> top > projects > sentences > docs > PMC:1447668 > annotations
sentences Home  

PMC:1447668 JSONTXT 23 Projects

Annnotations TAB TSV DIC JSON TextAE

Id Subject Object Predicate Lexical cue
T113 0-82 Sentence denotes Foxp3 Represses Retroviral Transcription by Targeting Both NF-κB and CREB Pathways
T1 0-123 Sentence denotes Foxp3 Represses Retroviral Transcription by Targeting Both NF-κB and CREB Pathways Foxp3 Suppresses NF-κB and CREB Pathways
T2 126-134 Sentence denotes Abstract
T114 135-320 Sentence denotes Forkhead box (Fox)/winged-helix transcription factors regulate multiple aspects of immune responsiveness and Foxp3 is recognized as an essential functional marker of regulatory T cells.
T3 135-320 Sentence denotes Forkhead box (Fox)/winged-helix transcription factors regulate multiple aspects of immune responsiveness and Foxp3 is recognized as an essential functional marker of regulatory T cells.
T115 321-439 Sentence denotes Herein we describe downstream signaling pathways targeted by Foxp3 that may negatively impact retroviral pathogenesis.
T4 321-439 Sentence denotes Herein we describe downstream signaling pathways targeted by Foxp3 that may negatively impact retroviral pathogenesis.
T116 440-615 Sentence denotes Overexpression of Foxp3 in HEK 293T and purified CD4+ T cells resulted in a dose-dependent and time-dependent decrease in basal levels of nuclear factor-κB (NF-κB) activation.
T5 440-615 Sentence denotes Overexpression of Foxp3 in HEK 293T and purified CD4+ T cells resulted in a dose-dependent and time-dependent decrease in basal levels of nuclear factor-κB (NF-κB) activation.
T117 616-858 Sentence denotes Deletion of the carboxyl-terminal forkhead (FKH) domain, critical for nuclear localization and DNA-binding activity, abrogated the ability of Foxp3 to suppress NF-κB activity in HEK 293T cells, but not in Jurkat or primary human CD4+ T cells.
T6 616-858 Sentence denotes Deletion of the carboxyl-terminal forkhead (FKH) domain, critical for nuclear localization and DNA-binding activity, abrogated the ability of Foxp3 to suppress NF-κB activity in HEK 293T cells, but not in Jurkat or primary human CD4+ T cells.
T118 859-1075 Sentence denotes We further demonstrate that Foxp3 suppressed the transcription of two human retroviral promoters (HIV-1 and human T cell lymphotropic virus type I [HTLV-I]) utilizing NF-κB-dependent and NF-κB-independent mechanisms.
T7 859-1075 Sentence denotes We further demonstrate that Foxp3 suppressed the transcription of two human retroviral promoters (HIV-1 and human T cell lymphotropic virus type I [HTLV-I]) utilizing NF-κB-dependent and NF-κB-independent mechanisms.
T119 1076-1193 Sentence denotes Examination of the latter identified the cAMP-responsive element binding protein (CREB) pathway as a target of Foxp3.
T8 1076-1193 Sentence denotes Examination of the latter identified the cAMP-responsive element binding protein (CREB) pathway as a target of Foxp3.
T120 1194-1495 Sentence denotes Finally, comparison of the percent Foxp3+CD4+CD25+ T cells to the HTLV-I proviral load in HTLV-I-infected asymptomatic carriers and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis suggested that high Foxp3 expression is associated with low proviral load and absence of disease.
T9 1194-1495 Sentence denotes Finally, comparison of the percent Foxp3+CD4+CD25+ T cells to the HTLV-I proviral load in HTLV-I-infected asymptomatic carriers and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis suggested that high Foxp3 expression is associated with low proviral load and absence of disease.
T121 1496-1620 Sentence denotes These results suggest an expanded role for Foxp3 in regulating NF-κB- and CREB-dependent cellular and viral gene expression.
T10 1496-1620 Sentence denotes These results suggest an expanded role for Foxp3 in regulating NF-κB- and CREB-dependent cellular and viral gene expression.
T11 1622-1630 Sentence denotes Synopsis
T12 1631-1820 Sentence denotes Over the past several years, mounting evidence has shown that immune tolerance in healthy individuals can be maintained by a population of T lymphocytes known as regulatory T cells (Tregs).
T13 1821-1960 Sentence denotes As a component of this system, a protein known as Foxp3 has been shown to be absolutely required for the development and function of Tregs.
T14 1961-2219 Sentence denotes While Foxp3 plays an important role in maintaining immune tolerance by blocking T cell proliferation and production of inflammatory proteins known as cytokines, little is known about the molecular mechanisms that are used by Foxp3 to accomplish these events.
T15 2220-2489 Sentence denotes The present study expands our understanding of how Foxp3 maintains a check on inappropriate immune responses by demonstrating that Foxp3 can block activation of key inducible proteins such as nuclear factor κB (NF-κB) and cAMP-responsive element binding protein (CREB).
T16 2490-2988 Sentence denotes Since NF-κB and CREB are integrally involved in controlling cell cycle progression, inflammatory cytokine production, and the replication of numerous viruses at the level of transcription, understanding the mechanisms by which Foxp3 functions to regulate cellular and viral gene expression may aid in the discovery of therapeutic approaches designed to rescue the expression and/or function of Foxp3, which have been found to be deficient in several autoimmune diseases and virus-induced disorders.
T17 2990-3002 Sentence denotes Introduction
T1143 3003-3260 Sentence denotes Immunological tolerance to self-antigens is the result of the deletion of self-reactive T lymphocytes in the thymus (central tolerance) and suppression of the activation of potentially self-reactive T lymphocytes in the periphery (peripheral tolerance) [1].
T18 3003-3260 Sentence denotes Immunological tolerance to self-antigens is the result of the deletion of self-reactive T lymphocytes in the thymus (central tolerance) and suppression of the activation of potentially self-reactive T lymphocytes in the periphery (peripheral tolerance) [1].
T1144 3261-3380 Sentence denotes Suppression of pathogenic T cell responses is mediated by naturally arising CD4+CD25+ T regulatory cells (Tregs) [2,3].
T19 3261-3380 Sentence denotes Suppression of pathogenic T cell responses is mediated by naturally arising CD4+CD25+ T regulatory cells (Tregs) [2,3].
T1145 3381-3571 Sentence denotes Deficiencies in Treg development and function have been linked to the severe autoimmune disorder known as immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) [4].
T20 3381-3571 Sentence denotes Deficiencies in Treg development and function have been linked to the severe autoimmune disorder known as immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) [4].
T1146 3572-4025 Sentence denotes In addition, recent studies have provided strong evidence that dysregulation of Treg development and/or function may be a significant factor in the pathogenesis of several autoimmune disorders (e.g., multiple sclerosis [5], myasthenia gravis [6], and type 1 diabetes [7]) and virus-induced immunologic disorders (e.g., human T lymphotropic virus type I [HTLV-I]-associated myelopathy/tropical spastic paraparesis [HAM/TSP], and HIV-induced AIDS [8–10]).
T21 3572-4025 Sentence denotes In addition, recent studies have provided strong evidence that dysregulation of Treg development and/or function may be a significant factor in the pathogenesis of several autoimmune disorders (e.g., multiple sclerosis [5], myasthenia gravis [6], and type 1 diabetes [7]) and virus-induced immunologic disorders (e.g., human T lymphotropic virus type I [HTLV-I]-associated myelopathy/tropical spastic paraparesis [HAM/TSP], and HIV-induced AIDS [8–10]).
T1147 4026-4244 Sentence denotes The transcription factor Foxp3 is a 431-amino acid (48-kDa) protein expressed at very high levels in CD4+CD25hi T cells and has previously been shown to be absolutely critical for Treg development and function [11–14].
T22 4026-4244 Sentence denotes The transcription factor Foxp3 is a 431-amino acid (48-kDa) protein expressed at very high levels in CD4+CD25hi T cells and has previously been shown to be absolutely critical for Treg development and function [11–14].
T1148 4245-4643 Sentence denotes Foxp3 contains a proline-rich amino-terminal domain reported to function as a nuclear factor of activated T cells (NF-AT) and nuclear factor-κB (NF-κB) binding domain, a central region containing a zinc finger and leucine zipper potentially important for protein-protein interactions, and a carboxyl-terminal forkhead (FKH) domain required for nuclear localization and DNA-binding activity [14–16].
T23 4245-4643 Sentence denotes Foxp3 contains a proline-rich amino-terminal domain reported to function as a nuclear factor of activated T cells (NF-AT) and nuclear factor-κB (NF-κB) binding domain, a central region containing a zinc finger and leucine zipper potentially important for protein-protein interactions, and a carboxyl-terminal forkhead (FKH) domain required for nuclear localization and DNA-binding activity [14–16].
T1149 4644-4959 Sentence denotes Functional inactivation of Foxp3 by genetic mutations affecting the Foxp3 coding region, as demonstrated in IPEX, or repression of Foxp3 expression by the HTLV-I-encoded transactivator protein Tax, as recently reported in patients with HAM/TSP, results in loss of regulatory activity in CD4+CD25hi T cells [4,8,17].
T24 4644-4959 Sentence denotes Functional inactivation of Foxp3 by genetic mutations affecting the Foxp3 coding region, as demonstrated in IPEX, or repression of Foxp3 expression by the HTLV-I-encoded transactivator protein Tax, as recently reported in patients with HAM/TSP, results in loss of regulatory activity in CD4+CD25hi T cells [4,8,17].
T1150 4960-5123 Sentence denotes Although it is clear that Foxp3 regulates T cell proliferation and cytokine production, very little is known concerning the molecular mechanisms of Foxp3 function.
T25 4960-5123 Sentence denotes Although it is clear that Foxp3 regulates T cell proliferation and cytokine production, very little is known concerning the molecular mechanisms of Foxp3 function.
T1151 5124-5528 Sentence denotes The first evidence to indicate how Foxp3 promotes the development and function of regulatory T cells came from a report by Ziegler and colleagues [16], which suggested that Foxp3 could inhibit transcriptional activation by physically interacting with forkhead binding sites located immediately adjacent to critical cis-acting NF-AT binding sites found in various cytokine promoters (e.g., IL-2 promoter).
T26 5124-5528 Sentence denotes The first evidence to indicate how Foxp3 promotes the development and function of regulatory T cells came from a report by Ziegler and colleagues [16], which suggested that Foxp3 could inhibit transcriptional activation by physically interacting with forkhead binding sites located immediately adjacent to critical cis-acting NF-AT binding sites found in various cytokine promoters (e.g., IL-2 promoter).
T1152 5529-5704 Sentence denotes That study also demonstrated that Foxp3 could repress activation of a synthetic reporter vector containing an SV40 promoter and three tandem copies of a forkhead binding site.
T27 5529-5704 Sentence denotes That study also demonstrated that Foxp3 could repress activation of a synthetic reporter vector containing an SV40 promoter and three tandem copies of a forkhead binding site.
T1153 5705-5898 Sentence denotes These results provided additional evidence suggesting that Foxp3 transcriptional repression was mediated by binding in a sequence-specific manner to promoters containing forkhead binding sites.
T28 5705-5898 Sentence denotes These results provided additional evidence suggesting that Foxp3 transcriptional repression was mediated by binding in a sequence-specific manner to promoters containing forkhead binding sites.
T1154 5899-6249 Sentence denotes A recent study by Bettelli and colleagues [15] further demonstrated that Foxp3 could inhibit NF-AT as well as NF-κB activation, although the mechanism of suppression was shown to involve direct protein-protein interactions between NF-AT or NF-κB and Foxp3 rather than binding of Foxp3 to promoter elements adjacent to cis-acting NF-AT or NF-κB sites.
