CD46 Activation Regulates miR-150–Mediated Control of GLUT1 Expression and Cytokine Secretion in Human CD4⁺ T Cells Free

T helper type 1 cells are proinflammatory effector cells characterized by their production of IFN-γ and expression of transcription factor T-bet (1), and they function to protect the host against intracellular pathogens. However, excessive Th1 responses can lead to pathological tissue damage, and inappropriately induced or unregulated Th1 responses can contribute to autoimmunity. The role of IL-10 in negative regulation of Th1 responses has been highlighted by lethal outcomes of IL-10 knockout or neutralization in toxoplasma or trypanosome mouse infection models, associated with hyperproduction of type I cytokines (2, 3). In these infections, IL-10 is predominantly produced by Th1 cells, and blocking this negative feedback leads to increased pathogen clearance, but also to immune-mediated damage to the host (4–6). In mice, IL-10 secretion from Th1 cells can be induced following repeated Ag stimulation (7), and a similar Th1/IL-10 negative feedback also develops in humans after repeated Ag exposure (8). IL-12–induced STAT4 activation (9) and subsequent IFN-γ and IL-2 (10) production are prerequisites for optimal development of these regulatory Th1 cells, ensuring that Th1-derived IL-10 negative feedback only develops during or after the peak of the response, therefore contributing to contraction.

A mechanism for induction of IL-10 secretion from human Th1 cells has been found with the discovery that CD46 (MCP) acts as a T cell costimulatory receptor (11) and is crucial for Th1 induction and production of the proinflammatory cytokine IFN-γ (12). Although essential for this initial induction of proinflammatory Th1 responses, CD46 stimulation in the presence of high IL-2 also mediates the subsequent coinduction of IL-10 in Th1 cells and their “switch” into a (self)regulatory phenotype (13). It has been hypothesized that the CD46-driven induction of IL-10 may be one strategy behind the targeting of CD46 by pathogens such as Neisseria meningitidis and measles virus (14). CD46 is expressed on all human nucleated cells but is absent in murine somatic tissues (15), meaning that murine-based immunological studies do not contribute to the understanding of the role of CD46 in human T cell responses. As a complement inhibitor, CD46 binds C3b and acts as cofactor for its inactivation by the serine protease factor I. C3b can be generated in an autocrine fashion by activated T cells, and it has also been identified as a ligand for the T cell costimulatory function of CD46 (13, 16). This activation-associated T cell–derived generation of C3b, as well as dependence of human CD28-costimulated Th1 responses on CD46/C3, leads to a model of autocrine CD46 signaling functioning downstream of CD28 during human Th1 cell activation.

The clinical importance of CD46-mediated regulation of Th1 responses is supported by the altered expression of CD46 isoforms in T cells from multiple sclerosis patients (17), and by the failure of T cells from patients with rheumatoid arthritis to develop the full IL-10–secreting regulatory phenotype upon sustained CD46 costimulation, compared with healthy controls (10). It is therefore of interest to investigate and potentially therapeutically harness the mechanisms by which this mode of immunoregulation functions. The downstream molecular effector pathways are still incompletely mapped, and we have focused on CD46-mediated alterations in microRNA (miRNA) expression. miRNAs have important roles as regulators of immune cell differentiation and function (18) and, more specifically, have been shown to affect T cell regulation, development, signaling, and metabolism (19–21). Several miRNAs have high specificity of expression in lymphocytes, and their expression is required for normal lymphocyte function. We found that CD46 signaling in CD4⁺ T cells leads to a strong reduction in miRNA (miR)-150 levels, and we then identified miR-150 targets, which include regulators of T cell metabolism and cytokine secretion. Furthermore, miR-150 is required for IL-10 secretion from CD46-stimulated Th1 cells. We therefore highlight the role of miR-150 in CD46-induced Th1 activation and regulation.

