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Id Subject Object Predicate Lexical cue
T77 0-92 Sentence denotes Runx3 and T-box proteins cooperate to establish the transcriptional program of effector CTLs
T1 0-92 Sentence denotes Runx3 and T-box proteins cooperate to establish the transcriptional program of effector CTLs
T2 95-103 Sentence denotes Abstract
T78 104-225 Sentence denotes Activation of naive CD8+ T cells with antigen induces their differentiation into effector cytolytic T lymphocytes (CTLs).
T3 104-225 Sentence denotes Activation of naive CD8+ T cells with antigen induces their differentiation into effector cytolytic T lymphocytes (CTLs).
T79 226-390 Sentence denotes CTLs lyse infected or aberrant target cells by exocytosis of lytic granules containing the pore-forming protein perforin and a family of proteases termed granzymes.
T4 226-390 Sentence denotes CTLs lyse infected or aberrant target cells by exocytosis of lytic granules containing the pore-forming protein perforin and a family of proteases termed granzymes.
T80 391-676 Sentence denotes We show that effector CTL differentiation occurs in two sequential phases in vitro, characterized by early induction of T-bet and late induction of Eomesodermin (Eomes), T-box transcription factors that regulate the early and late phases of interferon (IFN) γ expression, respectively.
T5 391-676 Sentence denotes We show that effector CTL differentiation occurs in two sequential phases in vitro, characterized by early induction of T-bet and late induction of Eomesodermin (Eomes), T-box transcription factors that regulate the early and late phases of interferon (IFN) γ expression, respectively.
T81 677-779 Sentence denotes In addition, we demonstrate a critical role for the transcription factor Runx3 in CTL differentiation.
T6 677-779 Sentence denotes In addition, we demonstrate a critical role for the transcription factor Runx3 in CTL differentiation.
T7 780-922 Sentence denotes Runx3 regulates Eomes expression as well as expression of three cardinal markers of the effector CTL program: IFN-γ, perforin, and granzyme B.
T82 780-1136 Sentence denotes Runx3 regulates Eomes expression as well as expression of three cardinal markers of the effector CTL program: IFN-γ, perforin, and granzyme B. Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs.
T8 923-1136 Sentence denotes Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs.
T828 1138-1413 Sentence denotes Naive CD8+ T cells differentiate into effector CTLs with the ability to lyse antigen-bearing target cells by exocytosis of lytic granules containing perforin and granzymes, and to produce inflammatory cytokines such as IFN-γ and TNF upon restimulation through the TCR (1, 2).
T9 1138-1413 Sentence denotes Naive CD8+ T cells differentiate into effector CTLs with the ability to lyse antigen-bearing target cells by exocytosis of lytic granules containing perforin and granzymes, and to produce inflammatory cytokines such as IFN-γ and TNF upon restimulation through the TCR (1, 2).
T829 1414-1547 Sentence denotes In vivo experiments have elucidated many critical parameters governing the development and evolution of primary CTL responses (3, 4).
T10 1414-1547 Sentence denotes In vivo experiments have elucidated many critical parameters governing the development and evolution of primary CTL responses (3, 4).
T11 1548-1723 Sentence denotes In this study, we have used in vitro systems such as those developed to study CD4+ T cell differentiation to define the molecular basis of effector CTL differentiation (5, 6).
T830 1548-1867 Sentence denotes In this study, we have used in vitro systems such as those developed to study CD4+ T cell differentiation to define the molecular basis of effector CTL differentiation (5, 6). The T-box transcription factors Eomesodermin (Eomes) and T-bet are needed for important aspects of effector and memory CTL differentiation (7).
T12 1724-1867 Sentence denotes The T-box transcription factors Eomesodermin (Eomes) and T-bet are needed for important aspects of effector and memory CTL differentiation (7).
T831 1868-2053 Sentence denotes In uninfected mice, compound deletion of the Tbx21 (encoding T-bet) and eomesodermin genes is associated with a selective loss of CD8+ T cells with an IL-2Rβ–high, memory phenotype (8).
T13 1868-2053 Sentence denotes In uninfected mice, compound deletion of the Tbx21 (encoding T-bet) and eomesodermin genes is associated with a selective loss of CD8+ T cells with an IL-2Rβ–high, memory phenotype (8).
T832 2054-2263 Sentence denotes Mice deficient for both T-bet and Eomes in T cells have impaired expression of cytolytic mediators, manifest poor cytolytic activity, and fail to control acute lymphocytic choriomeningitis virus infection (9).
T14 2054-2263 Sentence denotes Mice deficient for both T-bet and Eomes in T cells have impaired expression of cytolytic mediators, manifest poor cytolytic activity, and fail to control acute lymphocytic choriomeningitis virus infection (9).
T15 2264-2617 Sentence denotes Nevertheless, the specific roles of T-bet and Eomes in clonal expansion and CTL differentiation have not yet been resolved: in particular, it is not known whether these transcription factors function redundantly to control effector CD8+ T cell differentiation, and whether they do so directly by targeting specific effector cytokine and cytolytic genes.
T833 2264-2761 Sentence denotes Nevertheless, the specific roles of T-bet and Eomes in clonal expansion and CTL differentiation have not yet been resolved: in particular, it is not known whether these transcription factors function redundantly to control effector CD8+ T cell differentiation, and whether they do so directly by targeting specific effector cytokine and cytolytic genes. Runx proteins, a family of three DNA-binding transcription factors, control thymocyte differentiation and the CD4/CD8 lineage decision (10–13).
T16 2618-2761 Sentence denotes Runx proteins, a family of three DNA-binding transcription factors, control thymocyte differentiation and the CD4/CD8 lineage decision (10–13).
T834 2762-2906 Sentence denotes Runx3 and perforin mRNA are expressed by double-positive (DP) thymocytes and CD8+ single-positive (SP) thymocytes but not in CD4+ SP cells (14).
T17 2762-2906 Sentence denotes Runx3 and perforin mRNA are expressed by double-positive (DP) thymocytes and CD8+ single-positive (SP) thymocytes but not in CD4+ SP cells (14).
T835 2907-3155 Sentence denotes Although Runx3 is not expressed in naive CD4+ T cells, its expression is up-regulated during Th1 cell differentiation, and Runx3 influences Th1 cell differentiation and function through direct regulation of the Il4 and Ifng cytokine genes (15, 16).
T18 2907-3155 Sentence denotes Although Runx3 is not expressed in naive CD4+ T cells, its expression is up-regulated during Th1 cell differentiation, and Runx3 influences Th1 cell differentiation and function through direct regulation of the Il4 and Ifng cytokine genes (15, 16).
T836 3156-3314 Sentence denotes In contrast, all three Runx proteins are expressed in mature CD8+ T cells (10, 12), and Runx3-deficient CD8+ T cells show reduced cytolytic activity (12, 13).
T19 3156-3314 Sentence denotes In contrast, all three Runx proteins are expressed in mature CD8+ T cells (10, 12), and Runx3-deficient CD8+ T cells show reduced cytolytic activity (12, 13).
T20 3315-3393 Sentence denotes We therefore tested whether Runx3 influenced cytolytic T cell differentiation.
T837 3315-3505 Sentence denotes We therefore tested whether Runx3 influenced cytolytic T cell differentiation. In this report, we show that Runx3 and T-box factors synergistically regulate CTL differentiation and function.
T21 3394-3505 Sentence denotes In this report, we show that Runx3 and T-box factors synergistically regulate CTL differentiation and function.
T838 3506-3703 Sentence denotes T-bet is induced quickly upon TCR stimulation and is required for early programming of cytokine production (17), whereas Eomes is induced later during differentiation and sustains IFN-γ expression.
T22 3506-3703 Sentence denotes T-bet is induced quickly upon TCR stimulation and is required for early programming of cytokine production (17), whereas Eomes is induced later during differentiation and sustains IFN-γ expression.
T839 3704-3878 Sentence denotes Runx3 is required for Eomes and perforin expression, and both Eomes and Runx3 bind at the Prf1 locus; in contrast, perforin expression is unaffected in T-bet–deficient cells.
T23 3704-3878 Sentence denotes Runx3 is required for Eomes and perforin expression, and both Eomes and Runx3 bind at the Prf1 locus; in contrast, perforin expression is unaffected in T-bet–deficient cells.
T840 3879-4021 Sentence denotes T cells lacking Runx3 show decreased expression of IFN-γ and granzyme B, and Runx3 also binds the promoter regions of the Ifng and Gzmb genes.
T24 3879-4021 Sentence denotes T cells lacking Runx3 show decreased expression of IFN-γ and granzyme B, and Runx3 also binds the promoter regions of the Ifng and Gzmb genes.
T841 4022-4265 Sentence denotes Collectively, these results provide evidence for a complex transcriptional network in which Runx3 is a primary regulator of Gzmb expression but synergizes with T-bet and Eomes, respectively, to promote transcription of the Ifng and Prf1 genes.
T25 4022-4265 Sentence denotes Collectively, these results provide evidence for a complex transcriptional network in which Runx3 is a primary regulator of Gzmb expression but synergizes with T-bet and Eomes, respectively, to promote transcription of the Ifng and Prf1 genes.
