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T cell activation requires signaling through the T cell receptor (TCR) and costimulatory molecules such as CD28. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post transcriptionally and are also known to be involved in lymphocyte development and function. Here we set out to examine potential roles of miRNAs in T cell activation by using genome-wide expression profiling to identify miRNAs differentially regulated following T cell activation. One of the miRNAs up-regulated after T cell activation, miR-214, was predicted to be capable of targeting Pten based on bioinformatics and reports suggesting that it targets Pten in ovarian tumor cells. Up-regulation of miR-214 in T cells inversely correlated with PTEN levels. In vivo, transcripts containing the 3' untranslated region (3' UTR) of Pten including the miR-214 target sequence were negatively regulated after T cell activation, and forced expression of miR-214 in T cells led to increased proliferation after stimulation. Blocking CD28 signaling in vivo prevented miR-214 up-regulation in alloreactive T cells. Stimulation of T cells through the TCR alone was not sufficient to result in upregulation of miR-214. Thus, costimulation dependent up-regulation of miR-214 promotes T cell activation by targeting the negative regulator Pten. Thus, the requirement for T cell costimulation is in part related to its ability to regulate expression of miRNAs that control T cell activation.
T cell activation is a highly regulated process that requires the coordination of several sequential events that function to drive T cells to a differentiated state. Activation requires signaling through the T cell receptor (TCR) upon recognition of peptide-MHC complexes on the surface of antigen presenting cells, and the delivery of co-stimulatory signals. The CD28 co-stimulatory pathway plays a central role in activating signaling pathways, such as the PI3K pathway, that promote T cell survival, cytokine production and differentiation (1). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) controls T cell activation and maintains self-tolerance by negatively regulating signaling pathways that lead to activation (2). PTEN negatively regulates the AKT signaling pathway by dephosphorylating phosphatidylinositol 3,4,5-triphosphate (PIP3), a second messenger generated by PI3K that promotes the recruitment of 3-phosphoinositide-dependent protein kinase 1 (PDK1) to the TCR signaling complex. Costimulation through CD28 overcomes negative regulation of TCR signaling by PTEN supporting the notion that costimulation is needed to overcome the function of PTEN and allow for T cell activation (3). However, the mechanisms leading to costimulation dependent regulation of PTEN remain unclear.
MicroRNAs (miRNAs) are small endogenous noncoding RNAs that are approximately 21–22 nucleotides in length (4, 5). Most genes encoding miRNAs are transcribed by RNA polymerase II (Pol II), although some can be transcribed by Pol III (6–8). Primary transcripts encoding miRNAs are processed through a series of steps into pre-miRNA stem-loops of approximately 60 nucleotides by a miRNA processing complex that includes the RNase II enzyme Drosha and its partner DGCR8 (Pasha) (9, 10). Pre-miRNAs are transported to the nucleus by Exportin-5 in a RAS-related nuclear protein-guanosine triphosphate (GTP)-dependent manner and then further processed into a 21-22 nucleotide duplex by the RNase II enzyme Dicer. The functional miRNA strand is then loaded into the RNA-induced silencing complex (RISC). Mature miRNAs then guide the RISC complex to complementary target genes and repress gene expression by destabilizing the target mRNA or by repressing translation. miRNAs play a key role in several disease processes and development through their ability to control gene expression post transcriptionally.
Evolutionary conserved microRNAs (miRNAs) exist in all species analyzed to date, many of which have been shown to regulate vertebrate development and implicated in the development of cancer (10–16). miRNAs also play a key role in lymphocyte development and function (17–19). Given the emerging role of miRNAs in the control of lymphocyte development and function we set out to examine potential roles of miRNAs in T cell activation. PTEN is targeted by multiple miRNAs including miR-21 in human hepatocellular cancers (20), miR-26a in human glioma tumors (21), miR-216a and miR-217 in glomerular mesangial cells (22), and miRNA cluster 17–92 contributing to lymphomagenesis (23). It therefore appears that PTEN can be controlled by multiple miRNAs, however, the ability of a given miRNA to regulate PTEN appears to be cell-type specific. miRNAs that control PTEN in T cells have not been defined.