All primary cells were purified from fresh peripheral blood collected from healthy volunteers according to the permission of the local Ethics Committee in Lund and with informed, written consent. Blood was taken using EDTA-coated vacuum tubes, diluted in PBS EDTA at room temperature, and PBMCs were purified using Lymphoprep (Axis Shield) according to the manufacturer’s instructions. CD4⁺ T cells were then purified using positive selection magnetic cell sorting (Miltenyi Biotec) and purity (>95%) was verified by staining with allophycocyanin-labeled anti-CD4 (ImmunoTools). Cells were washed and resuspended in RPMI 1640 (Invitrogen) with 10% FCS and 50 U/ml IL-2 (Immunotools), and 3.5 × 10⁵ cells were plated out per well in 48-well plates and coated overnight with 2 μg/ml anti-CD3 (OKT3, BD Biosciences) and 2 μg/ml of either anti-CD28 (CD28.1, BD Biosciences) or anti-CD46 (Tra2-10, Sheffield University hybridoma biobank, Sheffield, U.K.).

Anti–CD25-FITC, anti–CD69-allophycocyanin, and anti–CD46-PE (ImmunoTools) were used to assess CD Ag expression. Fc-CD46 and Fc-CD55 were expressed in Chinese hamster ovary cells and purified on protein A columns as described in Harris et al. (22). For flow cytometry of purified T cells, viable cells were gated by exclusion of cells stained with fluorophore-labeled annexin V (ImmunoTools).

Cytokine secretion was measured using Miltenyi Biotec flow cytometry cytokine capture kits for IL-10 and IFN-γ, according to the manufacturer’s instructions. Alternatively, cytokines in supernatant were measured by ELISA (PeproTech/Mabtech).

RNA was purified using an miRNeasy Micro kit (Qiagen). RNA integrity was verified by automated electrophoresis (Experion RNA analyzer, Bio-Rad), and purity was assessed by 260:230 and 260:280 nm absorbance ratios. Cut-offs of 1.95 for 260:280 nm and 1.4 for the 260:230 nm ratios were used, as recommended by the microarray supplier.

T cells were collected from four healthy donors using heparin anti-coagulant, diluted with equal amounts of PBS EDTA, then layered over Lymphoprep and spun to collect PBMCs. CD4⁺ T cells were purified and plated out for stimulation as described above. Purity was assessed by flow cytometry and was in all cases ≥95%. RNA was purified from a set of unactivated cells before plating, to represent resting, for 0 h time point samples. For early activation samples, CD28 or CD46 costimulated cells were harvested 2 h after plating. After 36 h, the remaining CD46-costimulated cells were harvested and labeled for IL-10 or IFN-γ secretion using Miltenyi Biotec cytokine secretion kits. Cells were sorted into separate cytokine-secreting populations (IFN-γ⁺/IL-10^–, IFN-γ⁺/IL-10⁺, and IFN-γ^–/IL-10⁺) using a BD Biosciences FACS machine.

RNA from separate populations was labeled using the Hy3 miRNA power labeling kit (Exiqon) and hybridized to an locked nucleic acid (LNA)-based miRCURY miRNA microarray slides (Exiqon, version 11) using a Maui hybridization chamber (BioMicrosystems). Spot signals were acquired with an Agilent array scanner and GenePix Pro 4.1 was used for annotation (mirBase 14.0), signal processing, and quantification using default background subtraction. The arrays were normalized using the global locally weighted scatterplot smoothing (Lowess) regression algorithm. To determine expression changes, significant analysis of microarrays (23) was performed as implemented in MeV v4.3 for multi-class without using the fold-change criterion (24). Briefly, each four treatment replicates were grouped together, forming six classes, and a δ value of 0.102 corresponding to a strict mean false discovery rate of 0% was set. Validation of significant miRNA expression changes was performed as described below. Normalized and preprocessed array data were deposited to ArrayExpress (https://www.ebi.ac.uk/arrayexpress/) with accession no. E-MTAB-2658.

Array validation and mRNA/miRNA quantification were carried out using specific assays from Applied Biosystems, according to the manufacturer’s instructions. For mature miRNA quantification, cDNA was produced using miR-specific hairpin primers, then quantitative PCR (qPCR) was run using an Applied Biosystems 7900HT qPCR machine. β₂-Microglobulin, cyclophilin A, and TATA-box–binding protein were used as mRNA endogenous controls, whereas RNU-44 and RNU-38B were used for miRNA endogenous controls. Normalization using geometric means of multiple endogenous controls were performed according to the geNorm method (25). Relative expression levels of genes were compared using one-way or two-way ANOVA.

