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2_test

{"project":"2_test","denotations":[{"id":"19139168-17195845-59674050","span":{"begin":245,"end":247},"obj":"17195845"},{"id":"19139168-17195845-59674051","span":{"begin":2569,"end":2571},"obj":"17195845"},{"id":"19139168-17242680-59674052","span":{"begin":2573,"end":2575},"obj":"17242680"},{"id":"19139168-10544201-59674053","span":{"begin":3638,"end":3640},"obj":"10544201"},{"id":"19139168-17222571-59674054","span":{"begin":3719,"end":3721},"obj":"17222571"},{"id":"19139168-17646406-59674055","span":{"begin":3797,"end":3799},"obj":"17646406"},{"id":"19139168-17981204-59674056","span":{"begin":3893,"end":3895},"obj":"17981204"},{"id":"19139168-16273099-59674057","span":{"begin":4085,"end":4086},"obj":"16273099"},{"id":"19139168-11786644-59674058","span":{"begin":4212,"end":4214},"obj":"11786644"},{"id":"19139168-12464175-59674059","span":{"begin":7124,"end":7126},"obj":"12464175"},{"id":"19139168-12464175-59674060","span":{"begin":7253,"end":7255},"obj":"12464175"},{"id":"19139168-12796513-59674061","span":{"begin":7395,"end":7397},"obj":"12796513"}],"text":"Runx3 controls multiple aspects of the CTL differentiation program, in part through induction of Eomes\nBecause Runx3 is highly expressed in peripheral CD8+ T cells, and because of the T-bet-Runx3 cooperation we observed earlier in CD4+ T cells (15), we examined the role of Runx3 in effector CTL differentiation. We isolated CD8+ T cells from Runx3−/− (KO) mice of the outbred ICR background and their WT Runx3+/+ littermates by positive selection with anti-CD8 magnetic beads (Figs. S1 and S2, available at http://www.jem.org/cgi/content/full/jem.20081242/DC1). Strikingly, Runx3−/− CD8+ T cells were strongly impaired in their ability to differentiate into effector CTLs, as judged by expression of perforin, granzyme B, and IFN-γ (Fig. 3). Compared with WT T cells, perforin mRNA and protein expression were essentially undetectable in Runx3−/− T cells at day 6 of culture (Fig. 3, A and B). Runx3−/− T cells also had no detectable Eomes expression; in contrast, T-bet expression was unimpaired (Fig. 3 A). Furthermore, Runx3 was required for maximal production of IFN-γ, but not TNF or IL-2, by CD8+ T cells restimulated at day 6 (Fig. 3 C).\nFigure 3. Key role for Runx3 in effector CTL differentiation. (A) Western analysis of Runx3, Eomes, T-bet, and perforin expression in Runx3+/+ versus Runx3−/− CD8+ SP T cells differentiated for 6 d. β-Actin was used as a loading control. (B) Northern blot analysis of Prf1 mRNA expression in Runx3+/+ versus Runx3−/− CD8+ T cells differentiated for 6 d. β–Actin was used as a loading control. (C) Expression of granzyme B, IFN-γ, TNF, and IL-2 by resting or restimulated (6 h) Runx3+/+ versus Runx3−/− CD8+ SP T cells differentiated for 6 d. The vertical gray line indicates the granzyme B MFI for WT GFP+ cells. Results in A–C are representative of two independent experiments. (D) ChIP analysis of binding of endogenous Runx3 and Eomes to the Prf1 locus. Enrichment of the indicated genomic regions was evaluated by real-time PCR of DNA from immunoprecipitated and input chromatin. The data are the means of duplicate measurements from two chromatin preparations from two independent CD8+ T cell differentiations. The efficiency of recovery of input for the −1-kb region of Prf1 was 0.97% for the Runx3 ChIP and 0.5% for the Eomes ChIP.We previously reported that Th1 cell differentiation was regulated through a feed-forward loop in which T-bet is up-regulated early and induces Runx3, after which T-bet and Runx3 cooperate to induce IFN-γ and silence IL-4, thus promoting stable differentiation toward the Th1 lineage (15, 22). Because (a) Runx3 appeared necessary for Eomes induction (Fig. 3 A), (b) the kinetics of Eomes expression paralleled those of perforin expression (Fig. 2), and (c) overexpression of Eo-VP16 in either WT or T-bet–deficient T cells led to an increase in both perforin and IFN-γ expression (Fig. 2, B and D), we asked whether CTL differentiation was also potentially regulated by a feed-forward loop involving these same two classes of Runx and T-box transcription factors. Specifically, we asked whether Runx3, which was necessary for Eomes induction, then cooperated with Eomes to regulate transcription of the effector CTL markers perforin, IFN-γ, and granzyme B.