T29 5899-6249 Sentence denotes A recent study by Bettelli and colleagues [15] further demonstrated that Foxp3 could inhibit NF-AT as well as NF-κB activation, although the mechanism of suppression was shown to involve direct protein-protein interactions between NF-AT or NF-κB and Foxp3 rather than binding of Foxp3 to promoter elements adjacent to cis-acting NF-AT or NF-κB sites.
T1155 6250-6428 Sentence denotes Collectively, these data suggested that Foxp3 may function as a transcriptional repressor, potentially through the formation of both DNA-protein and protein-protein interactions.
T30 6250-6428 Sentence denotes Collectively, these data suggested that Foxp3 may function as a transcriptional repressor, potentially through the formation of both DNA-protein and protein-protein interactions.
T1156 6429-6749 Sentence denotes In the present study, we expanded upon these observations by defining additional requirements of Foxp3-mediated repression of NF-κB activation, and investigated whether Foxp3 could target additional signaling pathways by examining transcriptional activation of NF-κB-dependent and NF-κB-independent retroviral pathogens.
T31 6429-6749 Sentence denotes In the present study, we expanded upon these observations by defining additional requirements of Foxp3-mediated repression of NF-κB activation, and investigated whether Foxp3 could target additional signaling pathways by examining transcriptional activation of NF-κB-dependent and NF-κB-independent retroviral pathogens.
T1157 6750-6986 Sentence denotes The characterization of the molecular targets of Foxp3 and the mechanism(s) utilized by Foxp3 to support Treg development and function will aid in our understanding of the role Tregs play in the pathogenesis of human autoimmune disease.
T32 6750-6986 Sentence denotes The characterization of the molecular targets of Foxp3 and the mechanism(s) utilized by Foxp3 to support Treg development and function will aid in our understanding of the role Tregs play in the pathogenesis of human autoimmune disease.
T33 6988-6995 Sentence denotes Results
T3009 6997-7056 Sentence denotes Foxp3 Suppresses NF-κB Dependent Transcriptional Activation
T34 6997-7056 Sentence denotes Foxp3 Suppresses NF-κB Dependent Transcriptional Activation
T3010 7057-7385 Sentence denotes To ascertain the molecular mechanisms by which Foxp3 functions to promote the regulatory function of CD4+CD25hi T cells, we first confirmed the function of Foxp3 as a repressor of activation of NF-κB, previously implicated as a target of other forkhead/winged-helix family transcription factors (e.g., Foxj1 and Foxo3a) [19,20].
T35 7057-7385 Sentence denotes To ascertain the molecular mechanisms by which Foxp3 functions to promote the regulatory function of CD4+CD25hi T cells, we first confirmed the function of Foxp3 as a repressor of activation of NF-κB, previously implicated as a target of other forkhead/winged-helix family transcription factors (e.g., Foxj1 and Foxo3a) [19,20].
T3011 7386-7513 Sentence denotes We analyzed the effect of Foxp3 overexpression on NF-κB activation in HEK 293T cells in dose-response and time course analyses.
T36 7386-7513 Sentence denotes We analyzed the effect of Foxp3 overexpression on NF-κB activation in HEK 293T cells in dose-response and time course analyses.
T3012 7514-7845 Sentence denotes Transfection of HEK 293T cells with an NF-κB luciferase reporter vector in the presence or absence of increasing concentrations of a Foxp3 expression vector or a control vector (enhanced green fluorescent protein [EGFP]) was performed, and cells were harvested after 24 h to assay for luciferase activity and Foxp3 mRNA expression.
T37 7514-7845 Sentence denotes Transfection of HEK 293T cells with an NF-κB luciferase reporter vector in the presence or absence of increasing concentrations of a Foxp3 expression vector or a control vector (enhanced green fluorescent protein [EGFP]) was performed, and cells were harvested after 24 h to assay for luciferase activity and Foxp3 mRNA expression.
T3013 7846-8022 Sentence denotes Results indicated that as the concentration of Foxp3 transfected into cells increases (from 50 to 2,400 ng), the level of NF-κB activation decreases proportionally (Figure 1A).
T38 7846-8022 Sentence denotes Results indicated that as the concentration of Foxp3 transfected into cells increases (from 50 to 2,400 ng), the level of NF-κB activation decreases proportionally (Figure 1A).
T3014 8023-8114 Sentence denotes Foxp3 mRNA was also assayed to monitor activity of the Foxp3 expression vector (Figure 1B).
T39 8023-8114 Sentence denotes Foxp3 mRNA was also assayed to monitor activity of the Foxp3 expression vector (Figure 1B).
T3015 8115-8351 Sentence denotes Since NF-κB activation was partially affected by transfection of high concentrations of the control vector, we determined the fold inhibition of NF-κB activation by Foxp3 compared to the control vector at each concentration (Figure 1A).
T40 8115-8351 Sentence denotes Since NF-κB activation was partially affected by transfection of high concentrations of the control vector, we determined the fold inhibition of NF-κB activation by Foxp3 compared to the control vector at each concentration (Figure 1A).
T3016 8352-8481 Sentence denotes Fold inhibition of NF-κB activation was directly proportional to the level of Foxp3 mRNA expression detected by real-time RT-PCR.
T41 8352-8481 Sentence denotes Fold inhibition of NF-κB activation was directly proportional to the level of Foxp3 mRNA expression detected by real-time RT-PCR.
T3017 8482-8727 Sentence denotes To determine the level of Foxp3-mediated suppression of NF-κB activation over time, HEK 293T cells were transfected with an NF-κB luciferase reporter vector and an expression vector encoding Foxp3 or EGFP (control vector) and harvested over 4 d.
T42 8482-8727 Sentence denotes To determine the level of Foxp3-mediated suppression of NF-κB activation over time, HEK 293T cells were transfected with an NF-κB luciferase reporter vector and an expression vector encoding Foxp3 or EGFP (control vector) and harvested over 4 d.
T3018 8728-8829 Sentence denotes As shown in Figure 1C, NF-κB activation was suppressed by overexpression of Foxp3 at all time points.
T43 8728-8829 Sentence denotes As shown in Figure 1C, NF-κB activation was suppressed by overexpression of Foxp3 at all time points.
T3019 8830-9076 Sentence denotes Extending these results from established, in vitro HEK cell lines to primary human lymphocytes, overexpression of Foxp3 in purified CD4+ T cells from three healthy donors also down-regulated the steady-state level of NF-κB activation (Figure 1D).
T44 8830-9076 Sentence denotes Extending these results from established, in vitro HEK cell lines to primary human lymphocytes, overexpression of Foxp3 in purified CD4+ T cells from three healthy donors also down-regulated the steady-state level of NF-κB activation (Figure 1D).
T3020 9077-9289 Sentence denotes These results recapitulate those from Bettelli and colleagues [15] demonstrating that Foxp3 functions, in part, to block NF-κB-dependent transcription in human cell lines as well as in primary human CD4+ T cells.
T45 9077-9289 Sentence denotes These results recapitulate those from Bettelli and colleagues [15] demonstrating that Foxp3 functions, in part, to block NF-κB-dependent transcription in human cell lines as well as in primary human CD4+ T cells.
T46 9290-9346 Sentence denotes Figure 1 Foxp3 Suppresses NF-κB-Dependent Transcription
T47 9347-9649 Sentence denotes (A and B) HEK 293T cells (5 × 105) were seeded into six-well plates 1 d prior to transfection with an NF-κB luciferase reporter vector (200 ng) in the presence or absence of indicated concentrations of a Foxp3 expression vector or a control vector and internal reference plasmid (pGL4-TKhRluc2; 50 ng).
T48 9650-9764 Sentence denotes Cells were harvested 24 h posttransfection and analyzed for luciferase activity (A) and Foxp3 mRNA expression (B).
T49 9765-10044 Sentence denotes (C) HEK 293T cells (5 × 105) were seeded into six-well plates 1 d prior to transfection with an NF-κB luciferase reporter vector (200 ng) in the presence or absence of a Foxp3 expression vector or a control vector (1,500 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng).
T50 10045-10144 Sentence denotes Cells were harvested at 24, 48, 72, and 96 h posttransfection and analyzed for luciferase activity.
T51 10145-10361 Sentence denotes (D) CD4+ T cells (2 × 106) from three healthy donors were nucleofected with an NF-κB luciferase reporter vector (1,000 ng) and Foxp3 or control expression vectors (2,000 ng) and internal reference plasmid (1,000 ng).
T52 10362-10442 Sentence denotes Cells were harvested 24 h posttransfection and analyzed for luciferase activity.
T53 10443-10542 Sentence denotes Relative luciferase activity shown in A, C, and D was normalized to the internal reference control.
T4306 10544-10639 Sentence denotes The Carboxyl-Terminal FKH Domain Is Not Required for Suppression of NF-κB Activation in T Cells
T54 10544-10639 Sentence denotes The Carboxyl-Terminal FKH Domain Is Not Required for Suppression of NF-κB Activation in T Cells
T4307 10640-10917 Sentence denotes To define the requirements of Foxp3 with respect to inhibition of NF-κB-dependent transcription, we utilized a mutant of Foxp3 lacking the FKH domain (Figure 2A) [16], similar to the scurfy mutant Foxp3 of mice, and a mutant Foxp3 protein from a patient with IPEX [4,11,14,17].
T55 10640-10917 Sentence denotes To define the requirements of Foxp3 with respect to inhibition of NF-κB-dependent transcription, we utilized a mutant of Foxp3 lacking the FKH domain (Figure 2A) [16], similar to the scurfy mutant Foxp3 of mice, and a mutant Foxp3 protein from a patient with IPEX [4,11,14,17].
T4308 10918-11186 Sentence denotes Unlike full-length Foxp3, which localizes almost exclusively to the nucleus and can bind in a sequence-specific manner to forkhead binding sites, the ΔFKH mutant fails to localize to the nucleus and thus cannot interact with promoter elements or nuclear proteins [16].
T56 10918-11186 Sentence denotes Unlike full-length Foxp3, which localizes almost exclusively to the nucleus and can bind in a sequence-specific manner to forkhead binding sites, the ΔFKH mutant fails to localize to the nucleus and thus cannot interact with promoter elements or nuclear proteins [16].
T4309 11187-11382 Sentence denotes Therefore, we utilized the ΔFKH mutant to determine whether nuclear localization (or other function associated with the FKH domain) of Foxp3 was a prerequisite for inhibition of NF-κB activation.
T57 11187-11382 Sentence denotes Therefore, we utilized the ΔFKH mutant to determine whether nuclear localization (or other function associated with the FKH domain) of Foxp3 was a prerequisite for inhibition of NF-κB activation.
T4310 11383-11623 Sentence denotes Although Foxp3 interaction with NF-κB presumably takes place in the nucleus, it may also be possible for a cytoplasmic Foxp3 protein to bind to NF-κB in the cytoplasm and prevent localization to the nucleus following an activation stimulus.
T58 11383-11623 Sentence denotes Although Foxp3 interaction with NF-κB presumably takes place in the nucleus, it may also be possible for a cytoplasmic Foxp3 protein to bind to NF-κB in the cytoplasm and prevent localization to the nucleus following an activation stimulus.
T4311 11624-11781 Sentence denotes Overexpression of full-length Foxp3, but not of ΔFKH, was able to suppress activation of a cotransfected NF-κB reporter vector in HEK 293T cells (Figure 2B).
T59 11624-11781 Sentence denotes Overexpression of full-length Foxp3, but not of ΔFKH, was able to suppress activation of a cotransfected NF-κB reporter vector in HEK 293T cells (Figure 2B).
T4312 11782-11911 Sentence denotes Both Foxp3 and ΔFKH were expressed at very high levels following transfection as detected by real-time RT-PCR (unpublished data).