In order to examine the role of miRNAs in T cell activation and particularly control of PTEN, we performed genome-wide miRNA expression profiling. We identified a relatively limited number of miRNAs that were either up or down-regulated after T cell activation. One of the miRNAs shown to be up-regulated after T cell activation, miR-214, was predicted to be capable of targeting Pten based on bioinformatics and reports suggesting that it is capable of targeting Pten in tumor cells (24). Our data show that miR-214 targets Pten in activated T cells and that its expression leads to increased T cell proliferation. Furthermore, up-regulation of miR-214 was dependent on CD28 costimulation. Our results therefore suggest that costimulation through CD28 overcomes negative regulation of TCR signaling by allowing for increased expression of miR-214 which in turn regulates Pten expression. Thus, the requirement for T cell costimulation is in part related to its ability to regulate expression of miRNAs that control T cell activation.
All mice were obtained from Jackson Laboratories (Bar Harbor, ME) and housed using microisolator conditions in autoclaved cages and maintained on irradiated feed and autoclaved acidified drinking water. 4–6 week old female mice were used in all experiments. All experiments were performed in compliance with Institutional Guidelines.
Total T cells were purified by magnetic bead based cell sorting. CD4+ and CD8+ T cells were then purified by fluorescence-based cell sorting as described (25). T cells were activated by stimulating them with 2μg/mL anti-CD3 (2C11) and 1μg/mL CD28 (37.51) (ATCC, Manassas VA) in DMEM supplemented with L-glutamine, penicillin-streptomycin, sodium pyruvate (Mediatech, Manassas VA), nonessential amino acids (Invitrogen, Carlsbad CA) and 10% FCS (Hyclone, Brookfield WI).
Total RNA was extracted from unstimulated and stimulated (activated) T cells using the miRNeasy kit (Qiagen, Valencia CA) and used for microarray analysis as previously described (26). Total RNA from three separate preparations of unstimulated and activated T cells was pooled and used for microarray analysis. Microarray assays and statistical analysis were performed by LC Sciences (Houston, TX, www.lcsciences.com) using miRNA probe sequences from mmu-miRBase 11.0 (Sanger Institute, Cambridge, U.K.; http://microrna.sanger.ac.uk/sequences).
To generate MMP-LUC-3'-PTEN and MMP-LUC-MUT-3'-PTEN, oligonucleotides encoding the 3' UTR of Pten containing either the miR-214 target sequence or a mutated sequence respectively were cloned into pMIR-REPORT (Applied Biosystems/Ambion, Austin TX) downstream of luciferase. The fragment from each construct containing luciferase and either the miR-214 target sequence or mutated target was then cloned into the pMMP retroviral vector (kindly provided by Richard Mulligan, Harvard Medical School) to generate MMP-LUC-3'-PTEN or MMP-LUC-MUT-3'-PTEN. To generate pLL3.7-premiR-214 oligonucleotides encoding the pre-miR-214 sequence were cloned into the XhoI site of pLL3.7 (27) downstream of the U6 promoter. Vesicular stomatitis virus G (VSV-G) envelope protein pseudotyped viruses were prepared by packaging the retroviral or lentiviral vectors in 293T cells by transient transfection using the calcium phosphate method as previously described (28, 29). Functional titers of viral supernatants were determined in 293T cells by analyzing expression of GFP by flow cytometry.
HIO80 cells were obtained from Fox-Chase Cancer Center and grown in 199/MDCB 105 (1:1) medium supplemented with 5% FCS and 2μg/mL porcine insulin (Sigma, St. Louis MO). HIO80 cells were co-transfected with a 1:2 ratio of pMIR-Report to premiR-214-GFP or pLL3.7-GFP vector control constructs using FuGENE 6 (Roche, Indianapolis IN) according to manufacturer's specifications. Transfection efficiency was calculated by measuring GFP expression on a FacsCalibur flow cytometer (BD Biosciences, San Jose CA). Luciferase assays were performed 48 hours after transfection using SteadyLite reagent (Perkin-Elmer, Shelton CT) and measured on a Wallac microbeta Trilux luminometer.
Bone marrow cells were harvested from mice treated seven days prior with 5-fluorouracil and transduced as described previously (30). Bone marrow infections were performed at a multiplicity of infection of at least 1 for retroviruses or 5 for lentiviruses. 4×106 bone marrow cells were used to reconstitute lethally irradiated (11Gy) recipients.
