cells are also shown to induce robust CD8 + T cell responses by crosspresenting microbial and tumor antigens to CD8 + T cells [6].
Several groups have investigated unique gene expression patterns of cd T cells upon stimulation as hallmarks to distinguish them from ab T cells, but have reported finding relatively similar expression profiles thus far [7,8,9]. One of the most noteworthy findings was by Fahrer et al., who reported that cd and ab T cells show distinct expression patterns of both lipid metabolism and inflammatory genes upon Y. pseudotuberculosis infection [10]. These investigators reported that mRNA for several lipid metabolism genes were expressed only in the cd T cell samples. Another recent study reported that the response of cd T cells toward influenza virus was potently inhibited by blocking HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis, suggesting sterol metabolism may be important for the function of cd T cells [11].
Cholesterol maintains proper permeability and fluidity of the mammalian cell membrane to ensure cell growth and function. Cholesterol plays a role in mediating signal transduction by assisting the formation of lipid rafts, the specialized microdomains for organizing signaling molecules [12]. However, cholesterol levels must be properly regulated as excess sterol results in adverse effects on normal cell functions as well as the development of diseases such as atherosclerosis. Several studies have demonstrated that the homeostasis and functions of various T cell subsets are strongly linked to cellular and environmental cholesterol levels. Resting peripheral CD4 + T cells and the Th1 responses were both increased after in vivo cholesterol enrichment [13]. Coincidentally, we also reported that CD4 + T cells had increased intracellular cholesterol content and proliferative advantage in the absence of ABCG1, an cholesterol efflux transporter [14]. On the other hand, proliferation of NKT cells in response to aGalCer stimulation was lower in hypercholesterolemic ApoE 2/2 mice [15]. In this report, we provide novel evidence by which ab and cd T cells are differentially regulated by intracellular cholesterol content. We found that intracellular cholesterol levels are basally elevated in cd T cells and that this contributes to their ''primed for action'' phenotype by favoring TCR clustering and signaling.

Mice
C57BL/6J (000664) mice were purchased from The Jackson Laboratory. Mice were fed a standard rodent chow diet and housed in microisolator cages in a pathogen-free facility. All experiments followed guidelines of the La Jolla Institute for Allergy and Immunology Animal Care and Use Committee, and approval for use of rodents was obtained from the La Jolla Institute for Allergy and Immunology according to criteria outlined in the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health. Mice were euthanized by CO 2 inhalation.

Flow Cytometry
Spleens or peripheral lymph nodes (cervical, axillary, brachial, and inguinal) were excised and pushed through a 70-mm strainer. Red blood cells were lysed in RBC Lysis Buffer according to the manufacturer's protocol (BioLegend). All samples were collected in Dulbecco's PBS (Gibco) with 2 mM EDTA and were stored on ice during staining and analysis. 2-6610 6 cells were resuspended in 100 ml flow staining buffer [1% FBS plus 0.1% NaN 3 in PBS]. Fc receptors were blocked for 10 min and surface antigens were stained for 30 min at 4uC and washed twice with flow staining buffer. LIVE/DEAD Yellow Fixable Dead Cell Stain (Invitrogen) was used for analysis of viability, and forward-and side-scatter parameters were used for exclusion of doublets from analysis.
For measurement of intracellular lipids, after surface markers staining, cells were stained for an additional 15 min at room temperature with 5 ng/ml Nile Red (Enzo) and then washed twice.
For measurement of lipid rafts, cells were stained with Alexa Fluor 488-conjugated Cholera toxin subunit B (1/1000 dilution; Molecular Probes) together with other surface staining antibodies for 30 min at 4uC, and then washed twice.
Cellular fluorescence was assessed using LSR II flow cytometer (BD Biosciences) and percentages of subsets and median fluorescence intensity (MFI) were analyzed with FlowJo software (TreeStar version 9.2).
After several washes with PBS, the cell pellet was extracted with isopropanol containing 5-cholestane as internal standard. Total and free cholesterol content was determined by gas-liquid chromatography and normalized to cell numbers, as previously described [16]. Cholesteryl ester was calculated as (total cholesterol -free cholesterol) 61.67. Multiplying by 1.67 corrects for the average fatty acid mass that is lost during saponification.

Quantification of cell size
ab and cd T cells were isolated by cell sorting as described in the quantification of cholesterol content. Diameters of single cells were measured by Multisizer IV Coulter Counter (Beckman Coulter).