\nTo test this hypothesis, we used chromatin immunoprecipitation (ChIP) assays to ask whether Eomes and Runx3 bound regulatory regions of the Prf1, Ifng, and Gzmb genes (Fig. 3 D). Both proteins associated with gene regulatory regions in differentiated CTLs. Runx3 bound to the Prf1 and Gzmb transcription start sites (TSS); to a known IL-2 responsive enhancer located near −1 kb of the Prf1 gene (23); to the distal CTL-specific DNase I hypersensitive site 9 in the Prf1 locus (24); to the Ifng promoter near the TSS, as previously reported for Th1 cells (10); and to several DNase I hypersensitive sites in the Ifng locus (Fig. 3 D and not depicted) (25). Eomes bound primarily to the Prf1 TSS and the −1 kb enhancer; this binding was substantially greater than that observed at the promoter of the Il2rb gene, a known direct target of Eomes (8), and comparable to that observed at the Ifng TSS, a known target of T-box proteins in both Th1 and CD8+ T cells (Fig. 3 D) (17).\nTo determine whether Runx3 controlled the expression of CTL effector genes through its induction of Eomes, we retrovirally expressed Runx3 and Eo-VP16 in CD8+ T cells from Runx3−/− mice. Because of the limited number of CD8+ T cells in these mice, and because we saw no difference between Runx3−/− CD8+CD4− SP and CD8+CD4+ DP cells in our previous experiments, we used total Runx3−/− CD8+ T cells without further fractionation as recipients for retroviral transduction. Reconstitution of Runx3−/− CD8+ T cells with Runx3 restored expression of Eomes as well as perforin, granzyme B, and IFN-γ (Fig. 4, A and B). In addition, Runx3−/− T cells showed a compensatory up-regulation of Runx1, which was suppressed upon reconstitution with Runx3, indicating that Runx1 is a target of repression by Runx3. Notably, Eo-VP16 did not up-regulate perforin expression when expressed in Runx3−/− cells, even though it restored the capacity to induce IFN-γ expression upon TCR restimulation (Fig. 4, A and B). This result suggests strongly that perforin expression requires Runx3 and Eomes.\nFigure 4. Runx3 controls Eomes, perforin, granzyme B, and IFN-γ expression in effector CTLs. Runx3+/+ or Runx3−/− CD8+ T cells were activated and transduced with retroviruses bearing an empty IRES-GFP cassette (GFP) or also encoding Eomes-VP16 (Eo-VP16) or Myc-Runx3 (Runx3). The frequency of transduced cells in the cultures was equivalent for all constructs (∼75–90% GFP+ cells; not depicted). (A) Protein expression in whole-cell extracts (day 6) was analyzed by immunoblotting. Overexpression of Eomes-VP16 cannot be detected with the Eomes antibody, as the C-terminal epitope is within the region that has been replaced with the VP16 transactivation domain. (B) Expression of granzyme B and IFN-γ after culture for 6 d and restimulation for 4 h with PMA and ionomycin was determined by intracellular staining. The percentage of positively stained cells is shown above the gate; the mean fluorescence intensity (MFI) of granzyme B staining for the total population is shown below the gate. The vertical gray lines indicate the MFI for WT GFP+ cells. Results are representative of at least two independent experiments. (C) Schematic diagram of the transcriptional network involving Runx3 and T-box factors. T-bet is induced by TCR signals and is essential for early IFN-γ expression. Runx3 is present in naive CD8+ T cells and represses Runx1 and induces Eomes, perforin, granzyme B, and IFN-γ expression. Eomes may participate in sustaining late IFN-γ expression, whereas Runx3 and Eomes (but not T-bet) may cooperate to activate perforin expression. The dotted line indicates the partial effect of T-bet deficiency on Gzmb mRNA but not granzyme B protein expression.As expected from their defect in perforin and granzyme B expression, Runx3−/− CD8+ T cells showed defective cytolytic activity in a mixed lymphocyte reaction (12). However, TCR-stimulated Runx3−/− CD8+ cells were as effective as WT cells in killing tumor cells in a redirected CTL assay (12). Furthermore, CD8+ cells from the peritoneal cavity of Runx3−/− mice immunized with certain tumor cells effectively killed these targets (13). Therefore, although activation of the perforin/granzyme B machinery is defective in Runx3−/− CD8+ cells, these cells are not entirely devoid of cytolytic activity and could still effectively kill targets, possibly by alternative mechanisms such as the Fas–Fas ligand pathway."}