Real Time PCR using miRNA-specific stem-loop primers for reverse transcription and Taqman probes for mature murine miRNA was performed in accordance with manufacturers protocols (Applied Biosystems, Foster City, CA) and analyzed using an ABI 7900HT Real-Time PCR system. Data analysis was performed using the Applied Biosystems SDS Software package, version 2.2. Primers specific for snoRNA202 in mouse or RU48 in human cells were obtained from ABI and used as controls in all assays following the manufacturer's instructions. RQ was calculated by dividing miR-214 expression by the endogenous control. All assays were performed in triplicate.
Western blots were performed as previously described (31) using a PTEN specific rabbit monoclonal antibody 138G6 (Cell Signaling Technology, Cambridge MA). Mouse monoclonal antibody β-actin (C4) was used to detect β-actin as a loading control (Sigma, St. Louis MO). Western blots were quantified using ImageJ software version 10.2 (National Institute of Health, Bethesda MA).
Luciferase assays were performed using SteadyLite reagent (Perkin Elmer, Shelton CT) according to manufacturers instructions and analyzed on a Wallac microbeta Trilux luminometer.
To measure proliferation, purified T cells were labeled with with 0.6μM CellTrace Far Red DDAO for 6 min at room temperature. Labeled T cells (3×106) were activated for 72 hours. Cell proliferation was then analyzed by flow cytometry and analyzed using Flowjo software version 6.3 (Tree Star Inc, Ashland OR).
C57BL/6 mice were injected in the footpad with 2×107 BALB/c splenocytes. In some cases mice were also injected I.P. with 250μg CTLA4Ig. T cells were purified from draining popliteal lymph nodes and analyzed for miR-214 expression by RT-PCR. PTEN levels were assessed by Western blot.
All statistical calculations were performed using GraphPad Prism 4.0a software (GraphPad Software, San Diego CA). P values were determined using the Student's two-tailed t-test for independent samples. P values less than 0.05 were considered statistically significant.
To begin to examine the role of miRNAs in T cell activation we performed expression profiling on miRNA isolated from activated and resting T cells. T cells were purified from the spleens of C57BL/6 mice and stimulated with anti-CD3 and CD28 monoclonal antibodies for 72 hours (activated) or used to prepare miRNA without further stimulation (resting). A portion of each population was labeled with 5-(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) to monitor T cell activation based on proliferation. Approximately 89% of T cells stimulated with anti-CD3 and CD28 underwent proliferation indicating that they were activated (Data not shown). Genome-wide miRNA expression profiling of 599 murine miRNAs present in miRBase release 11 using μParaflo microfluidic biochip array technology revealed significant differences in the expression 145 miRNAs in unstimulated and activated T cells (Fig. 1, P<0.01). Of the miRNAs observed to be differentially expressed, 61 were up-regulated in activated T cells while 84 were down-regulated (Fig. 1). Narrowing our analysis to transcripts with mean signal intensities greater than 500 revealed 32 miRNAs that were up-regulated (Table I) and 43 that were down-regulated (Table II) in activated T cells.
Our array data revealed that miR-214 is significantly up-regulated upon T cell activation (P=5.63×10−6 between activated and resting, Table I). Bioinformatics analysis revealed that Pten is a potential target of miR-214 (32). Moreover, in human ovarian cancer cells, miR-214 has been shown to induce cell survival and resistance to chemotherapeutic agents by targeting PTEN (24). These observations led us to hypothesize that up-regulation of miR-214 following T cell activation may function to regulate PTEN expression thereby affecting T cell activation. To test this hypothesis we first set out to confirm that miR-214 is up-regulated in T cells upon activation by performing stem-loop Taqman© real-time PCR (PCR) assays. Consistent with our microarray data, we observed that miR-214 is significantly up-regulated in T cells after stimulation with anti-CD3 and CD28 (Fig. 2A). To further characterize the role of miR-214 in T cell proliferation we isolated CD4+ and