PCR array
Splenocytes were isolated from C57BL/6J mice. Untouched T cells were purified by Pan T Cell Isolation Kit II (Miltenyi Biotec), and were then separated into ab and cd T cells by TCRc/d + T Cell Isolation Kit (Miltenyi Biotec). Spleens from 6 mice were pooled per sample. The purity of cells was assessed by flow cytometry and was above 90%. Total RNA was collected with the RNeasy Plus Micro Kit according to the manufacturer's protocol (Qiagen). RNA purity and quantity was measured with a nanodrop spectrophotometer (Thermo Scientific). 150 ng RNA was used to for cDNA synthesis with RT 2 First Strand cDNA Kit (SABiosciences). Gene expression was detected using Lipoprotein Signaling & Cholesterol Metabolism PCR Array (PAMM-080, SABiosciences). Relative gene expression was analyzed using DDC t based fold-change calculations. Expression level of ab T cells was set as 1.

Quantitative RT-PCR
Magnetic beads purified ab and cd T cells from C57BL/6J splenocytes and total RNA extraction were stated above. Spleens from 3-4 mice were pooled per sample. 100 ng RNA was used for cDNA synthesis with iScript cDNA Synthesis Kit (Bio-Rad). mRNA expression was measured in real-time quantitative PCR using the following predesigned TaqMan gene expression assays (Applied Biosystems): Lcat (Mm00500505_m1), Trerf1 (Mm00553936_m1). Samples were assayed on a LightCycler 480 Real-Time PCR System (Roche). Threshold cycles (CT) were determined by an in-program algorithm assigning a fluorescence baseline based on readings prior to exponential amplification. Fold change in expression was calculated using the DDC t method with Gapdh as an endogenous control. Expression level of ab T cells was set as 1.

Confocal microscopy imaging
ab and cd T cells were isolated from female C57BL/6J mice using magnetic beads as stated in the PCR array section above. Cells were stained with Alexa Fluor 488-conjugated Cholera toxin subunit B (1/1000 dilution; Molecular Probes), washed twice with PBS, and spun unto glass slides. Slides were imaged with a FluoView FV10i confocal laser-scanning microscope (Olympus).

In vitro cholesterol loading and depletion
C57BL/6J splenocytes were stimulated with Dynabeads mouse T-activator CD3/CD28 beads (Invitrogen) at 1 bead: 1 cell ratio in the absence or presence of 1 mM methyl b-cyclodextrin (Sigma-Aldrich) to deplete cholesterol, or 10 or 20 mg/ml cholesterolloaded cyclodextrin (Sigma-Aldrich) to load cells with cholesterol for 2 or 4 hours. Cells were then removed from beads, stained for lipid rafts and activation markers, and analyzed by flow cytometry as described above.

In vitro T cell stimulation and proliferation assay
Splenocytes isolated from C57BL/6J mice were stained with 2.5 mM CFSE (Invitrogen) for 10 min at 37uC, and washed extensively. Cells were then plated at 5610 5 cells/well in a 96-well U-bottomed plate pre-coated with various concentrations of aCD3 antibody (BD Pharmingen). Soluble aCD28 antibody (BD Pharmingen) was added at 1 mg/ml. 24 hours later, cells were stained with surface markers, and CFSE dilution in ab and cd subsets was determined by flow cytometry. In some experiments, U0126 (Sigma-Aldrich) was added to cultures at 10 and 25 mM to inhibit ERK1/2 phosphorylation.

Statistical analysis
All results are expressed as mean 6 SEM. Results were analyzed by Student t test or ANOVA. Unpaired Student's t test and one-way analysis of variance were used for comparison of experimental groups. Statistical analysis is performed using GraphPad Prism software version 5.0b (GraphPad Software, Inc.). A probability value of less than 0.05 is considered significant.