T cells were washed in PBS, resuspended at 2 × 10⁶/ml in PBS with 0.2 μM CFSE (Sigma-Aldrich) at 37°C for 15 min, and then washed twice in culture medium before resuspension and plating for activation.

Purified CD4⁺ cells were transfected using a nucleofector 4D device (Lonza) using a primary cell kit with 100 nM of miR-150-specific LNA antagomiR (Exiqon), or control LNA. Cells were then rested overnight in 96-well plates in 10% RPMI 1640 before plating for activation as described above.

The 3′ untranslated regions (UTRs) of c-Myb (residues 1–1163) and solute carrier family 2, facilitated glucose transporter membrane (1SLC2A1; residues 11–854) were cloned into the pMIR-REPORT vector (a gift from Dr. Yvonne Ceder), which produces a firefly luciferase mRNA transcript with the cloned 3′ UTR. Human CD4⁺ T cell cDNA was used as a template for amplification of the c-Myb UTR using forward and reverse primers 5′-GCCGGCGACATTTCCAGAAAAGCATTATG-3′ and 5′-AAGCTTCCCAAATTACCAAGAACC-3′, respectively, and for amplification of the SLC2A1 UTR using forward and reverse primers 5′-GAGCTCATCACCAGCCCGGCCTG-3′ and 5′-GTTTAAACGTAAGTAACAGGAGTGAGGTG-3′, respectively. The amplicons contained all described miR-150 binding sites within the c-Myb UTR and the conserved miR-150 site in SLC2A1. PCR products were gel purified and cloned using the StrataClone Blunt cloning system (Agilent Technologies), amplified in DH5a cells, and then the c-Myb amplicon was removed by NaeI and HindIII double digestion, purified from the gel, and ligated into pMIR-REPORT opened with PmeI and HindIII digestion to create the pMIR c-Myb plasmid. The SLC2A1 insert was released by SacI and PmeI double digestion, purified from the gel, and then ligated into pMIR-REPORT opened by digestion with SacI and PmeI to create the pMIR–glucose transporter 1 (GLUT1) plasmid. Mutation of the SLC2A1 miR-150 binding site was then carried out using the QuikChange Lightning site-directed mutagenesis kit (Agilent Technologies), using forward and reverse primers 5′-CTGAGACCAGTTCGGTGCACTGGAGTG-3′ and 5′-CACTCCAGTGCACCGAACTGGTCTCAG-3′. Sequences of all UTR inserts were verified by Sanger sequencing (GATC Biotech). To perform luciferase assays, HEK293 cells were plated in 24-well plates for 24 h before cotransfection with 20 ng Renilla luciferase expression vector (a gift from Dr. Yvonne Ceder), 100 ng pMIR-REPORT plasmid, and either miR-150 mimic or negative control miRNA mimic (Dharmacon) to a final concentration of 50 nM, using DharmaFECT Duo transfection reagent (Dharmacon). After 24 h, cells were lysed and Renilla/firefly luciferase expression was assessed using the Dual-Glo luciferase assay (Promega) and a Wallac Victor² fluorometer (PerkinElmer).

cDNA oligomers consisting of human pre–miR-150, flanked by 30 additional nucleotides from the pri-miRNA sequence and BamHI/XhoI sticky ends, were synthesized (Eurofins MWG Operon) and hybridized together in hybridization buffer (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA). Phosphorylation at the 5′ end was carried out using polynucleotide kinase (Invitrogen) before the dsDNA was ligated into the pcDNA3 expression vector opened with BamHI/XhoI (Fermentas) and verified by sequencing. Jurkat cells were transfected using the 4D-Nucleofector machine and stable clones were selected using G418 (Life Technologies).