T26 4267-4289 Sentence denotes RESULTS AND DISCUSSION
T2695 4291-4352 Sentence denotes A cell culture system to monitor effector CTL differentiation
T27 4291-4352 Sentence denotes A cell culture system to monitor effector CTL differentiation
T2696 4353-4477 Sentence denotes We used a simple cell culture system to examine the kinetics of effector gene expression during CD8+ T cell differentiation.
T28 4353-4477 Sentence denotes We used a simple cell culture system to examine the kinetics of effector gene expression during CD8+ T cell differentiation.
T2697 4478-4713 Sentence denotes Naive CD8+ T cells from P14 TCR transgenic mice were activated for 2 d with anti-CD3 and anti-CD28 or with splenic APCs in the presence of Gp33 peptide, and were cultured in media containing 100 U/ml of recombinant human IL-2 (rhIL-2).
T29 4478-4713 Sentence denotes Naive CD8+ T cells from P14 TCR transgenic mice were activated for 2 d with anti-CD3 and anti-CD28 or with splenic APCs in the presence of Gp33 peptide, and were cultured in media containing 100 U/ml of recombinant human IL-2 (rhIL-2).
T2698 4714-4994 Sentence denotes We used TCR transgenic mice for these experiments because they provide a reliable source of CD8+ T cells that are truly naive; however, we chose not to stimulate cells with antigen in most experiments so as to avoid contamination with proteins and nucleic acids derived from APCs.
T30 4714-4994 Sentence denotes We used TCR transgenic mice for these experiments because they provide a reliable source of CD8+ T cells that are truly naive; however, we chose not to stimulate cells with antigen in most experiments so as to avoid contamination with proteins and nucleic acids derived from APCs.
T2699 4995-5216 Sentence denotes There were only minor differences in gene expression during differentiation induced by antigen/APC versus anti-CD3/anti-CD28, and the major conclusions presented in this report are the same for both activating conditions.
T31 4995-5216 Sentence denotes There were only minor differences in gene expression during differentiation induced by antigen/APC versus anti-CD3/anti-CD28, and the major conclusions presented in this report are the same for both activating conditions.
T2700 5217-5318 Sentence denotes Under our culture conditions, activated CD8+ T cells expanded exponentially and accumulated for >8 d.
T32 5217-5318 Sentence denotes Under our culture conditions, activated CD8+ T cells expanded exponentially and accumulated for >8 d.
T2701 5319-5467 Sentence denotes We limited our analysis to the first 6–8 d after activation, a period that coincides with clonal expansion of CD8+ T cells after activation in vivo.
T33 5319-5467 Sentence denotes We limited our analysis to the first 6–8 d after activation, a period that coincides with clonal expansion of CD8+ T cells after activation in vivo.
T3367 5469-5558 Sentence denotes Distinct expression kinetics of perforin and granzyme B during CTL development in culture
T34 5469-5558 Sentence denotes Distinct expression kinetics of perforin and granzyme B during CTL development in culture
T3368 5559-5706 Sentence denotes Our experiments revealed clear differences in the kinetics of perforin, granzyme B, and cytokine expression during CD8+ T cell activation (Fig. 1).
T35 5559-5706 Sentence denotes Our experiments revealed clear differences in the kinetics of perforin, granzyme B, and cytokine expression during CD8+ T cell activation (Fig. 1).
T3369 5707-5809 Sentence denotes Naive T cells showed detectable expression of perforin mRNA as well as perforin protein (Fig. 1, A–D).
T36 5707-5809 Sentence denotes Naive T cells showed detectable expression of perforin mRNA as well as perforin protein (Fig. 1, A–D).
T3370 5810-6037 Sentence denotes Relative to its expression in naive T cells, perforin (Prf1) mRNA expression did not increase appreciably at day 2 but showed a reproducible decrease at day 4, followed by robust reexpression between days 4 and 8 (Fig. 1, A–D).
T37 5810-6037 Sentence denotes Relative to its expression in naive T cells, perforin (Prf1) mRNA expression did not increase appreciably at day 2 but showed a reproducible decrease at day 4, followed by robust reexpression between days 4 and 8 (Fig. 1, A–D).
T3371 6038-6325 Sentence denotes In contrast, granzyme B (Gzmb) mRNA was low or undetectable in naive T cells but was strongly up-regulated by day 2 after stimulation and increased progressively until day 6 (Fig. 1, A and B); similarly, granzyme B protein was expressed by day 4 and remained high until day 6 (Fig. 1 E).
T38 6038-6325 Sentence denotes In contrast, granzyme B (Gzmb) mRNA was low or undetectable in naive T cells but was strongly up-regulated by day 2 after stimulation and increased progressively until day 6 (Fig. 1, A and B); similarly, granzyme B protein was expressed by day 4 and remained high until day 6 (Fig. 1 E).
T3372 6326-6535 Sentence denotes As expected, a small fraction of naive T cells expressed the cytokines IFN-γ and TNF in response to stimulation, and this capacity increased significantly in differentiated cells (Fig. 1 E; see also Fig. 2 A).
T39 6326-6535 Sentence denotes As expected, a small fraction of naive T cells expressed the cytokines IFN-γ and TNF in response to stimulation, and this capacity increased significantly in differentiated cells (Fig. 1 E; see also Fig. 2 A).
T40 6536-6545 Sentence denotes Figure 1.
T18355 6546-6745 Sentence denotes Kinetics of gene expression during CD8+ T cell differentiation. (A) Kinetics of Prf1, Gzmb, Tbx21 (T-bet), and Eomes mRNA expression in differentiating P14 CD8+ T cells analyzed by Northern blotting.
T41 6546-6745 Sentence denotes Kinetics of gene expression during CD8+ T cell differentiation. (A) Kinetics of Prf1, Gzmb, Tbx21 (T-bet), and Eomes mRNA expression in differentiating P14 CD8+ T cells analyzed by Northern blotting.
T18356 6746-6793 Sentence denotes RNA from day 7 Th1 cells was used as a control.
T42 6746-6793 Sentence denotes RNA from day 7 Th1 cells was used as a control.
T18357 6794-7005 Sentence denotes Sizes of mRNA transcripts are indicated. (B) Quantification of relative mRNA amounts by phosphorimager analysis. (C) Kinetics of protein expression in differentiating P14 CD8+ T cells analyzed by immunoblotting.
T43 6794-7005 Sentence denotes Sizes of mRNA transcripts are indicated. (B) Quantification of relative mRNA amounts by phosphorimager analysis. (C) Kinetics of protein expression in differentiating P14 CD8+ T cells analyzed by immunoblotting.
T18358 7006-7166 Sentence denotes Sizes of protein bands are indicated. (D) Relative protein amounts quantified from the Western blots. (E) Intracellular staining for granzyme B, IFN-γ, and TNF.
T44 7006-7166 Sentence denotes Sizes of protein bands are indicated. (D) Relative protein amounts quantified from the Western blots. (E) Intracellular staining for granzyme B, IFN-γ, and TNF.
T18359 7167-7246 Sentence denotes Granzyme B staining was specific relative to an isotype control (not depicted).
T45 7167-7246 Sentence denotes Granzyme B staining was specific relative to an isotype control (not depicted).
T18360 7247-7470 Sentence denotes Cells were restimulated with PMA and ionomycin for 4 h. (F) FACS-based assay to measure cytolytic activity of P14 CD8+ T cells against EL4 targets loaded with 0 (−) or 1 (+) μM Gp33 peptide (effector-to-target ratio = 5:1).
T46 7247-7470 Sentence denotes Cells were restimulated with PMA and ionomycin for 4 h. (F) FACS-based assay to measure cytolytic activity of P14 CD8+ T cells against EL4 targets loaded with 0 (−) or 1 (+) μM Gp33 peptide (effector-to-target ratio = 5:1).
T18361 7471-7603 Sentence denotes Percentage of Annexin V+ (apoptotic) target cells in the CD8-negative EL4 target population (dot plots) was determined (histograms).
T47 7471-7603 Sentence denotes Percentage of Annexin V+ (apoptotic) target cells in the CD8-negative EL4 target population (dot plots) was determined (histograms).
T18362 7604-7759 Sentence denotes Cytolytic activity was blocked by incubation with 2 mM EGTA (not depicted), confirming involvement of the granule exocytosis (perforin–granzyme B) pathway.
T48 7604-7759 Sentence denotes Cytolytic activity was blocked by incubation with 2 mM EGTA (not depicted), confirming involvement of the granule exocytosis (perforin–granzyme B) pathway.
T18363 7760-7844 Sentence denotes Data are representative of at least five (A–E) or three (F) independent experiments.
T49 7760-7978 Sentence denotes Data are representative of at least five (A–E) or three (F) independent experiments.We evaluated antigen-dependent cytolytic function in a short-term assay in which target cell death was measured within 2 h (Fig. 1 F).
T3373 7844-7978 Sentence denotes We evaluated antigen-dependent cytolytic function in a short-term assay in which target cell death was measured within 2 h (Fig. 1 F).
T3374 7979-8122 Sentence denotes By limiting the duration of TCR stimulation, this strategy minimizes cytolysis secondary to new gene expression during the period of the assay.
T50 7979-8122 Sentence denotes By limiting the duration of TCR stimulation, this strategy minimizes cytolysis secondary to new gene expression during the period of the assay.
T3375 8123-8349 Sentence denotes Naive T cells did not display significant cytolytic function in this short-term assay (unpublished data), most likely because they express immature (unprocessed) forms of perforin and lack the capacity to degranulate (18, 19).