CD8+ T cells and examined miR-214 expression at various time points following stimulation with anti-CD3 and CD28. miR-214 is rapidly up-regulated in both CD4+ and CD8+ T cells upon stimulation (Fig. 2B). Further analysis revealed that miR-214 is up-regulated in naive and memory CD4+ and CD8+ T cells after stimulation with anti-CD3 and CD28 (Fig. 3A). However, up-regulation of miR-214 was observed to be significantly higher in CD4+CD62hiCD44hi memory T cells than naïve CD4+ T cells, peaking at 48 hours after stimulation (Fig. 3B). In contrast, miR-214 appeared to be up-regulated more rapidly in naïve (Ly6cloCD44lo) CD8+ T cells when compared with memory (Ly6chiCD44hi) CD8+ T cells (Fig. 3B), however at 72 hours, expression of miR-214 was the same in each group. Up-regulation of miR-214 inversely correlated with levels of PTEN mRNA. PTEN mRNA levels were substantially reduced in both CD4+ and CD8+ T cells at all time points analyzed (Fig 4A). Up-regulation of miR-214 inversely correlated with levels of PTEN protein in naïve and memory CD4+ T cells (Fig. 4B). This effect was observed to a lesser extent in naïve CD8+ T cells (Fig. 4B). Down regulation of PTEN was not observed in memory CD8+ T cells until 72 hours (Fig. 4B), a point at which we observed miR-214 to be significantly up-regulated (Fig. 3B). At 72 hours, levels of Pten mRNA were decreased in both CD4 and CD8 cells (Fig. 4B), correlating with an increase in miR-214 (Fig. 2B) and a decrease in PTEN protein levels (Fig. 4B)
MicroRNAs function by targeting messenger RNA. Bioinformatics analysis suggested that the 3'UTR of Pten contains a miR-214 targeting site. To examine whether the 3'UTR of Pten is regulated during T cell activation we generated retroviruses in which the 3'UTR of Pten containing the miR-214 target sequence was cloned down-stream of luciferase (MMP-LUC-3'-PTEN). We also generated retroviruses in which the 3'UTR of Pten containing a mutated miR-214 target sequence was cloned down-stream of luciferase (MMP-LUC-MUT-3'-PTEN). To validate lentiviral constructs, HIO80 cells, which do not express endogenous miR-214, were transfected with MMP-LUC-3'-PTEN or MMP-LUC-MUT-3'-PTEN. The resulting lines were then were transfected with either pLL3.7-premiR-214 or pLL3.7 lentiviral vectors. 48 hours later expression of miR-214 was assessed by PCR (Fig. 5A). Cells transfected with premiR-214 expressed significantly more miR-214 when compared with cells transfected with control plasmid. Pre-miR214 activity was then examined based on luciferase activity. Expression of pre-miR-214 significantly decreased the level of luciferase activity in cells transfected with MMP-LUC-3'-PTEN (Fig. 5B). A reduction in luciferase activity was not observed in cells transfected with or MMP-LUC-MUT-3'-PTEN (Fig. 5B). Expression of miR-214 also led to a decrease in PTEN protein expression in HIO80 cells when compared with cells transfected with control plasmid alone (Fig. 5C), confirming that our lentivirally encoded miR-214 was capable of down-regulating PTEN expression.
To test the function of miR-214 in vivo, bone marrow was harvested from 3–4 week old female C57BL/6 mice treated 7 days prior with 150mg/kg 5-fluorouracil (5-FU) and transduced with either MMP-LUC-3'-PTEN or MMP-LUC-MUT-3'-PTEN and then used to reconstituted lethally irradiated C57BL/6 mice as previously described (30). When hematopoietic reconstitution was complete, mice reconstituted with either MMP-LUC-3'-PTEN or MMP-LUC-MUT-3'-PTEN transduced bone marrow were sacrificed and T cells were isolated. Stimulation of T cells from mice receiving MMP-LUC-3'-PTEN transduced bone marrow with anti-CD3 and CD28 led to a significant decrease in luciferase activity when compared to the level of activity observed in unstimulated T cells (Fig. 6A). We did not observe a decrease in luciferase activity in T cells isolated from mice receiving MMP-LUC-MUT-3'-PTEN transduced bone marrow following stimulation with anti-CD3 and CD28 (Fig. 6B), however we did observe a trend toward increased luciferase activity which is most likely the result of T cell proliferation. These data suggest that upon T cell activation, transcripts containing the 3'UTR of Pten matching the miR-214 seed sequence are targeted and down-regulated.