For Western blot analyses, cell lysates were separated using 10% SDS-PAGE gels under reducing conditions, transferred to polyvinylidene difluoride membranes with the Trans-Blot Turbo system (Bio-Rad), and membranes were blocked using fish gelatin (Nordic Naturals). Anti–c-Myb (Millipore) and anti–β-actin (Abcam) were used in combination with goat anti-mouse HRP secondary Ab (Dako), whereas anti-GLUT1 (Millipore) was used in combination with goat anti-rabbit HRP (Dako), and proteins were visualized using ECL reagents (Santa Cruz Biotechnology) and a ChemiDoc CCD camera (Bio-Rad). The small RNA Northern blot protocol was adapted from Kim et al. (26). Briefly, 100–500 ng total RNA was separated in denaturing urea 15% PAGE gels in TBE buffer before electrophoretic transfer to positively charged nylon membranes (Amersham Biosciences). RNA was cross-linked to the membrane in a UV chamber (Stratagene) and then blocked using ULTRAhyb (Ambion) for 1 h at 50°C. Double digoxigenin (DIG)–labeled LNA probe (1 nM, Exiqon) diluted in ULTRAhyb was incubated overnight at 50°C before the membrane was washed in 0.2× SSC buffer, followed by probing using anti–DIG-HRP Ab (Roche) in combination with DIG wash and block buffer kit (Roche) and detection by ECL reagents.

Jurkat cells were preincubated for 1 h in prewarmed PBS supplemented with 0.9 mM MgCl₂, 0.5 mM CaCl₂, and 25 mM glucose and then spun and resuspended at 2 × 10⁶/ml in 500 μl of the same solution spiked with 30 μCi/ml [³H]d-glucose (PerkinElmer) and incubated rotating at 37°C for 10 min. The incubation was then stopped by addition of ice-cold buffer and immediate washing. After three washes, the cell pellet was lysed and [³H]d-glucose uptake was counted using a beta counter (Wallac) and normalized to total protein content as assessed by BCA assay (Pierce). A control of uptake at a 0 time point was used by addition of [³H]d-glucose to cells on ice followed by immediate washing.

Studies of CD46 signaling have identified interactions of the intracellular domain of CD46 with SPAK kinase, α-E-catenin, and modulation of linker for activation of T cells activation and inducible cAMP early repressor/cAMP response element modulator nuclear translocation (10, 12, 27). However, CD46 signaling’s effect on miRNA expression, which is critical to normal T cell differentiation (28), is completely unexplored. Therefore, we investigated miRNA markers of T cell activation following CD46 activation. To study the molecular signatures engaged by CD46 activation of human CD4⁺ T cells, we first established the CD46-driven generation of proinflammatory IFN-γ⁺ and “switched” IL-10–secreting (self)regulatory Th1 cells. Peripheral blood CD4⁺ T cells were purified and stimulated in 48-well plates with plate-bound Abs against CD3 in combination with either CD28 or CD46. After 40 h of activation in the presence of IL-2, flow cytometry was used with cytokine capture staining reagents to quantify proportions of cells secreting IFN-γ, IL-10, or both cytokines (Fig. 1A). As previously described, CD46 costimulation leads to an increased proportion of cells that secrete IL-10, whereas CD28-costimulated cells lead to a measureable but substantially smaller population of IL-10⁺ cells (Fig. 1B).

To investigate the influence of miRNAs on CD46-induced cytokine secretion, we isolated the three cytokine-secreting populations that develop after CD46 stimulation (IFN-γ⁺, IL-10⁺, and an intermediate IFN-γ⁺IL-10⁺ population) (13) at 36 h and compared their miRNA profiles. To verify the integrity of the cell-sorted cytokine-secreting populations, mRNA coding for IFNG and IL-10 were quantified by qPCR (Fig. 1C). Additionally, the Th1 profile of the cells was verified by T-bet/GATA3 qPCR, which demonstrated that these cells were T-bet⁺ but GATA3⁻ (Fig. 1D). RNA was also purified from resting CD4⁺ T cells and either CD3 plus CD28– or CD3 plus CD46–stimulated T cells (2 h activation time). The miRNA expression profiles of these populations were then assessed using miRNA arrays covering >840 human miRNAs. Expression data of miRNAs that were differentially expressed between any of the populations are shown in Fig. 1E, and they mostly identified expression differences between the resting or early activated T cells, as well as cytokine-secreting cells, purified after 36 h. Interestingly, the most strongly upregulated miRNAs in the activated cytokine-secreting populations were mature products of pre–miR-886, which clustered together with activation marker miR-155. Pre–miR-886 has since been classified as a small noncoding RNA regulating activation of protein kinase R (29). We have since determined that this RNA is upregulated during both CD28 and CD46 costimulation (although much more strongly in response to CD46) and regulates protein kinase R activity in human T cells (A.M. Blom, L. Lind, M. Qayyam, and B.C. King, manuscript in preparation).