pmc-enju-pas

bionlp-st-ge-2016-coref

{"project":"bionlp-st-ge-2016-coref","denotations":[{"id":"T9873","span":{"begin":5025,"end":5032},"obj":"Antecedent"},{"id":"T9874","span":{"begin":6982,"end":6987},"obj":"Anaphor"},{"id":"T9875","span":{"begin":5277,"end":5282},"obj":"Antecedent"},{"id":"T9876","span":{"begin":5287,"end":5292},"obj":"Antecedent"},{"id":"T9867","span":{"begin":3421,"end":3434},"obj":"Anaphor"},{"id":"T9868","span":{"begin":3334,"end":3339},"obj":"Antecedent"},{"id":"T9869","span":{"begin":3344,"end":3349},"obj":"Antecedent"},{"id":"T9870","span":{"begin":4905,"end":4910},"obj":"Anaphor"},{"id":"T9871","span":{"begin":4898,"end":4903},"obj":"Antecedent"},{"id":"T9872","span":{"begin":5119,"end":5121},"obj":"Anaphor"}],"relations":[{"id":"R8041","pred":"boundBy","subj":"T9867","obj":"T9868"},{"id":"R8042","pred":"boundBy","subj":"T9867","obj":"T9869"},{"id":"R8045","pred":"boundBy","subj":"T9870","obj":"T9871"},{"id":"R8046","pred":"boundBy","subj":"T9872","obj":"T9873"},{"id":"R8048","pred":"boundBy","subj":"T9874","obj":"T9875"},{"id":"R8049","pred":"boundBy","subj":"T9874","obj":"T9876"}],"namespaces":[{"prefix":"_base","uri":"https://bionlp.dbcls.jp/ontology/ge.owl#"}],"text":"Runx3 controls multiple aspects of the CTL differentiation program, in part through induction of Eomes\nBecause Runx3 is highly expressed in peripheral CD8+ T cells, and because of the T-bet-Runx3 cooperation we observed earlier in CD4+ T cells (15), we examined the role of Runx3 in effector CTL differentiation. We isolated CD8+ T cells from Runx3−/− (KO) mice of the outbred ICR background and their WT Runx3+/+ littermates by positive selection with anti-CD8 magnetic beads (Figs. S1 and S2, available at http://www.jem.org/cgi/content/full/jem.20081242/DC1). Strikingly, Runx3−/− CD8+ T cells were strongly impaired in their ability to differentiate into effector CTLs, as judged by expression of perforin, granzyme B, and IFN-γ (Fig. 3). Compared with WT T cells, perforin mRNA and protein expression were essentially undetectable in Runx3−/− T cells at day 6 of culture (Fig. 3, A and B). Runx3−/− T cells also had no detectable Eomes expression; in contrast, T-bet expression was unimpaired (Fig. 3 A). Furthermore, Runx3 was required for maximal production of IFN-γ, but not TNF or IL-2, by CD8+ T cells restimulated at day 6 (Fig. 3 C).\nFigure 3. Key role for Runx3 in effector CTL differentiation. (A) Western analysis of Runx3, Eomes, T-bet, and perforin expression in Runx3+/+ versus Runx3−/− CD8+ SP T cells differentiated for 6 d. β-Actin was used as a loading control. (B) Northern blot analysis of Prf1 mRNA expression in Runx3+/+ versus Runx3−/− CD8+ T cells differentiated for 6 d. β–Actin was used as a loading control. (C) Expression of granzyme B, IFN-γ, TNF, and IL-2 by resting or restimulated (6 h) Runx3+/+ versus Runx3−/− CD8+ SP T cells differentiated for 6 d. The vertical gray line indicates the granzyme B MFI for WT GFP+ cells. Results in A–C are representative of two independent experiments. (D) ChIP analysis of binding of endogenous Runx3 and Eomes to the Prf1 locus. Enrichment of the indicated genomic regions was evaluated by real-time PCR of DNA from immunoprecipitated and input chromatin. The data are the means of duplicate measurements from two chromatin preparations from two independent CD8+ T cell differentiations. The efficiency of recovery of input for the −1-kb region of Prf1 was 0.97% for the Runx3 ChIP and 0.5% for the Eomes ChIP.We previously reported that Th1 cell differentiation was regulated through a feed-forward loop in which T-bet is up-regulated early and induces Runx3, after which T-bet and Runx3 cooperate to induce IFN-γ and silence IL-4, thus promoting stable differentiation toward the Th1 lineage (15, 22). Because (a) Runx3 appeared necessary for Eomes induction (Fig. 3 A), (b) the kinetics of Eomes expression paralleled those of perforin expression (Fig. 2), and (c) overexpression of Eo-VP16 in either WT or T-bet–deficient T cells led to an increase in both perforin and IFN-γ expression (Fig. 2, B and D), we asked whether CTL differentiation was also potentially regulated by a feed-forward loop involving these same two classes of Runx and T-box transcription factors. Specifically, we asked whether Runx3, which was necessary for Eomes induction, then cooperated with Eomes to regulate transcription of the effector CTL markers perforin, IFN-γ, and granzyme B.