T51 8123-8349 Sentence denotes Naive T cells did not display significant cytolytic function in this short-term assay (unpublished data), most likely because they express immature (unprocessed) forms of perforin and lack the capacity to degranulate (18, 19).
T3376 8350-8523 Sentence denotes Even after activation for 2 or 4 d, the cells showed poor cytolytic activity (Fig. 1 F), in striking contrast to their capacity for efficient cytokine production (Fig. 1 E).
T52 8350-8523 Sentence denotes Even after activation for 2 or 4 d, the cells showed poor cytolytic activity (Fig. 1 F), in striking contrast to their capacity for efficient cytokine production (Fig. 1 E).
T3377 8524-8679 Sentence denotes Only cells cultured until day 6 displayed robust cytotoxicity, as judged by their ability to induce apoptosis in a large number of target cells (Fig. 1 F).
T53 8524-8679 Sentence denotes Only cells cultured until day 6 displayed robust cytotoxicity, as judged by their ability to induce apoptosis in a large number of target cells (Fig. 1 F).
T3378 8680-8993 Sentence denotes These results show that after a strong priming stimulus through TCRs and co-stimulatory receptors in vitro, granzyme B expression and the ability to produce effector cytokines are programmed early, whereas perforin expression and cytolytic function are induced later, during the phase of clonal expansion in IL-2.
T54 8680-8993 Sentence denotes These results show that after a strong priming stimulus through TCRs and co-stimulatory receptors in vitro, granzyme B expression and the ability to produce effector cytokines are programmed early, whereas perforin expression and cytolytic function are induced later, during the phase of clonal expansion in IL-2.
T3379 8994-9124 Sentence denotes Therefore, the two major effector functions of CTL, cytokine production and cytolytic activity, are not intrinsically coregulated.
T55 8994-9124 Sentence denotes Therefore, the two major effector functions of CTL, cytokine production and cytolytic activity, are not intrinsically coregulated.
T4758 9126-9200 Sentence denotes Distinct kinetics of T-bet and Eomes expression during CTL differentiation
T56 9126-9200 Sentence denotes Distinct kinetics of T-bet and Eomes expression during CTL differentiation
T4759 9201-9404 Sentence denotes The T-box transcription factors T-bet and Eomes have been linked to the regulation of genes encoding effector cytokines (e.g., Ifng) and genes important for cytolytic function (e.g., Prf1 and GzmB) (20).
T57 9201-9404 Sentence denotes The T-box transcription factors T-bet and Eomes have been linked to the regulation of genes encoding effector cytokines (e.g., Ifng) and genes important for cytolytic function (e.g., Prf1 and GzmB) (20).
T4760 9405-9518 Sentence denotes We investigated the kinetics of expression of these transcription factors in our in vitro cultures (Fig. 1, A–D).
T58 9405-9518 Sentence denotes We investigated the kinetics of expression of these transcription factors in our in vitro cultures (Fig. 1, A–D).
T4761 9519-9741 Sentence denotes T-bet mRNA and protein were not detectable in naive CD8+ T cells, but were strongly induced upon TCR priming (day 2) and remained expressed through day 6 of differentiation (Fig. 1, A and C; quantified in Fig. 1, B and D).
T59 9519-9741 Sentence denotes T-bet mRNA and protein were not detectable in naive CD8+ T cells, but were strongly induced upon TCR priming (day 2) and remained expressed through day 6 of differentiation (Fig. 1, A and C; quantified in Fig. 1, B and D).
T4762 9742-9952 Sentence denotes In contrast, Eomes expression was low or undetectable at both the mRNA and protein levels in naive CD8+ T cells, and TCR priming in culture had only a modest effect on its expression at day 2 (Fig. 1, A and C).
T60 9742-9952 Sentence denotes In contrast, Eomes expression was low or undetectable at both the mRNA and protein levels in naive CD8+ T cells, and TCR priming in culture had only a modest effect on its expression at day 2 (Fig. 1, A and C).
T4763 9953-10051 Sentence denotes Strong induction of Eomes mRNA and protein was only observed at day 4 and later (Fig. 1, A and C).
T61 9953-10051 Sentence denotes Strong induction of Eomes mRNA and protein was only observed at day 4 and later (Fig. 1, A and C).
T4764 10052-10262 Sentence denotes T-bet mRNA expression slightly preceded the expression of GzmB mRNA; similarly, Eomes mRNA and protein were expressed ∼1 d ahead of the reexpression of perforin mRNA and protein, respectively (Fig. 1, B and D).
T62 10052-10262 Sentence denotes T-bet mRNA expression slightly preceded the expression of GzmB mRNA; similarly, Eomes mRNA and protein were expressed ∼1 d ahead of the reexpression of perforin mRNA and protein, respectively (Fig. 1, B and D).
T4765 10263-10436 Sentence denotes This detailed kinetic analysis suggested that, under our culture conditions, T-bet and Eomes contribute to distinct aspects of gene transcription during CTL differentiation.
T63 10263-10436 Sentence denotes This detailed kinetic analysis suggested that, under our culture conditions, T-bet and Eomes contribute to distinct aspects of gene transcription during CTL differentiation.
T4766 10437-10633 Sentence denotes T-bet is required early for IFN-γ production, and our data suggested that Eomes might not function during this early period but rather might contribute later to the control of perforin expression.
T64 10437-10633 Sentence denotes T-bet is required early for IFN-γ production, and our data suggested that Eomes might not function during this early period but rather might contribute later to the control of perforin expression.
T4767 10634-10883 Sentence denotes Our data seemed most consistent with a model in which TCR signals induce T-bet, which in turn induces IFN-γ (17) and possibly granzyme B; subsequently, Eomes is induced during the period of clonal expansion in IL-2 and activates perforin expression.
T65 10634-10883 Sentence denotes Our data seemed most consistent with a model in which TCR signals induce T-bet, which in turn induces IFN-γ (17) and possibly granzyme B; subsequently, Eomes is induced during the period of clonal expansion in IL-2 and activates perforin expression.
T6109 10885-10958 Sentence denotes Perforin and granzyme B expression are not appreciably regulated by T-bet
T66 10885-10958 Sentence denotes Perforin and granzyme B expression are not appreciably regulated by T-bet
T6110 10959-11141 Sentence denotes To test the model outlined in the previous paragraph directly, we compared the expression of IFN-γ, perforin, and granzyme B in CD8+ T cells from WT and Tbx21 (T-bet)-deficient mice.
T67 10959-11141 Sentence denotes To test the model outlined in the previous paragraph directly, we compared the expression of IFN-γ, perforin, and granzyme B in CD8+ T cells from WT and Tbx21 (T-bet)-deficient mice.
T6111 11142-11241 Sentence denotes As expected (17, 21), naive Tbx21−/− CD8+ T cells produced IFN-γ poorly upon activation (Fig. 2 A).
T68 11142-11241 Sentence denotes As expected (17, 21), naive Tbx21−/− CD8+ T cells produced IFN-γ poorly upon activation (Fig. 2 A).
T6112 11242-11424 Sentence denotes Notably, this deleterious effect of T-bet deficiency was only observed in differentiating CD8+ T cells until day 4 of culture but was almost completely mitigated by day 6 (Fig. 2 A).
T69 11242-11424 Sentence denotes Notably, this deleterious effect of T-bet deficiency was only observed in differentiating CD8+ T cells until day 4 of culture but was almost completely mitigated by day 6 (Fig. 2 A).
T6113 11425-11532 Sentence denotes This most likely reflected compensation by Eomes, which was strongly induced between days 4 and 6 (Fig. 1).
T70 11425-11532 Sentence denotes This most likely reflected compensation by Eomes, which was strongly induced between days 4 and 6 (Fig. 1).
T6114 11533-11666 Sentence denotes In contrast, T-bet–deficient T cells cultured for 6 d showed no defect in perforin mRNA expression (Fig. 2 B, compare lanes 1 and 4).
T71 11533-11666 Sentence denotes In contrast, T-bet–deficient T cells cultured for 6 d showed no defect in perforin mRNA expression (Fig. 2 B, compare lanes 1 and 4).
T6115 11667-11873 Sentence denotes We consistently observed a modest reduction in GzmB mRNA in T-bet–deficient T cells (Fig. 2 B, compare lanes 1 and 4), which did not translate into a decrease in expression of granzyme B protein (Fig. 2 C).
T72 11667-11873 Sentence denotes We consistently observed a modest reduction in GzmB mRNA in T-bet–deficient T cells (Fig. 2 B, compare lanes 1 and 4), which did not translate into a decrease in expression of granzyme B protein (Fig. 2 C).
T73 11874-11883 Sentence denotes Figure 2.
T19200 11884-12062 Sentence denotes Regulation of perforin, granzyme B, and IFN-γ expression by T-bet and Eomes in differentiating CTLs. (A) IFN-γ expression by WT (Tbx21+/+) and T-bet–deficient (Tbx21−/−) T cells.
T74 11884-12062 Sentence denotes Regulation of perforin, granzyme B, and IFN-γ expression by T-bet and Eomes in differentiating CTLs. (A) IFN-γ expression by WT (Tbx21+/+) and T-bet–deficient (Tbx21−/−) T cells.