To further examine the role of miR-214 in T cell activation, the sequence encoding pre-miR-214 was cloned into the lentiviral vector pLL3.7 (27) to generate pLL3.7-premiR-214. In this vector pre-miR-214 is expressed under the control of the U6 promoter and GFP is expressed under the control of the CMV promoter. VSV-G pseudotyped viral particles were then produced using either the pLL3.7-premiR-214 lentiviral construct or pLL3.7 alone. Bone marrow was then harvested from C57BL/6 mice and transduced with either pLL3.7-premiR-214 or pLL3.7 control virus and used to reconstitute lethally irradiated C57BL/6 mice. When hematopoietic reconstitution was complete, splenocytes were harvested from reconstituted mice, stained with 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) succinimidyl ester (DDAO-SE) and stimulated with anti-CD3 and CD28 for 72 hours. We then gated on GFP+ CD4+ or CD8+ T cells and examined proliferation based on DDAO-SE dye dilution. We found that GFP+ T cells derived from pLL3.7-premiR-214 virally transduced progenitors showed enhanced proliferation when compared to GFP+ T cells from mice receiving pLL3.7 control transduced bone marrow (Fig. 7A–B). These data indicate that expression of miR-214 in T cells increases their capacity to proliferate upon stimulation.
To understand if costimulation is required for miR-214 up-regulation in T cells, we injected fully allogeneic BALB/c splenocytes into the footpads of untreated C57BL/6 mice or C57BL/6 mice that had been treated with CTLA4-Ig. Five days later, T cells from popliteal lymph nodes draining the injected footpads were isolated and miR-214 expression was analyzed by PCR. Expression of miR-214 was significantly increased in T cells isolated from mice injected with allogeneic cells compared to C57BL/6 controls (Fig. 8A). Treatment with CTLA4-Ig resulted in a significant reduction in miR-214 expression in T cells from mice injected with allogeneic cells. Analysis of PTEN levels by Western blotting revealed an inverse correlation between miR-214 expression and PTEN. T cells from mice injected with allogeneic cells exhibited relatively low levels of PTEN when compared with control T cells from C57BL/6 mice (Fig. 8B). T cells from mice treated with CTLA4-Ig that were injected with allogeneic cells exhibited levels of PTEN that were similar to that observed in control T cells from C57BL/6 mice (Fig. 7B). To confirm that miR-214 is up-regulated in a co-stimulation dependent fashion, we purified T cells from the spleens of C57BL/6 mice, and stimulated them with either anti-CD3 monoclonal antibody alone, or anti-CD3 in conjuction with anti-CD28. After 48 hours, levels of miR-214 had substantially increased in T cells stimulated with anti-CD3 and CD28 (Fig. 8C). In contrast, levels of miR-214 in T cells stimulated with anti-CD3 alone remained similar to those in control unstimulated T cells (Fig. 8C). Together, these results suggest that miR-214 is up-regulated in T cells upon antigen encounter in a CD28 dependent fashion.
Several recent reports have shown that miRNAs play an important role in T cell development and function (33–36). We set out to identify miRNAs that regulate T cell activation. Using genome-wide miRNA expression profiling we identified miRNAs that are differentially expressed in unstimulated and activated T cells. The data obtained from our microarray analysis led us to examine potential targets of miRNAs up-regulated after T cell activation, focusing on potential targets known to play a role in T cell activation. Based on bioinformatics, miR-214 has been suggested to target PTEN (32). Moreover, in human ovarian cancer cells, miR-214 has been shown to induce cell survival and resistance to chemotherapeutic agents by targeting PTEN (24). Because of the importance of PTEN in controlling T cell activation we therefore set out to examine whether miR-214 expression following T cell activation may play a role in regulating Pten. We reasoned that up-regulation of miR-214 following T cell activation may serve to down-regulate PTEN thereby promoting T cell activation. Based on our array data, miR-214 was up-regulated approximately 16 fold following activation with anti-CD3 and CD28. PCR analysis revealed that miR-214 is rapidly up-regulated after stimulation of either CD4 or CD8 T cells with anti-CD3 and CD28. Up-regulation of miR-214 inversely correlated with both levels of PTEN mRNA and protein in both naïve T cells and memory CD4+ T cells. Luciferase reporter assays formally demonstrated that the 3'UTR of PTEN matching the miR-214 seed sequence is targeted in activated T cells. Functionally, expression of miR-214 in T cells increased their capacity to proliferate upon stimulation. Moreover, co-stimulatory blockade using CTLA4-Ig resulted in decrease in miR-214 expression in alloreactive T cells suggesting that miR-214 is up-regulated in T cells upon antigen encounter in a CD28 dependent fashion. Together these results support the idea that miR-214 plays a role in controlling T cell activation through its ability to target Pten. While miR-214 has been shown to be important in development, muscle cell differentiation, cardiac hypertrophy and protection from apoptosis (37–41) to our knowledge a role for miR-214 in T cells has not been shown.