Array data showed strong upregulation of miR-155 and downregulation of miR-150 after CD46-mediated costimulation. To compare this effect with CD28, purified CD4⁺ T cells were stimulated with either CD3/CD28 or CD3/CD46 and subsequent miRNA expression was evaluated by qPCR. miR-155, which is upregulated in activated lymphocytes and is necessary for their normal function (30), was more highly upregulated in cells costimulated with CD46 in comparison with CD28 (Fig. 2A), whereas miR-150 was more strongly downregulated in CD46-costimulated cells (Fig. 2B). A known target of miR-150 is the transcription factor c-Myb (31), and in negative correlation to miR-150 downregulation, c-Myb was most strongly upregulated in CD46-stimulated T cells (Fig. 2C), showing a functional outcome of stronger miR-150 downregulation after CD46 costimulation.

miR-155 upregulation is strongly associated with T cell activation, and we therefore investigated whether CD46 costimulation led to increased activation compared with CD28. Cell activation was assessed by measurement of CD25 (the high-affinity IL-2R α-chain) and CD69 expression after overnight stimulation, demonstrating that CD46 costimulation resulted in higher proportions of activated cells (Fig. 2D, 2E). However, despite increased levels of miR-155, we did not detect measurable decreases in the negative regulators of T cell activation, CTLA4, or suppressor of cytokine signaling 1, which have been described as targets of miR-155 (32, 33) (Fig. 2F). Both of these genes are upregulated during activation, and CTLA4 upregulation was significantly higher after CD46 compared with CD28 costimulation, also reflecting the greater costimulatory potential of CD46. Additionally, a significantly lower number of CD4⁺ T cells remained undivided 90 h after CD28 costimulation as compared with CD46 costimulation (Fig. 2G). Collectively, these data support a significantly stronger costimulatory potential of CD46 when compared with CD28. This was not due to the preferential CD46-mediated activation and expansion of a specific T cell subset, because CD46 costimulation also resulted in increased CD25 upregulation in purified naive CD4⁺ T cells, compared with CD28 (Fig. 2H). Titration experiments show that this is not due to a difference in affinity of the anti-CD28 and -CD46 Abs (data not shown).

T cell activation results in rapid proliferation, and costimulation is a requirement for the associated upregulation of glucose metabolism that is needed for increased energy expenditure (34). CD28 activation in mice, and particularly CD46 costimulation in human CD4⁺ T cells, has been shown to be critical in the upregulation of the high-affinity cell surface GLUT1, encoded by the SLC2A1 gene, leading to a subsequent increase in glycolytic glucose consumption (35, 36). We have already identified that CD46 gives a stronger costimulatory signal in terms of the outcomes of activation and proliferation, and as such it is expected that there would also be a greater energy requirement. Positions 573–580 of the 3′ UTR of SLC2A1 mRNA contains an 8-mer match to the seed sequence of miR-150, which is conserved among several mammalian species, including chimpanzee and rhesus macaque (but not rat or mouse), and we therefore investigated whether the greater miR-150 downregulation associated with CD46 stimulation may result in enhanced upregulation of GLUT1 expression.

We generated stably transfected Jurkat clones that overexpress mature miR-150, as confirmed by Northern blot (Fig. 3A). miR-150 expression was within physiological levels as assessed by qPCR (not shown), and it was functional as confirmed by 52 ± 19% downregulation of c-Myb protein in these cells (Fig. 3B; quantified in Fig. 3C). Compared to cells carrying the empty vector alone, Jurkat cells expressing miR-150 had significantly lower levels of both the SLC2A1 transcript (Fig. 3D) and the encoded GLUT1 protein (Fig. 3E; quantified in Fig. 3F, 56 ± 27% downregulation). These same cells were also less efficient at glucose uptake in comparison with controls (Fig. 3G), verifying the functional significance of the measured GLUT1 downregulation.