T19201 12063-12239 Sentence denotes Naive CD8+ T cells, or cells activated and cultured for 4 or 6 d, were restimulated with PMA and ionomycin for 6 h, and IFN-γ expression was assessed by intracellular staining.
T75 12063-12239 Sentence denotes Naive CD8+ T cells, or cells activated and cultured for 4 or 6 d, were restimulated with PMA and ionomycin for 6 h, and IFN-γ expression was assessed by intracellular staining.
T19202 12240-12528 Sentence denotes Numbers show the percentage of IFN-γ+ cells. (B) Northern blot analysis of Prf1 and GzmB mRNA expression in WT or T-bet–deficient CD8+ T cells activated and either left uninfected (uninf) or transduced with retroviruses expressing Eomes-VP16 (Eo-VP16) or an empty IRES-GFP cassette (GFP).
T76 12240-12528 Sentence denotes Numbers show the percentage of IFN-γ+ cells. (B) Northern blot analysis of Prf1 and GzmB mRNA expression in WT or T-bet–deficient CD8+ T cells activated and either left uninfected (uninf) or transduced with retroviruses expressing Eomes-VP16 (Eo-VP16) or an empty IRES-GFP cassette (GFP).
T19203 12529-12581 Sentence denotes Total cellular RNA was analyzed on day 6 of culture.
T77 12529-12581 Sentence denotes Total cellular RNA was analyzed on day 6 of culture.
T19204 12582-12992 Sentence denotes The frequency of transduced cells in the cultures was equivalent for both constructs (∼65–70% GFP+ cells; not depicted). (C) Granzyme B and IFN-γ expression by Tbx21+/+ and Tbx21−/− T cells analyzed in restimulated cells that had been cultured for 5 d. (D) IFN-γ production by cells transduced with Eo-VP16 or control (GFP) retroviruses (RV) measured on day 4 after 6 h of restimulation with PMA and ionomycin.
T78 12582-12992 Sentence denotes The frequency of transduced cells in the cultures was equivalent for both constructs (∼65–70% GFP+ cells; not depicted). (C) Granzyme B and IFN-γ expression by Tbx21+/+ and Tbx21−/− T cells analyzed in restimulated cells that had been cultured for 5 d. (D) IFN-γ production by cells transduced with Eo-VP16 or control (GFP) retroviruses (RV) measured on day 4 after 6 h of restimulation with PMA and ionomycin.
T19205 12993-13042 Sentence denotes Numbers show the percentage of GFP+ IFN-γ+ cells.
T79 12993-13042 Sentence denotes Numbers show the percentage of GFP+ IFN-γ+ cells.
T19206 13043-13130 Sentence denotes Results are representative of three (A and C) or two (B and D) independent experiments.
T80 13043-13436 Sentence denotes Results are representative of three (A and C) or two (B and D) independent experiments.To examine the role of Eomes, we transduced naive CD8+ T cells from WT and Tbx21−/− mice with retroviruses containing internal ribosome entry site (IRES)–GFP that were either empty or encoded a strongly transactivating version of Eomes (Eo-VP16) (8), and expanded them for 6 d under our culture conditions.
T6116 13130-13436 Sentence denotes To examine the role of Eomes, we transduced naive CD8+ T cells from WT and Tbx21−/− mice with retroviruses containing internal ribosome entry site (IRES)–GFP that were either empty or encoded a strongly transactivating version of Eomes (Eo-VP16) (8), and expanded them for 6 d under our culture conditions.
T6117 13437-13587 Sentence denotes Eo-VP16, but not the empty GFP retrovirus, increased perforin expression in both WT and T-bet–deficient CD8+ T cells (Fig. 2 B, lanes 2, 3, 5, and 6).
T81 13437-13587 Sentence denotes Eo-VP16, but not the empty GFP retrovirus, increased perforin expression in both WT and T-bet–deficient CD8+ T cells (Fig. 2 B, lanes 2, 3, 5, and 6).
T6118 13588-13711 Sentence denotes As expected, Eo-VP16 also rescued the early defect in IFN-γ production observed in T-bet–deficient CD8+ T cells (Fig. 2 D).
T82 13588-13711 Sentence denotes As expected, Eo-VP16 also rescued the early defect in IFN-γ production observed in T-bet–deficient CD8+ T cells (Fig. 2 D).
T6119 13712-13900 Sentence denotes However, Eo-VP16 did not induce GzmB mRNA expression in either WT or T-bet–deficient cells; thus, the partial T-bet dependence of GzmB mRNA expression cannot be compensated for by Eo-VP16.
T83 13712-13900 Sentence denotes However, Eo-VP16 did not induce GzmB mRNA expression in either WT or T-bet–deficient cells; thus, the partial T-bet dependence of GzmB mRNA expression cannot be compensated for by Eo-VP16.
T7962 13902-14004 Sentence denotes Runx3 controls multiple aspects of the CTL differentiation program, in part through induction of Eomes
T84 13902-14004 Sentence denotes Runx3 controls multiple aspects of the CTL differentiation program, in part through induction of Eomes
T7963 14005-14214 Sentence denotes Because 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.
T85 14005-14214 Sentence denotes Because 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.
T86 14215-14385 Sentence denotes 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.
T7964 14215-14464 Sentence denotes 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).
T87 14386-14464 Sentence denotes S1 and S2, available at http://www.jem.org/cgi/content/full/jem.20081242/DC1).
T7965 14465-14644 Sentence denotes 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).
T88 14465-14644 Sentence denotes 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).
T7966 14645-14796 Sentence denotes 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).
T89 14645-14796 Sentence denotes 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).
T7967 14797-14911 Sentence denotes Runx3−/− T cells also had no detectable Eomes expression; in contrast, T-bet expression was unimpaired (Fig. 3 A).
T90 14797-14911 Sentence denotes Runx3−/− T cells also had no detectable Eomes expression; in contrast, T-bet expression was unimpaired (Fig. 3 A).
T7968 14912-15047 Sentence denotes 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).
T91 14912-15047 Sentence denotes 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).
T92 15048-15057 Sentence denotes Figure 3.
T20175 15058-15589 Sentence denotes 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.
T93 15058-15589 Sentence denotes 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.
T20176 15590-15660 Sentence denotes The vertical gray line indicates the granzyme B MFI for WT GFP+ cells.
T94 15590-15660 Sentence denotes The vertical gray line indicates the granzyme B MFI for WT GFP+ cells.
T20177 15661-15804 Sentence denotes Results in A–C are representative of two independent experiments. (D) ChIP analysis of binding of endogenous Runx3 and Eomes to the Prf1 locus.
T95 15661-15804 Sentence denotes Results in A–C are representative of two independent experiments. (D) ChIP analysis of binding of endogenous Runx3 and Eomes to the Prf1 locus.
T20178 15805-15931 Sentence denotes Enrichment of the indicated genomic regions was evaluated by real-time PCR of DNA from immunoprecipitated and input chromatin.
T96 15805-15931 Sentence denotes Enrichment of the indicated genomic regions was evaluated by real-time PCR of DNA from immunoprecipitated and input chromatin.
T20179 15932-16063 Sentence denotes The data are the means of duplicate measurements from two chromatin preparations from two independent CD8+ T cell differentiations.
T97 15932-16063 Sentence denotes The data are the means of duplicate measurements from two chromatin preparations from two independent CD8+ T cell differentiations.
T20180 16064-16186 Sentence denotes 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.
T98 16064-16479 Sentence denotes 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).
T7969 16186-16479 Sentence denotes 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).
T7970 16480-16950 Sentence denotes 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.
T99 16480-16950 Sentence denotes 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.
T7971 16951-17143 Sentence denotes 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.
T100 16951-17143 Sentence denotes 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.
T7972 17144-17322 Sentence denotes To 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).
T101 17144-17322 Sentence denotes To 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).
T7973 17323-17400 Sentence denotes Both proteins associated with gene regulatory regions in differentiated CTLs.
T102 17323-17400 Sentence denotes Both proteins associated with gene regulatory regions in differentiated CTLs.
T7974 17401-17799 Sentence denotes 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).
T103 17401-17799 Sentence denotes 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).
T7975 17800-18118 Sentence denotes 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).
T104 17800-18118 Sentence denotes 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).
T7976 18119-18305 Sentence denotes To 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.
T105 18119-18305 Sentence denotes To 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.
T7977 18306-18588 Sentence denotes 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.
T106 18306-18588 Sentence denotes 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.
T7978 18589-18730 Sentence denotes 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).
T107 18589-18730 Sentence denotes 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).
T7979 18731-18917 Sentence denotes 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.
T108 18731-18917 Sentence denotes 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.
T7980 18918-19114 Sentence denotes 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).
T109 18918-19114 Sentence denotes 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).
T7981 19115-19195 Sentence denotes This result suggests strongly that perforin expression requires Runx3 and Eomes.
T110 19115-19195 Sentence denotes This result suggests strongly that perforin expression requires Runx3 and Eomes.
T111 19196-19205 Sentence denotes Figure 4.
T20952 19206-19288 Sentence denotes Runx3 controls Eomes, perforin, granzyme B, and IFN-γ expression in effector CTLs.
T112 19206-19288 Sentence denotes Runx3 controls Eomes, perforin, granzyme B, and IFN-γ expression in effector CTLs.
T20953 19289-19471 Sentence denotes 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).