\nTo test this hypothesis, we used chromatin immunoprecipitation (ChIP) assays to ask whether Eomes and Runx3 bound regulatory regions of the Prf1, Ifng, and Gzmb genes (Fig. 3 D). Both proteins associated with gene regulatory regions in differentiated CTLs. Runx3 bound to the Prf1 and Gzmb transcription start sites (TSS); to a known IL-2 responsive enhancer located near −1 kb of the Prf1 gene (23); to the distal CTL-specific DNase I hypersensitive site 9 in the Prf1 locus (24); to the Ifng promoter near the TSS, as previously reported for Th1 cells (10); and to several DNase I hypersensitive sites in the Ifng locus (Fig. 3 D and not depicted) (25). Eomes bound primarily to the Prf1 TSS and the −1 kb enhancer; this binding was substantially greater than that observed at the promoter of the Il2rb gene, a known direct target of Eomes (8), and comparable to that observed at the Ifng TSS, a known target of T-box proteins in both Th1 and CD8+ T cells (Fig. 3 D) (17).\nTo determine whether Runx3 controlled the expression of CTL effector genes through its induction of Eomes, we retrovirally expressed Runx3 and Eo-VP16 in CD8+ T cells from Runx3−/− mice. Because of the limited number of CD8+ T cells in these mice, and because we saw no difference between Runx3−/− CD8+CD4− SP and CD8+CD4+ DP cells in our previous experiments, we used total Runx3−/− CD8+ T cells without further fractionation as recipients for retroviral transduction. Reconstitution of Runx3−/− CD8+ T cells with Runx3 restored expression of Eomes as well as perforin, granzyme B, and IFN-γ (Fig. 4, A and B). In addition, Runx3−/− T cells showed a compensatory up-regulation of Runx1, which was suppressed upon reconstitution with Runx3, indicating that Runx1 is a target of repression by Runx3. Notably, Eo-VP16 did not up-regulate perforin expression when expressed in Runx3−/− cells, even though it restored the capacity to induce IFN-γ expression upon TCR restimulation (Fig. 4, A and B). This result suggests strongly that perforin expression requires Runx3 and Eomes.\nFigure 4. Runx3 controls Eomes, perforin, granzyme B, and IFN-γ expression in effector CTLs. Runx3+/+ or Runx3−/− CD8+ T cells were activated and transduced with retroviruses bearing an empty IRES-GFP cassette (GFP) or also encoding Eomes-VP16 (Eo-VP16) or Myc-Runx3 (Runx3). The frequency of transduced cells in the cultures was equivalent for all constructs (∼75–90% GFP+ cells; not depicted). (A) Protein expression in whole-cell extracts (day 6) was analyzed by immunoblotting. Overexpression of Eomes-VP16 cannot be detected with the Eomes antibody, as the C-terminal epitope is within the region that has been replaced with the VP16 transactivation domain. (B) Expression of granzyme B and IFN-γ after culture for 6 d and restimulation for 4 h with PMA and ionomycin was determined by intracellular staining. The percentage of positively stained cells is shown above the gate; the mean fluorescence intensity (MFI) of granzyme B staining for the total population is shown below the gate. The vertical gray lines indicate the MFI for WT GFP+ cells. Results are representative of at least two independent experiments. (C) Schematic diagram of the transcriptional network involving Runx3 and T-box factors. T-bet is induced by TCR signals and is essential for early IFN-γ expression. Runx3 is present in naive CD8+ T cells and represses Runx1 and induces Eomes, perforin, granzyme B, and IFN-γ expression. Eomes may participate in sustaining late IFN-γ expression, whereas Runx3 and Eomes (but not T-bet) may cooperate to activate perforin expression. The dotted line indicates the partial effect of T-bet deficiency on Gzmb mRNA but not granzyme B protein expression.As expected from their defect in perforin and granzyme B expression, Runx3−/− CD8+ T cells showed defective cytolytic activity in a mixed lymphocyte reaction (12). However, TCR-stimulated Runx3−/− CD8+ cells were as effective as WT cells in killing tumor cells in a redirected CTL assay (12). Furthermore, CD8+ cells from the peritoneal cavity of Runx3−/− mice immunized with certain tumor cells effectively killed these targets (13). Therefore, although activation of the perforin/granzyme B machinery is defective in Runx3−/− CD8+ cells, these cells are not entirely devoid of cytolytic activity and could still effectively kill targets, possibly by alternative mechanisms such as the Fas–Fas ligand pathway."}

