Caenorhabditis elegans DAF-16 transcription factor is critical for diverse biological processes, particularly longevity and stress resistance. Disruption of the DAF-2 signaling cascade promotes DAF-16 activation, and confers resistance to killing by pathogenic bacteria, such as Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis. However, daf-16 mutants exhibit similar sensitivity to these bacteria as wild-type animals, suggesting that DAF-16 is not normally activated by these bacterial pathogens. In this report, we demonstrate that DAF-16 can be directly activated by fungal infection and wounding in wild-type animals, which is independent of the DAF-2 pathway. Fungal infection and wounding initiate the Gαq signaling cascade, leading to Ca2+ release. Ca2+ mediates the activation of BLI-3, a dual-oxidase, resulting in the production of reactive oxygen species (ROS). ROS then activate DAF-16 through a Ste20-like kinase-1/CST-1. Our results indicate that DAF-16 in the epidermis is required for survival after fungal infection and wounding. Thus, the EGL-30-Ca2+-BLI-3-CST-1-DAF-16 signaling represents a previously unknown pathway to regulate epidermal damage response.The  Drechmeria coniospora and Clonostachys rosea, infect the epidermis of the roundworm Caenorhabditis elegans by producing conidia. Here we demonstrated that the DAF-16/FOXO transcription factor in the epidermis has a direct role in C. elegans defense against fungal infection and physical injury. We found that the EGL-30/EGL-8/IP3/ITR-1 signaling pathway triggers epidermal Ca2+ release through IP3 and its receptor ITR-1 after fungal infection. Ca2+ release induces the production of reactive oxygen species (ROS) by activating a dual-oxidase BLI-3. ROS in turn mediate DAF-16 activation in a Ste20-like kinase-1/CST-1-dependent manner. Thus, DAF-16 could act in a cell-autonomous way in the epidermis as an active regulator of immune responses to fungal infection and physical injury.In the natural environment, animals encounter different pathogens. Thus, different tissues within an organism must develop specific immune systems for survival. The epidermis acts as a physical barrier and represents a first line of defense against infection and physical injury in a variety of animals. Natural nematophagous fungi, such as  Caenorhabditis elegans as a model host have revealed the involvement of evolutionarily conserved signaling pathways in the innate immune response to microbial infection and injury, including the DAF-2/DAF-16 insulin-like signaling pathway C. elegans DAF-2 is orthologous to the mammalian insulin/insulin-like growth factor-1 receptor daf-2 mutants exhibit increased resistance to pathogenic bacteria, such as Pseudomonas aeruginosa and Staphylococcus aureusdaf-2 mutants is suppressed by mutations in daf-16, suggesting a crucial role for DAF-16 in innate immunity against bacteria Drosophila and human tissues, FOXOs also induce the expression of a variety of antimicrobial peptides, such as drosomycin and defensins All organisms are in constant contacts with a variety of microorganisms. The innate immune system in hosts provides the first line of defense against these microorganisms. During the last decade, studies using P. aeruginosa, S. aureus, Enterococcus faecalis and Salmonella enterica, daf-16 mutants are not significantly more susceptible than wild-type worms to the killing mediated by these bacteria daf-16 by RNAi in wild-type worms does not affect susceptibility to P. aeruginosa PA14, intestinal-specific knock-down of daf-16 leads to enhanced susceptibility to PA14 Escherichia coli (EPEC) and Bacillus thuringiensis induce DAF-16 nuclear translocation, respectively daf-16 mutants are more sensitive to the two bacterial pathogens Although DAF-16 is involved in immune responses to pathogenic bacteria including P. aeruginosa, S. aureus, E. faecalis, S. enterica, and human pathogenic yeast Candida albicans infect the nematode intestine Drechmeria coniospora and Clonostachys rosea, infect the epidermis of nematode, leading to epidermal cell damage C. elegans infected with D. coniosporaD. coniospora-upregulated genes and DAF-16 target genes. These findings prompted us to examine the role of DAF-16 in the innate immune response to fungal infection. After exposure of C. elegans to D. coniospora and C. rosea, we found that daf-16 mutants were more susceptible than wild-type worms to killing by fungi. Further studies indicated that fungal infections resulted in the activation of DAF-16 as a consequence of the production of reactive oxygen species (ROS). Similar results were obtained with nematodes subjected to physical injury. Our data demonstrate that DAF-16 can act in a tissue-specific way in the epidermis as an active regulator of immune responses to fungal infection and physical injury.Pathogenic bacteria, including D. coniospora infection D. coniospora are also targets of DAF-16 (P<0.0001). To further confirm these results, we randomly selected eight of these genes and determined their expression by qPCR , DAF-16 nuclear localization was observed (daf-16(mu86) mutants, after treatment with spiny balls  also induced DAF-16 nuclear translocation predominately both in the hypodermis and the intestine without fungal infection  mutants after infection by D. coniospora and C. rosea. We found that daf-16(mu86) mutants exhibited enhanced susceptibility to killing by D. coniospora  animals were more sensitive than wild-type animals to physical injury  promoter D. coniospora infection and physical injury in wild-type animals , a fluorescent dye that has been used to detect the ROS levels in C. elegansAccumulating evidence suggests that the levels of ROS in tissues are induced in response to physical wounding in human epithelial keratinocytes C. elegans, there are two DUOX homologs. BLI-3/Ce-DUOX-1 is the major enzyme responsible for the production of ROS bli-3 and the standard feeding RNAi with construct based on bli-3 result in a severe blistered phenotype bli-3 in a 1/10 dilution reduced more than 50% bli-3 mRNA levels  mediate ROS production during wound responses in zebra fish larvae and Drosophila embryos ny balls , indicatectively . Similarny balls . Taken tbli-3 RNAi enhanced susceptibility to killing by D. coniospora and spiny balls (daf-16(mu86) background, knock-down of bli-3 by RNAi did not cause an increase in susceptibility to D. coniospora and spiny balls compared to daf-16(mu86) mutants alone. bli-3 is mainly expressed in the epidermis of nematodes bli-3 function only in the adult epidermis. As expected, after D. coniospora infection and treatment with spiny balls, epidermal-specific RNAi of bli-3 significantly suppressed the production of ROS  encodes a protein that lacks the peroxidase domain, but retains its ability to produce ROS. bli-3(e767) mutants exhibited similar sensitivity to killing by D. coniospora and spiny balls as did wild-type animals  family of enzymes, which hydrolyze phosphatidylinositol 4,5-diphosphate (PIP2) to produce inositol 1,4,5-trisphosphate (IP3) and diacylglycerol 2+ release after needle wounding 2+ release after D. coniospora infection. We used worms overexpressing N-terminal IP3 binding domains (“IP3 sponges” (cz12690)) in the epidermis itr-1(sa73) mutants  shifted the survival curve to mimic the daf-16(mu86) phenotype after D. coniospora infection (daf-16(mu86); itr-1(sa73) double mutants was similar to that of daf-16(mu86) mutants. In C. elegans, itr-1 is expressed in many tissues, including the epidermis itr-1 significantly reduced the survival of worms after infection of D. coniospora and treatment with spiny ball  or egl-8(n488) mutants after infection of D. coniospora and treatment with spiny balls  or egl-8(n488) mutants compared to control worms after infection of D. coniospora and treatment with spiny balls. Similarly, mutations in egl-30 or egl-8 significantly suppressed the expression of Psod-3::GFP induced by Drechmeria coniospora and spiny balls  and egl-8(n488) mutants were more sensitive than wild-type worms to killing by D. coniospora  phenotype. daf-16(mu86); egl-30(n686) or daf-16(mu86); egl-8(n488) double mutants were indistinguishable from daf-16(mu86) for sensitivity to D. coniospora  and egl-8(n488) mutants to D. coniospora infection and physical injury, respectively  mediates oxidative stress-induced activation of FOXO transcription factors f DAF-16 , but didny balls . These rcst-1 by RNAi reduced the survival of nematodes after D. coniospora infection (daf-16(mu86);cst-1 RNAi was comparable to that of daf-16(mu86) mutants  mutants after D. coniospora infection and treatment with spiny balls  and similarity (61%) to the human DUOX1, casting doubt as to whether calcium binding is required for C. elegans BLI-3 function Drosophila DUOX1 also shares a relatively low identity (42%) and similarity (66%) to the human DUOX1. Because ROS-producing Drosophila DUOX1 enzymatic activity depends on intracellular Ca2+ through binding to the EF-hand domains 2+ modulates the enzymatic activity of C. elegans BLI-3.Impairment in release of CaP. aeruginosa suppresses the activity of DAF-16 by activating DAF-2 insulin-like signaling daf-16 by RNAi completely inhibits the ability of CST-1 to extend life span in C. elegans, whether CST-1 activates DAF-16 under oxidative stress remains unclear. Our data indicate that ROS mediates activation of DAF-16 in response to epidermal damage in a CST-dependent manner. These results support a model in which the evolutionarily conserved MST/CST pathway functions in parallel with the insulin signaling pathway to regulate FOXO/DAF-16 by oxidative stress The PI3K-Akt-FOXO signaling pathway is evolutionarily conserved from nematodes to mammals C. elegansnlp genes encoding antimicrobial peptides (AMPs) in response to fungal challenge and needle wounding 2+ release through IP3 and its receptor ITR-1 after wounding 2+ upon fungal infection and physical injury. Since epidermal DAF-16 is required for innate immune response to fungal infection and physical injury, it is an important immune effector of EGL-30-Ca2+ in the epidermis. However, mutations in daf-16 do not alter the expression of AMPs induced by fungal infection, which is consistent with the observation that the EGL-30-Ca2+ pathway appears not to be involved in the up-regulation of AMPs after wounding The epidermis forms a protective barrier against physical damage and pathogen entry Drosophila and human tissues Because FOXOs are conserved from worms to humans, it is of great interest to investigate whether FOXOs are involved in epidermal innate immunity in other species . FOXOs have been shown to mediate the induction of antimicrobial peptides, such as defensins, both in 2+/BLI-3/ROS/CST-1 signaling represents a novel pathway to regulate DAF-16 activity , daf-16(mu86), bli-3(e767), itr-1(sa73), bar-1(ga80), egl-30(n686), egl-8(n488), ins-7(ok1573), TJ356-daf-16::gfp), muIs84 (Psod-3::gfp), NR222 (rde-1(ne219); kzIs9); and NR350 (rde-1(ne219); kzIs20) were kindly provided by the Caenorhabditis Genetics Center (CGC). The CZ13896 (Pcol-19-GCaMP (juIs319)), CZ12690 (Pcol-19-Superspronge (juEx3052)), and CZ15386 ) strains were kindly provided by Dr. Andrew D. Chisholm . The strain GR1353 (daf-2(e1370) III; mgIs41[daf-16::gfp]) and the strain for intestinal-specific RNAi (sid-1(qt9); Is[vha-6pr::sid-1]; Is[sur-5pr::GFPNLS]) were kindly provided by Dr. Gary Ruvkun .The following Mutants and transgenic strains were backcrossed three times into the N2 strain used in the laboratory. All strains were maintained on nematode growth media (NGM) and fed with E. coli strain OP50.C. elegans growth at 20°C Standard conditions were used for D. coniospora: 50–60 L4 nematodes were transferred to fresh plates seeded