T113 19289-19471 Sentence denotes 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).
T20954 19472-19677 Sentence denotes 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.
T114 19472-19677 Sentence denotes 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.
T20955 19678-20010 Sentence denotes 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.
T115 19678-20010 Sentence denotes 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.
T20956 20011-20189 Sentence denotes 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.
T116 20011-20189 Sentence denotes 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.
T20957 20190-20249 Sentence denotes The vertical gray lines indicate the MFI for WT GFP+ cells.
T117 20190-20249 Sentence denotes The vertical gray lines indicate the MFI for WT GFP+ cells.
T20958 20250-20405 Sentence denotes Results are representative of at least two independent experiments. (C) Schematic diagram of the transcriptional network involving Runx3 and T-box factors.
T118 20250-20405 Sentence denotes Results are representative of at least two independent experiments. (C) Schematic diagram of the transcriptional network involving Runx3 and T-box factors.
T20959 20406-20482 Sentence denotes T-bet is induced by TCR signals and is essential for early IFN-γ expression.
T119 20406-20482 Sentence denotes T-bet is induced by TCR signals and is essential for early IFN-γ expression.
T20960 20483-20604 Sentence denotes Runx3 is present in naive CD8+ T cells and represses Runx1 and induces Eomes, perforin, granzyme B, and IFN-γ expression.
T120 20483-20604 Sentence denotes Runx3 is present in naive CD8+ T cells and represses Runx1 and induces Eomes, perforin, granzyme B, and IFN-γ expression.
T20961 20605-20750 Sentence denotes Eomes may participate in sustaining late IFN-γ expression, whereas Runx3 and Eomes (but not T-bet) may cooperate to activate perforin expression.
T121 20605-20750 Sentence denotes Eomes may participate in sustaining late IFN-γ expression, whereas Runx3 and Eomes (but not T-bet) may cooperate to activate perforin expression.
T20962 20751-20867 Sentence denotes The dotted line indicates the partial effect of T-bet deficiency on Gzmb mRNA but not granzyme B protein expression.
T122 20751-21030 Sentence denotes 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).
T7982 20867-21030 Sentence denotes 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).
T7983 21031-21159 Sentence denotes However, TCR-stimulated Runx3−/− CD8+ cells were as effective as WT cells in killing tumor cells in a redirected CTL assay (12).
T123 21031-21159 Sentence denotes However, TCR-stimulated Runx3−/− CD8+ cells were as effective as WT cells in killing tumor cells in a redirected CTL assay (12).
T7984 21160-21301 Sentence denotes Furthermore, CD8+ cells from the peritoneal cavity of Runx3−/− mice immunized with certain tumor cells effectively killed these targets (13).
T124 21160-21301 Sentence denotes Furthermore, CD8+ cells from the peritoneal cavity of Runx3−/− mice immunized with certain tumor cells effectively killed these targets (13).
T7985 21302-21577 Sentence denotes 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.
T125 21302-21577 Sentence denotes 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.
T11889 21579-21685 Sentence denotes Runx3 and T-box factors control a complex program of transcriptional regulation during CTL differentiation
T126 21579-21685 Sentence denotes Runx3 and T-box factors control a complex program of transcriptional regulation during CTL differentiation
T11890 21686-21864 Sentence denotes Collectively, these data provide evidence that Runx3, together with T-box factors, orchestrates a complex program of transcriptional regulation in differentiating CTL (Fig. 4 C).
T127 21686-21864 Sentence denotes Collectively, these data provide evidence that Runx3, together with T-box factors, orchestrates a complex program of transcriptional regulation in differentiating CTL (Fig. 4 C).
T11891 21865-21927 Sentence denotes Runx3 is present in naive CD8+ T cells before activation (12).
T128 21865-21927 Sentence denotes Runx3 is present in naive CD8+ T cells before activation (12).
T11892 21928-22030 Sentence denotes It represses Runx1 and has a positive role in the induction of Eomes, granzyme B, perforin, and IFN-γ.
T129 21928-22030 Sentence denotes It represses Runx1 and has a positive role in the induction of Eomes, granzyme B, perforin, and IFN-γ.
T11893 22031-22161 Sentence denotes Runx3 binds to promoters and putative regulatory regions of the latter three genes, suggesting a direct effect on gene expression.
T130 22031-22161 Sentence denotes Runx3 binds to promoters and putative regulatory regions of the latter three genes, suggesting a direct effect on gene expression.
T11894 22162-22271 Sentence denotes Additional experiments are needed to determine whether Eomes and Runx1 are also direct target genes of Runx3.
T131 22162-22271 Sentence denotes Additional experiments are needed to determine whether Eomes and Runx1 are also direct target genes of Runx3.
T11895 22272-22373 Sentence denotes Surprisingly, Runx3 contributed to the optimal expression of TNF, IL-2, and IFN-γ at day 4 (Fig. S2).
T132 22272-22373 Sentence denotes Surprisingly, Runx3 contributed to the optimal expression of TNF, IL-2, and IFN-γ at day 4 (Fig. S2).
T11896 22374-22541 Sentence denotes For TNF and IL-2, the requirement for Runx3 subsides by day 6 (Fig. 3 C), possibly because of compensation by Runx1, which is derepressed in Runx3−/− cells (Fig. 4 A).
T133 22374-22541 Sentence denotes For TNF and IL-2, the requirement for Runx3 subsides by day 6 (Fig. 3 C), possibly because of compensation by Runx1, which is derepressed in Runx3−/− cells (Fig. 4 A).
T11897 22542-22685 Sentence denotes Runx3 continues to be required for IFN-γ expression even at day 6, perhaps because of its role in the induction of Eomes expression (Fig. 4 A).
T134 22542-22685 Sentence denotes Runx3 continues to be required for IFN-γ expression even at day 6, perhaps because of its role in the induction of Eomes expression (Fig. 4 A).
T11898 22686-22955 Sentence denotes An unexpected finding was that the two T-box transcription factors, T-bet and Eomes, are up-regulated with very different kinetics in CD8+ T cells under our culture conditions and have nonredundant roles in the subsequent expression of key effector proteins (Fig. 4 C).
T135 22686-22955 Sentence denotes An unexpected finding was that the two T-box transcription factors, T-bet and Eomes, are up-regulated with very different kinetics in CD8+ T cells under our culture conditions and have nonredundant roles in the subsequent expression of key effector proteins (Fig. 4 C).
T11899 22956-23116 Sentence denotes T-bet is needed early to confer on activated CD8+ T cells the competence to produce IFN-γ upon restimulation, but its function is less important at later times.
T136 22956-23116 Sentence denotes T-bet is needed early to confer on activated CD8+ T cells the competence to produce IFN-γ upon restimulation, but its function is less important at later times.
T11900 23117-23274 Sentence denotes Eomes, which is induced late and functions downstream of Runx3, may substitute for T-bet in promoting the acute expression of IFN-γ in restimulated CTLs (8).
T137 23117-23274 Sentence denotes Eomes, which is induced late and functions downstream of Runx3, may substitute for T-bet in promoting the acute expression of IFN-γ in restimulated CTLs (8).
T11901 23275-23531 Sentence denotes Indeed, T-bet and Eomes both contribute to perforin expression in NK cells (8, 26), and Eomes induces granzyme B as effectively as T-bet in developing Th2 cells (7); thus, the relative roles of these T-box transcription factors vary depending on cell type.
T138 23275-23531 Sentence denotes Indeed, T-bet and Eomes both contribute to perforin expression in NK cells (8, 26), and Eomes induces granzyme B as effectively as T-bet in developing Th2 cells (7); thus, the relative roles of these T-box transcription factors vary depending on cell type.
T11902 23532-23680 Sentence denotes Surprisingly, however, Eomes and T-bet appeared nonredundant in their ability to induce two other markers of CTL function, Prf1 and Gzmb (Fig. 4 C).
T139 23532-23680 Sentence denotes Surprisingly, however, Eomes and T-bet appeared nonredundant in their ability to induce two other markers of CTL function, Prf1 and Gzmb (Fig. 4 C).
T11903 23681-23912 Sentence denotes Rather, T-bet and Eomes were involved in regulating granzyme B and perforin expression, respectively: up-regulation of T-bet and Eomes mRNA and protein closely preceded up-regulation of Gzmb and Prf1 mRNA and protein, respectively.
T140 23681-23912 Sentence denotes Rather, T-bet and Eomes were involved in regulating granzyme B and perforin expression, respectively: up-regulation of T-bet and Eomes mRNA and protein closely preceded up-regulation of Gzmb and Prf1 mRNA and protein, respectively.
T11904 23913-24078 Sentence denotes T-bet had no role in perforin expression under our culture conditions, and Eo-VP16 did not affect granzyme B expression when expressed in T-bet−/− or Runx3−/− cells.
T141 23913-24078 Sentence denotes T-bet had no role in perforin expression under our culture conditions, and Eo-VP16 did not affect granzyme B expression when expressed in T-bet−/− or Runx3−/− cells.
T11905 24079-24438 Sentence denotes Because conventional Eomes-deficient mice die before precursor cells can be isolated for bone marrow transfers (27) and because T cells conditionally deficient in Eomes have only recently been described (9), we were unable to introduce Runx3 into Eomes-deficient CD8+ T cells to test formally whether Eomes cooperated with Runx3 to induce perforin expression.