T60 11782-11911 Sentence denotes Both Foxp3 and ΔFKH were expressed at very high levels following transfection as detected by real-time RT-PCR (unpublished data).
T4313 11912-12060 Sentence denotes These data appear to suggest that the carboxyl-terminal FKH domain is critically important for Foxp3 to down-regulate NF-κB-dependent transcription.
T61 11912-12060 Sentence denotes These data appear to suggest that the carboxyl-terminal FKH domain is critically important for Foxp3 to down-regulate NF-κB-dependent transcription.
T4314 12061-12227 Sentence denotes However, NF-κB activation was blocked to a similar extent by both full-length Foxp3 and ΔFKH in Jurkat T cells (Figure 2C) and primary human CD4+ T cells (Figure 2D).
T62 12061-12227 Sentence denotes However, NF-κB activation was blocked to a similar extent by both full-length Foxp3 and ΔFKH in Jurkat T cells (Figure 2C) and primary human CD4+ T cells (Figure 2D).
T4315 12228-12390 Sentence denotes Western blot analysis of NF-κB p65 expression demonstrated that Foxp3 and ΔFKH does not block NF-κB activation at the level of p65 protein expression (Figure 2E).
T63 12228-12390 Sentence denotes Western blot analysis of NF-κB p65 expression demonstrated that Foxp3 and ΔFKH does not block NF-κB activation at the level of p65 protein expression (Figure 2E).
T4316 12391-12610 Sentence denotes These results are very interesting with respect to Foxp3 function, because they suggest that the carboxyl-terminal FKH domain, and possibly nuclear localization, are dispensable for Foxp3 function in T cell populations.
T64 12391-12610 Sentence denotes These results are very interesting with respect to Foxp3 function, because they suggest that the carboxyl-terminal FKH domain, and possibly nuclear localization, are dispensable for Foxp3 function in T cell populations.
T4317 12611-12741 Sentence denotes Alternative interpretations may include the possibility that the localization of ΔFKH differ between epithelial cells and T cells.
T65 12611-12741 Sentence denotes Alternative interpretations may include the possibility that the localization of ΔFKH differ between epithelial cells and T cells.
T4318 12742-12843 Sentence denotes In either case, these results suggest a cell type-specific mechanism of action for this Foxp3 mutant.
T66 12742-12843 Sentence denotes In either case, these results suggest a cell type-specific mechanism of action for this Foxp3 mutant.
T67 12844-12952 Sentence denotes Figure 2 FKH Domain of Foxp3 Is Required to Inhibit NF-κB Activation in HEK 293T Cells but Not CD4+ T Cells
T68 12953-13098 Sentence denotes (A) Schematic representation of human Foxp3, including proline-rich (P-P-P), zinc finger (ZNF), leucine zipper (ZIP), and forkhead (FKH) domains.
T69 13099-13193 Sentence denotes Mutations within the Foxp3 gene associated with IPEX have been previously described [4,14,43].
T70 13194-13309 Sentence denotes The FKH domain contains a nuclear localization signal (NLS), which is required for Foxp3 expression in the nucleus.
T71 13310-13689 Sentence denotes (B and C) HEK 293T cells (5 × 105) (B) were seeded into six-well plates 1 d prior to transfection or Jurkat T cells (1 × 106) (C) were seeded into six-well plates on the day of transfection with an NF-κB luciferase reporter vector (200 ng) in the presence or absence of a Foxp3, ΔFKH, or control expression vector (1,000 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng).
T72 13690-13773 Sentence denotes Cells were harvested at 24 h posttransfection and analyzed for luciferase activity.
T73 13774-13852 Sentence denotes Relative luciferase activity was normalized to the internal reference control.
T74 13853-14083 Sentence denotes (D) CD4+ T cells (2 × 106) from healthy donors were nucleofected with an NF-κB reporter plasmid (2,000 ng), together with either a Foxp3, ΔFKH, or control expression vector (2,000 ng), and an internal reference plasmid (1,000 ng).
T75 14084-14164 Sentence denotes Cells were harvested 24 h posttransfection and analyzed for luciferase activity.
T76 14165-14243 Sentence denotes Relative luciferase activity was normalized to the internal reference control.
T77 14244-14285 Sentence denotes Results from one healthy donor are shown.
T78 14286-14567 Sentence denotes (E) Foxp3, ΔFKH, and NF-κB p65 expression in whole-cell extracts (20 μg) derived from HEK 293T cells transfected with a control expression vector (EGFP), Foxp3, or ΔFKH expression vectors were analyzed by Western blot analysis. β-actin expression was analyzed as a loading control.
T5628 14569-14652 Sentence denotes Foxp3 Suppresses HIV-1 Gene Expression in Part through Blocking Activation of NF-κB
T79 14569-14652 Sentence denotes Foxp3 Suppresses HIV-1 Gene Expression in Part through Blocking Activation of NF-κB
T5629 14653-14874 Sentence denotes If Foxp3 functions as a repressor of NF-κB-dependent gene expression, then we hypothesized that Foxp3 overexpression could selectively down-regulate transcription from promoters previously shown to be responsive to NF-κB.
T80 14653-14874 Sentence denotes If Foxp3 functions as a repressor of NF-κB-dependent gene expression, then we hypothesized that Foxp3 overexpression could selectively down-regulate transcription from promoters previously shown to be responsive to NF-κB.
T5630 14875-15113 Sentence denotes To address this question, we examined the transcriptional activation of the HIV-1 LTR, which contains two tandem cis-acting NF-κB binding sites located between positions −102 and −81 with respect to the transcription initiation site [21].
T81 14875-15113 Sentence denotes To address this question, we examined the transcriptional activation of the HIV-1 LTR, which contains two tandem cis-acting NF-κB binding sites located between positions −102 and −81 with respect to the transcription initiation site [21].
T5631 15114-15220 Sentence denotes NF-κB plays a crucial role in regulating gene expression directed from the HIV-1 LTR in CD4+ T cells [21].
T82 15114-15220 Sentence denotes NF-κB plays a crucial role in regulating gene expression directed from the HIV-1 LTR in CD4+ T cells [21].
T5632 15221-15454 Sentence denotes Overexpression of full-length Foxp3, but not ΔFKH, in HEK 293T cells was able to inhibit basal activation of the HIV-1 LTR (Figure 3A), similar to what was previously demonstrated with the synthetic NF-κB reporter vector (Figure 2B).
T83 15221-15454 Sentence denotes Overexpression of full-length Foxp3, but not ΔFKH, in HEK 293T cells was able to inhibit basal activation of the HIV-1 LTR (Figure 3A), similar to what was previously demonstrated with the synthetic NF-κB reporter vector (Figure 2B).
T5633 15455-15564 Sentence denotes Furthermore, HIV-1 LTR activation was suppressed by full-length Foxp3 and ΔFKH in Jurkat T cells (Figure 3B).
T84 15455-15564 Sentence denotes Furthermore, HIV-1 LTR activation was suppressed by full-length Foxp3 and ΔFKH in Jurkat T cells (Figure 3B).
T5634 15565-15843 Sentence denotes To demonstrate that Foxp3-mediated HIV-1 LTR repression was associated with interactions with NF-κB bound to the HIV-1 LTR, we compared basal activation of the HIV-1 LTR or an identical HIV-1 LTR lacking the NF-κB sites located between −102 and −81 (HIV-1 Δ-κB LTR) (Figure 3C).
T85 15565-15843 Sentence denotes To demonstrate that Foxp3-mediated HIV-1 LTR repression was associated with interactions with NF-κB bound to the HIV-1 LTR, we compared basal activation of the HIV-1 LTR or an identical HIV-1 LTR lacking the NF-κB sites located between −102 and −81 (HIV-1 Δ-κB LTR) (Figure 3C).
T5635 15844-16011 Sentence denotes This mutant HIV-1 LTR construct exhibited reduced levels of transcription compared to the parental HIV-1 LTR in purified healthy donor CD4+ T cells (unpublished data).
T86 15844-16011 Sentence denotes This mutant HIV-1 LTR construct exhibited reduced levels of transcription compared to the parental HIV-1 LTR in purified healthy donor CD4+ T cells (unpublished data).
T5636 16012-16280 Sentence denotes However, directly comparing the effect of Foxp3 overexpression on the activation of these two viral promoters demonstrated that Foxp3 was more capable of suppressing transcriptional activation of the HIV-1 LTR (Figure 3D) compared to the mutated HIV-1 LTR (Figure 3E).
T87 16012-16280 Sentence denotes However, directly comparing the effect of Foxp3 overexpression on the activation of these two viral promoters demonstrated that Foxp3 was more capable of suppressing transcriptional activation of the HIV-1 LTR (Figure 3D) compared to the mutated HIV-1 LTR (Figure 3E).
T5637 16281-16418 Sentence denotes These results suggest that Foxp3 down-regulation of HIV-1 LTR activation was mediated at least in part by cis-acting NF-κB binding sites.
T88 16281-16418 Sentence denotes These results suggest that Foxp3 down-regulation of HIV-1 LTR activation was mediated at least in part by cis-acting NF-κB binding sites.
T5638 16419-16579 Sentence denotes Residual levels of inhibition of the HIV-1 Δ-κB LTR by Foxp3 may be due to NF-AT binding sites located upstream of the NF-κB sites within the HIV-1 LTR [22,23].
T89 16419-16579 Sentence denotes Residual levels of inhibition of the HIV-1 Δ-κB LTR by Foxp3 may be due to NF-AT binding sites located upstream of the NF-κB sites within the HIV-1 LTR [22,23].
T90 16580-16638 Sentence denotes Figure 3 Foxp3 Inhibits Basal Activation of the HIV-1 LTR
T91 16639-17022 Sentence denotes (A and B) HEK 293T cells (5 × 105) (A) were seeded into six-well plates 1 d prior to transfection or Jurkat T cells (1 × 106) (B) were seeded into six-well plates on the day of transfection with an HIV-1 LTR luciferase reporter vector (200 ng) in the presence or absence of a Foxp3, ΔFKH, or control expression vector (1,000 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng).
T92 17023-17106 Sentence denotes Cells were harvested at 24 h posttransfection and analyzed for luciferase activity.
T93 17107-17219 Sentence denotes (C) Schematic representation of HIV-1 LTR luciferase reporter constructs pHIV-1 LTR-luc and pHIV-1 Δ-κB LTR-luc.
T94 17220-17497 Sentence denotes (D and E) CD4+ T cells (2 × 106) from two healthy donors were nucleofected with either parental HIV-1 LTR (D) or HIV-1 LTR lacking NF-κB binding sites (at −102 to −81) (E) (1,000 ng), Foxp3 or control expression vectors (2,000 ng), and an internal reference plasmid (1,000 ng).
T95 17498-17578 Sentence denotes Cells were harvested 24 h posttransfection and analyzed for luciferase activity.
T96 17579-17681 Sentence denotes Relative luciferase activity shown in A, B, D, and E was normalized to the internal reference control.
T6788 17683-17750 Sentence denotes The Transactivation Functions of HTLV-I Tax Are Suppressed by Foxp3
T97 17683-17750 Sentence denotes The Transactivation Functions of HTLV-I Tax Are Suppressed by Foxp3
T6789 17751-17946 Sentence denotes Previous studies by Bettelli and colleagues have demonstrated that Foxp3 can repress both the basal levels of NF-κB activation as well as tumor necrosis factor-α-stimulated NF-κB activation [15].
T98 17751-17946 Sentence denotes Previous studies by Bettelli and colleagues have demonstrated that Foxp3 can repress both the basal levels of NF-κB activation as well as tumor necrosis factor-α-stimulated NF-κB activation [15].
T6790 17947-18084 Sentence denotes Our next step was to determine whether Foxp3 could also suppress the activation of NF-κB caused by a strong viral transactivator protein.