Results
Cholesterol metabolism-related genes are differentially regulated in ab and cd T cells Cholesterol is essential for maintaining proper permeability and fluidity in the cell membrane, and its level is tightly regulated within the cell. T cell proliferation is strongly linked to cellular cholesterol level and the regulation of genes involved in cholesterol metabolism [17]. To investigate whether cholesterol metabolism is differentially regulated in ab and cd T cell subsets, we examined the expression profiles of cholesterol-related pathway genes in ab and cd T cells from C57BL/6J mice with a pathway-based microarray. The result showed that many genes involved in esterification (Acat1, 2 and Lcat) or utilization (Cyp39a1, Cyp46a1, Trerf1) of free cholesterol were upregulated in cd T cells. The expressions of Pcsk9, which downregulates LDLR posttranslationally [18], and Abca1, the main efflux transporter for cholesterol, were up-regulated in cd T cells as well (Fig. 1A). Genes relatively abundant and with significant fold-changes were confirmed in Taqman-based quantitative RT-PCR assays. Consistent with the PCR array results, the cd T cells had a 1.6 fold increase in Abca1, a 3.4 fold increase in Pcsk9, a 2 fold increase in Acat1, a 4 fold increase in Acat2, a 2 fold increase in Lcat, and a 2.1 fold increase in Trerf1, compared to ab T cells (Fig. 1B). These array data suggest that cd T cells express effort to lower free intracellular cholesterol levels by up-regulating genes involving in sterol efflux, esterification or other utilization pathways, possibly in response to higher intracellular cholesterol content.
Cholesterol content and lipid rafts are differentially regulated in ab and cd T cells To investigate whether lipid and cholesterol levels are different in ab and cd T cells, we used Nile Red as a probe to detect neutral lipids (consisting mostly of cholesteryl ester and triglyceride [19,20]). cd T cells showed a significant increase in Nile Red staining compared to ab T cells, indicating that cd T cells contained more neutral lipid ( Fig. 2A). We next specifically measured cholesterol levels in the two subsets. Resting ab and cd T cells were purified from C57BL/6J mice, and the intracellular cholesterol content in each subset was measured by gas chromatography. Interestingly, we found that the total intracellular cholesterol content was significantly higher in cd T cells (0.3960.03 vs 0.2260.00 mg/10 6 cells in ab T cells, P,0.05). Levels of free cholesterol seemed higher in the cd T cells, but the difference did not reach statistical significance. Cholesteryl ester levels were also higher in cd T cells (0.1860.05 vs 0.0160.04 mg/ 10 6 cells in ab T cells, P,0.05) (Fig. 2B). The increase in cholesteryl ester content correlates with the increase in intracellular neutral lipids observed by Nile Red staining of cd T cells. Average cell diameter appeared to be the same for ab and cd T cells (6.3960.05 vs 6.4560.02 mm in ab T cells, P = 0.222), hence the increased intracellular cholesterol level was not due to a larger cell size of cd T cells (Fig. 2C).
Cholesterol is an indispensible component of membrane lipid rafts, which modulate TCR signaling by clustering proteins involved in TCR signaling. Perturbation of lipid rafts has been shown to interfere with T cell activation [21,22,23]. To learn whether the increase in cholesterol levels in cd T cells impacted their membrane lipid raft content, fluorescently-labeled CT-B, which binds to an essential raft-associated protein, GM1, was used to quantify lipid rafts on the plasma membrane [14,24]. Approximately 2-fold more CT-B bound to the plasma membrane of cd compared to ab cells, suggesting an increase in plasma membrane lipid raft content in cd cells (Fig. 2D). Microscopic images of CT-B staining also clearly showed more aggregation of lipid rafts on the plasma membranes of cd cells (Fig. 2E). Aggregation of lipid rafts and raft-associated proteins is positively associated with TCR signaling and T cell activation [23]. Taken together, these data suggest that higher cholesterol content in cd cells results in increased membrane lipid raft content, which could in turn, change TCR signaling in the cd T cells.