T142 24079-24438 Sentence denotes Because conventional Eomes-deficient mice die before precursor cells can be isolated for bone marrow transfers (27) and because T cells conditionally deficient in Eomes have only recently been described (9), we were unable to introduce Runx3 into Eomes-deficient CD8+ T cells to test formally whether Eomes cooperated with Runx3 to induce perforin expression.
T11906 24439-24677 Sentence denotes Collectively, our data are consistent with a transcriptional network in which preexisting Runx3 cooperates with the induced T-box factors T-bet and Eomes and IL-2Rβ signals (unpublished data) to orchestrate CTL differentiation (Fig. 4 C).
T143 24439-24677 Sentence denotes Collectively, our data are consistent with a transcriptional network in which preexisting Runx3 cooperates with the induced T-box factors T-bet and Eomes and IL-2Rβ signals (unpublished data) to orchestrate CTL differentiation (Fig. 4 C).
T144 24678-25062 Sentence denotes Our data recall the “feed-forward” interaction between T-bet and Runx3 that we previously described in CD4+ (Th1) T cells (15) but are distinct in two respects: in differentiating Th1 cells, T-bet is induced by TCR signals and IFN-γ, and in turn induces Runx3 (15), whereas in differentiating CD8+ T cells, preexisting Runx3 is required to induce the T-box transcription factor Eomes.
T11907 24678-25275 Sentence denotes Our data recall the “feed-forward” interaction between T-bet and Runx3 that we previously described in CD4+ (Th1) T cells (15) but are distinct in two respects: in differentiating Th1 cells, T-bet is induced by TCR signals and IFN-γ, and in turn induces Runx3 (15), whereas in differentiating CD8+ T cells, preexisting Runx3 is required to induce the T-box transcription factor Eomes. Whole-genome experiments in these and other systems will be required to establish whether cooperation between T-box and Runx family transcription factors is a general feature of cellular differentiation programs.
T145 25063-25275 Sentence denotes Whole-genome experiments in these and other systems will be required to establish whether cooperation between T-box and Runx family transcription factors is a general feature of cellular differentiation programs.
T146 25277-25298 Sentence denotes MATERIALS AND METHODS
T13821 25300-25324 Sentence denotes Antibodies and reagents.
T147 25300-25324 Sentence denotes Antibodies and reagents.
T13822 25325-25508 Sentence denotes The following antibodies used for intracellular or surface stains were obtained from eBioscience: anti–IL-2, anti–IFN-γ, anti-TNF, anti–granzyme B, anti-CD8, anti-CD25, and anti-CD44.
T148 25325-25508 Sentence denotes The following antibodies used for intracellular or surface stains were obtained from eBioscience: anti–IL-2, anti–IFN-γ, anti-TNF, anti–granzyme B, anti-CD8, anti-CD25, and anti-CD44.
T13823 25509-25541 Sentence denotes Anti-CD69 was purchased from BD.
T149 25509-25541 Sentence denotes Anti-CD69 was purchased from BD.
T13824 25542-25678 Sentence denotes For ChIP experiments, the anti-Eomes antibody was obtained from Abcam and the anti-Runx3 antibody was produced by the Groner laboratory.
T150 25542-25678 Sentence denotes For ChIP experiments, the anti-Eomes antibody was obtained from Abcam and the anti-Runx3 antibody was produced by the Groner laboratory.
T13825 25679-25825 Sentence denotes The following antibodies were used for immunoblotting: antiperforin (Abcam), anti-Eomes (Abcam), and anti–Pol-II (Santa Cruz Biotechnology, Inc.).
T151 25679-25825 Sentence denotes The following antibodies were used for immunoblotting: antiperforin (Abcam), anti-Eomes (Abcam), and anti–Pol-II (Santa Cruz Biotechnology, Inc.).
T152 25826-25863 Sentence denotes The T-bet antibody was provided by L.
T13826 25826-25919 Sentence denotes The T-bet antibody was provided by L. Glimcher (Harvard School of Public Health, Boston, MA).
T153 25864-25919 Sentence denotes Glimcher (Harvard School of Public Health, Boston, MA).
T13827 25920-25998 Sentence denotes The following reagents were used for the experiments presented in this report:
T154 25920-25998 Sentence denotes The following reagents were used for the experiments presented in this report:
T13828 25999-26173 Sentence denotes Annexin V–FITC Apoptosis Detection Kit (BD), CD8 Negative Isolation Kit (Invitrogen), CD8 MicroBeads (Miltenyi Biotec), and SYBR Green PCR Core Reagents (Applied Biosystems).
T155 25999-26173 Sentence denotes Annexin V–FITC Apoptosis Detection Kit (BD), CD8 Negative Isolation Kit (Invitrogen), CD8 MicroBeads (Miltenyi Biotec), and SYBR Green PCR Core Reagents (Applied Biosystems).
T13829 26174-26308 Sentence denotes The Gp33 peptide (KAVYNFATC) was synthesized by the Tufts University Core Facility, and 10 mM of stock solutions was prepared in DMSO.
T156 26174-26308 Sentence denotes The Gp33 peptide (KAVYNFATC) was synthesized by the Tufts University Core Facility, and 10 mM of stock solutions was prepared in DMSO.
T14376 26310-26356 Sentence denotes Isolation and culture of primary CD8+ T cells.
T157 26310-26356 Sentence denotes Isolation and culture of primary CD8+ T cells.
T14377 26357-26584 Sentence denotes CD8+ T cells from 4–8-wk-old Tcra−/− × P14 TCR transgenic (Taconic), C57BL/6J WT, or Tbx21−/− (The Jackson Laboratory) mice were purified (>95% purity) by negative selection (Invitrogen) from pooled spleen and lymph node cells.
T158 26357-26584 Sentence denotes CD8+ T cells from 4–8-wk-old Tcra−/− × P14 TCR transgenic (Taconic), C57BL/6J WT, or Tbx21−/− (The Jackson Laboratory) mice were purified (>95% purity) by negative selection (Invitrogen) from pooled spleen and lymph node cells.
T14378 26585-26693 Sentence denotes CD8+ T cells from Runx3−/− mice on the ICR background were purified by positive selection (Miltenyi Biotec).
T159 26585-26693 Sentence denotes CD8+ T cells from Runx3−/− mice on the ICR background were purified by positive selection (Miltenyi Biotec).
T160 26694-26899 Sentence denotes All mice were maintained in specific pathogen-free barrier facilities and used according to protocols approved by the Immune Disease Institute and the Harvard Medical School Animal Care and Use Committees.
T14379 26694-27122 Sentence denotes All mice were maintained in specific pathogen-free barrier facilities and used according to protocols approved by the Immune Disease Institute and the Harvard Medical School Animal Care and Use Committees. For stimulation, purified CD8+ T cells were cultured at 106 cells/ml (10 ml) in T25 flasks coated with 1 μg/ml each of anti-CD3 (clone 2C11) and anti-CD28 (clone 37.51) by pretreatment with 300 μg/ml goat anti–hamster IgG.
T161 26900-27122 Sentence denotes For stimulation, purified CD8+ T cells were cultured at 106 cells/ml (10 ml) in T25 flasks coated with 1 μg/ml each of anti-CD3 (clone 2C11) and anti-CD28 (clone 37.51) by pretreatment with 300 μg/ml goat anti–hamster IgG.
T14380 27123-27276 Sentence denotes After 48 h, cells were removed from the TCR stimulation and recultured at a concentration of 5 × 105 cells/ml in media supplemented with 100 U/ml rhIL-2.
T162 27123-27276 Sentence denotes After 48 h, cells were removed from the TCR stimulation and recultured at a concentration of 5 × 105 cells/ml in media supplemented with 100 U/ml rhIL-2.
T14381 27277-27413 Sentence denotes Every 24 h, viable cells were counted and readjusted to 5 × 105 cells/ml with fresh media containing the corresponding amount of rhIL-2.
T163 27277-27413 Sentence denotes Every 24 h, viable cells were counted and readjusted to 5 × 105 cells/ml with fresh media containing the corresponding amount of rhIL-2.
T15043 27415-27460 Sentence denotes Isolation of CD8+ T cells from Runx3−/− mice.
T164 27415-27460 Sentence denotes Isolation of CD8+ T cells from Runx3−/− mice.
T15044 27461-27544 Sentence denotes Runx3-deficient T cells fail to silence CD4 expression normally (Fig. S1) (12, 13).
T165 27461-27544 Sentence denotes Runx3-deficient T cells fail to silence CD4 expression normally (Fig. S1) (12, 13).
T15045 27545-27717 Sentence denotes We therefore further fractionated the positively selected CD8+ T cells from Runx3 KO mice into CD8+CD4− SP or CD8+CD4+ DP cells by separation using anti-CD4 magnetic beads.
T166 27545-27717 Sentence denotes We therefore further fractionated the positively selected CD8+ T cells from Runx3 KO mice into CD8+CD4− SP or CD8+CD4+ DP cells by separation using anti-CD4 magnetic beads.
T15046 27718-27877 Sentence denotes This yielded a Runx3 KO SP “enriched” population that contained 75% CD8+CD4− cells and a KO DP enriched population that contained 85% CD8+CD4+ cells (Fig. S1).