To confirm the active target site of miR-150, the 3′ UTR of SLC2A1 was cloned into a luciferase reporter vector, which was then subjected to targeted mutagenesis to alter the seed site and disrupt miR-150 binding (Fig. 4A). We found that although miR-150 caused a trend for increased luciferase signal in the control reporter vector, it significantly reduced the amount of luciferase signal for the transcript carrying the SLC2A1 3′ UTR (Fig. 4B). The positive control miR-150 target, carrying the c-Myb 3′ UTR, was more strongly downregulated, possibly due to this sequence carrying multiple miR-150 seed sites (31). Mutating residues within the SLC2A1 miR-150 seed binding site prevented regulation by miR-150, returning luciferase signal to that of the control, confirming the identified miR-150 binding site. Consistent with miR-150 regulation of SLC2A1 translation, CD46 costimulation leads to a greater upregulation of SLC2A1 transcript (Fig. 4C), compared with CD28 costimulation. Finally, nucleofection of primary human CD4⁺ T cells with miR-150–locked nucleic acid (LNA) antisense oligomers also resulted in a significant increase in the SCL2A1 mRNA, as detected by qPCR (Fig. 4D). Taken together, these results confirm miR-150 regulation of the SLC2A1 transcript in human T cells.

Previous experiments have shown that soluble forms of CD46 can compete with cell surface CD46 for activating ligands and impede CD46-mediated T cell signaling (10). To investigate physiological roles of autocrine CD46 signaling, we inhibited CD46 ligation during T cell activation (usually mediated by T cell–derived C3b or Jagged1 expression) by administration of a soluble Fc-CD46 fusion protein. Addition of Fc-CD46 to Jurkat cells blocked Ab binding of cell surface CD46 (Fig. 5A), demonstrating the ability of Fc-CD46 to competitively inhibit binding of ligands. Treatment of T cells during CD3 and CD28 stimulation with Fc-CD46, but not Fc-tagged CD55 (another complement regulator expressed on T cells), reduced the upregulation of activation marker CD25 (Fig. 5B), confirming that auto- or juxtacrine CD46 signaling is involved downstream of CD28 costimulation during T cell activation. Having already established that CD46 signaling leads to accelerated downregulation of miR-150 expression, we also confirmed that addition of Fc-CD46 as a soluble competitor of autocrine CD46 signaling prevented miR-150 downregulation (Fig. 5C). We then assessed expression of the miR-150 target transcripts c-Myb and SLC2A1 in CD4⁺ T cells activated via CD3/CD28 in the presence of the blocking Fc-CD46. After overnight activation, cells treated with Fc-CD46 had lower expression of c-Myb (Fig. 5D) and SLC2A1 (Fig. 5E) transcripts, consistent with a higher miR-150 level caused by interruption of autocrine CD46 signaling. These data show the importance of autocrine CD46 signaling during CD28-costimulated T cell activation, and they imply a role for CD46-induced regulation of miR-150 targets downstream of CD28. Blocking of CD46 signaling by addition of Fc-CD46 also inhibited secretion of IL-10, but not IFN-γ, from cells stimulated during 36 h by anti-CD3 and anti-CD28 (Fig. 5F), consistent with the known role of CD46 in induction of Th1 IL-10 secretion downstream of CD28 (13). We therefore instigated further investigation of the function of CD46-mediated signaling, and in particular downstream miRNA expression, in the cytokine-secreting subpopulations of CD46-activated Th1 T cells.

The array data were analyzed to identify miRNAs, which were differentially expressed between the three CD46-induced cytokine-secreting populations (Fig. 6A). Differential expression of miR-150 was verified by qPCR (Fig. 6B), revealing a significantly increased expression in the regulatory IL-10⁺ cells compared with IFN-γ⁺ and IFN-γ⁺/IL-10⁺ cells. To assess the function of elevated miR-150 in IL-10⁺ feedback, we used a specific LNA inhibitor. After transfection of primary T cells with miR-150 antisense LNA, there was a statistically nonsignificant trend for reduction of IL-10 mRNA in cells stimulated for 60 h via CD3 and CD46 (Fig. 6C). However, this translated into a significant reduction in IL-10 protein secretion as measured by ELISA, whereas IFN-γ secretion was unaffected (Fig. 6D). This was confirmed in separate time course experiments (Fig. 6E). Overall activation of cells, as measured by CD69 upregulation, was not affected (Fig. 6F), nor was proliferation (not shown). These results identify miR-150 as a positive regulator of IL-10 cytokine secretion in Th1 cells.