T99 17947-18084 Sentence denotes Our next step was to determine whether Foxp3 could also suppress the activation of NF-κB caused by a strong viral transactivator protein.
T6791 18085-18210 Sentence denotes HTLV-I encodes a multifunctional transactivator protein, Tax, capable of activating both the NF-κB and CREB pathways [24–29].
T100 18085-18210 Sentence denotes HTLV-I encodes a multifunctional transactivator protein, Tax, capable of activating both the NF-κB and CREB pathways [24–29].
T6792 18211-18429 Sentence denotes Since the HTLV-I Tax protein can function at multiple levels in both the cytoplasm and the nucleus to stimulate activation of NF-κB [28,29], we hypothesized that overexpression of Foxp3 may interfere with this process.
T101 18211-18429 Sentence denotes Since the HTLV-I Tax protein can function at multiple levels in both the cytoplasm and the nucleus to stimulate activation of NF-κB [28,29], we hypothesized that overexpression of Foxp3 may interfere with this process.
T6793 18430-18604 Sentence denotes However, since Tax-dependent HTLV-I gene expression is independent of NF-κB [18], we also hypothesized that Foxp3 would not affect Tax-dependent activation of the HTLV-I LTR.
T102 18430-18604 Sentence denotes However, since Tax-dependent HTLV-I gene expression is independent of NF-κB [18], we also hypothesized that Foxp3 would not affect Tax-dependent activation of the HTLV-I LTR.
T6794 18605-18767 Sentence denotes To test these hypotheses, we overexpressed HTLV-I Tax, full-length Foxp3, and/or ΔFKH in HEK 293T cells cotransfected with an HTLV-I LTR or NF-κB reporter vector.
T103 18605-18767 Sentence denotes To test these hypotheses, we overexpressed HTLV-I Tax, full-length Foxp3, and/or ΔFKH in HEK 293T cells cotransfected with an HTLV-I LTR or NF-κB reporter vector.
T6795 18768-18878 Sentence denotes As shown in Figure 4A, HTLV-I Tax strongly up-regulated NF-κB-dependent transcriptional activation (~60-fold).
T104 18768-18878 Sentence denotes As shown in Figure 4A, HTLV-I Tax strongly up-regulated NF-κB-dependent transcriptional activation (~60-fold).
T6796 18879-19001 Sentence denotes Interestingly, overexpression of Foxp3, but not ΔFKH, suppressed Tax-mediated activation of NF-κB-dependent transcription.
T105 18879-19001 Sentence denotes Interestingly, overexpression of Foxp3, but not ΔFKH, suppressed Tax-mediated activation of NF-κB-dependent transcription.
T6797 19002-19268 Sentence denotes These observations further suggest that the carboxyl-terminal FKH domain is required for inhibiting activation of NF-κB in the presence of Tax in HEK 293T cells, strikingly similar to the requirements of Foxp3 inhibition of basal NF-κB activation shown in Figure 2B.
T106 19002-19268 Sentence denotes These observations further suggest that the carboxyl-terminal FKH domain is required for inhibiting activation of NF-κB in the presence of Tax in HEK 293T cells, strikingly similar to the requirements of Foxp3 inhibition of basal NF-κB activation shown in Figure 2B.
T6798 19269-19609 Sentence denotes Transactivation of the HTLV-I LTR was stimulated about 55-fold by overexpression of Tax (Figure 4B), while transfection of Foxp3 suppressed Tax-dependent HTLV-I LTR activation, although HTLV-I LTR activation in the presence or absence of Tax is independent of NF-κB or NF-AT (another transcriptional activator known to interact with Foxp3).
T107 19269-19609 Sentence denotes Transactivation of the HTLV-I LTR was stimulated about 55-fold by overexpression of Tax (Figure 4B), while transfection of Foxp3 suppressed Tax-dependent HTLV-I LTR activation, although HTLV-I LTR activation in the presence or absence of Tax is independent of NF-κB or NF-AT (another transcriptional activator known to interact with Foxp3).
T6799 19610-19756 Sentence denotes Furthermore, overexpression of ΔFKH also led to suppression of HTLV-I transactivation by Tax to a similar extent as full-length Foxp3 (Figure 4B).
T108 19610-19756 Sentence denotes Furthermore, overexpression of ΔFKH also led to suppression of HTLV-I transactivation by Tax to a similar extent as full-length Foxp3 (Figure 4B).
T6800 19757-19944 Sentence denotes The suppressive effects shown in Figure 4A and 4B were not the result of Foxp3 down-regulating the expression of the transfected Tax plasmid as determined by real-time RT-PCR (Figure 4C).
T109 19757-19944 Sentence denotes The suppressive effects shown in Figure 4A and 4B were not the result of Foxp3 down-regulating the expression of the transfected Tax plasmid as determined by real-time RT-PCR (Figure 4C).
T6801 19945-20242 Sentence denotes These results strongly suggest that Foxp3 interacts with transcriptional regulators in addition to NF-κB and NF-AT, and that the carboxyl-terminal FKH domain, and therefore localization to the nucleus, are not required for inhibition of Tax-mediated HTLV-I LTR activation (even in HEK 293T cells).
T110 19945-20242 Sentence denotes These results strongly suggest that Foxp3 interacts with transcriptional regulators in addition to NF-κB and NF-AT, and that the carboxyl-terminal FKH domain, and therefore localization to the nucleus, are not required for inhibition of Tax-mediated HTLV-I LTR activation (even in HEK 293T cells).
T111 20243-20331 Sentence denotes Figure 4 Transactivation Functions of HTLV-I Tax Are Blocked by Overexpression of Foxp3
T112 20332-20646 Sentence denotes (A and B) HEK 293T cells (5 × 105) were seeded into six-well plates 1 d prior to transfection with NF-κB (A) or HTLV-I LTR (B) luciferase reporter vectors (200 ng) in the presence or absence of an HTLV-I Tax, Foxp3, ΔFKH, or control expression vector (500 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng).
T113 20647-20730 Sentence denotes Cells were harvested at 24 h posttransfection and analyzed for luciferase activity.
T114 20731-20809 Sentence denotes Relative luciferase activity was normalized to the internal reference control.
T115 20810-21031 Sentence denotes (C) Expression of HTLV-I Tax in (A) and (B) was measured by quantitative RT-PCR (TaqMan) to demonstrate that Foxp3 did not suppress Tax transactivation of NF-κB and the HTLV-I LTR by down-regulating the expression of Tax.
T116 21032-21356 Sentence denotes (D) HEK 293T cells (5 × 105) were seeded into six-well plates 1 d prior to transfection with a Gal4 luciferase reporter vector (200 ng) in the presence or absence of a Gal4-BD or Gal4-BD-Tax expression vector (500 ng), Foxp3, ΔFKH, or control expression vector (500 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng).
T117 21357-21440 Sentence denotes Cells were harvested at 24 h posttransfection and analyzed for luciferase activity.
T118 21441-21519 Sentence denotes Relative luciferase activity was normalized to the internal reference control.
T6802 21522-21784 Sentence denotes To determine whether Foxp3 inhibited the transactivation functions of Tax by directly associating with this viral protein, we generated an expression vector in which HTLV-I Tax was fused in-frame to the carboxyl terminus of the Gal4 DNA-binding domain (Gal4-BD).
T119 21522-21784 Sentence denotes To determine whether Foxp3 inhibited the transactivation functions of Tax by directly associating with this viral protein, we generated an expression vector in which HTLV-I Tax was fused in-frame to the carboxyl terminus of the Gal4 DNA-binding domain (Gal4-BD).
T6803 21785-21981 Sentence denotes This Gal4-BD-Tax fusion protein activated transcription of a synthetic promoter containing five Gal4 binding sites, while Gal4-BD was insufficient to stimulate transcription by itself (Figure 4D).
T120 21785-21981 Sentence denotes This Gal4-BD-Tax fusion protein activated transcription of a synthetic promoter containing five Gal4 binding sites, while Gal4-BD was insufficient to stimulate transcription by itself (Figure 4D).
T6804 21982-22246 Sentence denotes Transactivation of the Gal4-resposive promoter by Gal4-BD-Tax remained relatively unaffected by overexpression of either EGFP (control), Foxp3, or ΔFKH, suggesting that Foxp3 does not repress Tax transactivation by directly interfacing with the HTLV-I Tax protein.
T121 21982-22246 Sentence denotes Transactivation of the Gal4-resposive promoter by Gal4-BD-Tax remained relatively unaffected by overexpression of either EGFP (control), Foxp3, or ΔFKH, suggesting that Foxp3 does not repress Tax transactivation by directly interfacing with the HTLV-I Tax protein.
T6805 22247-22482 Sentence denotes To confirm that Foxp3 had a direct effect on HTLV-I replication, we transfected HEK 293T cells with a well-characterized HTLV-I infectious molecular clone (termed ACH) [30] in the presence of full-length Foxp3, ΔFKH, or control vector.
T122 22247-22482 Sentence denotes To confirm that Foxp3 had a direct effect on HTLV-I replication, we transfected HEK 293T cells with a well-characterized HTLV-I infectious molecular clone (termed ACH) [30] in the presence of full-length Foxp3, ΔFKH, or control vector.
T6806 22483-22644 Sentence denotes ACH has been previously shown to direct the expression of viral antigens, produce infectious virus, and transform CD4+ T cells both in vitro and in vivo [31,32].
T123 22483-22644 Sentence denotes ACH has been previously shown to direct the expression of viral antigens, produce infectious virus, and transform CD4+ T cells both in vitro and in vivo [31,32].
T6807 22645-22771 Sentence denotes After 24 h, the amount of viral antigen expression, in this case Tax mRNA, was detected by a sensitive real-time RT-PCR assay.
T124 22645-22771 Sentence denotes After 24 h, the amount of viral antigen expression, in this case Tax mRNA, was detected by a sensitive real-time RT-PCR assay.
T6808 22772-23014 Sentence denotes As illustrated in Figure 5, the level of Tax mRNA synthesized from ACH was down-regulated in the presence of Foxp3 compared to the level produced in the presence of the control vector. ΔFKH did not have a discernable affect on Tax expression.
T125 22772-23014 Sentence denotes As illustrated in Figure 5, the level of Tax mRNA synthesized from ACH was down-regulated in the presence of Foxp3 compared to the level produced in the presence of the control vector. ΔFKH did not have a discernable affect on Tax expression.
T6809 23015-23135 Sentence denotes These data indicate that Foxp3 is capable of repressing the expression of Tax from an infectious HTLV-I molecular clone.
T126 23015-23135 Sentence denotes These data indicate that Foxp3 is capable of repressing the expression of Tax from an infectious HTLV-I molecular clone.
T127 23136-23233 Sentence denotes Figure 5 Foxp3 Inhibits the Expression of HTLV-I Tax Directed from an Infectious Molecular Clone
T128 23234-23471 Sentence denotes HEK 293T cells (5 × 105) were seeded into six-well plates 1 d prior to transfection in the absence or presence of an HTLV-I infectious molecular clone (pACH; 800 ng) and either a control (EGFP), Foxp3, or ΔFKH expression vector (800 ng).
T129 23472-23567 Sentence denotes Expression of HTLV-I Tax from the molecular clone was measured by quantitative RT-PCR (TaqMan).
T9031 23569-23647 Sentence denotes Increased Foxp3 Protein Expression Is Associated with Low HTLV-I Proviral Load
T130 23569-23647 Sentence denotes Increased Foxp3 Protein Expression Is Associated with Low HTLV-I Proviral Load
T9032 23648-23880 Sentence denotes Since Foxp3 is expressed almost exclusively within CD4+CD25+ T cells, a major viral reservoir for HTLV-I [33], it was important to determine whether there was an association between Foxp3 and HTLV-I replication in infected patients.