with heat-killed E. coli OP50, with ∼1.0×108D. coniospora spores at 25°C. Killing assays with C. rosea: ∼1.0×108 spores of C. rosea were inoculated onto plates containing heat-killed E. coli OP50 for 1–2 days at 28°C, and the infection experiments were started by adding 50–60 nematodes to each plate at 25°C. The number of living worms was counted by using a light microscope at time intervals. Immobile nematodes unresponsive to touch were scored as dead. One-sided rank log tests were used to the statistical significance of the differences between treatments.Killing assays with P. aeruginosa: P. aeruginosa PA14  was cultured in Luria broth (LB), then seeded on slow-killing plates, which contain modified NGM (0.35% instead of 0.25% peptone). PA14 was incubated first for 24 h at 37°C and then for 24 h at 25°C. The infection experiments were started by adding 50–60 nematodes to each plate at 25°C. Killing assays with S. aureus: S. aureus ATCC 25923  was cultured in tryptic soy broth , then seeded on plates containing modified NGM (0.35% instead of 0.25% peptone). The infection experiments were started by adding 50–60 nematodes to each plate at 25°C.Killing assays with 5 per ml. 100 µl of the spiny ball suspension was thoroughly added to on plates containing modified NGM with heat-killed E. coli OP50. The infection experiments were started by adding 50–60 nematodes to each plate at 25°C. Mobile and immobile nematodes were counted every 12 h after their addition.Spiny balls were purified by a previously described method (sid-1(qt9); Is[vha-6pr::sid-1]; Is[sur-5pr::GFPNLS]) was used in intestine-specific RNAi , and the strain NR350 was used in muscular-specific RNAi.RNAi bacterial strains containing targeting genes were obtained from the Ahringer RNAi library After 12 h of fungal infection or treatment with spiny balls, the worms were immediately mounted in M9 onto microscope slides. The slides were viewed using a Zeiss Axioskop 2 plus fluorescence microscope  with a digit camera. The status of DAF-16 localization was categorized as cytosolic localization, nuclear localization when localization is observed throughout the entire body from head to tail, or intermediate localization when there is a visible nuclear localization but one not as complete as nuclear Total RNA from worms was isolated using Trizol reagent . Random-primed cDNAs were generated by RT of the total RNA samples using a standard protocol. A real-time-PCR analysis was performed with the ABI Prism 7000 Sequence Detection system  using SYBR Premix-ExTag . β-Tubulin was used for internal control. The primers used for PCR are listed in dpy-7 and clo-19 genes have shown to be expressed in the epidermis Pdpy-7:daf-16 fusion gene was chemically synthesized, and obtained from Generay Biotech Co. . The DNA fragment contains a 436 bp of dpy-7 promoter fragment , a 1530 bp of the daf-16 cDNA, a 729 bp of the GFP cDNA and a 234 bp of the 3′-UTR of unc-54. The Pcol-19:egl-30 fusion gene was constructed as follows. A 2838 bp of col-19 promoter fragment was obtained by PCR on C. elegans genomic DNA using primers 5′-GCT CTA GAG CAT CGT CAC ATT CTG TCT-3′ and 5′-TCC CCC GGG GGC TTT CCA TCG TCT CC-3′ followed by XbaI and SmaI digestion. The fragment was inserted into XbaI and SmaI digested pPD95.79, resulting in plasmid pPDegl. A 1116 bp fragment of egl-30 cDNA was amplified by PCR on C. elegans genomic DNA using primers 5′- TCC CCC GGG TTG TTC TAT TCG CTG GCT T-3′ and 5′-GGG GTA CCC CAA GTT GTA CTC CTT CAG ATT AT-3′ followed by SmaI and KpnI digestion. The fragment was inserted into SmaI and KpnI digested pPDegl vector.The Pdpy-7:daf-16 fusion gene fragment or the vector containing Pcol-19::egl-30 fusion gene was co-injected with rol-6 plasmid (pRF4) into gonads of wild-type and egl-30(n686) animals by standard techniques Pdyp-7::daf-16 or Pcol-19::egl-30 were confirmed in prior to each pathogenesis assay.The The ROS levels were detected by 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) as a probe as described previously with modifications 2+ in the epidermis, GCaMP fluorescence imaged was obtained using confocal microscopy (Zeiss LSM-510) with a 40×objective, as described previously 0) and induction fluorescence (Ft) were obtained by averaging fluorescence in five ROIs in the epidermis then subtracting the average of five ROIs in the background before and after fungal infection or injury. GCaMP fluorescence was normalized to an internal control, Pcol-19-tdTomato. The change in fluorescence ΔF was expressed as the ratio of change with respect to the baseline [(Ft–F0)/F0]. Raw data from fluorescent microscopy were then analyzed using ImageJ.To analyze CaDifferences in survival rates were analyzed using the log-rank test. Differences in gene expression, distribution of DAF-16, and fluorescence intensity were assessed by performing a one-way ANOVA followed by a Student-Newman-Keuls test. Data were analyzed using SPSS11.0 software (SPSS Inc.). To test for significant overlap between different gene lists, a Fisher's exact test was used.Table S1The target genes of DAF-16 up-regulated by D. coniospora infection. When compared DAF-16 target genes to published microarray analysis of gene expression induced by D. coniospora infection, 48 of the genes up-regulated by D. coniospora were found to be targets of DAF-16.(DOC)Click here for additional data file.Table S2The primers are used for real-time PCR. This table lists all primers for real-time PCR analysis.(DOC)Click here for additional data file.Text S1Supporting Figures. Figure S1. Fungal infection and mutation in daf-12 induce DAF-16 nuclear translocation. (A) Wild-type worms in the absence of D. coniospora. (B) Wild-type worms were exposed to D. coniospora for 12 h. (C) daf-2 mutants were growth under normal conditions without D. coniospora. Figure S2. Both epidermal- and intestinal-specific knock-down of daf-16 by RNAi suppress the expression of DAF-16 target genes. (A) qPCR analysis of expression of DAF-16 target genes in NR222 strains (CTR), CTR 24 h after D. coniospora infection (CTR+DC), and CTR subjected to daf-16 RNAi after D. coniospora infection . (B) qPCR analysis of expression of the intestinal-specific RNAi strain sid-1(qt9);Is[sur-5::GFP]; alxIs7[VHA-6p::SID-1::SL2::GFP] (CTR), CTR 24 h after D. coniospora infection (CTR+DC), and CTR subjected to daf-16 RNAi after D. coniospora infection . *P<0.05, CTR+DC relative to CTR+DC+ daf-16 RNAi. Figure S3. Genetic loss of ins-7 has no effect on DAF-16 translocation and the survival of worms after D. coniospora infection and physical injury. (A) Mutation in ins-7(ok1573) did not influence the nuclear accumulation of DAF-16 after D. coniospora infection (DC) and treatment with spiny balls (SB). (B and C) Mutation in ins-7(ok1573) did not affect the survival of nematodes after D. coniospora infection (B) and treatment with spiny balls (C). Figure S4. DAF-16 is required for resistance to fungal infection. (A) daf-16(mu86) mutants were sensitive to C. rosea infection. (B–C) daf-16 RNAi reduced the survival rate of nematodes exposed to D. coniospora (B) and C. rosea (C). P<0.001 relative to wild-type animals. Figure S5. Epidermal-specific knock-down of daf-16 reduces the expression of daf-16 in the hypodermis. (A) NR222 strains were exposed to D. coniospora for 12 h. (B) NR222 strains subjected to daf-16 RNAi were exposed to D. coniospora for 12 h. Figure S6. Muscular-specific daf-16 RNAi has no effect on sensitivity to D. coniospora infection and spiny balls. (A) NR350 strains were exposed to D. coniospora. (B) NR350 strains were exposed to spiny balls. After 12 h of treatment, the DAF-16::GFP expression pattern was observed. Figure S7. Elevated daf-16 expression in the epidermis confers resistance to fungal infection and physical injury. Overexpression of daf-16 under epidermal (dpy-7) promoter increased survival of nematodes after D. coniospora infection (A) and treatment of spiny balls (B). Figure S8. Knock-down of bli-3 in a 1/10 dilution results in a decrease in the expression of bli-3. qPCR analysis of bli-3 expression in WT worms subjected to bli-3 RNAi in a 1/10 dilution. Figure S9. BLI-3 in the epidermis is required for ROS production, DAF-16 nuclear accumulation, and resistance to fungal infection and physical injury. (A) Epidermal-specific RNAi of bli-3 in a 1/10 dilution significantly reduced the production of ROS induced by D. coniospora (DC) or spiny balls (SB). (B) Epidermal-specific RNAi of bli-3 in a 1/10 dilution significantly reduced DAF-16 nuclear accumulation induced by D. coniospora or spiny balls. (C–D) Epidermal-specific RNAi of bli-3 in a 1/10 dilution significantly reduced the survival rate of nematodes exposed to D. coniospora (C) or spiny balls (D). (E–F) Intestinal-specific RNAi of bli-3 in a 1/10 dilution did not influence the survival of nematodes after D. coniospora infection (E) and treatment with spiny balls (F). Figure S10. The peroxidase activity of BLI-3 is not crucial for resistance to fungal infection and physical injury. (A–B) The mutant bli-3(e767) encoding a protein that lacks the peroxidase domain exhibited similar sensitivity to killing by D. coniospora infection (A) and spiny balls (B) as wild-type animals. Figure S11. A disturbance of IP3 signaling reduces the survival of worms after fungal infection and physical injury. (A–B) Worms overexpressing IP3 sponges (cz12690) in the epidermis exhibited enhanced susceptibility to killing by D. coniospora (A) and spiny balls (B). Figure S12. ITR-1 in the epidermis is required for resistance to fungal infection and physical injury. (A–B) Epidermal-specific RNAi of itr-1 significantly reduced the survival rate of worms after D. coniospora infection (A) or treatment with spiny balls (B). (C–D) Intestinal-specific knock-down of itr-1 did not influence the survival of nematodes after D. coniospora infection (C) and treatment with spiny balls (D). Figure S13. Epidermal-specific, rather than intestinal-specific, knockdown of egl-30 or egl-8 suppresses innate immunity. (A–B) Epidermal-specific knock-down of egl-30 or egl-8 by RNAi reduced the survival of nematodes exposure to D. coniospora infection (A) and spiny balls (B). (C–D) Intestinal-specific knock-down of egl-30 or egl-8 did not influence the survival of nematodes after D. coniospora infection (C) and treatment with spiny balls (D). Figure S14. EGL-30 and EGL-8 in the epidermis are required for resistance to fungal infection and physical injury. (A–B) Epidermal-specific expression of egl-30 rescued immune-deficient phenotypes in egl-30(n686) mutants to D. coniospora infection (A) and treatment with spiny balls (B). (C–D) Epidermal-specific expression of egl-8 (cz15896) rescued immune-deficient phenotypes in egl-8(n488) mutants to D. coniospora infection (C) and treatment with spiny balls (D). Figure S15. cst-1 RNAi significantly reduces the expression of cst-1. After worms were subjected to cst-1 RNAi,the expression of cst-1 was determined by qPCR. Figure S16. CST is not required for ROS production induced by fungal infection and physical injury.cst-1 RNAi did not influence the production of ROS induced by D. coniospora (DC) and spiny balls (SB). Figure S17. CST-1 in the intestine is not required for resistance to fungal infection and physical injury. (A–B) Intestinal-specific knock-down of cst-1 by RNAi did not affect the survival rate of nematodes after D. coniospora infection (A) and treatment with spiny balls (B). Figure S18. BAR-1 is not involved in DAF-16 activation upon fungal infection and physical injury. Mutations in bar-1(ga80) did not influence the nuclear accumulation of DAF-16 after D. coniospora infection (DC) and treatment with spiny balls (SB).(PDF)Click here for additional data file.