As miRNAs have now emerged as key players in the regulation of cellular functions, we have assessed whether CD46 costimulation impacts on this important immune-regulative system in T cells. Indeed, CD46-mediated signals not only induce a specific miRNA landscape early upon T cell activation, but the levels of several specific miRNAs are then subsequently modulated during the CD46-induced Th1 switching phase. CD46 has dual roles in T cell activation, being required both for initiation of Th1 responses and also for inducing regulation in the form of Th1-derived IL-10 secretion. We have found evidence for CD46-induced miRNA expression patterns that affect both of these phases of the Th1 T cell response. miR-150 is expressed most abundantly in lymphocytes, and it has an important role in regulating c-Myb expression in B cell development (31). c-Myb itself is a transcription factor with prolific binding sites across the genome (37), but it has been shown to interact with GATA3 in induction and maintenance of Th2 responses (38). We found c-Myb to be upregulated during T cell activation, in concordance to a general downregulation in miR-150. Regulation of gene expression by miRNAs is an area of growing complexity, with modes of action determined at transcriptional and posttranscriptional levels (39), in manners that may or may not affect targets at the mRNA level. The action of a miRNA on any specific target can also depend on the relative levels of competing endogenous target mRNAs (40), as well as the expression of noncoding regulatory “sponge” RNAs, which bind to and neutralize miRNAs in a sequence-specific manner (41). This therefore makes it hard to predict outcomes at the protein level from RNA data alone (42). This, in addition to the lack of a representative animal model for the immune functions of CD46, as well as ample evidence for its role in human disease (10, 12, 14, 43), makes the delineation of the molecular pathways of CD46 signaling in disease both an important and challenging task.

During activation, we found that CD46 is a potent costimulatory receptor, leading to more rapid upregulation of miR-155 compared with CD28 costimulation. miR-155 is necessary for normal immune function (30), and it becomes upregulated during activation. T cell activation leads to rapid growth and proliferation and therefore places a great metabolic demand on the cell. Glycolysis upregulation is a hallmark of T cell activation, and it is already known that costimulation is required for GLUT1 upregulation and an increase in glucose uptake, which is a limiting step in T cell activation (34, 35). GLUT1 is the most important T cell glucose transporter, having been found essential for both activation and effector function (44). It is also now known that CD46 signaling acts downstream of CD28 costimulation during T cell activation and is strictly required for initiation of Th1 responses (12). When we and others block signaling using soluble forms of CD46 that competitively inhibit interaction of cell surface CD46 with autocrine stimulatory ligands, T cell activation is diminished (45).

We have recently shown that increased glycolysis upon T cell activation, which is specifically required for Th1 induction (46), is dependent on CD46 signaling (36), and that CD28 activation alone is not sufficient to induce the high levels of GLUT1 (and linker for activation of T cells 1) expression, which mediate increased glucose and amino acid influx. Our finding that CD46 signaling leads to a rapid downregulation of miR-150 expression is tied into this by the finding that miR-150 targets the SLC2A1 transcript encoding the glucose transporter GLUT1. We have experimentally confirmed the conserved miR-150 seed site at positions 573–580 of the SLC2A1 3′ UTR and shown that the SLC2A1 transcript is more abundant in T cell populations with lower miR-150 levels, and that CD46 costimulation results in lower miR-150 expression and higher SLC2A1/GLUT1. Additionally, blocking autocrine CD46 costimulation limits miR-150 downregulation and resulted in decreased SLC2A1 levels and limited activation. This reveals a novel level of control of cellular metabolism by CD46 during T cell activation, via regulation of miR-150 expression, and, in turn, control of GLUT1-dependent glycolysis.