T131 23648-23880 Sentence denotes Since Foxp3 is expressed almost exclusively within CD4+CD25+ T cells, a major viral reservoir for HTLV-I [33], it was important to determine whether there was an association between Foxp3 and HTLV-I replication in infected patients.
T9033 23881-24133 Sentence denotes We therefore quantitated the Foxp3 protein expression in CD4+CD25+ T cells by flow cytometry and the HTLV-I proviral load (a surrogate marker of viral replication) by real-time PCR from eight patients with HAM/TSP and eight asymptomatic carriers (ACs).
T132 23881-24133 Sentence denotes We therefore quantitated the Foxp3 protein expression in CD4+CD25+ T cells by flow cytometry and the HTLV-I proviral load (a surrogate marker of viral replication) by real-time PCR from eight patients with HAM/TSP and eight asymptomatic carriers (ACs).
T9034 24134-24187 Sentence denotes The data from this analysis is summarized in Table 1.
T133 24134-24187 Sentence denotes The data from this analysis is summarized in Table 1.
T9035 24188-24380 Sentence denotes As expected, patients with HAM/TSP exhibited significantly higher proviral loads (indicated as HTLV-I proviral DNA copies/100 cells) (34.68 ± 23.19) compared to ACs (4.75 ± 5.47) (p = 0.0008).
T134 24188-24380 Sentence denotes As expected, patients with HAM/TSP exhibited significantly higher proviral loads (indicated as HTLV-I proviral DNA copies/100 cells) (34.68 ± 23.19) compared to ACs (4.75 ± 5.47) (p = 0.0008).
T9036 24381-24553 Sentence denotes The percentage of Foxp3+ cells within the CD4+CD25+ T cell population was significantly greater in ACs (43.23 ± 12.95) than in HAM/TSP patients (18.59 ± 5.77) (p = 0.0033).
T135 24381-24553 Sentence denotes The percentage of Foxp3+ cells within the CD4+CD25+ T cell population was significantly greater in ACs (43.23 ± 12.95) than in HAM/TSP patients (18.59 ± 5.77) (p = 0.0033).
T9037 24554-24673 Sentence denotes These data suggest that high levels of Foxp3 protein expression are associated with reduced HTLV-I replication in vivo.
T136 24554-24673 Sentence denotes These data suggest that high levels of Foxp3 protein expression are associated with reduced HTLV-I replication in vivo.
T9038 24674-24872 Sentence denotes They also support our recent reports that high proviral loads, which have been shown to correlate with high Tax mRNA in HTLV-I-infected patients, are associated with reduced Foxp3 expression [8,34].
T137 24674-24872 Sentence denotes They also support our recent reports that high proviral loads, which have been shown to correlate with high Tax mRNA in HTLV-I-infected patients, are associated with reduced Foxp3 expression [8,34].
T138 24873-24950 Sentence denotes Table 1 Expression of Foxp3 in CD4+CD25+ T Cells in HTLV-I-Infected Patients
T9931 24952-25008 Sentence denotes CREB Is a Target for Transcriptional Repression by Foxp3
T139 24952-25008 Sentence denotes CREB Is a Target for Transcriptional Repression by Foxp3
T9932 25009-25265 Sentence denotes Although Foxp3 could down-regulate Tax-dependent transactivation of the HTLV-I LTR (Figure 4B) and inhibit Tax expression from an infectious molecular clone (Figure 5), Foxp3 failed to modulate Tax function in the absence of the viral promoter (Figure 4D).
T140 25009-25265 Sentence denotes Although Foxp3 could down-regulate Tax-dependent transactivation of the HTLV-I LTR (Figure 4B) and inhibit Tax expression from an infectious molecular clone (Figure 5), Foxp3 failed to modulate Tax function in the absence of the viral promoter (Figure 4D).
T9933 25266-25423 Sentence denotes These results led us to hypothesize that Foxp3 acts on HTLV-I gene expression by interacting with proteins important for driving HTLV-I LTR activity in vivo.
T141 25266-25423 Sentence denotes These results led us to hypothesize that Foxp3 acts on HTLV-I gene expression by interacting with proteins important for driving HTLV-I LTR activity in vivo.
T9934 25424-25597 Sentence denotes Previous studies have demonstrated that the Tax-responsive elements within the HTLV-I LTR play a crucial role in driving Tax-mediated transactivation of the HTLV-I LTR [18].
T142 25424-25597 Sentence denotes Previous studies have demonstrated that the Tax-responsive elements within the HTLV-I LTR play a crucial role in driving Tax-mediated transactivation of the HTLV-I LTR [18].
T9935 25598-25798 Sentence denotes The Tax-responsive elements have been shown to resemble CREB binding sites, bind CREB in vitro and in vivo, and facilitate HTLV-I LTR activation both in the presence and in the absence of Tax [35,36].
T143 25598-25798 Sentence denotes The Tax-responsive elements have been shown to resemble CREB binding sites, bind CREB in vitro and in vivo, and facilitate HTLV-I LTR activation both in the presence and in the absence of Tax [35,36].
T9936 25799-26128 Sentence denotes Ching and colleagues [18] demonstrated that addition of a dominant-negative CREB expression vector resulted in nearly complete inhibition of Tax-mediated activation of the HTLV-I LTR, while blocking NF-κB activation by addition of a dominant-negative IKKβ expression vector had no effect on Tax transactivation of the HTLV-I LTR.
T144 25799-26128 Sentence denotes Ching and colleagues [18] demonstrated that addition of a dominant-negative CREB expression vector resulted in nearly complete inhibition of Tax-mediated activation of the HTLV-I LTR, while blocking NF-κB activation by addition of a dominant-negative IKKβ expression vector had no effect on Tax transactivation of the HTLV-I LTR.
T9937 26129-26262 Sentence denotes Therefore, we hypothesized that Foxp3 may inhibit Tax transactivation of the HTLV-I LTR via disruption of the CREB signaling pathway.
T145 26129-26262 Sentence denotes Therefore, we hypothesized that Foxp3 may inhibit Tax transactivation of the HTLV-I LTR via disruption of the CREB signaling pathway.
T9938 26263-26465 Sentence denotes To test this possibility, HEK 293T cells were transfected with an HTLV-I LTR or synthetic CREB reporter vector along with a control expression vector (EGFP) or expression vectors encoding Foxp3 or ΔFKH.
T146 26263-26465 Sentence denotes To test this possibility, HEK 293T cells were transfected with an HTLV-I LTR or synthetic CREB reporter vector along with a control expression vector (EGFP) or expression vectors encoding Foxp3 or ΔFKH.
T9939 26466-26689 Sentence denotes As shown in Figure 6A, Foxp3 down-regulated basal activation of the HTLV-I LTR and transcription of a synthetic CREB reporter vector, suggesting that Foxp3 down-regulates HTLV-I LTR activation by targeting the CREB pathway.
T147 26466-26689 Sentence denotes As shown in Figure 6A, Foxp3 down-regulated basal activation of the HTLV-I LTR and transcription of a synthetic CREB reporter vector, suggesting that Foxp3 down-regulates HTLV-I LTR activation by targeting the CREB pathway.
T9940 26690-26874 Sentence denotes Deletion of the FKH domain of Foxp3 dampened the suppressive effect of Foxp3, but did not completely abrogate suppression, as is seen with NF-κB-responsive promoters in HEK 293T cells.
T148 26690-26874 Sentence denotes Deletion of the FKH domain of Foxp3 dampened the suppressive effect of Foxp3, but did not completely abrogate suppression, as is seen with NF-κB-responsive promoters in HEK 293T cells.
T9941 26875-27046 Sentence denotes Like NF-κB activation, CREB transcriptional activation was also suppressed by expression of Foxp3, and to a similar extent ΔFKH, in healthy donor CD4+ T cells (Figure 6B).
T149 26875-27046 Sentence denotes Like NF-κB activation, CREB transcriptional activation was also suppressed by expression of Foxp3, and to a similar extent ΔFKH, in healthy donor CD4+ T cells (Figure 6B).
T9942 27047-27158 Sentence denotes Similarly, Foxp3 and ΔFKH also repressed basal HTLV-I LTR activation in primary human CD4+ T cells (Figure 6C).
T150 27047-27158 Sentence denotes Similarly, Foxp3 and ΔFKH also repressed basal HTLV-I LTR activation in primary human CD4+ T cells (Figure 6C).
T9943 27159-27252 Sentence denotes To our knowledge, this is the first evidence implicating CREB as a molecular target of Foxp3.
T151 27159-27252 Sentence denotes To our knowledge, this is the first evidence implicating CREB as a molecular target of Foxp3.
T9944 27253-27490 Sentence denotes As observed with NF-κB activation, ΔFKH was a more potent inhibitor of CREB activation in CD4+ T cells than in HEK 293T cells, further indicating that a cell type-specific mechanism of action may govern the function of this Foxp3 mutant.
T152 27253-27490 Sentence denotes As observed with NF-κB activation, ΔFKH was a more potent inhibitor of CREB activation in CD4+ T cells than in HEK 293T cells, further indicating that a cell type-specific mechanism of action may govern the function of this Foxp3 mutant.
T153 27491-27544 Sentence denotes Figure 6 Foxp3 Inhibits CREB-Dependent Transcription
T154 27545-27835 Sentence denotes (A) HEK 293T cells (5 × 105) were seeded into six-well plates 1 d prior to transfection with a CREB or an HTLV-I LTR luciferase reporter vector (200 ng) in the presence or absence of a Foxp3, ΔFKH, or control expression vector (500 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng).
T155 27836-28105 Sentence denotes (B) Purified CD4+ T cells (2 × 106) from two healthy donors were transfected with a CREB luciferase reporter vector (2,000 ng) in the presence or absence of a Foxp3, ΔFKH, or control expression vector (2,000 ng) and internal reference plasmid (pGL4-TKhRluc2; 1,000 ng).
T156 28106-28378 Sentence denotes (C) Purified CD4+ T cells (2 × 106) from healthy donors were transfected with an HTLV-I LTR luciferase reporter vector (2,000 ng) in the presence or absence of a Foxp3, ΔFKH, or control expression vector (2,000 ng) and internal reference plasmid (pGL4-TKhRluc2; 1,000 ng).
T157 28379-28705 Sentence denotes (D) HEK 293T cells (5 × 105) were seeded into six-well plates 1 d prior to transfection with a Gal4 luciferase reporter vector (200 ng) in the presence or absence of Gal4-BD, Gal4-BD-CREB-1, or Gal4-BD-c-Jun (500 ng) along with Foxp3 or control expression vector (500 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng).
T158 28706-28809 Sentence denotes Cells from A, B, C, and D were harvested at 24 h posttransfection and analyzed for luciferase activity.
T159 28810-28888 Sentence denotes Relative luciferase activity was normalized to the internal reference control.
T160 28889-29167 Sentence denotes (E) Foxp3, ΔFKH, and CREB-1 expression in whole-cell extracts (20 μg) derived from HEK 293T cells transfected with a control expression vector (EGFP), Foxp3, or ΔFKH expression vectors were analyzed by Western blot analysis. β-actin expression was analyzed as a loading control.
T9945 29170-29435 Sentence denotes To determine whether Foxp3 functioned by directly signaling through CREB, we utilized expression vectors encoding CREB-1 or c-Jun (a member of the activator protein 1 family of transcription factors) fused in-frame to the Gal4-BD (Gal4-BD-CREB-1 and Gal4-BD-c-Jun).
T161 29170-29435 Sentence denotes To determine whether Foxp3 functioned by directly signaling through CREB, we utilized expression vectors encoding CREB-1 or c-Jun (a member of the activator protein 1 family of transcription factors) fused in-frame to the Gal4-BD (Gal4-BD-CREB-1 and Gal4-BD-c-Jun).