Persistent PDA remains a common clinical presentation in preterm infants. We have already shown that high shunt volume increases LVO and infaScans were performed at 3T using pediatric and extremities coils. Infants were scanned with ear protection, routine monitoring and without sedation/anesthesia. Optimized 2D SSFP short axis 10-slice stacks[27 control infants median(range) corrected-GA 34+4(28+3-39+3)weeks, weight at scan 1730(790-3050)grams and 12 PDA infants (determined by echo prior to MRI) corrected-GA 29+5(27+3-36+1)weeks, weight at scan 1100(660-2400)grams were scanned. Shunt volume ranged from 7-74% of LVO[Although LVO, EDV and LVmass are significantly increased in PDA infants, EF and %thick are not. In addition none of the PDA infants are over the Starlings curve. This would suggest that function is maintained in these enlarged hearts.MRC UK studentship


Caenorhabditis elegans, mutants of the conserved insulin/IGF-1 signalling (IIS) pathway are long-lived and stress resistant due to the altered expression of DAF-16 target genes such as those involved in cellular defence and metabolism. The three Δ9 desaturase genes, fat-5, fat-6 and fat-7, are included amongst these DAF-16 targets, and it is well established that Δ9 desaturase enzymes play an important role in survival at low temperatures. However, no assessment of cold tolerance has previously been reported for IIS mutants. We demonstrate that long-lived age-1(hx546) mutants are remarkably resilient to low temperature stress relative to wild type worms, and that this is dependent upon daf-16. We also show that cold tolerance following direct transfer to low temperatures is increased in wild type worms during the facultative, daf-16 dependent, dauer stage. Although the cold tolerant phenotype of age-1(hx546) mutants is predominantly due to the Δ9 desaturase genes, additional transcriptional targets of DAF-16 are also involved. Surprisingly, survival of wild type adults following a rapid temperature decline is not dependent upon functional daf-16, and cellular distributions of a DAF-16::GFP fusion protein indicate that DAF-16 is not activated during low temperature stress. This suggests that cold-induced physiological defences are not specifically regulated by the IIS pathway and DAF-16, but expression of DAF-16 target genes in IIS mutants and dauers is sufficient to promote cross tolerance to low temperatures in addition to other forms of stress.In  Caenorhabditis elegansDrosophila melanogasterMus musculusGenetic modifications which promote longevity are associated with increased resistance to environmental stress in model organisms ranging from yeast to mice. These longevity and stress-resistant phenotypes can be induced by disrupting conserved signal transduction pathways which respond to changes in nutrient availability and/or other environmental conditions  are known to play a key role in preventing and repairing damage induced by high temperatures C. elegans Δ9 desaturase genes fat-5, fat-6 and fat-7 is regulated by the IIS pathway and DAF-16 fat-6 has been identified as a direct target of DAF-16 daf-16-dependent, dauer stage age-1(hx546) mutant and wild type C. elegans at low temperatures, and assessed the contribution of Δ9 desaturase genes to cold tolerance in each genotype.At favourable growth temperatures, expression of the three age-1(hx546) mutant adults following direct transfer from 20°C to 4°C±0.5°C. This is substantially below the thermal range for wild type growth and activity of 15°C–25°C age-1(hx546) mutants relative to wild type adults under these conditions , with mean survival times increased by approximately 85% (daf-16(mu86) null mutants and in age-1(hx546); daf-16(mu86) double mutants following direct transfer from 20°C to 4°C±0.5°C. No significant differences in survival were observed among wild type, daf-16(mu86) mutants and age-1(hx546); daf-16(mu86) double mutants under these conditions (daf-16 is required for increased cold tolerance of age-1(hx546) mutants, but does not contribute to survival of wild type adults at low temperatures.To establish if insulin/IGF-1 signalling plays a role in cold tolerance, survival times were compared between young wild type (N2) and tely 85% . This innditions . This sudaf-169 desaturase genes are up-regulated in wild type dauers, we also examined resistance to low temperature stress during this stage. Over 90% of dauers survived at 4°C±0.5°C until after all fed wild type and age-1(hx546) mutant adults had died (age-1(hx546) mutant adults is lower than in wild type dauers under these conditions (z = 8.85 p<0.001).Dauer formation is partially regulated by the IIS pathway and is dependent upon had died . This inC. elegans Δ9 desaturase enzymes exhibit slight differences in substrate specificity, with FAT-5 primarily converting palmitic acid to palmitoleic acid, whilst FAT-6 and FAT-7 primarily convert stearic acid to oleic acid fat-6 or fat-7 induce compensatory responses in the expression of the remaining Δ9 desaturase genes, and mutants that are defective for single Δ9 desaturase genes display no obvious phenotype 9 desaturase enzymes contribute to cold tolerance in age-1(hx546) mutants, the expression of multiple Δ9 desaturase genes was simultaneously eliminated or suppressed using a combination of loss-of-function mutations and RNA interference (RNAi).The fat-6(tm331); fat-7(wa36) double mutant had dramatically reduced survival times at 4°C in the wild type  and age-1(hx546) mutant  backgrounds relative to respective controls (age-1(hx546) mutant background, suggesting that fat-6 and fat-7 play an important role in the increased cold tolerance of these long-lived mutants. The age-1(hx546); fat-6(tm331); fat-7(wa36) triple mutants were moderately more sensitive to low temperatures than wild type worms , with mean survival times reduced by almost 14%. However, fat-6(tm331); fat-7(wa36) individuals with an age-1 mutant background were more cold tolerant than those with a wild type background , with mean survival times remaining approximately 43% greater. This suggests that changes induced by the age-1(hx546) mutant allele which promote cold tolerance involve another gene or genes, in addition to fat-6 and fat-7.The controls . Whilst fat-5 in fat-6(tm331) mutants reduced mean survival times at 4°C  by almost 24%, and RNAi of fat-5 in fat-7(wa36) mutants reduced mean survival times at 4°C  by 30% ; fat-6(tm331) double mutants  by approximately 33%, and in age-1(hx546); fat-7(wa36) double mutants  by 38%, relative to age-1(hx546) mutant controls (fat-6(tm331) or fat-7(wa36) desaturase mutants with an age-1(hx546) mutant background subjected to fat-5 RNAi remained considerably more cold tolerant than equivalent individuals in a wild type background (fat-6(tm331) mutants: z = 7.12, p<0.001, fat-7(wa36) mutants: z = 7.49, p<0.001). In both cases mean survival times remained approximately 52% greater in the age-1(hx546) mutant background.Relative to wild type controls, RNAi of ) by 30% . Similarcontrols . However9 desaturase genes cause embryonic lethality 9 desaturase genes had been reduced or eliminated, RNAi was used in two different strategies. First, fat-5 expression was reduced in fat-6(tm331); fat-7(wa36) double mutants and in age-1(hx546); fat-6(tm331); fat-7(wa36) triple mutants. Second, fat-6 and fat-7 expression was reduced in fat-5(tm420) mutants and in age-1(hx546); fat-5(tm420) double mutants. As fat-6 and fat-7 have approximately 84% nucleotide homology, the expression of both genes is suppressed when either is targeted by RNAi Simultaneous mutations in all three Δ9 desaturase genes was reduced or eliminated, survival times at 4°C were reduced dramatically in both the wild type and age-1(hx546) mutant backgrounds relative to controls (fat-5 in fat-6(tm331); fat-7(wa36) double mutants reduced mean survival times  by 67%, and RNAi of fat-6 and fat-7 in fat-5(tm420) mutants reduced mean survival times  by approximately 58%. Relative to age-1 mutant controls, RNAi of fat-5 in age-1(hx546); fat-6(tm331); fat-7(wa36) triple mutants reduced mean survival times  by almost 69%, and RNAi of fat-6 and fat-7 in age-1(hx546); fat-5(tm420) double mutants reduced mean survival times  by 62%. In both genetic backgrounds, fat-6(tm331); fat-7(wa36) mutants subjected to fat-5 RNAi appeared more sensitive to cold than fat-5(tm420) mutants subjected to fat-6/fat-7 RNAi. However, these differences were not statistically significant and, even if real, may simply reflect reduced efficiency of RNAi relative to loss-of-function mutations. Importantly, individuals with an age-1(hx546) mutant background remained more cold tolerant than equivalent individuals with a wild type background even when the expression of all three Δ9 desaturase genes had been knocked out or knocked down. When fat-5 expression was suppressed by RNAi in age-1(hx546); fat-6(tm331); fat-7(wa36) triple mutants, mean survival times were higher than in fat-6(tm331); fat-7(wa36) double mutants  by approximately 75%. Similarly, mean survival times remained greater in age-1(hx546); fat-5(tm420) double mutants on fat-6/fat-7 RNAi plates than in fat-5(tm420) mutants on fat-6/fat-7 RNAi plates  by 66%. These results again suggest that Δ9 desaturase genes play an important role in the cold tolerant phenotype of age-1(hx546) mutants, but indicate that additional genes are also involved.When the function of all three Δcontrols . CompareD. melanogaster strains C. elegans genotypes, we compared recovery times at room temperature (∼22°C), following 6 hours exposure to 4°C±0.5°C, among a representative set of strains; wild type (N2), age-1(hx546) mutants, fat-6(tm331); fat-7(wa36) double mutants and age-1(hx546); fat-6(tm331); fat-7(wa36) triple mutants. Considerable variation in cold coma recovery times was observed among the different genotypes (age-1(hx546) mutants resumed locomotion more rapidly than wild type worms after removal from the low temperature stress , with mean recovery times reduced by almost 27%, differences in recovery times between the two genotypes were less apparent than differences in survival following prolonged periods at 4°C. However, the fat-6(tm331); fat-7(wa36) double mutant had substantially delayed recovery times in the wild type  and age-1(hx546) mutant  backgrounds relative to respective controls. Mean recovery times were prolonged by 74% in the wild type background and by almost 76% in the age-1(hx546) mutant background. FAT-6 and/or FAT-7 thus promote recovery of locomotion in both genotypes following exposure to low temperatures. Although recovery was delayed in age-1(hx546); fat-6(tm331); fat-7(wa36) triple mutants relative to the wild type strain , these worms recovered more rapidly than fat-6(tm331); fat-7(wa36) double mutants with a background that was wild type for age-1 , with mean recovery times reduced by approximately 26%. These results reinforce the findings from the survival experiments  mutants, and exemplify the use of cold coma recovery assays to assess variation in cold tolerance among C. elegans genotypes.