Cholesterol depletion reduces the activated phenotype of cd T cells To confirm that the increased cholesterol content of cd cells results in enrichment of membrane lipid rafts, which in turn would likely impact TCR signaling and activation, we manipulated cholesterol levels in ab and cd T cells and examined their lipid raft content and activation status in vitro. Examination of surface markers shows cd T cells possess an activated status in vivo (increased percentages of CD44 hi CD62L 2 and CD69 + cells) (Fig. 3A-B). The enhanced activation of cd T cells again suggests that these cells are more ''primed for action'' at baseline or resting.  ab and cd T cells from C57BL/6J spleen were stimulated with aCD3/CD28 antibodies in the absence or presence of methyl bcyclodextrin (MbCD) to remove cellular cholesterol [25]. Lipid raft content and T cells activation markers were assessed by flow cytometry. Compared to ab T cells, cd T cells have a higher lipid raft content in the absence of MbCD (P,0.05). In cells treated with MbCD, the lipid raft content was lowered to similar levels in both ab and cd T cells (Fig. 3C). After incubation with MbCD for 4 hours, we observed a reduction in percentages of CD44 hi CD62L 2 cd cells, but not ab cells (Fig. 3D). With 4 hours of MbCD treatment, we found a slight but not significant reduction of early T cell activation marker CD69 (Fig. 3E). However, at 2 hours of cholesterol depletion, CD69 + cells were significantly lowered in only the cd cells (P,0.01) (Fig. 3E, inset). We reasoned that since CD69 is one of the earliest expressed markers in T cell activation [26], its expression pattern may reflect T cell activation status early upon stimulation. We also used cholesterol-loaded cyclodextrin to enhance cellular cholesterol content in ab and cd T cells [27,28]. Interestingly, we found that the percentage of CD44 hi CD62L 2 cells was significantly increased in ab T cells in a dose-dependent manner with cholesterol addition. On the other hand, activated cd T cells were gradually reduced with excess cholesterol, possibly due to oversaturation of membrane cholesterol (Fig. 3F). The fact that ab T cells, which had less cellular cholesterol at baseline, became activated with cholesterol loading supports the idea that increased cholesterol content leads to the primed phenotype in T cells.
cd T cells are more proliferative and show enhanced basal ERK1/2 phosphorylation cd T cells play a critical role in the initial stages of various pathophysiological conditions. Compared to conventional ab T cells, cd T cells respond much faster when faced with microbial and tumor invasions [4,29]. Indeed, we found a significant increase in BrdU incorporation in cd T cells (2.6760.09% of total cd + ) from C57BL/6J mouse spleen compared to ab T cells (0.7960.02% of total ab f+ , P,0.001) (Fig. 4A). Together with the increased expression of activation markers (Fig. 3A-B), our data suggesting that cd T cells possess a more ''primed for action'' status at baseline in the absence of any external stimulation.
Next, we wanted to investigate the molecular mechanisms by which cholesterol stimulates cd T cell activation. Because the accumulated cholesterol favors lipid raft formation, which could modulate TCR signaling, we first considered the possibility that cd T cells had a lower TCR threshold [23]. To examine this hypothesis, we stimulated resting ab and cd T cells with a fixed concentration of soluble aCD28 and an increasing concentration of plate-bound aCD3 antibodies, and measured proliferation using CFSE dilution [14]. We found that cd T cells are more proliferative at the higher aCD3 concentration (Fig. 4B, top). However, when the log scaling of aCD3 concentration versus percentage of maximum response was plotted to determine the EC 50 values, we did not see a significant difference in TCR threshold between ab and cd T cells (Fig. 4B, lower). Our data sugest that an enhanced TCR signaling strength, but not a lower TCR threshold, likely accounts for the cholesterol-induced activation and proliferation in cd T cells. We also noticed that with no or lower levels of aCD3 stimulation, membrane CD3 expression was significantly higher in cd T cells compared to ab T cells. This again indicates a stronger TCR signaling in the cd T cells basally or with low levels of stimulation. However, the expression of CD3 was downregulated and gradually became indifferent in both ab and cd T cells under stronger stimulation (Fig. 4C) To determine whether basal TCR signaling is indeed increased in the cd T cells, we stimulated purified resting ab and cd T cells with aCD3 and aCD28 antibodies, and examined the phosphorylation status of ERK1/2, a well-studied TCR downstream effector for proliferation [30,31]. We found an increase in basal ERK1/2 phosphorylation in unstimulated cd T cells, compared to almost no basal phosphorylation in ab T cells (Fig. 4D). The enhanced ERK1/2 phosphorylation likely accounts for the increased proliferation and activation of cd T cells in the resting status ( Fig. 3A-B, 4A).
Enhanced TCR signaling and ERK1/2 phosphorylation lead to T cell hyperplasia. To confirm that enhanced ERK1/2 signaling accounts, at least in part, for the hyperproliferative phenotype of cd T cells, we performed an in vitro proliferation assay in the presence of an ERK1/2 inhibitor, U0126. Indeed, U0126 incubation significantly inhibited cd T cell proliferation in a dose-dependent manner. However, under the same condition, the overall growth inhibition effect was not significant in the ab T cells (Fig. 4E). The results provide further evidence that enhanced ERK1/2 signaling contributes to the hyperproliferative phenotype of cd T cells.