bionlp-st-ge-2016-spacy-parsed

UBERON-AE

{"project":"UBERON-AE","denotations":[{"id":"T7584","span":{"begin":7291,"end":7308},"obj":"http://purl.obolibrary.org/obo/UBERON_0001179"}],"text":"Runx3 controls multiple aspects of the CTL differentiation program, in part through induction of Eomes\nBecause Runx3 is highly expressed in peripheral CD8+ T cells, and because of the T-bet-Runx3 cooperation we observed earlier in CD4+ T cells (15), we examined the role of Runx3 in effector CTL differentiation. We isolated CD8+ T cells from Runx3−/− (KO) mice of the outbred ICR background and their WT Runx3+/+ littermates by positive selection with anti-CD8 magnetic beads (Figs. S1 and S2, available at http://www.jem.org/cgi/content/full/jem.20081242/DC1). Strikingly, Runx3−/− CD8+ T cells were strongly impaired in their ability to differentiate into effector CTLs, as judged by expression of perforin, granzyme B, and IFN-γ (Fig. 3). Compared with WT T cells, perforin mRNA and protein expression were essentially undetectable in Runx3−/− T cells at day 6 of culture (Fig. 3, A and B). Runx3−/− T cells also had no detectable Eomes expression; in contrast, T-bet expression was unimpaired (Fig. 3 A). Furthermore, Runx3 was required for maximal production of IFN-γ, but not TNF or IL-2, by CD8+ T cells restimulated at day 6 (Fig. 3 C).\nFigure 3. Key role for Runx3 in effector CTL differentiation. (A) Western analysis of Runx3, Eomes, T-bet, and perforin expression in Runx3+/+ versus Runx3−/− CD8+ SP T cells differentiated for 6 d. β-Actin was used as a loading control. (B) Northern blot analysis of Prf1 mRNA expression in Runx3+/+ versus Runx3−/− CD8+ T cells differentiated for 6 d. β–Actin was used as a loading control. (C) Expression of granzyme B, IFN-γ, TNF, and IL-2 by resting or restimulated (6 h) Runx3+/+ versus Runx3−/− CD8+ SP T cells differentiated for 6 d. The vertical gray line indicates the granzyme B MFI for WT GFP+ cells. Results in A–C are representative of two independent experiments. (D) ChIP analysis of binding of endogenous Runx3 and Eomes to the Prf1 locus. Enrichment of the indicated genomic regions was evaluated by real-time PCR of DNA from immunoprecipitated and input chromatin. The data are the means of duplicate measurements from two chromatin preparations from two independent CD8+ T cell differentiations. The efficiency of recovery of input for the −1-kb region of Prf1 was 0.97% for the Runx3 ChIP and 0.5% for the Eomes ChIP.We previously reported that Th1 cell differentiation was regulated through a feed-forward loop in which T-bet is up-regulated early and induces Runx3, after which T-bet and Runx3 cooperate to induce IFN-γ and silence IL-4, thus promoting stable differentiation toward the Th1 lineage (15, 22). Because (a) Runx3 appeared necessary for Eomes induction (Fig. 3 A), (b) the kinetics of Eomes expression paralleled those of perforin expression (Fig. 2), and (c) overexpression of Eo-VP16 in either WT or T-bet–deficient T cells led to an increase in both perforin and IFN-γ expression (Fig. 2, B and D), we asked whether CTL differentiation was also potentially regulated by a feed-forward loop involving these same two classes of Runx and T-box transcription factors. Specifically, we asked whether Runx3, which was necessary for Eomes induction, then cooperated with Eomes to regulate transcription of the effector CTL markers perforin, IFN-γ, and granzyme B.