‘Cold coma’ recovery times have been used to characterise variability in cold tolerance among different enotypes . Althougeriments  concernidaf-16 does not appear to be required for cold tolerance in wild type adults, we asked specifically if the DAF-16 transcription factor becomes activated at low temperatures. Cellular distributions of a DAF-16::GFP fusion protein were categorised on a scale ranging from 1 , representing minimal DAF-16 activity, to 4 , representing maximal DAF-16 activity (fat-7 expression has been reported in wild type C. elegans within 3 hours of transfer from 25°C to 10°C age-1(+) adults at 20°C, accumulation of DAF-16::GFP was observed in the nuclei of age-1(hx546) mutants  individuals displayed categories 3 or 4, p<0.001) (age-1(hx546) mutant adults at 4°C , no significant change in the subcellular localisation of DAF-16::GFP was observed in age-1(+) C. elegans at this temperature relative to controls maintained at 20°C  mutants clearly overlap with the mechanism(s) required for cold resistance.As functional activity . Distribp<0.001) . Althoug at 20°C . These oage-1(hx546) mutants have increased cold tolerance relative to wild type C. elegans when transferred directly from an optimal growth temperature to 4°C during early adulthood. As activity and reproduction completely arrest at this temperature, survival at 4°C does not simply reflect longevity under optimal conditions. We have also established for the first time that dauer larvae can survive for prolonged periods at low temperatures compared to adults which have undergone development in favourable growth conditions, which may represent a survival strategy in seasonal temperate environments.It is well established that mutations which disrupt insulin/IGF-1 signalling promote longevity and stress resistance in diverse evolutionary lineages. However, no assessment of cold tolerance has previously been reported in any IIS mutant of any species, despite this presumably being an important component of life history in many wild populations. We have demonstrated that age-1(hx546) mutants and dauers may arise because these worms are physiologically prepared for the stress, at least with regard to the threat to membrane function, through changes in membrane lipid composition brought about by the up-regulation of Δ9 desaturase genes. Both a cold tolerant phenotype reduction and a prolongation of cold coma recovery time arise when Δ9 desaturase expression is experimentally disrupted in age-1(hx546) mutants. Tolerance of 4°C appeared reduced to a greater extent when both the fat-6 and fat-7 Δ9 desaturase genes were defective as compared to when fat-5 and fat-6 or fat-5 and fat-7 expression was eliminated or suppressed. Consistent with this, Brock et al.fat-6(tm331); fat-7(wa36) double mutants also displayed additional defects, including reduced fat storage, slow growth, reduced fecundity and a high proportion of embryonic lethality, despite a more than 40-fold increase in fat-5 expression age-1(hx546) mutant than the wild type background, but the age-1 mutation still conferred some elevation of cold tolerance. This suggests that while the cold tolerance of age-1(hx546) mutants is principally due to the Δ9 desaturase genes, additional genes contribute to the phenotype. Mutations in age-1 may cause changes in the expression of other genes encoding enzymes involved in lipid metabolism that may also affect membrane fluidity The cold tolerant phenotype of daf-2(e1370) mutants and dauers age-1(hx546) mutants than in the wild type age-1(hx546) mutants than in wild type C. elegans9 desaturase enzymes to enhance the preservation of membrane function at low temperatures in age-1(hx546) mutants.IIS mutants and dauers differ from wild type adults in other aspects of metabolism which may be involved in cold tolerance. For instance, genes involved in the synthesis of trehalose are up-regulated in et al.C. elegans following a period of acclimation at 10°C is not exclusively dependent upon membrane lipid composition, and that additional mechanisms must contribute to cold acclimation and cold tolerance in poikilothermic animals. Heat-shock proteins enhance cold tolerance in S. cerevisiaeage-1(hx546) mutants and dauers than in wild type adults age-1(hx546) mutants.Murray daf-16(mu86) mutants are as sensitive to low temperatures as wild type adults. Additionally, no activation of DAF-16, as revealed by nuclear localisation of DAF-16::GFP, was observed when individuals with a wild type background were exposed to low temperatures. This suggests that insulin/IGF-1 signalling and DAF-16 activation are not required for the expression of genes in physiological defence against cold-induced damage. RNAi of fat-6 and fat-7 also increase resistance to high temperatures in wild type C. elegans independently of daf-16In contrast to high temperature and oxidative stress daf-16::gfp fusion gene et al.C. elegans strain most accurately portrays changes in subcellular distribution of DAF-16 under different thermal conditions, cold tolerance in wild type C. elegans has also been shown here to be daf-16 independent.Using a line transgenic for a Pseudomonas aeruginosa infection daf-16, yet functional daf-16 is not required for wild type levels of resistance to these conditions daf-16 independent mechanisms which target effector genes with expression up or down regulated in IIS mutants and dauers may be involved in mediating the cold-induced stress response. Several transcription factors, in addition to DAF-16, are known to regulate Δ9 desaturase gene expression, including the nuclear hormone receptors NHR-49 and NHR-80, and SBP-1, the C. elegans homologue of sterol-regulatory-element-binding-protein (SREBP) transcription factors, key regulators of lipid homeostasis in mammals C. elegans. One or more of these transcription factors or yet another transcription factor may well contribute to cold tolerance in age-1(hx546) mutants, including through the activation of the desaturase genes. However, as the increased cold tolerant phenotype of age-1(hx546) mutants is entirely dependent upon functional daf-16, all the additional resistance to cold stress exhibited by the age-1(hx546) mutants appears to be mediated by DAF-16 activity.DAF-16::GFP translocates to the nucleus in response to high temperatures, oxidative stress and starvation C. elegans. Such a relationship has previously been observed in D. melanogaster lines selected for increased longevity age-1(hx546) mutants promotes cold tolerance, Broughton et al.D. melanogaster. However, the ablated flies displayed reduced levels of the cryoprotectant trehalose in the hemolymph, while trehalose levels are up-regulated in C. elegans IIS mutants and dauer larvae We have established for the first time that an association between longevity and resistance to low temperatures exists in C. elegans populations that experience rapid fluctuations in temperature. In some invertebrates diapause is the primary mechanism for over-winter survival, and is induced in response to changes in temperature and/or photoperiod , CF1038 daf-16(mu86), BX107 fat-5(tm420), BX106 fat-6(tm331), BX153 fat-7(wa36), BX160 fat-6(tm331); fat-5(tm420), BX110 fat-7(wa36); fat-5(tm420), BX156 fat-6(tm331); fat-7(wa36) and TJ356 N2; zIs356 . Long-lived age-1(hx546) mutants were utilized due to their ability to develop and reproduce at temperatures which induce developmental arrest in other IIS mutants. Crosses between age-1(hx546) mutants and additional strains were confirmed by PCR using gene specific primers listed in E. coli strain HB101. Strains were maintained at 20°C until cold tolerance assays commenced.The following genotypes were obtained from the Caenorhabditis Genetics Centre: N2 Bristol (wild type), TJ1052 C. elegans fed on HT115 bacteria containing an empty RNAi plasmid vector (pL4440) were used as negative controls.RNA interference (RNAi) was induced by feeding using clones from the Ahringer library. RNAi experiments were performed on NGM plates supplemented with Ampicillin, Tetracycline and IPTG as previously described Age-synchronised adults, on the first day of reproduction, were transferred directly from 20°C to 4°C±0.5°C on plates containing approximately 20 individuals each. Survival was monitored regularly, following 20–30 minutes recovery at room temperature, until all individuals had died. If survival status was not immediately apparent, worms were gently touched with a platinum wire to stimulate a response.Dauer larvae were transferred directly from 20°C to 4°C±0.5°C approximately 24 hours after populations had become starved. Survival status was monitored in 100 individuals, divided among 5 NGM plates, until all non-dauer controls had died.Age-synchronised adults, on the first day of reproduction, were transferred from 20°C to 4°C±0.5°C for 6 hours on NGM plates containing 10 individuals each. After the stress period, recovery times were monitored at room temperature (approximately 22°C). Individuals were considered to have recovered when they began to move spontaneously around the NGM plates. Recovery times were recorded in 100 individuals per genotype over 2 separate blocks.Cellular distributions of a DAF-16::GFP fusion protein were compared among well fed young adults which had been maintained at the control temperature (20°C) or exposed to 4°C±0.5°C for 6 hours. DAF-16::GFP localisation was categorised from 1–4, where 1 represents an unlocalised distribution throughout the cells and 2, 3 and 4 represent increasingly nuclear distributions, in 100–120 individuals per genotype and temperature treatment over 3 separate blocks. Worms were anaesthetized with 5 mM levamisole and epifluorescence imaging was performed using a Leica DMR HC confocal microscope.All analyses were performed in R version 2.10.1 For survival analysis, a small number of individuals which had died from causes other than exposure to low temperatures (e.g. rupturing of the vulva) were removed from the analysis. Each survival experiment was analysed independently using an accelerated failure time parametric regression model with a Weibull error distribution. These models were selected based upon visual inspection of the plotted residuals following analyses using a variety of different probability distributions. They were considered to be more robust than non-parametric survival models because the latter typically have greatest power when the hazard ratios (relative risks of death) between two covariates are proportional, and this was not the case with our data. However, to assess the generality of the results obtained from the accelerated failure time models, we also analysed the survival experiments using non-parametric weighted cox regression models, which provide unbiased average hazard ratio estimates rather than assuming proportional hazards. We obtained qualitatively very similar results from the two sets of analyses, but provide only the z-scores derived from the accelerated failure time models in the results section.Cold coma recovery times were compared among genotypes using a generalised linear mixed effects model with a gamma error distribution. The model was fitted using the penalised quasi-likelihood (PQL) method DAF-16::GFP intracellular localisation scores were compared between genotypes and among temperature treatments using an ordinal multinomial continuation-ratio logit model Table S1Sequences of oligonucleotide primer pairs used in PCRs on genomic DNA to confirm age-1, fat-5, fat-6, fat-7 and daf-16 genotypes.(DOCX)Click here for additional data file.