T9946 29436-29670 Sentence denotes As shown in Figure 6D, activation of a Gal4-responsive reporter vector by Gal4-BD-CREB-1 was down-regulated by Foxp3 compared to control vector (EGFP), indicating that Foxp3 functions by directly or indirectly interacting with CREB-1.
T162 29436-29670 Sentence denotes As shown in Figure 6D, activation of a Gal4-responsive reporter vector by Gal4-BD-CREB-1 was down-regulated by Foxp3 compared to control vector (EGFP), indicating that Foxp3 functions by directly or indirectly interacting with CREB-1.
T9947 29671-29848 Sentence denotes However, Foxp3 failed to markedly affect transcriptional activation of Gal4-BD-c-Jun (c-Jun has also been demonstrated to bind to the HTLV-I LTR) and Gal4-BD-Tax (see Figure 5).
T163 29671-29848 Sentence denotes However, Foxp3 failed to markedly affect transcriptional activation of Gal4-BD-c-Jun (c-Jun has also been demonstrated to bind to the HTLV-I LTR) and Gal4-BD-Tax (see Figure 5).
T9948 29849-30041 Sentence denotes Importantly, the mechanism of Foxp3-mediated inhibition of CREB-dependent transcription was not due to a block in CREB-1 protein expression, as determined by Western blot analysis (Figure 6E).
T164 29849-30041 Sentence denotes Importantly, the mechanism of Foxp3-mediated inhibition of CREB-dependent transcription was not due to a block in CREB-1 protein expression, as determined by Western blot analysis (Figure 6E).
T9949 30042-30326 Sentence denotes Although these results demonstrate that Foxp3 functions as a co-repressor of CREB activation (in addition to NF-κB and NF-AT), we were unable to detect a direct physical interaction between CREB-1 and Foxp3 by coimmunoprecipitation or mammalian two-hybrid analysis (unpublished data).
T165 30042-30326 Sentence denotes Although these results demonstrate that Foxp3 functions as a co-repressor of CREB activation (in addition to NF-κB and NF-AT), we were unable to detect a direct physical interaction between CREB-1 and Foxp3 by coimmunoprecipitation or mammalian two-hybrid analysis (unpublished data).
T9950 30327-30534 Sentence denotes Therefore, our data suggest that Foxp3 may interfere with CREB signaling at an upstream event, such as phosphorylation of CREB or recruitment/function of coactivator proteins CREB-binding protein (CBP)/p300.
T166 30327-30534 Sentence denotes Therefore, our data suggest that Foxp3 may interfere with CREB signaling at an upstream event, such as phosphorylation of CREB or recruitment/function of coactivator proteins CREB-binding protein (CBP)/p300.
T12322 30536-30623 Sentence denotes Foxp3 Antagonizes CREB Transcriptional Activation by Disrupting Coactivator Recruitment
T167 30536-30623 Sentence denotes Foxp3 Antagonizes CREB Transcriptional Activation by Disrupting Coactivator Recruitment
T12323 30624-30948 Sentence denotes Stimulation of CREB-dependent transcription by reagents that activate adenylate cyclase and increase cAMP levels (e.g., forskolin) increase the transactivation potential of CREB through phosphorylation of serine 133 by protein kinase A, which permits binding and recruitment of coactivators CBP/p300 to the promoter [37,38].
T168 30624-30948 Sentence denotes Stimulation of CREB-dependent transcription by reagents that activate adenylate cyclase and increase cAMP levels (e.g., forskolin) increase the transactivation potential of CREB through phosphorylation of serine 133 by protein kinase A, which permits binding and recruitment of coactivators CBP/p300 to the promoter [37,38].
T12324 30949-31060 Sentence denotes Phosphorylation of serine 133 does not, however, affect the DNA-binding activity of CREB in most cases [39–41].
T169 30949-31060 Sentence denotes Phosphorylation of serine 133 does not, however, affect the DNA-binding activity of CREB in most cases [39–41].
T12325 31061-31175 Sentence denotes Addition of forskolin to HEK 293T cells stimulated activation of a CREB reporter vector about 65 fold (Figure 7A).
T170 31061-31175 Sentence denotes Addition of forskolin to HEK 293T cells stimulated activation of a CREB reporter vector about 65 fold (Figure 7A).
T12326 31176-31281 Sentence denotes Overexpression of Foxp3 was capable of down-regulating forskolin-induced CREB transcriptional activation.
T171 31176-31281 Sentence denotes Overexpression of Foxp3 was capable of down-regulating forskolin-induced CREB transcriptional activation.
T12327 31282-31565 Sentence denotes The functional interaction between Foxp3 and CREB did not affect the DNA-binding activity of CREB-1, but did show a modest decrease in activating transcription factor 2 (ATF-2) DNA-binding activity in the presence of forskolin as determined by transcription factor ELISA (Figure 7B).
T172 31282-31565 Sentence denotes The functional interaction between Foxp3 and CREB did not affect the DNA-binding activity of CREB-1, but did show a modest decrease in activating transcription factor 2 (ATF-2) DNA-binding activity in the presence of forskolin as determined by transcription factor ELISA (Figure 7B).
T173 31566-31661 Sentence denotes Figure 7 Foxp3 Antagonizes CREB Activation by Blocking Recruitment of Coactivator Protein p300
T174 31662-31958 Sentence denotes (A and B) HEK 293T cells (5 × 105) were seeded into six-well plates 1 d prior to transfection with a CREB or an HTLV-I LTR luciferase reporter vector (200 ng) in the presence or absence of a Foxp3, DFKH, or control expression vector (500 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng).
T175 31959-32038 Sentence denotes Forskolin (10 μM) was added to the appropriate reactions 20 h posttransfection.
T176 32039-32158 Sentence denotes Reactions were assayed 24 h posttransfection for luciferase activity (A) and CREB-1 and ATF-2 DNA-binding activity (B).
T177 32159-32467 Sentence denotes (C) HEK 293T cells (5 × 105) were seeded into six-well plates 1 d prior to transfection with a Gal4 luciferase reporter vector (200 ng) in the presence or absence of Gal4-BD-CREB-1 (500 ng) along with a control, Foxp3, or p300 expression vector (500 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng).
T178 32468-32551 Sentence denotes Cells were harvested at 24 h posttransfection and analyzed for luciferase activity.
T179 32552-32643 Sentence denotes Relative luciferase activity in (A and C) was normalized to the internal reference control.
T180 32644-32848 Sentence denotes (D) Whole-cell extracts from HEK 293T cells transfected with a control (EGFP) or Foxp3 expression vector together with a p300-HA expression vector were immunoprecipitated with anti-HA monoclonal antibody.
T181 32849-32996 Sentence denotes Proteins were then separated by SDS-PAGE and immunoblotted with anti-Foxp3 (ab10563) to detect immunoprecipitates and with anti-HA for the lysates.
T12328 32999-33164 Sentence denotes While Foxp3 has been shown to bind to and repress activation of both NF-κB and NF-AT, exactly how Foxp3 functions to bring about this affect has not been elucidated.
T182 32999-33164 Sentence denotes While Foxp3 has been shown to bind to and repress activation of both NF-κB and NF-AT, exactly how Foxp3 functions to bring about this affect has not been elucidated.
T12329 33165-33435 Sentence denotes To determine how Foxp3 blocks CREB-dependent transcription, we examined whether Foxp3 was capable of (1) disrupting the recruitment of coactivator proteins and/or (2) preventing phosphorylation of CREB at serine 133 (which is a prerequisite for coactivator recruitment).
T183 33165-33435 Sentence denotes To determine how Foxp3 blocks CREB-dependent transcription, we examined whether Foxp3 was capable of (1) disrupting the recruitment of coactivator proteins and/or (2) preventing phosphorylation of CREB at serine 133 (which is a prerequisite for coactivator recruitment).
T12330 33436-33580 Sentence denotes Since both of these events are required for CREB-dependent gene expression, we hypothesized that Foxp3 may affect CREB activation at both steps.
T184 33436-33580 Sentence denotes Since both of these events are required for CREB-dependent gene expression, we hypothesized that Foxp3 may affect CREB activation at both steps.
T12331 33581-33863 Sentence denotes To determine whether Foxp3 can disrupt the function/recruitment of the coactivator protein p300, we introduced a Gal4 reporter vector and a Gal4-BD-CREB-1 expression vector into HEK 293T cells in the absence or presence of Foxp3, p300, and/or control expression vectors (Figure 7C).
T185 33581-33863 Sentence denotes To determine whether Foxp3 can disrupt the function/recruitment of the coactivator protein p300, we introduced a Gal4 reporter vector and a Gal4-BD-CREB-1 expression vector into HEK 293T cells in the absence or presence of Foxp3, p300, and/or control expression vectors (Figure 7C).
T12332 33864-33959 Sentence denotes As expected, p300 overexpression stimulated transcription of the Gal4-BD-CREB-1 fusion protein.
T186 33864-33959 Sentence denotes As expected, p300 overexpression stimulated transcription of the Gal4-BD-CREB-1 fusion protein.
T12333 33960-34128 Sentence denotes Foxp3, again, repressed basal levels of Gal4-BD-CREB-1 activation by more than 2-fold, while effectively neutralizing Gal4-BD-CREB-1 activation in the presence of p300.
T187 33960-34128 Sentence denotes Foxp3, again, repressed basal levels of Gal4-BD-CREB-1 activation by more than 2-fold, while effectively neutralizing Gal4-BD-CREB-1 activation in the presence of p300.
T12334 34129-34268 Sentence denotes We next analyzed the effect of Foxp3 on phosphorylation of CREB at serine 133 in forskolin-treated HEK 293T cells by Western blot analysis.
T188 34129-34268 Sentence denotes We next analyzed the effect of Foxp3 on phosphorylation of CREB at serine 133 in forskolin-treated HEK 293T cells by Western blot analysis.
T12335 34269-34419 Sentence denotes Overexpression of Foxp3 failed to reduce the detectable levels of CREB phosphorylation using a phosphospecific antibody for CREB-1 (unpublished data).
T189 34269-34419 Sentence denotes Overexpression of Foxp3 failed to reduce the detectable levels of CREB phosphorylation using a phosphospecific antibody for CREB-1 (unpublished data).
T12336 34420-34647 Sentence denotes However, when we attempted to determine whether Foxp3 could physically interact with the coactivator protein p300, we found that p300 immunoprecipitated Foxp3 when both proteins were overexpressed in HEK 293T cells (Figure 7D).
T190 34420-34647 Sentence denotes However, when we attempted to determine whether Foxp3 could physically interact with the coactivator protein p300, we found that p300 immunoprecipitated Foxp3 when both proteins were overexpressed in HEK 293T cells (Figure 7D).
T12337 34648-34875 Sentence denotes Collectively, these results suggest that Foxp3 antagonizes CREB-dependent gene expression by directly interacting with coactivator p300 and interfering with its function and/or recruitment to CREB-responsive promoter sequences.
T191 34648-34875 Sentence denotes Collectively, these results suggest that Foxp3 antagonizes CREB-dependent gene expression by directly interacting with coactivator p300 and interfering with its function and/or recruitment to CREB-responsive promoter sequences.
T192 34877-34887 Sentence denotes Discussion
T14397 34888-35017 Sentence denotes In the present study, we show that Foxp3 functions as a potent repressor of NF-κB- and CREB-dependent transcriptional activation.
T193 34888-35017 Sentence denotes In the present study, we show that Foxp3 functions as a potent repressor of NF-κB- and CREB-dependent transcriptional activation.
T14398 35018-35150 Sentence denotes Furthermore, the carboxyl-terminal FKH domain appears to be dispensable for mediating these effects, at least in T cell populations.