T167 27718-27877 Sentence denotes This yielded a Runx3 KO SP “enriched” population that contained 75% CD8+CD4− cells and a KO DP enriched population that contained 85% CD8+CD4+ cells (Fig. S1).
T15047 27878-28033 Sentence denotes The cells were stimulated with anti-CD3+ anti-CD28 for 2 d before removing them from the TCR stimulus and culturing them in media containing 100 U/ml IL-2.
T168 27878-28033 Sentence denotes The cells were stimulated with anti-CD3+ anti-CD28 for 2 d before removing them from the TCR stimulus and culturing them in media containing 100 U/ml IL-2.
T15048 28034-28182 Sentence denotes As previously reported, TCR-induced proliferation of Runx3−/− CD8+ T cells was severely impaired, irrespective of CD4 expression (Fig. S1) (12, 13).
T169 28034-28182 Sentence denotes As previously reported, TCR-induced proliferation of Runx3−/− CD8+ T cells was severely impaired, irrespective of CD4 expression (Fig. S1) (12, 13).
T15049 28183-28333 Sentence denotes However, the Runx3−/− cells showed cell-surface expression patterns indicative of activated cells, including up-regulation of CD25 and CD69 (Fig. S1).
T170 28183-28333 Sentence denotes However, the Runx3−/− cells showed cell-surface expression patterns indicative of activated cells, including up-regulation of CD25 and CD69 (Fig. S1).
T15050 28334-28570 Sentence denotes As expected from their ability to up-regulate CD25, Runx3−/− CD8+ T cells responded to IL-2 supplementation after day 2 and efficiently expanded until day 6 of the culture period, albeit at slower rates compared with WT cells (Fig. S1).
T171 28334-28570 Sentence denotes As expected from their ability to up-regulate CD25, Runx3−/− CD8+ T cells responded to IL-2 supplementation after day 2 and efficiently expanded until day 6 of the culture period, albeit at slower rates compared with WT cells (Fig. S1).
T15051 28571-29023 Sentence denotes Although a fraction of the KO DP cells silenced CD4 expression after activation, the ratio of SP/DP cells in each enriched population remained constant thereafter, and we did not observe any major differences between these two populations throughout the culture period, indicating that in terms of effector CTL differentiation and under our culture conditions, Runx3−/− CD8+ T cells that also coexpress CD4 are indistinguishable from those that do not.
T172 28571-29023 Sentence denotes Although a fraction of the KO DP cells silenced CD4 expression after activation, the ratio of SP/DP cells in each enriched population remained constant thereafter, and we did not observe any major differences between these two populations throughout the culture period, indicating that in terms of effector CTL differentiation and under our culture conditions, Runx3−/− CD8+ T cells that also coexpress CD4 are indistinguishable from those that do not.
T173 29024-29110 Sentence denotes The data presented in Fig. S2 are from Runx3 KO SP cells, whereas those shown in Figs.
T15052 29024-29153 Sentence denotes The data presented in Fig. S2 are from Runx3 KO SP cells, whereas those shown in Figs. 3 and 4 are from total Runx3 KO CD8 cells.
T174 29111-29153 Sentence denotes 3 and 4 are from total Runx3 KO CD8 cells.
T16126 29155-29185 Sentence denotes FACS-based cytotoxicity assay.
T175 29155-29185 Sentence denotes FACS-based cytotoxicity assay.
T16127 29186-29421 Sentence denotes To measure cytotoxicity, EL4 thymoma target cells were loaded with 0 or 1 μM Gp33 peptide for 2 h before a 2-h coincubation with P14 CD8+ T cells at the effector-to-target ratios indicated in the figures in 96-well round-bottom plates.
T176 29186-29421 Sentence denotes To measure cytotoxicity, EL4 thymoma target cells were loaded with 0 or 1 μM Gp33 peptide for 2 h before a 2-h coincubation with P14 CD8+ T cells at the effector-to-target ratios indicated in the figures in 96-well round-bottom plates.
T16128 29422-29521 Sentence denotes After the coincubation period, cells were stained with Annexin V–FITC and anti-CD8–allophycocyanin.
T177 29422-29521 Sentence denotes After the coincubation period, cells were stained with Annexin V–FITC and anti-CD8–allophycocyanin.
T16129 29522-29702 Sentence denotes Data analysis was performed with FlowJo software (Tree Star, Inc.); EL4 target cells (CD8-negative events) were gated, and the percentage of Annexin V+ target cells was determined.
T178 29522-29702 Sentence denotes Data analysis was performed with FlowJo software (Tree Star, Inc.); EL4 target cells (CD8-negative events) were gated, and the percentage of Annexin V+ target cells was determined.
T16373 29704-29741 Sentence denotes Cytokine and surface marker staining.
T179 29704-29741 Sentence denotes Cytokine and surface marker staining.
T16374 29742-29960 Sentence denotes To assess cytokine production, cells were restimulated with 10 nM PMA + 1 μM ionomycin for 6 h (unless indicated otherwise in the figures), and intracellular cytokine stains were performed as previously described (28).
T180 29742-29960 Sentence denotes To assess cytokine production, cells were restimulated with 10 nM PMA + 1 μM ionomycin for 6 h (unless indicated otherwise in the figures), and intracellular cytokine stains were performed as previously described (28).
T16375 29961-30351 Sentence denotes To detect expression of surface molecules, cells were washed in PBS, resuspended in FACS wash buffer (3% FBS, 0.1% sodium azide, 30 mM Hepes, 1× PBS) containing the antibodies indicated in the figures at previously optimized concentrations, incubated for 15 min at room temperature (RT), washed, and resuspended in 2% formaldehyde fixative solution before acquisition on a FACSCalibur (BD).
T181 29961-30351 Sentence denotes To detect expression of surface molecules, cells were washed in PBS, resuspended in FACS wash buffer (3% FBS, 0.1% sodium azide, 30 mM Hepes, 1× PBS) containing the antibodies indicated in the figures at previously optimized concentrations, incubated for 15 min at room temperature (RT), washed, and resuspended in 2% formaldehyde fixative solution before acquisition on a FACSCalibur (BD).
T16647 30353-30401 Sentence denotes Retroviral transduction of primary CD8+ T cells.
T182 30353-30401 Sentence denotes Retroviral transduction of primary CD8+ T cells.
T16648 30402-30574 Sentence denotes For transduction experiments, viral supernatants were generated by calcium phosphate transfection of Phoenix cells and concentration by overnight centrifugation at 6,000 g.
T183 30402-30574 Sentence denotes For transduction experiments, viral supernatants were generated by calcium phosphate transfection of Phoenix cells and concentration by overnight centrifugation at 6,000 g.
T16649 30575-30805 Sentence denotes At ∼42 h after the initial TCR activation of 106 CD8+ T cells per well in 12-well plates, the culture media was removed and replaced with complete media supplemented with 8 μg/ml polybrene containing fresh plus concentrated virus.
T184 30575-30805 Sentence denotes At ∼42 h after the initial TCR activation of 106 CD8+ T cells per well in 12-well plates, the culture media was removed and replaced with complete media supplemented with 8 μg/ml polybrene containing fresh plus concentrated virus.
T16650 30806-30904 Sentence denotes The plates were centrifuged at 700 g for 1 h at RT before returning to 37°C for an additional 5 h.
T185 30806-30904 Sentence denotes The plates were centrifuged at 700 g for 1 h at RT before returning to 37°C for an additional 5 h.
T186 30905-30996 Sentence denotes Retroviral constructs for Eomes-VP16 and the MIG control empty vector were a gift from S.L.
T16651 30905-31055 Sentence denotes Retroviral constructs for Eomes-VP16 and the MIG control empty vector were a gift from S.L. Reiner (University of Pennsylvania, Philadelphia, PA) (8).
T187 30997-31055 Sentence denotes Reiner (University of Pennsylvania, Philadelphia, PA) (8).
T16948 31057-31089 Sentence denotes ChIP and real-time PCR analysis.
T188 31057-31089 Sentence denotes ChIP and real-time PCR analysis.
T16949 31090-31336 Sentence denotes 20 × 106 CD8+ T cells per immunoprecipitation were fixed by adding a 1/10th volume of fixation solution (11.1% formaldehyde, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM Hepes) to 1 volume of culture media and were incubated for 10 or 30 min at RT.
T189 31090-31336 Sentence denotes 20 × 106 CD8+ T cells per immunoprecipitation were fixed by adding a 1/10th volume of fixation solution (11.1% formaldehyde, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM Hepes) to 1 volume of culture media and were incubated for 10 or 30 min at RT.
T16950 31337-31395 Sentence denotes Fixation was stopped with 120 mM glycine on ice for 5 min.
T190 31337-31395 Sentence denotes Fixation was stopped with 120 mM glycine on ice for 5 min.
T16951 31396-31615 Sentence denotes Fixed cells were washed 2× with cold PBS, 1× with cold solution I (10 mM Tris [pH 7.5], 10 mM EDTA, 0.5 mM EGTA, 1% Triton X-100), and 1× with cold solution II (10 mM Tris [pH 7.5], 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl).
T191 31396-31615 Sentence denotes Fixed cells were washed 2× with cold PBS, 1× with cold solution I (10 mM Tris [pH 7.5], 10 mM EDTA, 0.5 mM EGTA, 1% Triton X-100), and 1× with cold solution II (10 mM Tris [pH 7.5], 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl).