However, CD4+ Th’s signals that govern CTL survival and functional memory are still not completely understood. In this study, we assessed the role of CD4+ Th cells with acquired antigen-presenting machineries in determining CTL fates. We utilized an adoptive co-transfer into CD4+ T cell-sufficient or -deficient mice of OTI CTLs and OTII Th cells or Th cells with various gene deficiencies pre-stimulated in vitro by ovalbumin (OVA)-pulsed dendritic cell (DCova). CTL survival was kinetically assessed in these mice using FITC-anti-CD8 and PE-H-2Kb/OVA257-264 tetramer staining by flow cytometry. We show that by acting via endogenous CD40L and IL-2, and acquired peptide-MHC-I (pMHC-I) complex signaling, CD4+ Th cells enhance survival of transferred effector CTLs and their differentiation into the functional memory CTLs capable of protecting against highly-metastasizing tumor challenge. Moreover, RT-PCR, flow cytometry and Western blot analysis demonstrate that increased survival of CD4+ Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic  molecules. Taken together, our results reveal a previously unexplored mechanistic role for CD4+ Th cells in programming CTL survival and memory recall responses. This knowledge could also aid in the development of efficient adoptive CTL cancer therapy.Involvement of CD4 Following recognition of foreign antigen (Ag), they undergo 3 distinct phases of immune responses  or CD4+ T cell-deficient  mice. We showed that Th cells enhanced CTL survival and their differentiation into functional memory pool, leading to CTL-mediated immune protection against highly metastasizing tumor challenge. In the effector/contraction phase, the inherent ability of CD4+ Th cells to provide IL-2 and CD40L and the low level of acquired pMHC-I complexes was found to be critical for endowing CTLs with survival, and memory development advantages. The down-stream molecular mechanisms underlying the prolonged survival of Th-helped CTLs were also systematically assessed.Here, we generated effector Th cells and CTLs by All the animal experiments were performed as per the guidelines approved by the University Committee on Animal Care and Supply, University of Saskatchewan. Protocol Number: 20100027.b (AF6-88.5), Iab (KH74), CD80 (16-10A1), CD40 (3/23), CD40L (MR1), CD54 (3E2), CD62L (MEL-14), CD69 (H1.2F3), TRAIL (N2B2), PD-1 (J43) and IFN-γ (XMG1.2), and streptavidin-PE-Texas-Red, PE-Cy5 or -FITC were purchased from BD-Biosciences or eBiolegend. The biotin-anti-IL-7Rα (A7R34) and -peptide/MHC-I (pMHC-I) (25-D1.16) Abs were purchased from eBioscience. The FITC-anti-perforin (CB5.4) Ab was obtained from Alexis Chemicals. The FITC-anti-CD8 (KT15) Ab and H-2Kb/OVA257-264 tetramer were obtained from Beckman Coulter. The OVA-transfected, mouse malignant melanoma (BL6-10OVA) and EL4 (EG7) cell lines were cultured as described previously [b and Iab was obtained from American Type Culture Collection. The age-matched wild-type (WT) C57BL/6 , OVA257-264 and OVA323-329-specific TCR-transgenic OTI and OTII, H-2Kb-/- and Iab-/- mice on WT B6 background were purchased from Jackson Laboratory. The OTII/CD40L-/- and OTII/IL-2-/- mice were generated by backcrossing designated gene-deficient mice with OTII mice [The biotin- and fluorescent dye-labeled (FITC or PE) antibodies (Abs) specific for CD4 (GK1.5), CD11c (HL3), CD44 (IM7), H-2Kb AF6-8.5, Iab b-/- mice were referred as DCova and DCova(Kb-/-), respectively.Bone-marrow-derived, DCova from WT mice were generated by culturing bone marrow cells for 6 days in medium containing IL-4 (20 ng/ml) and GM-CSF (20 ng/ml) and pulsing with 0.1 mg/mL OVA overnight at 37°C as described previously . OVA-pul+ and CD8+ T cells were isolated from WT, OTII or OTI splenocytes as previously described [+ T (effector CTL) and Th cells, the OTI CD8+ or OTII CD4+ T cells  were respectively cultured with irradiated  DCova  at 1:4 ratio for three days as previously described [+ and CD8+ T cells were purified by Ficoll-Paque (Sigma-Aldrich) separation followed by using CD4 and CD8 MACS microbeads (Beckman Coulter) and termed effector CD4+ Th cells and CTLs, respectively. The naïve, effector CTL and Th cells were stained with panel of cell-, naïve-, activation- or memory-specific markers, and characterized phenotypically by flow cytometry. The Th cells derived from OTII/CD40L-/- and OTII/IL-2-/- mice were termed Th(CD40L-/-) and Th(IL-2-/-), respectively. DCova(Kb-/-)-stimulated Th cells without acquired pMHC-I molecules were referred as Th(pMHC-I-/-). For cytokine profiling, effector CTL and Th or Th(IL-2-/-) were respectively re-stimulated with irradiated EG7 and OVAII -pulsed LB27, and culture supernatants were assessed using cytokine ELISA kits (R&D Systems) from previous description with few modifications [+ T cells were co-cultured with irradiated DCova in the presence or absence of naïve OTII CD4+ T cells for 3 days. For priming studies, the activated CD8+ T cells were purified by Ficoll-Paque separation and negative selection using anti-CD4 (L3T4) paramagnetic beads , and termed effector CTLs and CTLs with help in in vitro priming, respectively.The naïve CD4escribed . The actications . Naive O6 effector CTLs alone that received help during priming period or 5x106 effectors CTLs with or without 2x106 Th cells  were i.v. transferred WT mice. Similarly, effector CTLs alone that received help during priming period or effectors CTLs with or without effector Th, Th(IL-2-/-), Th(CD40L-/-), and Th (pMHC-I-/-) cells (2x106each) were i.v. transferred to Iab-/- mice. After confirming equal engraftment on the following day, the effector CTL survival was monitored 6 or 60 days later in peripheral blood by staining with H-2Kb/OVA257-264 tetramer and FITC-anti-CD8 Ab (tetramer assay) [6 DCova 60 days later and monitored for the expansion of memory CTLs on 4th day by tetramer assay. To phenotypically characterize effector or memory CTLs, the blood samples were collected 6 or 60 days later, and stained for tetramer assay along with panel of biotin-conjugated Abs specific for effector or memory markers, and streptavidin-PE-Texas Red.Approximately 5x106 effector CTLs alone that received help during priming period or 5x106 effectors CTLs with or without 2x106 Th cells  were i.v. transferred WT and Iab-/- mice. In addition, to understand mechanism of CD4+ T cells, some Iab-/- mice groups were also transferred with effector CTLs with effector Th, Th(IL-2-/-), Th(CD40L-/-) or Th(pMHC-I-/-) cells (2x106each). Hundred days later, all the groups were i.v. challenged with highly-metastasizing BL6-10OVA tumor cells (0.5x106/mice), monitored for protection as described previously, and sacrificed later to determine the numbers of surface black tumor colonies in the lungs [To assess functional effect of memory CTLs, approximately 5x10he lungs .th day of tumor challenge, the splenocytes of helped or unhelped mice were re-stimulated with OVAI  and subjected to intracellular IFN-γ staining (BD-Biosciences) as described previously [On the 24eviously .b-/- mice were i.v. injected with effector CTLs (5x106) with or without effector CD4+ Th (2x106) cells. The blood and lymphoid organs were collected 16 days later, and processed to remove RBCs. The T lymphocytes were enriched in nylon wool columns (C & A Scientific). After labeling T lymphocytes with H-2Kb/OVA257-264 tetramer and anti-PE microbeads (Miltenyi Biotec Inc), a highly purified population of OVA-specific CTLs was obtained by positive selection by passing in 2 separate MACS columns sequentially. The purified CTLs from WT or Iab-/- mice, which were transferred with CTLs plus effector CD4+ Th cells were termed “helped CTLs” in WT or Iab-/- mice, whereas the purified CTLs from Iab-/- mice transferred with only CTLs were termed “unhelped CTLs” in Iab-/- mice.The WT and Iab-/- and unhelped CTLs in Iab-/-) was examined using RT2ProfilerTM PCR array (SuperArray Bioscience). Total RNA was isolated using RNeasy extraction kit (Qiagen) and reverse transcribed using RT2 First Strand Kit (SuperArray Bioscience). The mRNA expression of each gene in array system was performed using StepOnePlus thermocycler (Applied Biosystems) and analyzed using Hprt1, Gapdh, and β-actin as internal controls in web-based software as per manufacturer’s instructions.The expression of pathway-focused panel of 84 genes related to apoptosis in the above three groups of CTLs (helped CTLs in WT or Iab-/- and unhelped CTLs in Iab-/-) were further subjected to quantitative RT-PCR (qRT-PCR) for validation of the array results. Sequence-specific primers for β-actin, Bcl-2, FasL and TRAIL were previously described [TRAIL receptor, Nfκb1, Bcl10, Akt1, Caspase-4 and -7 were given in supplementary information , β-actin (13E5), Bcl10 (C79F1), Akt1 (2H10), phospho-Akt1 (S473), cleaved Caspase-3 (Asp175) and -7 (Asp198) , NF-κB-p65 (C-20), phospho-NF-κB-p65 (Ser536) (Santa Cruz Biotechnology) and NFATc1 (7A6) (BD Biosciences). The blots were incubated with goat anti-rabbit IRDyeR800/680CW, and band densities were quantified using ODYSSEY densitometer (LI-COR Bioscience).t-test or analysis of variance, respectively. p<0.05 was considered statistically significant.The statistical analysis was performed using Graphpad Prism-3.0. The results are presented as mean ± SD. The statistical significance between two or more groups was analyzed by Student’s b, CD40 and CD80 and OVA-specific pMHC-I , activation marker (CD69) and effector molecule (perforin), but not IL-7Rα or CD62L [6cells/24hrs) and