T194 35018-35150 Sentence denotes Furthermore, the carboxyl-terminal FKH domain appears to be dispensable for mediating these effects, at least in T cell populations.
T14399 35151-35344 Sentence denotes This observation may become important in light of recent reports suggesting that Foxp3 expression in thymic epithelial cells was crucial for directing development of T cells in the thymus [42].
T195 35151-35344 Sentence denotes This observation may become important in light of recent reports suggesting that Foxp3 expression in thymic epithelial cells was crucial for directing development of T cells in the thymus [42].
T14400 35345-35599 Sentence denotes Interestingly, the majority of the genetic mutations associated with IPEX, a severe autoimmune disorder caused by functional inactivation of Foxp3, map to the carboxyl-terminal FKH domain or the leucine zipper domain in the central region of the protein.
T196 35345-35599 Sentence denotes Interestingly, the majority of the genetic mutations associated with IPEX, a severe autoimmune disorder caused by functional inactivation of Foxp3, map to the carboxyl-terminal FKH domain or the leucine zipper domain in the central region of the protein.
T14401 35600-35710 Sentence denotes Only one mutation associated with IPEX to date has been mapped to the amino-terminal proline-rich region [43].
T197 35600-35710 Sentence denotes Only one mutation associated with IPEX to date has been mapped to the amino-terminal proline-rich region [43].
T14402 35711-36104 Sentence denotes It is possible that the FKH domain has a complex tertiary structure that is particularly sensitive to misfolding caused by genetic mutations and that an intact FKH domain is absolutely critical for promoting Foxp3 function in the nucleus, whereas the structure of the amino-terminal proline-rich region may tolerate certain mutations as long as the NF-κB/NF-AT binding motif remains unaltered.
T198 35711-36104 Sentence denotes It is possible that the FKH domain has a complex tertiary structure that is particularly sensitive to misfolding caused by genetic mutations and that an intact FKH domain is absolutely critical for promoting Foxp3 function in the nucleus, whereas the structure of the amino-terminal proline-rich region may tolerate certain mutations as long as the NF-κB/NF-AT binding motif remains unaltered.
T14403 36105-36156 Sentence denotes This motif may also include the zinc finger domain.
T199 36105-36156 Sentence denotes This motif may also include the zinc finger domain.
T14404 36157-36319 Sentence denotes A logical region that may be targeted by the amino-terminal proline-rich region of Foxp3 is the Rel homology domain found in both NF-κB and NF-AT family proteins.
T200 36157-36319 Sentence denotes A logical region that may be targeted by the amino-terminal proline-rich region of Foxp3 is the Rel homology domain found in both NF-κB and NF-AT family proteins.
T14405 36320-36527 Sentence denotes A region that may also be important with respect to Foxp3 function is the leucine zipper domain, as demonstrated by the number of mutations associated with IPEX that have been mapped in this region of Foxp3.
T201 36320-36527 Sentence denotes A region that may also be important with respect to Foxp3 function is the leucine zipper domain, as demonstrated by the number of mutations associated with IPEX that have been mapped in this region of Foxp3.
T14406 36528-36679 Sentence denotes The role of this domain in Foxp3 function remains uncharacterized, but may play a role in dimer formation as it does in other Foxp family members [44].
T202 36528-36679 Sentence denotes The role of this domain in Foxp3 function remains uncharacterized, but may play a role in dimer formation as it does in other Foxp family members [44].
T14407 36680-36932 Sentence denotes Because the pathogenesis of a number of retroviral-induced immunologic disorders such as HIV-1/AIDS and HTLV-I/HAM/TSP have been associated with dysregulation of Foxp3 expression [8,45], we also examined the role of Foxp3 in retroviral gene expression.
T203 36680-36932 Sentence denotes Because the pathogenesis of a number of retroviral-induced immunologic disorders such as HIV-1/AIDS and HTLV-I/HAM/TSP have been associated with dysregulation of Foxp3 expression [8,45], we also examined the role of Foxp3 in retroviral gene expression.
T14408 36933-37239 Sentence denotes HIV-1 LTR activation in CD4+ T cells is critically dependent on two tandem NF-κB sites located between nucleotide positions −102 and −81 within the HIV-1 enhancer region, whereas HTLV-I LTR activation in the presence or absence of the HTLV-I-encoded transactivator protein Tax is independent of NF-κB [18].
T204 36933-37239 Sentence denotes HIV-1 LTR activation in CD4+ T cells is critically dependent on two tandem NF-κB sites located between nucleotide positions −102 and −81 within the HIV-1 enhancer region, whereas HTLV-I LTR activation in the presence or absence of the HTLV-I-encoded transactivator protein Tax is independent of NF-κB [18].
T14409 37240-37344 Sentence denotes To our knowledge for the first time, Foxp3 was shown to have a direct effect on HIV-1 LTR transcription.
T205 37240-37344 Sentence denotes To our knowledge for the first time, Foxp3 was shown to have a direct effect on HIV-1 LTR transcription.
T14410 37345-37480 Sentence denotes Deletion of the NF-κB sites within the HIV-1 enhancer region reduced the responsiveness of the HIV-1 LTR to Foxp3-mediated suppression.
T206 37345-37480 Sentence denotes Deletion of the NF-κB sites within the HIV-1 enhancer region reduced the responsiveness of the HIV-1 LTR to Foxp3-mediated suppression.
T14411 37481-37672 Sentence denotes In addition, the FKH domain of Foxp3 was required for this inhibitory effect in HEK 293T cells, but not in Jurkat T cells, similar to Foxp3-mediated suppression of a synthetic NF-κB reporter.
T207 37481-37672 Sentence denotes In addition, the FKH domain of Foxp3 was required for this inhibitory effect in HEK 293T cells, but not in Jurkat T cells, similar to Foxp3-mediated suppression of a synthetic NF-κB reporter.
T14412 37673-37946 Sentence denotes The direct effect of Foxp3 down-regulating HIV-1 gene expression correlates well with recently reported evidence indicating that higher regulatory activity of CD4+CD25+ T cells from HIV-1-infected patients was associated with lower HIV-1 viral loads in these patients [46].
T208 37673-37946 Sentence denotes The direct effect of Foxp3 down-regulating HIV-1 gene expression correlates well with recently reported evidence indicating that higher regulatory activity of CD4+CD25+ T cells from HIV-1-infected patients was associated with lower HIV-1 viral loads in these patients [46].
T14413 37947-38102 Sentence denotes Foxp3 also affected two well-known functions of HTLV-I Tax: transactivation of the NF-κB pathway and, most surprisingly, transactivation of the HTLV-I LTR.
T209 37947-38102 Sentence denotes Foxp3 also affected two well-known functions of HTLV-I Tax: transactivation of the NF-κB pathway and, most surprisingly, transactivation of the HTLV-I LTR.
T14414 38103-38213 Sentence denotes Transactivation of the HTLV-I LTR by Tax involves the interaction of ATF/CREB factors with Tax in the nucleus.
T210 38103-38213 Sentence denotes Transactivation of the HTLV-I LTR by Tax involves the interaction of ATF/CREB factors with Tax in the nucleus.
T14415 38214-38387 Sentence denotes Binding of Tax enhances ATF/CREB dimerization and promotes assembly of Tax-ATF/CREB complexes onto specific sequences in the viral promoter known as Tax-responsive elements.
T211 38214-38387 Sentence denotes Binding of Tax enhances ATF/CREB dimerization and promotes assembly of Tax-ATF/CREB complexes onto specific sequences in the viral promoter known as Tax-responsive elements.
T14416 38388-38544 Sentence denotes This series of steps allows Tax to recruit coactivator proteins CBP/p300 to the viral promoter and facilitate a high level of viral gene expression [24–26].
T212 38388-38544 Sentence denotes This series of steps allows Tax to recruit coactivator proteins CBP/p300 to the viral promoter and facilitate a high level of viral gene expression [24–26].
T14417 38545-38753 Sentence denotes Transactivation of the NF-κB pathway by Tax was inhibited by overexpression of full-length Foxp3, but not ΔFKH, as seen with basal activation of the HIV-1 LTR and a synthetic NF-κB reporter in HEK 293T cells.
T213 38545-38753 Sentence denotes Transactivation of the NF-κB pathway by Tax was inhibited by overexpression of full-length Foxp3, but not ΔFKH, as seen with basal activation of the HIV-1 LTR and a synthetic NF-κB reporter in HEK 293T cells.
T14418 38754-38924 Sentence denotes However, Tax-mediated transactivation of the HTLV-I LTR was inhibited by overexpression of both full-length Foxp3 as well as ΔFKH in both HEK 293T cells and CD4+ T cells.
T214 38754-38924 Sentence denotes However, Tax-mediated transactivation of the HTLV-I LTR was inhibited by overexpression of both full-length Foxp3 as well as ΔFKH in both HEK 293T cells and CD4+ T cells.
T14419 38925-39177 Sentence denotes We demonstrated that Foxp3 did not directly affect the functioning of Tax, but rather Foxp3 targeted the transcription factors required for Tax transactivation (i.e., NF-κB and a then-unknown cellular factor, which we identified in this study as CREB).
T215 38925-39177 Sentence denotes We demonstrated that Foxp3 did not directly affect the functioning of Tax, but rather Foxp3 targeted the transcription factors required for Tax transactivation (i.e., NF-κB and a then-unknown cellular factor, which we identified in this study as CREB).
T14420 39178-39294 Sentence denotes The negative effect of Foxp3 on HTLV-I gene expression was confirmed utilizing an HTLV-I infectious molecular clone.
T216 39178-39294 Sentence denotes The negative effect of Foxp3 on HTLV-I gene expression was confirmed utilizing an HTLV-I infectious molecular clone.
T14421 39295-39552 Sentence denotes Importantly, we demonstrated that HTLV-I-infected individuals with the highest levels of Foxp3 protein expression within the CD4+CD25+ T cells population exhibited lower proviral loads than did individuals with the lowest levels of Foxp3 protein expression.
T217 39295-39552 Sentence denotes Importantly, we demonstrated that HTLV-I-infected individuals with the highest levels of Foxp3 protein expression within the CD4+CD25+ T cells population exhibited lower proviral loads than did individuals with the lowest levels of Foxp3 protein expression.
T14422 39553-39779 Sentence denotes Previous studies have demonstrated that the HTLV-I proviral load directly correlates with HTLV-I Tax mRNA load, the frequency of immunopathogenic virus-specific CD8+ T cells, and disease severity in patients with HAM/TSP [47].
T218 39553-39779 Sentence denotes Previous studies have demonstrated that the HTLV-I proviral load directly correlates with HTLV-I Tax mRNA load, the frequency of immunopathogenic virus-specific CD8+ T cells, and disease severity in patients with HAM/TSP [47].
T14423 39780-39916 Sentence denotes These results have important implications on the utility of Foxp3 in controlling viral gene expression and thus pathogenesis of HAM/TSP.
T219 39780-39916 Sentence denotes These results have important implications on the utility of Foxp3 in controlling viral gene expression and thus pathogenesis of HAM/TSP.
T14424 39917-40210 Sentence denotes Therefore, Foxp3 becomes an attractive target for the development of novel therapeutic applications directed at modulating the expression of this important regulatory protein, especially in light of recent observations that the expression of Foxp3 can also be down-regulated by HTLV-I Tax [8].
T220 39917-40210 Sentence denotes Therefore, Foxp3 becomes an attractive target for the development of novel therapeutic applications directed at modulating the expression of this important regulatory protein, especially in light of recent observations that the expression of Foxp3 can also be down-regulated by HTLV-I Tax [8].
T14425 40211-40428 Sentence denotes As the activation of the HTLV-I LTR depends primarily on ATF/CREB proteins (whether in the presence or the absence of Tax), we investigated whether Foxp3 could interact with this additional cellular signaling pathway.