T16952 31616-31900 Sentence denotes After washes, cell pellets were resuspended at 40 × 106 cells/ml in ChIP lysis buffer (150 mM NaCl, 25 mM Tris [pH 7.5], 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate plus protease and phosphatase inhibitors), and chromatin was sheared with a sonicator to yield 0.5–1-kb DNA fragments.
T192 31616-31900 Sentence denotes After washes, cell pellets were resuspended at 40 × 106 cells/ml in ChIP lysis buffer (150 mM NaCl, 25 mM Tris [pH 7.5], 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate plus protease and phosphatase inhibitors), and chromatin was sheared with a sonicator to yield 0.5–1-kb DNA fragments.
T16953 31901-32110 Sentence denotes After preclearing the sheared chromatin with protein A–sepharose beads and removing 5% as input chromatin, immunoprecipitation was performed by adding optimized antibody amounts (per 20 × 106 cell equivalents:
T193 31901-32110 Sentence denotes After preclearing the sheared chromatin with protein A–sepharose beads and removing 5% as input chromatin, immunoprecipitation was performed by adding optimized antibody amounts (per 20 × 106 cell equivalents:
T16954 32111-32278 Sentence denotes 2.5 μg anti-Eomes, 1:100 dilution anti-Runx3), followed by overnight incubation at 4°C; protein A–sepharose beads were added for the last 3 h of the incubation period.
T194 32111-32278 Sentence denotes 2.5 μg anti-Eomes, 1:100 dilution anti-Runx3), followed by overnight incubation at 4°C; protein A–sepharose beads were added for the last 3 h of the incubation period.
T16955 32279-32513 Sentence denotes Beads were washed 2× with RIPA buffer (50 mM Tris [pH 8], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% deoxycholate), 1× with high salt buffer (50 mM Tris [pH 8], 500 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS), and 1× with TE buffer.
T195 32279-32513 Sentence denotes Beads were washed 2× with RIPA buffer (50 mM Tris [pH 8], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% deoxycholate), 1× with high salt buffer (50 mM Tris [pH 8], 500 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS), and 1× with TE buffer.
T16956 32514-32618 Sentence denotes After the last wash, DNA was eluted by resuspending the beads in elution buffer (1% SDS, 100 mM NaHCO3).
T196 32514-32618 Sentence denotes After the last wash, DNA was eluted by resuspending the beads in elution buffer (1% SDS, 100 mM NaHCO3).
T16957 32619-32824 Sentence denotes Both input and ChIP chromatin were then treated with RNase A (5 μg total) for 1 h at 37°C, followed by the addition of proteinase K (100 μg total) and overnight incubation at 65°C to reverse cross-linking.
T197 32619-32824 Sentence denotes Both input and ChIP chromatin were then treated with RNase A (5 μg total) for 1 h at 37°C, followed by the addition of proteinase K (100 μg total) and overnight incubation at 65°C to reverse cross-linking.
T16958 32825-32977 Sentence denotes DNA was then purified with QIAquick columns (Gel Extraction Kit; QIAGEN) according to the manufacturer's instructions and resuspended in a 50-μl volume.
T198 32825-32977 Sentence denotes DNA was then purified with QIAquick columns (Gel Extraction Kit; QIAGEN) according to the manufacturer's instructions and resuspended in a 50-μl volume.
T16959 32978-33218 Sentence denotes For real-time PCR detection of immunoprecipitated targets using the SYBR Green PCR Kit, a standard curve was obtained with serial dilutions of input DNA for each sample, and 1 μl ChIP DNA was used per PCR reaction (performed in duplicates).
T199 32978-33218 Sentence denotes For real-time PCR detection of immunoprecipitated targets using the SYBR Green PCR Kit, a standard curve was obtained with serial dilutions of input DNA for each sample, and 1 μl ChIP DNA was used per PCR reaction (performed in duplicates).
T16960 33219-33315 Sentence denotes Melt curves and agarose gels were analyzed to ensure amplification of specific target sequences.
T200 33219-33315 Sentence denotes Melt curves and agarose gels were analyzed to ensure amplification of specific target sequences.
T16961 33316-33428 Sentence denotes Refer to Table S1 (available at http://www.jem.org/cgi/content/full/jem.20081242/DC1) for a list of primer sets.
T201 33316-33428 Sentence denotes Refer to Table S1 (available at http://www.jem.org/cgi/content/full/jem.20081242/DC1) for a list of primer sets.
T16962 33429-33611 Sentence denotes The data are presented as the number of immunoprecipitated target sequences relative to input chromatin, assuming two copies of target sequence per cell equivalent used for the ChIP.
T202 33429-33611 Sentence denotes The data are presented as the number of immunoprecipitated target sequences relative to input chromatin, assuming two copies of target sequence per cell equivalent used for the ChIP.
T17563 33613-33648 Sentence denotes Northern and Western blot analyses.
T203 33613-33648 Sentence denotes Northern and Western blot analyses.
T17564 33649-33733 Sentence denotes RNA isolation and Northern blot analysis was performed as previously described (29).
T204 33649-33733 Sentence denotes RNA isolation and Northern blot analysis was performed as previously described (29).
T17565 33734-33955 Sentence denotes In brief, 10 μg of total RNA was loaded per lane and transferred to positively charged nylon membranes (Hybond-N+; GE Healthcare), which was confirmed by ethidium bromide staining of ribosomal RNA species on the membrane.
T205 33734-33955 Sentence denotes In brief, 10 μg of total RNA was loaded per lane and transferred to positively charged nylon membranes (Hybond-N+; GE Healthcare), which was confirmed by ethidium bromide staining of ribosomal RNA species on the membrane.
T17566 33956-34120 Sentence denotes Membranes were hybridized with 1 ng/ml α-[32P]dCTP–labeled trichloroacetic acid precipitable probe in ExpressHyb hybridization buffer (Clontech Laboratories, Inc.).
T206 33956-34120 Sentence denotes Membranes were hybridized with 1 ng/ml α-[32P]dCTP–labeled trichloroacetic acid precipitable probe in ExpressHyb hybridization buffer (Clontech Laboratories, Inc.).
T17567 34121-34260 Sentence denotes All cDNA probes were confirmed to have the appropriate single-copy specificity under these conditions using genomic Southern blot analysis.
T207 34121-34260 Sentence denotes All cDNA probes were confirmed to have the appropriate single-copy specificity under these conditions using genomic Southern blot analysis.
T17568 34261-34320 Sentence denotes Band intensities were acquired by phosphorimaging analysis.
T208 34261-34320 Sentence denotes Band intensities were acquired by phosphorimaging analysis.
T17569 34321-34694 Sentence denotes For Western analysis, whole-cell protein lysates were obtained from CD8+ T cells at the time points indicated in the figures during clonal expansion in 100 U/ml IL-2 with lysis buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 10% glycerol, 5 mM EDTA, 1% NP-40) by resuspending samples in 10 μl per 106 cells and incubating on ice for 30 min in the presence of protease inhibitors.
T209 34321-34694 Sentence denotes For Western analysis, whole-cell protein lysates were obtained from CD8+ T cells at the time points indicated in the figures during clonal expansion in 100 U/ml IL-2 with lysis buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 10% glycerol, 5 mM EDTA, 1% NP-40) by resuspending samples in 10 μl per 106 cells and incubating on ice for 30 min in the presence of protease inhibitors.
T17570 34695-34837 Sentence denotes Immunoblot analysis was performed with the antibodies indicated in the figures after SDS-PAGE (10–30 μg of total protein was loaded per well).
T210 34695-34837 Sentence denotes Immunoblot analysis was performed with the antibodies indicated in the figures after SDS-PAGE (10–30 μg of total protein was loaded per well).
T17571 34838-35021 Sentence denotes Quantification of detected protein was performed with an Intelligent Dark Box unit (LAS-3000; Fujifilm) and normalized for loading with the amount of RNA Pol-II detected in each lane.
T211 34838-35021 Sentence denotes Quantification of detected protein was performed with an Intelligent Dark Box unit (LAS-3000; Fujifilm) and normalized for loading with the amount of RNA Pol-II detected in each lane.
T18121 35023-35052 Sentence denotes Online supplemental material.
T212 35023-35052 Sentence denotes Online supplemental material.
T18122 35053-35134 Sentence denotes Fig. S1 shows the characterization of peripheral CD8+ T cells from Runx3−/− mice.
T213 35053-35134 Sentence denotes Fig. S1 shows the characterization of peripheral CD8+ T cells from Runx3−/− mice.
T18123 35135-35231 Sentence denotes Fig. S2 shows effector protein expression by Runx3 WT and KO cells at day 4 of in vitro culture.
T214 35135-35231 Sentence denotes Fig. S2 shows effector protein expression by Runx3 WT and KO cells at day 4 of in vitro culture.
T18124 35232-35297 Sentence denotes Primer sequences used for ChIP experiments are shown in Table S1.
T215 35232-35297 Sentence denotes Primer sequences used for ChIP experiments are shown in Table S1.
T18125 35298-35396 Sentence denotes Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20081242/DC1.
T216 35298-35396 Sentence denotes Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20081242/DC1.
T217 35398-35420 Sentence denotes Supplementary Material
T218 35421-35450 Sentence denotes [Supplemental Material Index]