TNF-α (1.15±0.13ng/mL/106cells/24hrs), but not IL-4 or IL-10 cytokine, indicating a functional effector CTL phenotype. DCova-stimulated CD4+ T cells displayed their subset marker (CD4) and activation marker (CD69) and a low level of acquired pMHC-I. These cells secreted IFN-γ (2.1±0.35ng/mL/106cells/24hrs) and IL-2 (2.75±0.47ng/mL/106cells/24hrs), but not IL-4 and IL-10 cytokine, confirming a type 1 T helper (Th1) cell phenotype [In line with previous observations , OVA-pulc pMHC-I S1a. Folmination  , corroborating previous observations [+ T cell-deficient mice (P<0.05). Furthermore, increasing the dose of Th cells from 2x106 to 5x106 cells per mouse resulted in corresponding increase of memory CTL pool in both WT and CD4+ T cell-deficient mice (P<0.01)  compared to unhelped CTLs (3.9%)  (Since a reduced CTL memory pool was observed in the absence of CD4b-/-)-stimulated Th(pMHC-I-/-) cells failed to display these molecules (-/-) and Th(IL-2-/-) cells failed to display CD40L expression or IL-2 secretion (-/-), Th(IL-2-/-) or Th(pMHC-I-/-) cells were co-transferred with effector CTLs into CD4+ T cell-deficient mice, and CTLs were monitored for their survival and memory development. During the memory stage, effector CTLs transferred alone or with Th(CD40L-/-), Th(IL-2-/-) or Th(pMHC-I-/-) failed to survive, and expand upon boosting (P<0.01 versus Th cell-helped CTLs) , Th(CD40L-/-) or Th(pMHC-I-/-) cells were transferred to Iab-/- mice as indicated. Hundred days later, all the groups were challenged with highly-metastasizing BL6-10OVA tumor cells. In line with the above recall responses, helped CTLs that received help during or after priming completely protected WT mice against tumor challenge , Th(CD40L-/-) or Th(IL-2-/-) cells failed to get protection, and had considerably increased tumor colonies of varying sizes in lungs  were transferred WT mice. Similarly, effector CTLs alone that received help during priming period or effectors CTLs with or without effector Th, Th. Among the up-regulated genes, Caspase family genes, Casp-7, -2 and -3, TNF family members Tnfrsf1a and Trp53, which mediate apoptosis induction, were prominent. Interestingly, 34 genes were found to be significantly down-regulated, of which 24 were pro-survival and 10 were pro-apoptotic genes. Some of the most prominent pro-survival genes down-regulated include Akt1, Api5, Bag1, Bcl-2, Birc3 and 5, Nfκb1, Nol3, Pak7, Pim3, Traf1, and Zc3hc1. The down-regulation of some pro-apoptotic genes, although surprising, may represent compensatory mechanisms that occur in cells experiencing various stresses in an attempt to prevent cell death. Second, in contrast to the situation observed in unhelped CTLs, there was a notable shift in the gene-expression profile in helped CTLs, favoring their survival . Interestingly, a considerable overlap was observed in the up- and down-regulated genes in helped CTLs derived from WT and CD4+ T cell-deficient mice  and down-regulation of the proapoptotic  genes  in comparison to helped CTLs in either Iab-/- or B6 mice   might have played some role in subsequent survival of helped CTLs as IL-7Rα is known to influence CTL survival [-/-), Th(IL-2-/-) or Th(pMHC-I-/-) failed to survive, and expand upon boosting. In contrast, helped memory CTLs expanded up to only 2 fold from their basal levels, reaching almost 5.8±1.5% of the total CD8+ T cell population, reflecting the contribution of endogenous CD4+ T cell help at the time of boost [+ T cells derived from adoptively transferred CD4+ Th cells may also rescue functional recall responses. Therefore, the failure of recall responses in mouse groups with transfer of CD4+ Th(IL-2-/-) and Th(CD40L-/-) cells may also be a result of lacking help IL-2 and CD40L signaling [b-/- mice expressed inhibitory PD-1 molecule, which is consistent with another recent report by Fuse et al, showing that helpless memory CTLs upregulated PD-1 expression and failed in recall responses [+ Th cells rescued CTL from AICD via CD4+ Th cell-to-CD8+ T cell contact [+ Th cell’s CD40L and IL-2 signaling affect CTL phenotype and memory development is still unknown. Conducting experiments to address this question is underway in our laboratory. Furthermore, how CD4+ Th cell’s helper signals such as IL-2 and CD40L are delivered to cognate CD8+ T cells in vivo to govern CTL survival leading to functional memories is also not well described.Identifying specific factors derived from helper CD4research . In the  studied ,37. Tanc studied –8 and ILtor CTLs , and protor CTLs . It has survival ,40, thussurvival . In thissurvival ,43. Duriof boost . In addiignaling ,13. In tesponses . These oesponses ,13. The  contact ,13. In a+ T cells acquired Ag-presenting machinery such as pMHC-I complexes during DC stimulation [+ T cell help to cognate CD8+ T cells. Our recent two photon imaging study provides a strong in vivo evidence, showing that CD4+ Th cells with acquired pMHC-I, but not CD4+ Th cells without acquired pMHC-I are capable of directly interacting with cognate naïve CD8+ T cells in vivo [+ Th cells with acquired pMHC-I can also directly interact with effector CD8+ T cells in vivo to deliver helper signaling. Therefore, the results of this study strongly support the recent emerging notion of the substantial role of Th cells in rescuing effector CTLs from AICD and in promoting the functional memory generation [in vivo.We previously demonstrated that CD4mulation ,21,27. Tmulation . The acq in vivo . This evneration ,41,46. Pneration . It is v+ T cell-provided help was shown to be required for effector CTL survival via the regulation of TRAIL and Bcl-xL [+ T cell help effects on CTL fates. Our data show that the poor survival of unhelped CTLs correlates well with the down-regulation of key survival genes, which mediate signals both at nuclear and cytoplasmic levels, that are required for the activation of the pro-survival NF-κB and attenuation of the pro-apoptotic JNK pathways [+ T cells. A few genes up-regulated above naïve T cell levels, including Akt1, Xiap, CD40lg and Traf1, might have played a leading role in the effector CTL survival. The serine–threonine kinase Akt1, for example, executes potent antiapoptotic effects by inhibiting a cohort of cell death-inducing molecules and activating multiple anti-apoptotic factors [L [+ T cells, leading to efficient CTL survival [+ T cells lacking CD4+ T cell help express TRAIL and die by TRAIL-mediated cell death [+ T cells also express TRAIL, indicating that TRAIL-mediated extrinsic apoptotic pathway appears to be another key mediator in the contraction of adoptively transferred effector CTLs.Previous reports  togetherd Bcl-xL ,9,14. Inpathways . In contpathways ,49. Inte factors ,51. Akt-ctors [L ,52,53. Msurvival . Our datsurvival . It was ll death . In this+ T cell therapy refers to an immunotherapy approach in which tumor-infiltrating lymphocytes (TILs) isolated from tumor specimen are activated and expanded in vitro to large quantities, using anti-CD3/CD28 antibodies and IL-2, and then transferred back to the cancer patients [in vitro-expanded tumor-infiltrating CD8+ T cells, has achieved some degree of success [+ T cell-deficient environment, typically associated with primary cancer therapies [+ T cells promoted CTL survival and tumor localization via rendering tumor environment permissive in the course of adoptive CTL therapy [Adoptive CD8patients . Alternapatients –61. To d success ,63. Howe success ,63 due therapies ,33. It h therapy . TherefoTaken together, our results reveal a previously unexplored mechanism of Th cell action in programming CTL responses. The observed Th cell-effector CTL cooperation could explain why memory CTLs, generated under cognate Th cell help, obtain significant survival and recall advantages. Moreover, this knowledge could aid in the development of efficient adoptive CTL cancer therapeutics.Figure S1+ (effector Th) and CD8+ (effector CTL) T cells (solid thick lines) were stained with a panel of Abs specific for acquired pMHC I and markers for T cell subsets, naïve, activation/maturation or memory T cells as indicated. The cells were then analyzed by flow cytometry. One representative of the two independent experiments is shown. b. DCova and Th cells or effector CTLs were stained with PE-anti-CD11c and FITC-anti-CD4 or FITC-anti-CD8 Abs. The value in the panel represents the mean%±(SD) of CD4highCD11clow (left) or CD8highCD11clow (right) cells in the total population of CD4high Th or CD8high effector CTLs, respectively. One representative figure of three independent experiments is shown.a. DCova, naïve and DCova-stimulated CD4Click here for additional data file.Figure S26 effector CTLs with Th cells (2x106) or without Th cells were adoptively co-transferred to CD4+ T cell-sufficient or CD4+ T cell-deficient mice. On the 16th day, the transferred helped or unhelped effector CTLs were purified from blood and lymphoid organs of CD4+ T cell-sufficient or CD4+ T cell-deficient mice by positive selection using PE-H-2Kb/OVA257-264 tetramer and anti-PE microbeads. The purified CTLs were stained with FITC-anti-CD8 Ab and analyzed for purity by flow cytometry. The data represent mean% ±(S.D) and are cumulative of three independent experiments with two to six mice per group.Approximately 5x10Click here for additional data file.Table S1Click here for additional data file.Table S2A. Top genes uniquely up-regulated above 3 fold. B. Top genes uniquely down-regulated below 3 fold.Click here for additional data file.