Discussion
ab and cd T cells share many common features, yet are functionally and metabolically distinct. For example, expression of activation markers, cytokines, and TCR signaling pathways are very similar in the two subsets [32,33,34]. However, cd T cells are negative for both CD4 and CD8, and are MHC-independent in antigen recognition. Their rapid responses upon antigen encounter also distinguish them from ab T cells. cd cells are thought to be involved in innate immunity whereas ab cells are involved in adaptive immunity [2,3,4]. To date, very little is known about how antigen-naïve cd T cells hold the ''primed for action'' status and are able to become activated and proliferative in such a short period of time. In this report, we provide novel insights for the distinct phenotypes and differential regulation of ab and cd T cells. We showed that, in accordance with previous reports [35,36], cd T cells proliferate faster and are more activated than ab T cells at resting conditions (Fig. 3A-B and 4A). We found that cd T cells have significantly higher intracellular neutral lipid and cholesterol content ( Fig. 2A-B). Furthermore, we found that the increased cholesterol content in cd T cells results in an increase in lipid raft formation (Fig. 2D-E), which favors TCR clustering and signaling. The enhanced TCR signaling is demonstrated by elevated ERK1/2 phosphorylation, proliferation, and activation marker expression in cd T cells (Fig. 3A-B, 4A, D). Thus, cd T cells are kept in a ''primed for action'' status at a resting state, and are ready to undergo rapid proliferation and activation. We show that cholesterol, at least in part, modulates their function.
One of the best-studied functions of lipid rafts is the regulation of TCR signaling. By clustering TCR molecules and their associated factors, lipid rafts provide a concentrated and stable signaling micro-domain for TCR-dependent T cell activation [22,23,37]. Cholesterol, a key component of lipid rafts, is indispensible for TCR-dependent functions, and the modulation of cholesterol levels tightly correlates with the strength of TCR signaling. For example, cholesterol depletion of lymphocytes results in reduced raft domains and subsequent dysregulated T cell signaling [38]. On the other hand, a recent study showed that enriching membrane cholesterol by squalene enhances the responses of CD4 + T cells in vivo [13]. The hyper-activation of T cells in the autoimmune disease systemic lupus erythematosus is likely caused by high cholesterol-induced lowering of the TCR threshold [21]. Due to the increase in cholesterol content in cd cells, we initially hypothesized that their hyper-active phenotype was caused by a lowered TCR threshold [23]. However, to our surprise, we found that the TCR thresholds were similar in ab and cd T cells in vitro (Fig. 4B). The higher membrane CD3 expression of cd T cells under no or low stimulation suggested an enhanced basal TCR strength in these cells (Fig. 4C). The enhanced TCR signaling is confirmed by increased level of ERK1/2 phosphorylation in cd T cells at the resting state (Fig. 4D). Thus, our data supports the model that the increased intracellular cholesterol content favors lipid raft formation and enhanced TCR signaling strength in cd T cells. Our data also provide direct evidence linking high intracellular cholesterol level to the activation of cd T cells. MbCD-mediated cholesterol depletion lowers the lipid raft content of cd to the same level as ab T cells, and significantly attenuated the expression of activation markers in cd T cells (Fig. 3C-E). On the other hand, increasing cellular cholesterol enhanced the activation status of ab T cells (Fig. 3F). The cholesterol depletion and loading studies support the notion that cellular cholesterol content directly and positively impacts T cell activation.
Our data provides novel evidence that elevated cellular cholesterol content contributes to rapid proliferation and activation of cd T cells, which are well-recognized hallmarks of these cells [35]. However, the mechanism by which cholesterol accumulates in cd T cells requires further investigation. More studies are underway to explore what factors/signaling pathways are involved in this process. cd T cells are potent producers of The difference in expression of early activation marker CD69 was not statistically significant in both ab or cd T cells after MbCD treatment for 4 hours. However, the reduction of CD69 was significant in cd T cells at 2 hours (inset) Results were averaged from 8 mice. Results were shown in mean 6 SEM. (F) C57BL/6J splenocytes were stimulated with