\nTo test this hypothesis, we used chromatin immunoprecipitation (ChIP) assays to ask whether Eomes and Runx3 bound regulatory regions of the Prf1, Ifng, and Gzmb genes (Fig. 3 D). Both proteins associated with gene regulatory regions in differentiated CTLs. Runx3 bound to the Prf1 and Gzmb transcription start sites (TSS); to a known IL-2 responsive enhancer located near −1 kb of the Prf1 gene (23); to the distal CTL-specific DNase I hypersensitive site 9 in the Prf1 locus (24); to the Ifng promoter near the TSS, as previously reported for Th1 cells (10); and to several DNase I hypersensitive sites in the Ifng locus (Fig. 3 D and not depicted) (25). Eomes bound primarily to the Prf1 TSS and the −1 kb enhancer; this binding was substantially greater than that observed at the promoter of the Il2rb gene, a known direct target of Eomes (8), and comparable to that observed at the Ifng TSS, a known target of T-box proteins in both Th1 and CD8+ T cells (Fig. 3 D) (17).\nTo determine whether Runx3 controlled the expression of CTL effector genes through its induction of Eomes, we retrovirally expressed Runx3 and Eo-VP16 in CD8+ T cells from Runx3−/− mice. Because of the limited number of CD8+ T cells in these mice, and because we saw no difference between Runx3−/− CD8+CD4− SP and CD8+CD4+ DP cells in our previous experiments, we used total Runx3−/− CD8+ T cells without further fractionation as recipients for retroviral transduction. Reconstitution of Runx3−/− CD8+ T cells with Runx3 restored expression of Eomes as well as perforin, granzyme B, and IFN-γ (Fig. 4, A and B). In addition, Runx3−/− T cells showed a compensatory up-regulation of Runx1, which was suppressed upon reconstitution with Runx3, indicating that Runx1 is a target of repression by Runx3. Notably, Eo-VP16 did not up-regulate perforin expression when expressed in Runx3−/− cells, even though it restored the capacity to induce IFN-γ expression upon TCR restimulation (Fig. 4, A and B). This result suggests strongly that perforin expression requires Runx3 and Eomes.\nFigure 4. Runx3 controls Eomes, perforin, granzyme B, and IFN-γ expression in effector CTLs. Runx3+/+ or Runx3−/− CD8+ T cells were activated and transduced with retroviruses bearing an empty IRES-GFP cassette (GFP) or also encoding Eomes-VP16 (Eo-VP16) or Myc-Runx3 (Runx3). The frequency of transduced cells in the cultures was equivalent for all constructs (∼75–90% GFP+ cells; not depicted). (A) Protein expression in whole-cell extracts (day 6) was analyzed by immunoblotting. Overexpression of Eomes-VP16 cannot be detected with the Eomes antibody, as the C-terminal epitope is within the region that has been replaced with the VP16 transactivation domain. (B) Expression of granzyme B and IFN-γ after culture for 6 d and restimulation for 4 h with PMA and ionomycin was determined by intracellular staining. The percentage of positively stained cells is shown above the gate; the mean fluorescence intensity (MFI) of granzyme B staining for the total population is shown below the gate. The vertical gray lines indicate the MFI for WT GFP+ cells. Results are representative of at least two independent experiments. (C) Schematic diagram of the transcriptional network involving Runx3 and T-box factors. T-bet is induced by TCR signals and is essential for early IFN-γ expression. Runx3 is present in naive CD8+ T cells and represses Runx1 and induces Eomes, perforin, granzyme B, and IFN-γ expression. Eomes may participate in sustaining late IFN-γ expression, whereas Runx3 and Eomes (but not T-bet) may cooperate to activate perforin expression. The dotted line indicates the partial effect of T-bet deficiency on Gzmb mRNA but not granzyme B protein expression.As expected from their defect in perforin and granzyme B expression, Runx3−/− CD8+ T cells showed defective cytolytic activity in a mixed lymphocyte reaction (12). However, TCR-stimulated Runx3−/− CD8+ cells were as effective as WT cells in killing tumor cells in a redirected CTL assay (12). Furthermore, CD8+ cells from the peritoneal cavity of Runx3−/− mice immunized with certain tumor cells effectively killed these targets (13). Therefore, although activation of the perforin/granzyme B machinery is defective in Runx3−/− CD8+ cells, these cells are not entirely devoid of cytolytic activity and could still effectively kill targets, possibly by alternative mechanisms such as the Fas–Fas ligand pathway."}

GO-BP

GO-MF