The objective of this study was to quantify microstructural remodeling in peri-infarcted and infarcted porcine myocardium using diffusion tensor MRI (DT-MRI) for the first time. High resolution ex vivo late gadolinium enhanced (LGE) MRI was used to segment the DT-MRI data into normal, peri-infarct and infarcted myocardium. LGE-MRI based segmentation produces regions with significantly different microstructural remodeling.T1-weighted late gadolinium enhanced (LGE) magnetic resonance imaging (MRI) is recognized as the “gold standard” for MRI based myocardial infarct mapping . PreviouAntero-septal infarctions in adult female porcine hearts (N=3) were achieved via micro-bead injection distal to the mid left anterior descending coronary artery. After 8-weeks, Gd-DTPA was injected (0.1mmol/kg) and allowed to circulate for 15 minutes before euthanizing. Normal adult porcine hearts (M=3) served as controls. The hearts were excised and LGE MRI (0.33x0.33x0.50mm resolution) began within 2 hours of sacrifice. Immediately afterwards, co-registered DT-MRI (1x1x3mm resolution) was performed. Myocardial voxels were segmented as normal, peri-infarct or infarct based on signal intensity (SI) thresholds of the LGE images for each heart. We defined bootstrapped histograms and medians with 95% confidence intervals (95%-CIs) of each DT invariant in order to make statistical comparisons of non-Gaussian datasets tractable. Remote myocardium in infarcted hearts was also compared to myocardium of control hearts.DT invariant medians and 95%-CIs for segmented myocardium in each heart are listed in Table LGE segmentation of DT-MRI data identifies regions of statistically significant microstructural remodeling in peri-infarcted and infarcted myocardium. Improved LGE segmentation methods hold promise for identifying regions of microstructural remodeling when DT-MRI is not available.Mahajan R01-HL084261; Shivkumar R01-HL084261 and R01-HL067647; Garfinkel P01-HL78931 (Core).


The generation of globally relevant HIV-1 immunogens mimicking native, trimeric Envelope (Env) structure, remains a major challenge for HIV-1 vaccine development. We identified a mosaic Env sequence, originally designed to optimize cellular immunologic coverage of global HIV-1 sequence diversity, which was capable of forming biochemically stable trimers. We assessed this mosaic Env gp140 trimer in guinea pig immunogenicity studies compared to our previously reported biochemically stable C97ZA012 (clade C) trimer.Stable Env gp140 trimer derived from a synthetic mosaic sequence was stabilized with the T4-fibritin C-terminal trimerization tag and produced in 293T cells via PEI transfection. Characterization was performed by Western blotting, size-exclusion chromatography and surface plasmon resonance (SPR). Guinea pigs were immunized three times with 100 µg of mosaic or clade C gp140 protein trimer in CpG/Emulsigen adjuvants. Antibody responses were determined by ELISA and TZM.bl neutralizing antibody assays.Stabilized mosaic gp140 trimer exhibited a single band by Western blotting, single peak by size-exclusion chromatography both after production, a freeze-thaw cycle and 7-day incubation at 4ºC. SPR analyses revealed mosaic gp140 binding with bNAb VRC01. The trimeric mosaic gp140 immunogen elicited high-titer antibodies in guinea pigs by ELISA and high-titer, cross-clade neutralization activity against tier 1 viruses. When compared to clade C gp140 trimer, NAb responses generated by mosaic gp140 trimer were 8.2- and 3.7-fold higher against clade B viruses  but were 10.3- and 46.7-fold lower against clade A and C viruses .A novel, foldon-stabilized mosaic gp140 trimer elicits high-titer binding antibodies as well as high-titer, cross-clade neutralization of tier 1 viruses. The profile of the NAbs elicited by the mosaic trimer differed from that elicited by the clade C trimer. Further exploration and refinement of this concept may contribute to the development of a globally relevant Env immunogen.


Plasmodium has a complex cell biology and it is essential to dissect the cell-signalling pathways underlying its survival within the host.2+ signalling in Plasmodium berghei and Plasmodium yoelii malaria parasites were investigated.Using the fluorescence resonance energy transfer (FRET) peptide substrate Abz-AIKFFARQ-EDDnp and Fluo4/AM, the effects of extracellular ATP on triggering proteolysis and CaP. yoelii and P. berghei. Addition of ATP  to isolated parasites previously loaded with Fluo4/AM in a Ca2+-containing medium led to an increase in cytosolic calcium. This rise was blocked by pre-incubating the parasites with either purinergic antagonists PPADS (50 μM), TNP-ATP (50 μM) or the purinergic blockers KN-62 (10 μM) and Ip5I (10 μM). Incubating P. berghei infected cells with KN-62 (200 μM) resulted in a changed profile of merozoite surface protein 1 (MSP1) processing as revealed by western blot assays. Moreover incubating P. berghei for 17 h with KN-62 (10 μM) led to an increase in rings forms  and a decrease in trophozoite forms .The protease activity was blocked in the presence of the purinergic receptor blockers suramin (50 μM) and PPADS (50 μM) or the extracellular and intracellular calcium chelators EGTA (5 mM) and BAPTA/AM , respectively for P. berghei protease(s) activity and that MSP1 is one target in this pathway.The data clearly show that purinergic signalling modulates  Plasmodium grows and develops inside red blood cells (RBCs), concomitant structural -3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinolinesulfonic acid ester), Triton X-100 and dihydroethidium were purchased from Sigma- Aldrich . BAPTA/acetoxymethyl ester (AM) and Fluo4/AM were bought from Molecular Probes Inc. . The peptide Abz-AIKFFARQ-EDDnp was analytical grade and synthesized according to Hirata, 1994. Analyses of Ca2+ concentration in P. berghei and P.yoelii Fluo4/AM labelled isolated parasites (108 cells ml-1) after addition of adenosine (10 and 15 μM)  in P. berghei or adenosine (10 μM)  in P. yoelii. P values were calculated by comparison with control data . Bar graphs represent means with SEM.Click here for file


We report our experience with 8 patients with thoraco-abdominal aneurysm repair in period of 30 months (June 2010 till December 2012), in MAGDALENA - Clinic for Cardiovascular Diseases, Medical University of Osijek.We conducted a retrospective observational analysis of our experience with different approaches and perfusion techniques in patients with thoraco-abdominal aneurysm.- Group 1: 3 patients: aortic cross-clamp, with left heart bypass, without selective organ perfusion- Group 2: 2 patients: aortic cross-clamp, without left heart bypass, selective organ perfusion- Group 3: 1 patient: aortic cross-clamp, with left heart bypass, selective organ perfusion- Group 4: 2 patients: aortic cross-clamp, without left heart bypass, without selective organ perfusionBetween June 2010 and December 2012, we performed 8 patients with zero mortality and one late post-operative complication, paraplegia after 48 hours. Left heart bypass duration 57 ± 14 minutes, aortic cross-clamp time duration 48 ± 13 minutes, median ICU stay was 95 hours, average hospital stay was 12 ± 3 days.Thoraco-abdominal aneurysm repair with appropriate choice of operative technique and possibility of using left heart bypass and/or selective organ protection provides good results by means of no mortality and low rate of postoperative complications and morbidity.


Activation of I-κB kinases (IKKs) and NF-κB by the human T lymphotrpic virus type 1(HTLV-1) trans-activator/oncoprotein, Tax, is thought to promote cell proliferation and transformation. Paradoxically, expression of Tax in most cells leads to drastic up-regulation of cyclin-dependent kinase inhibitors, p21CIP1/WAF1 and p27KIP1, which cause p53-/pRb-independent cellular senescence. Here we demonstrate that p21CIP1/WAF1-/p27KIP1-mediated senescence constitutes a checkpoint against IKK/NF-κB hyper-activation. Senescence induction by Tax is attenuated by mutations in Tax that reduce IKK/NF-κB activation and prevented by blocking NF-κB using a degradation-resistant mutant of I-κBα despite constitutive IKK activation. Small hairpin RNA-mediated knockdown indicates that RelA induces this senescence program by acting upstream of the anaphase promoting complex and RelB to stabilize p27KIP1 protein and p21CIP1/WAF1 mRNA respectively. Finally, we show that downregulation of NF-κB by the HTLV-1 anti-sense protein, HBZ, delay or prevent the onset of Tax-induced senescence. We propose that the balance between Tax and HBZ expression determines the outcome of HTLV-1 infection. Robust HTLV-1 replication and elevated Tax expression drive IKK/NF-κB hyperactivation and trigger senescence. HBZ, however, modulates Tax-mediated viral replication and NF-κB activation, thus allowing HTLV-1-infected cells to proliferate, persist, and evolve. Finally, inactivation of the senescence checkpoint can facilitate persistent NF-κB activation and leukemogenesis.