Mammalian microRNA's can act either through the degradation of their target mRNA, or through transcriptional blockade which affects the levels os protein produced, but not expression of mRNA. Our data indicates that levels of PTEN mRNA, which is targeted by miR-214, are substantially reduced in activated T cells. This suggests that miR-214 acts through degradation of PTEN mRNA in T cells.
Consistent with other reports, we observed that several miRNAs are either up or down-regulated in T cells following stimulation (42). However, in that report, analysis of expression levels by microarray led to the identification of relatively few miRNAs (miR-16, miR-142-3p, miR-142-5p, miR-150, miR-15b and let-7f) which are significantly down regulated in virus specific effector CD8+ cells compared to naïve T cells and relatively few miRNAs that are up-regulated. Our results significantly expand the number of miRNAs observed to be up or down-regulated as a consequence of T cell activation and confirm other reports related to the role of miRNAs in lymphoid cells. Consistent with our data is the observation that during differentiation of naïve T cells into Th1 or Th2 effectors miR-150, miR-26a and let-7d levels decrease and the observation that miR-150 expression in naïve T cells is rapidly down-regulated upon TCR engagement (43). Interestingly, miR-155 is up-regulated by the transcription factor FOXP3 and critical for T regulatory cell function (35). Our array data shows that miR-155 is up-regulated in T cells following TCR stimulation suggesting activation of T cells with anti-CD3 and CD28 enhances miRNAs required for Treg development (35).
We focused on miRNAs that might control Pten expression, because Pten plays a critical role in regulating T cell activation. While Pten has been shown to be regulated by miRNAs in a cell type-specific fashion, miRNAs that regulate Pten in T cells have not been defined. PTEN is targeted by miR-21 in human hepatocellular cancers (20), miR-26a in human glioma tumors (21), miR-216a and miR-217 in glomerular mesangial cells (22). Stimulation of T cells with anti-CD3 and CD28 resulted in the down-regulation miR-21 and miR-26a suggesting that these miRNAs may not be involved in regulation of Pten expression in T cells. miR-216a and miR-217 were not expressed in T cells. Expression of the miRNA 17–92 cluster has been shown to contribute to lymphoproliferative disease through its effects on Pten and Bim, and lead to enhanced survival and proliferation of T cells over expressing this miRNA cluster (23). miR-20a was the only member of the miR-17–92 cluster analyzed that was differentially expressed at significant levels in activated T cells however, this miRNA was down-regulated in activated T cells. This suggests that the miR-17–92 gene cluster is may not be responsible for down-modulation of Pten in activated T cells. We also observed that expression of miR-181a, which has previously been show to modulate T cell sensitivity by regulating multiple phosphatases (34), was down-regulated in activated T cells. This observation is consistent with the observation that primed T cells contain fewer copies of miR-181a when compared with naïve T cells.
It has previously been suggested that costimulation through CD28 is required for T cell activation in order to overcome negative regulation by PTEN (3). Our data suggest that stimulation through CD28 is required in order to up-regulate miR-214 which in turn targets the 3' UTR of Pten thereby reducing PTEN levels and promoting T cell activation. Our results therefore provide a link between the need for costimulation through CD28 and expression of a miRNA that decreases expression of a negative regulator of T cell activation. To our knowledge a link between costimulation through CD28, regulation of PTEN and miRNAs has not been previously described. This observation raises the possibility of manipulating miRNA expression for the purpose of altering T cell responsiveness.
The authors thank Drs. Mohamed Sayegh and Laurence Turka, as well as members of the Iacomini Laboratory for helpful discussions. We also thank Drs. Christoph Eicken (Head of Technical Services, LC Sciences, Houston TX) for suggestions related to microarrays and statistical interpretation of data, and Dr. Jin Q Chen for kindly providing HIO80 cells.
Peter Jindra was supported NIH training grant T32 AI070085 and is currently supported by an AST/Wyeth Basic Science Fellowship Grant. Jessamyn Bagley is supported by an American Heart Association Scientist Development Grant. This work was also supported by NIH grants 1R01 AI53666 and 1R01 AI070601 awarded to JI.