Cellular metabolism is thought to not only be important for T cell activation, but also subset differentiation (47). CD46 leads not only to potent T cell activation, but also to the development of an IL-10–secreting regulatory T cell population (13). These cells develop from the IFN-γ⁺ proinflammatory population. We found that in this population of CD46-induced Tr1-like cells, there was an increased level of miR-150 compared with the proinflammatory cytokine-secreting populations, meaning that the initial reduction of miR-150 in activated Th1 cells is partially reversed as the cells switch to a regulatory phenotype. GLUT1 and glycolysis also have a role in defining cytokine secretion and different T cell subsets. Inhibition of glycolysis has been found to compromise the ability of T cells to produce IFN-γ by freeing up GAPDH from the glycolysis pathway, when it is then able to play an alternative role by binding to the UTR of IFNG mRNA, limiting translation (46). This is of particular interest in the CD46-induced IL-10–secreting Th1 cells, which have higher levels of miR-150 compared with either IFN-γ– or IFN-γ/IL-10–secreting Th1 cells and have also undergone a switch to cease IFN-γ production. Lower rates of glycolysis are therefore linked to nonproduction of IFN-γ, consistent with the observation that regulatory T cells have a lower relative rate of glycolysis compared with effector T cells, relying instead on oxidative phosphorylation and lipid oxidation (48). It is therefore possible that increased miR-150 expression in the switched IL-10–secreting Th1 cells contributes to the shutdown of IFN-γ secretion. miR-150 likely regulates several targets relevant for the establishment and maintenance of the regulatory Th1 phenotype, typical of the described function of miRNAs, which can achieve regulation of phenotypes by inhibiting multiple targets within the same processes or pathways, with a cumulative effect (49, 50).

miR-150 also has known roles in cancer. It has previously been described that miR-150 regulates FLT3 in leukemia (51), but miR150 is also downregulated in mucosal-associated lymphoid tissue lymphoma, Burkitt lymphoma (52), Hodgkin lymphoma (53), and other B and T cell lymphoma cell lines (54) as well as primary human T and NK cell lymphomas (55, 56). Forced overexpression studies in lymphocytes lead to lower proliferation and increased apoptosis, mirroring the role for miR-150 downregulation as a requirement for initiation of T cell proliferation after activation. Aerobic glycolysis is also highly upregulated in cancer, and the loss of miR-150 expression in various lymphomas may play a role in upregulation of GLUT1 in the development of this disease.

We have not yet investigated the relationship between miR-150 and c-Myb in IL-10⁺ Th1 cells, although c-Myb has been previously identified as a marker of inducible regulatory T cells (57). The binding of c-Myb to promoters is also regulated by the availability of those sequences, which alter with chromatin structure state and even cell cycle phase (58). c-Myb may therefore be regulating different gene sets in the IL-10⁺IFN-γ⁻ subset as compared with the IFN-γ⁺ cells. Small interfering RNA–mediated knockdown of CD4⁺ c-Myb led to a general suppression of T cell activation, limiting IFN-γ secretion and development of the subsequent IL-10⁺ populations (data not shown), a result that, unfortunately, does not help to shed light on any specific function for c-Myb within the specific IL-10⁺IFN-γ⁻ regulatory subpopulation. The targeting of immune cell–expressed CD46 itself for therapy is complicated due to its differing roles at different phases of the response, as explained above, and its nearly ubiquitous expression on other nonimmune cell types. Better understanding of downstream effector systems may therefore offer improved opportunities to modulate this key immunological pathway. Recently, the targeting of T cell metabolism has been demonstrated to be a potentially effective means of therapy for modulating T cell–mediated autoimmunity (59), and an improved understanding of the roles of CD46-dependent miRNAs involved in T cell metabolism offer additional therapeutic opportunities. We therefore propose the highly lymphocyte-enriched miR-150 as a potential target for therapeutic intervention for modulation of IL-10 feedback and regulation of Th1 responses in infectious disease or autoimmunity.

We acknowledge the support of the Medical Research Council Centre for Transplantation. We are grateful to Dr. Yvonne Ceder for reagents and advice and to Liza Lind for technical assistance.

The authors have no financial conflicts of interest.