Using Runx3-ChIP-seq and transcriptome analysis of wild type vs. Runx3-/- primary cells we have now identified Runx3-regulated genes in the two cell types at both resting and IL-2-activated states. Runx3-bound genomic regions in both cell types were distantly located relative to gene transcription start sites and were enriched for RUNX and ETS motifs. Bound genomic regions significantly overlapped T-bet and p300-bound enhancer regions in Runx3-expressing Th1 helper cells. Compared to resting cells, IL-2-activated CD8+ T and NK cells contain three times more Runx3-regulated genes that are common to both cell types. Functional annotation of shared CD8+ T and NK Runx3-regulated genes revealed enrichment for immune-associated terms including lymphocyte activation, proliferation, cytotoxicity, migration and cytokine production, highlighting the role of Runx3 in CD8+ T and NK activated cells. The transcription factor Runx3 is highly expressed in CD8 These cytotoxic lymphocytes recognize foreign, infected or tumor cells and following activation undergo interleukin-dependent proliferation. They then kill target cells by releasing perforin and granzyme B-containing granules and cytokines such as tumor necrosis factor (TNF) and interferon gamma (Ifnγ) [The Runx transcription factor (TF) family consists of 3 highly conserved members Runx1-3) that function as key regulators of lineage specific gene expression in major developmental pathways [-3 that fa (Ifnγ) . Runx3 is highly expressed in mature CD8-TC and in NKC and plays an important role in their proliferation and activation -7. SeverRunx3 ChIP-seq was conducted using the highly specific in house polyclonal anti-Runx3 antibody Poly-G  Figure . Model-bCd4, silencer in first intron [Cd8, enhancers located between Cd8a and Cd8b1 [Ncr1 encoding the NKC lineage restricted receptor NKp46 [Klr regions in CD8-TC  of annotated genes, reveled that 75% and 55% of Runx3 peaks in CD8-TC and NKC, respectively, were located more than 5 kb away from the nearest TSS, and 19%-32%, respectively, were >50 kb away from a TSS . These rTGt/cGGt/c is considered the canonical RUNX motif [TGTGGT  genes in resting CD8-TC and NKC, respectively, with ~60% and ~40% of these genes being down-regulated and up-regulated, respectively, in both cell types  of IL-2-activated CD8-TC/NKC Runx3-bound regions with previously reported p300-bound  and T-be T cells , manifesDe novo motif finding analysis [+ T cells is quite low but induced in a T-bet-dependent manner upon activation and differentiation along the Th1 lineage [Ifnγ and Il4 in Th1 cells [analysis  revealed lineage ,27. Runxh1 cells . Runx3 ah1 cells ,28 and T-/- vs. WT) in IL-2 activated CD8-TC and NKC, respectively  of these genes in the same direction (up- or down-regulated) in both cell types (http://www.ingenuity.com), revealed enrichment for ontology terms including cellular growth and proliferation, cellular development, cell death and inflammatory response  were common to both cell types (p=2.7E-41), compared to 37 common genes expected by chance . This obll types  supportsll types  and 20 oll types . This frresponse . Taken t-/- CD8-TC and/or NKC are described in Fhl2 [Rab27b [Slc16a1 [Tnfrsf9 [Itgae [Kit [Myc [Pik3cg [Appl2 [Myb [Il2ra and Styk1 [Grap [Runx1 [Smad3 [Tspan32 [Inpp4b [A number of Runx3-regulated genes that could contribute to the impaired proliferation phenotype in IL-2-activated Runx3 in Fhl2 , Rab27b  [Rab27b , Slc16a1[Slc16a1 , Tnfrsf9[Tnfrsf9 , Itgae [9 [Itgae , Kit [35Kit [Myc , Pik3cg  [Pik3cg , Appl2 [g [Appl2 , Myb [39nd Styk1  were dowk1 [Grap , Runx1 [p [Runx1 , Smad3 [1 [Smad3 , Tspan32[Tspan32  and Inpp [Inpp4b . SeveralCombined ChIP-seq and gene expression analyses revealed that the number of Runx3-regulated genes in IL-2 activated CD8-TC and NKC was 1.5- and 2.2-fold, respectively, higher than at the cell resting state . Of the T cells develop from CLPs via Notch1-Bcl11b-dependent pathway  and lackRunx3 occupies thousands of genomic loci in resting and IL-2-activated CD8-TC and NKC, reflecting a common property of many TFs, including the other two RUNX family members Runx1 ,52 and RTGt/cGGt/c. Interestingly, the variant RUNX motifs were not distributed evenly in Runx3 peaks located at different genomic regions, rather the TGCGGt/c and TGTGGt/c variants were more prevalent at promoter and enhancer regions, respectively. These results indicated that Runx3 binds to DNA directly to canonical RUNX motifs. Indeed, de novo motif finding revealed that the most enriched motifs corresponded to these variant canonical RUNX motifs, as was also found for Runx1-bound regions in megakaryocytes [in vivo IL-15-activated NK cells.About 90% of Runx3 peaks in resting and IL-2-activated CD8-TC and NKC harbor at least one RUNX motif that conforms to the canonical DNA sequence ryocytes  and for de novo occupied regions that are unique to IL-2-activated cells are enriched for RUNX-RUNX and ETS-RUNX modules. These findings suggested that Runx3 and Ets family TFs cooperate in regulating common target genes in CD8-TC and NKC. This possibility is supported by observations that Runx family TFs [In addition to RUNX, Runx3-bound promoter and enhancer regions are enriched for ETS family motifs and mily TFs  includinmily TFs  and Runxmily TFs ,57 collamily TFs  that RUNEts family genes. For example, Runx3 repressed Ets1 and Ets2 in IL-2-activated CD8-TC and NKC  GSE50131). On the other hand, Runx3 enhanced expression of the Ets family genes Fli1, Gabpa and Gabpb1 in IL-2-activated NKC  in both CD8-TC and NKC [Klrk1, which encodes a co-stimulatory receptor for CD8-TC [Klrk1 is among Runx3-regulated genes in IL-2-activated CD8-TC that are down regulated in Runx3-/- cells  and FACS sorted to obtain DX5+ cells. Freshly isolated cells were defined as resting CD8-TC and NKC. CD8-TC (8x105 cells/ml) were activated for 2 days using anti-CD3e  coated plates plus soluble anti-CD28  as described [3 cells/ml and cultured for 4 days in the presence of 100u/ml recombinant human recombinant interleukin 2 (IL-2) . NKC were activated by culture for 7 days in the presence of 1000 units/ml IL-2 or mouse IL-15 . At the end of culture >98% of cells were CD3+CD8-TC and NKp46+ NKC. These experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Weizmann Institute of Science (Permit Number: 01190113-2). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.CD8-TC were isolated from spleen of WT ICR mice using anti-CD8 magnetic beads according to manufacturer instructions (BD Biosciences) yielding a highly enriched population (~95%) of CD3escribed . Cells w7 resting or IL-2-activated CD8-TC or NKC, was fragmented to an average size of ~200-400 bp by 22-40 cycles of sonication (30 sec each) in 15 ml tubes using the Bioraprtor UCD-200 sonicator . For ChIP, 40 ul of anti-RUNX3 [http://www.genomatix.de/solutions/genomatix-genome-analyzer.html) using the Bed-file tool at default parameters. ChIP was performed essentially as described . Brieflyti-RUNX3  or anti-ti-RUNX3  version ti-RUNX3  with defti-RUNX3  and [25,TGt/cGGt/c we used CisGenome (http://www.biostat.jhsph.edu/~hji/cisgenome/). Overrepresentation of each of the alternative RUNX motifs and RUNX-containing modules, in which the 2 TF motifs are up to 50 bp apart, was determined with Genomatix and denovo motif discovery in Runx3 peaks was carried out using MEME-ChIP [To find peaks with the consensus RUNX motif EME-ChIP . RNA and was isolated using the RNeasy Micro kit (Qiagen), according to manufacturer instructions. Purified RNA was reverse-transcribed, amplified and labeled with Affymetrix GeneChip whole transcript sense target labeling kit. Labeled cDNA  from WT and Runx3-/- CD8-TC and NKC was analyzed using Affymetrix mouse gene ST 1.0 and A430.2 microarrays, respectively, according to manufacturer instructions. Microarrays were scanned by GeneChip scanner 3000 7G and statistical analysis of data was performed using the Partek® Genomics Suite  software. CEL files (containing raw expression measurements) were imported to Partek GS and data was preprocessed and normalized using the RMA (Robust Multichip Average) algorithm  with GC -/- versus WT) and Runx3-bound genes was done according to Subramanian et al., 2005 [-/- versus WT expression values and statistical enrichment of Runx3-bound genes within the ranked list was evaluated.The association between 2 variable gene sets, such as differentially expressed genes and Runx3-bound genes, was tested using 2x2 contingency tables with R commander in R statistical software package. The significance of an overlap between 2 similar gene sets, such as Runx3-regulated genes, was carried as described in . GSEA anl., 2005 . All micTM RT kit (Qiagen). Two RT cDNA reactions were prepared for each RNA sample and then mixed to reduce variability. qPCR was performed using light cycler 480  with 480 SYBR Green I master (Roch) with Tm 61°C, using Actb as calibrator. Fold change between Runx3-/- and WT expression was calculated using Excel-based REST software. The primers used are listed in RNA was purified from IL-2-activated WT and Runx3-/- CD8-TC as described above and an equal amount of RNA from the 3 biological repeats was mixed and reverse transcribed with OmniscriptFigure S1Western analysis of Runx3 immunoprecipitation (IP). Anti-Runx3 antibody (Poly-G), but not non-immune serum (NIS), immunoprecipitated Runx3 (left panel) but not GAPDH (right panel) from whole cell extract (WCE) of spleen CD8-TC. Anti-Runx3 (Poly-G) Immunoprecipitated material was analyzed by Western blotting using monoclonal mouse monoclonal anti-Runx3 or anti-GAPDH antibody.(TIF)Click here for additional data file.Figure S2Denovo motif finding and RUNX-containing modules in Runx3-bound regions of IL-2-activated CD8-TC and NKC. Top 3 motifs in Runx3 occupied promoter (A) or enhancer (B) regions. (C) Enrichment of RUNX-containing modules in Runx3-bound enhancer regions in IL-2-activated CD8-TC and NKC. (TIF)Click here for additional data file.Figure S3-/- vs.Scatter plot comparing gene expression of IL-2-activated Runx3WT CD8-TC and NKC. -/- vs. WT cells (1.5-fold). Examples of Runx3-regulated genes are indicated. Red and blue dots mark up- or down-regulated genes, respectively, in Runx3(TIF)Click here for additional data file.Figure S4-/- vs.Quantitative RT-PCR analysis of 7 Runx3-regulated genes in IL-2-activated Runx3WT CD8-TC. -/- vs. WT showed the same pattern as in the microarray analysis. Data represent mean± SE of two independent assays. The 4 down-regulated and 3 up-regulated genes in Runx3(TIF)Click here for additional data file.Figure S5Ingenuity enriched ontology terms of Runx3-regulated genes common to IL-2-activated CD8-TC and NKC.(TIF)Click here for additional data file.Table S1Predicted biological functions of Runx3.(DOC)Click here for additional data file.Table S2-/- versus WT resting and IL-2-activated CD8-TC and NKC.Microarray differentially expressed genes in Runx3(XLS)Click here for additional data file.Table S3Lists of Runx3-regulated genes in resting and IL-2-activated CD8-TC and NKC.(XLS)Click here for additional data file.Table S4Lists of Runx3-regulated genes that are common to CD8-TC and NKC.(XLS)Click here for additional data file.Table S5Sequence of primers used for qPCR analysis.(DOC)Click here for additional data file.