{"project":"GO-MF","denotations":[{"id":"T10031","span":{"begin":1090,"end":1094},"obj":"http://purl.obolibrary.org/obo/GO_0005134"},{"id":"T10032","span":{"begin":3576,"end":3580},"obj":"http://purl.obolibrary.org/obo/GO_0005134"},{"id":"T10033","span":{"begin":2501,"end":2505},"obj":"http://purl.obolibrary.org/obo/GO_0005136"},{"id":"T10034","span":{"begin":3950,"end":3972},"obj":"http://purl.obolibrary.org/obo/GO_0035326"},{"id":"T10035","span":{"begin":3965,"end":3972},"obj":"http://purl.obolibrary.org/obo/GO_0005488"},{"id":"T10036","span":{"begin":7660,"end":7666},"obj":"http://purl.obolibrary.org/obo/GO_0005488"},{"id":"T20465","span":{"begin":1585,"end":1589},"obj":"http://purl.obolibrary.org/obo/GO_0005134"},{"id":"T20466","span":{"begin":1846,"end":1853},"obj":"http://purl.obolibrary.org/obo/GO_0005488"},{"id":"T21621","span":{"begin":5839,"end":5847},"obj":"http://purl.obolibrary.org/obo/GO_0003823"}],"text":"Runx3 controls multiple aspects of the CTL differentiation program, in part through induction of Eomes\nBecause Runx3 is highly expressed in peripheral CD8+ T cells, and because of the T-bet-Runx3 cooperation we observed earlier in CD4+ T cells (15), we examined the role of Runx3 in effector CTL differentiation. We isolated CD8+ T cells from Runx3−/− (KO) mice of the outbred ICR background and their WT Runx3+/+ littermates by positive selection with anti-CD8 magnetic beads (Figs. S1 and S2, available at http://www.jem.org/cgi/content/full/jem.20081242/DC1). Strikingly, Runx3−/− CD8+ T cells were strongly impaired in their ability to differentiate into effector CTLs, as judged by expression of perforin, granzyme B, and IFN-γ (Fig. 3). Compared with WT T cells, perforin mRNA and protein expression were essentially undetectable in Runx3−/− T cells at day 6 of culture (Fig. 3, A and B). Runx3−/− T cells also had no detectable Eomes expression; in contrast, T-bet expression was unimpaired (Fig. 3 A). Furthermore, Runx3 was required for maximal production of IFN-γ, but not TNF or IL-2, by CD8+ T cells restimulated at day 6 (Fig. 3 C).\nFigure 3. Key role for Runx3 in effector CTL differentiation. (A) Western analysis of Runx3, Eomes, T-bet, and perforin expression in Runx3+/+ versus Runx3−/− CD8+ SP T cells differentiated for 6 d. β-Actin was used as a loading control. (B) Northern blot analysis of Prf1 mRNA expression in Runx3+/+ versus Runx3−/− CD8+ T cells differentiated for 6 d. β–Actin was used as a loading control. (C) Expression of granzyme B, IFN-γ, TNF, and IL-2 by resting or restimulated (6 h) Runx3+/+ versus Runx3−/− CD8+ SP T cells differentiated for 6 d. The vertical gray line indicates the granzyme B MFI for WT GFP+ cells. Results in A–C are representative of two independent experiments. (D) ChIP analysis of binding of endogenous Runx3 and Eomes to the Prf1 locus. Enrichment of the indicated genomic regions was evaluated by real-time PCR of DNA from immunoprecipitated and input chromatin. The data are the means of duplicate measurements from two chromatin preparations from two independent CD8+ T cell differentiations. The efficiency of recovery of input for the −1-kb region of Prf1 was 0.97% for the Runx3 ChIP and 0.5% for the Eomes ChIP.We previously reported that Th1 cell differentiation was regulated through a feed-forward loop in which T-bet is up-regulated early and induces Runx3, after which T-bet and Runx3 cooperate to induce IFN-γ and silence IL-4, thus promoting stable differentiation toward the Th1 lineage (15, 22). Because (a) Runx3 appeared necessary for Eomes induction (Fig. 3 A), (b) the kinetics of Eomes expression paralleled those of perforin expression (Fig. 2), and (c) overexpression of Eo-VP16 in either WT or T-bet–deficient T cells led to an increase in both perforin and IFN-γ expression (Fig. 2, B and D), we asked whether CTL differentiation was also potentially regulated by a feed-forward loop involving these same two classes of Runx and T-box transcription factors. Specifically, we asked whether Runx3, which was necessary for Eomes induction, then cooperated with Eomes to regulate transcription of the effector CTL markers perforin, IFN-γ, and granzyme B.