T-activator CD3/CD28 beads in the presence or absence of 10 and 20 mg/ml cholesterol for 4 hours. The percentage of CD44 hi CD62L 2 cells was significantly increased in ab T cells in a dose-dependent manner of cholesterol addition. On the other hand, activated cd T cells were reduced with excess cholesterol. Results were averaged from 8 mice and shown in mean 6 SEM. * P,0.05; ** P,0.01; *** P,0.001; ns not statistically significant. doi:10.1371/journal.pone.0063746.g003 effector cytokines such as IL-17 and IFNc [39]. The fate of IL-17or IFNc-producing subsets is pre-determined by CD27 and other factors such as TGFb in the thymus [36,40,41]. The two subsets are differentially regulated and respond to different stimuli [42,43]. For example, antigen-naïve cd T cells predominantly produce IL-17 for neutrophil recruitment and resolution of early infection. On the other hand, antigen-experienced cd T cells produce IFNc at the site of infection [44]. Particularly, cd T cells are found to be the major source of IL-17 in naïve mice, and IL-17 production by cd cells can be further modulated in various pathological conditions including atherosclerosis [36,40,43,45,46]. Although cd cells have not been directly studied in atherosclerosis, given that atherosclerosis is driven by both cholesterol and T cells, It would be interesting to study whether cholesterol regulates cd subset determination, and whether cd T cells play a pivotal role in atherogenesis. For instance, increased cholesterol levels may favor the development of a specific cd subset or further regulate its responsiveness to stimuli. We plan to investigate whether the , and then stimulated with 0-10 mg/ml plate-bound aCD3 and 1 mg/ml soluble aCD28 for 24 hours. CFSE content in cells was measured by flow cytometry and graphed against aCD3 concentrations. Results were averaged from 5 mice, and shown in mean 6 SEM. Lower: Log scaling of aCD3 concentration against % of maximum response was used to calculate EC 50 . (C) CD3 expression in cd T cells was higher than in ab T cells under no or lower stimulation, but decreased more dramatically with stronger stimulation. The experimental condition was the same as in (B). (D) Western blotting shows high levels of phosphorylated ERK1/2 in cd T cells, relative to ab T cells, at baseline. ab and cd T cells were isolated from the same set of C57BL/6J mice via FACS sorting. Cells were either untreated, or incubated with aCD3 and aCD28 antibodies (20 mg/ml each) for 5 minutes and then with anti-hamster IgG (20 mg/ml) for additional 2 minutes at 37uC. Cells were lyzed, separated by SDS-PAGE, and immunoblotted with indicated antibodies. The image was representative of 3 independent experiments. (E) Inhibition of ERK1/2 significantly lowers proliferation in cd T cells in vitro. Splenocytes from C57BL/ 6J mice were labeled with CFSE (2.5 mM), and then stimulated with 1 mg/ml plate-bound aCD3 and 1 mg/ml soluble aCD28 for 24 hours, in the absence (vehicle) or presence of 10 mM and 25 mM U0126. CFSE dilution in cells was measured by flow cytometry, and used to calculate cell proliferation. Results were averaged from 7-8 mice, and shown in mean 6 SEM. ** P,0.01; *** P,0.001; ns not statistically significant. doi:10.1371/journal.pone.0063746.g004 development of cd T cell subsets, as well as the effectiveness and activation of these subsets are affected by elevating cholesterol levels in vivo.
In sum, cd T cells are more proliferative and activated than ab cells in vivo, suggesting cd cells are at a more ''primed for action'' status. The increased cholesterol content of cd T cells suggests that cd are able to reach the cholesterol checkpoint faster to allow for rapid proliferation and activation [17]. In addition, increased cholesterol levels in the cell resulted in a higher lipid raft content, which promoted cd T cell activation. Cholesterol depletion experiments confirmed that both lipid raft content and activation markers were perturbed in cholesterol-depleted cd T cells, thus further proving that intracellular cholesterol levels are key to cd activation. cd T cells were shown to have the same TCR threshold as ab T cells. However, ERK1/2, the key downstream effector of TCR signaling, is heavily phosphorylated in unstimulated cd T cells, and ERK1/2 inhibition effectively reduced cd T cell proliferation, suggesting that the ''primed for action'' phenotype of cd T cells is the result of enhanced TCR signaling strength due to increased cholesterol content. Taken together, our data provide a novel mechanism by which cholesterol differentially regulates the physiology of ab and cd T cell subsets.