\nTo test this hypothesis, we used chromatin immunoprecipitation (ChIP) assays to ask whether Eomes and Runx3 bound regulatory regions of the Prf1, Ifng, and Gzmb genes (Fig. 3 D). Both proteins associated with gene regulatory regions in differentiated CTLs. Runx3 bound to the Prf1 and Gzmb transcription start sites (TSS); to a known IL-2 responsive enhancer located near −1 kb of the Prf1 gene (23); to the distal CTL-specific DNase I hypersensitive site 9 in the Prf1 locus (24); to the Ifng promoter near the TSS, as previously reported for Th1 cells (10); and to several DNase I hypersensitive sites in the Ifng locus (Fig. 3 D and not depicted) (25). Eomes bound primarily to the Prf1 TSS and the −1 kb enhancer; this binding was substantially greater than that observed at the promoter of the Il2rb gene, a known direct target of Eomes (8), and comparable to that observed at the Ifng TSS, a known target of T-box proteins in both Th1 and CD8+ T cells (Fig. 3 D) (17).\nTo determine whether Runx3 controlled the expression of CTL effector genes through its induction of Eomes, we retrovirally expressed Runx3 and Eo-VP16 in CD8+ T cells from Runx3−/− mice. Because of the limited number of CD8+ T cells in these mice, and because we saw no difference between Runx3−/− CD8+CD4− SP and CD8+CD4+ DP cells in our previous experiments, we used total Runx3−/− CD8+ T cells without further fractionation as recipients for retroviral transduction. Reconstitution of Runx3−/− CD8+ T cells with Runx3 restored expression of Eomes as well as perforin, granzyme B, and IFN-γ (Fig. 4, A and B). In addition, Runx3−/− T cells showed a compensatory up-regulation of Runx1, which was suppressed upon reconstitution with Runx3, indicating that Runx1 is a target of repression by Runx3. Notably, Eo-VP16 did not up-regulate perforin expression when expressed in Runx3−/− cells, even though it restored the capacity to induce IFN-γ expression upon TCR restimulation (Fig. 4, A and B). This result suggests strongly that perforin expression requires Runx3 and Eomes.\nFigure 4. Runx3 controls Eomes, perforin, granzyme B, and IFN-γ expression in effector CTLs. Runx3+/+ or Runx3−/− CD8+ T cells were activated and transduced with retroviruses bearing an empty IRES-GFP cassette (GFP) or also encoding Eomes-VP16 (Eo-VP16) or Myc-Runx3 (Runx3). The frequency of transduced cells in the cultures was equivalent for all constructs (∼75–90% GFP+ cells; not depicted). (A) Protein expression in whole-cell extracts (day 6) was analyzed by immunoblotting. Overexpression of Eomes-VP16 cannot be detected with the Eomes antibody, as the C-terminal epitope is within the region that has been replaced with the VP16 transactivation domain. (B) Expression of granzyme B and IFN-γ after culture for 6 d and restimulation for 4 h with PMA and ionomycin was determined by intracellular staining. The percentage of positively stained cells is shown above the gate; the mean fluorescence intensity (MFI) of granzyme B staining for the total population is shown below the gate. The vertical gray lines indicate the MFI for WT GFP+ cells. Results are representative of at least two independent experiments. (C) Schematic diagram of the transcriptional network involving Runx3 and T-box factors. T-bet is induced by TCR signals and is essential for early IFN-γ expression. Runx3 is present in naive CD8+ T cells and represses Runx1 and induces Eomes, perforin, granzyme B, and IFN-γ expression. Eomes may participate in sustaining late IFN-γ expression, whereas Runx3 and Eomes (but not T-bet) may cooperate to activate perforin expression. The dotted line indicates the partial effect of T-bet deficiency on Gzmb mRNA but not granzyme B protein expression.As expected from their defect in perforin and granzyme B expression, Runx3−/− CD8+ T cells showed defective cytolytic activity in a mixed lymphocyte reaction (12). However, TCR-stimulated Runx3−/− CD8+ cells were as effective as WT cells in killing tumor cells in a redirected CTL assay (12). Furthermore, CD8+ cells from the peritoneal cavity of Runx3−/− mice immunized with certain tumor cells effectively killed these targets (13). Therefore, although activation of the perforin/granzyme B machinery is defective in Runx3−/− CD8+ cells, these cells are not entirely devoid of cytolytic activity and could still effectively kill targets, possibly by alternative mechanisms such as the Fas–Fas ligand pathway."}

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