T221 40211-40428 Sentence denotes As the activation of the HTLV-I LTR depends primarily on ATF/CREB proteins (whether in the presence or the absence of Tax), we investigated whether Foxp3 could interact with this additional cellular signaling pathway.
T14426 40429-40616 Sentence denotes While the DNA-binding activity of CREB is, in most cases, constitutive, the transactivation potential of CREB is regulated by the phosphorylation of CREB and recruitment of CBP/p300 [48].
T222 40429-40616 Sentence denotes While the DNA-binding activity of CREB is, in most cases, constitutive, the transactivation potential of CREB is regulated by the phosphorylation of CREB and recruitment of CBP/p300 [48].
T14427 40617-40819 Sentence denotes Our data demonstrate that Foxp3 interferes with the latter of these two processes and that the recruitment of the coactivator protein p300, and resulting transcriptional activation are blocked by Foxp3.
T223 40617-40819 Sentence denotes Our data demonstrate that Foxp3 interferes with the latter of these two processes and that the recruitment of the coactivator protein p300, and resulting transcriptional activation are blocked by Foxp3.
T14428 40820-40906 Sentence denotes This may be the result of the physical interaction we detected between Foxp3 and p300.
T224 40820-40906 Sentence denotes This may be the result of the physical interaction we detected between Foxp3 and p300.
T14429 40907-41164 Sentence denotes With respect to HTLV-I LTR activity, while full-length Foxp3 inhibited both basal and Tax-dependent transcription by ~50%, ΔFKH appeared less effective in suppressing basal activation (~25% inhibition) compared to Tax-dependent activation (~50% inhibition).
T225 40907-41164 Sentence denotes With respect to HTLV-I LTR activity, while full-length Foxp3 inhibited both basal and Tax-dependent transcription by ~50%, ΔFKH appeared less effective in suppressing basal activation (~25% inhibition) compared to Tax-dependent activation (~50% inhibition).
T14430 41165-41369 Sentence denotes The effect of ΔFKH on basal activation of the HTLV-I LTR in HEK 293T cells was very similar to that shown for a synthetic CREB reporter, suggesting that the FKH domain of Foxp3 is important at some level.
T226 41165-41369 Sentence denotes The effect of ΔFKH on basal activation of the HTLV-I LTR in HEK 293T cells was very similar to that shown for a synthetic CREB reporter, suggesting that the FKH domain of Foxp3 is important at some level.
T14431 41370-41539 Sentence denotes As observed with NF-κB activation, the Foxp3 mutant lacking the FKH domain was a stronger inhibitor of CREB activation in CD4+ T cells than in HEK 293T epithelial cells.
T227 41370-41539 Sentence denotes As observed with NF-κB activation, the Foxp3 mutant lacking the FKH domain was a stronger inhibitor of CREB activation in CD4+ T cells than in HEK 293T epithelial cells.
T14432 41540-41697 Sentence denotes Therefore, it appears that in CD4+ T cells, the FKH domain is dispensable for the proper functioning of Foxp3 with respect to both NF-κB and CREB activation.
T228 41540-41697 Sentence denotes Therefore, it appears that in CD4+ T cells, the FKH domain is dispensable for the proper functioning of Foxp3 with respect to both NF-κB and CREB activation.
T14433 41698-41872 Sentence denotes In summary, this is, to our knowledge, the first direct evidence implicating a role for the Treg-specific transcription factor Foxp3 in regulating retroviral gene expression.
T229 41698-41872 Sentence denotes In summary, this is, to our knowledge, the first direct evidence implicating a role for the Treg-specific transcription factor Foxp3 in regulating retroviral gene expression.
T14434 41873-41946 Sentence denotes In addition, we identify the CREB pathway as a molecular target of Foxp3.
T230 41873-41946 Sentence denotes In addition, we identify the CREB pathway as a molecular target of Foxp3.
T14435 41947-42365 Sentence denotes Since CREB has been shown to regulate multiple genes involved in transcription (e.g., JunD, c-Fos, signal transducer of activated T cells 3 [STAT3]), cell cycle (e.g., p15INK4b, cyclin A, cyclin D1), and immune regulation (e.g., IL-2, IL-6, T-cell receptor α) (reviewed in [48]), the findings presented in this report broaden the potential range of signaling pathways under the control of the regulatory protein Foxp3.
T231 41947-42365 Sentence denotes Since CREB has been shown to regulate multiple genes involved in transcription (e.g., JunD, c-Fos, signal transducer of activated T cells 3 [STAT3]), cell cycle (e.g., p15INK4b, cyclin A, cyclin D1), and immune regulation (e.g., IL-2, IL-6, T-cell receptor α) (reviewed in [48]), the findings presented in this report broaden the potential range of signaling pathways under the control of the regulatory protein Foxp3.
T232 42366-42524 Sentence denotes Our evidence stresses the importance of Foxp3 expression and Treg function in the development and maintenance of protective immunity against HIV-1 and HTLV-I.
T14436 42366-42656 Sentence denotes Our evidence stresses the importance of Foxp3 expression and Treg function in the development and maintenance of protective immunity against HIV-1 and HTLV-I. Based on recent findings, Foxp3 may limit HIV-1 and HTLV-I transcription by interfering with activation of NF-κB and CREB pathways.
T233 42525-42656 Sentence denotes Based on recent findings, Foxp3 may limit HIV-1 and HTLV-I transcription by interfering with activation of NF-κB and CREB pathways.
T14437 42657-43018 Sentence denotes However, observing that this inhibitory effect is not absolute, a low level of viral gene expression may persist in CD4+ T cells (in particular regulatory T cells, which are known reservoirs of HIV-1 and HTLV-I) and result in the accumulation of viral proteins that either stimulate NF-κB and/or CREB activation or directly inhibit Foxp3 expression or function.
T234 42657-43018 Sentence denotes However, observing that this inhibitory effect is not absolute, a low level of viral gene expression may persist in CD4+ T cells (in particular regulatory T cells, which are known reservoirs of HIV-1 and HTLV-I) and result in the accumulation of viral proteins that either stimulate NF-κB and/or CREB activation or directly inhibit Foxp3 expression or function.
T14438 43019-43206 Sentence denotes The imbalance of NF-κB and CREB activation caused by these viral gene products may be a crucial step in the pathogenesis of virus-induced immunological disorders such as AIDS and HAM/TSP.
T235 43019-43206 Sentence denotes The imbalance of NF-κB and CREB activation caused by these viral gene products may be a crucial step in the pathogenesis of virus-induced immunological disorders such as AIDS and HAM/TSP.
T14439 43207-43480 Sentence denotes Future studies will be directed at identifying and characterizing cellular proteins that interact with Foxp3 both in the nucleus and cytoplasm, in order to better address how Foxp3 functions to guide the development and function of regulatory T cells in health and disease.
T236 43207-43480 Sentence denotes Future studies will be directed at identifying and characterizing cellular proteins that interact with Foxp3 both in the nucleus and cytoplasm, in order to better address how Foxp3 functions to guide the development and function of regulatory T cells in health and disease.
T237 43482-43503 Sentence denotes Materials and Methods
T18722 43505-43518 Sentence denotes Cell culture.
T238 43505-43518 Sentence denotes Cell culture.
T18723 43519-43634 Sentence denotes HEK 293T cells were cultured in Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, California, United States).
T239 43519-43634 Sentence denotes HEK 293T cells were cultured in Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, California, United States).
T18724 43635-43728 Sentence denotes Jurkat T cells and primary human CD4+ T cells were cultured in RPMI-1640 medium (Invitrogen).
T240 43635-43728 Sentence denotes Jurkat T cells and primary human CD4+ T cells were cultured in RPMI-1640 medium (Invitrogen).
T18725 43729-43958 Sentence denotes Media were supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (Cambrex, East Rutherford, New Jersey, United States), and 10% fetal bovine serum (Atlanta Biologicals, Norcross, Georgia, United States).
T241 43729-43958 Sentence denotes Media were supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (Cambrex, East Rutherford, New Jersey, United States), and 10% fetal bovine serum (Atlanta Biologicals, Norcross, Georgia, United States).
T18920 43960-43990 Sentence denotes Patients and cell preparation.
T242 43960-43990 Sentence denotes Patients and cell preparation.
T18921 43991-44228 Sentence denotes PBMCs were prepared by centrifugation over Ficoll-Hypaque gradients (BioWhittaker, Walkersville, Maryland, United States) from eight HAM/TSP patients and eight ACs, and the cells were viably cryopreserved in liquid nitrogen until tested.
T243 43991-44228 Sentence denotes PBMCs were prepared by centrifugation over Ficoll-Hypaque gradients (BioWhittaker, Walkersville, Maryland, United States) from eight HAM/TSP patients and eight ACs, and the cells were viably cryopreserved in liquid nitrogen until tested.
T18922 44229-44284 Sentence denotes HAM/TSP was diagnosed according to WHO guidelines [49].
T244 44229-44284 Sentence denotes HAM/TSP was diagnosed according to WHO guidelines [49].
T18923 44285-44500 Sentence denotes HTLV-I seropositivity was determined by ELISA (Abbott Laboratories, Abbott Park, Illinois, United States), with confirmation by Western blot analysis (Genelabs Technologies, Redwood City, California, United States).
T245 44285-44500 Sentence denotes HTLV-I seropositivity was determined by ELISA (Abbott Laboratories, Abbott Park, Illinois, United States), with confirmation by Western blot analysis (Genelabs Technologies, Redwood City, California, United States).
T18924 44501-44643 Sentence denotes Blood samples were obtained after informed consent as part of a clinical protocol reviewed and approved by the NIH institutional review panel.
T246 44501-44643 Sentence denotes Blood samples were obtained after informed consent as part of a clinical protocol reviewed and approved by the NIH institutional review panel.
T19221 44645-44654 Sentence denotes Plasmids.
T247 44645-44654 Sentence denotes Plasmids.
T248 44655-44817 Sentence denotes Expression vectors encoding human Foxp3 (pCMV-Foxp3-IRES-EGFP) and human Foxp3 lacking the forkhead (FKH) domain (pCMV-ΔFKH-IRES-EGFP) were generous gifts from S.
T19222 44655-45209 Sentence denotes Expression vectors encoding human Foxp3 (pCMV-Foxp3-IRES-EGFP) and human Foxp3 lacking the forkhead (FKH) domain (pCMV-ΔFKH-IRES-EGFP) were generous gifts from S. Ziegler (Benaroya Research Institute). pEGFP-C2 was provided by I. Lipinski (NIDDK/NIH). pcDNA3 was provided by K. T. Jeang (NIAID/NIH). pCMV4-Tax was a generous gift from W. Greene (University of California San Francisco). pGL4-luc2 and pGL4-TKhRluc2 were purchased from Promega (Madison, Wisconsin, United States). pUC18 was purchased from Stratagene (La Jolla, California, United States).
T249 44818-44884 Sentence denotes Ziegler (Benaroya Research Institute). pEGFP-C2 was provided by I.
T250 44885-44932 Sentence denotes Lipinski (NIDDK/NIH). pcDNA3 was provided by K.
T251 44933-44935 Sentence denotes T.
T252 44936-44992 Sentence denotes Jeang (NIAID/NIH). pCMV4-Tax was a generous gift from W.
T253 44993-45209 Sentence denotes Greene (University of California San Francisco). pGL4-luc2 and pGL4-TKhRluc2 were purchased from Promega (Madison, Wisconsin, United States). pUC18 was purchased from Stratagene (La Jolla, California, United States).
T19223 45210-45462 Sentence denotes HIV-1 wt LTR and HIV-1 Δ-κB LTR luciferase reporter vectors were constructed by cloning the XhoI/HindIII LTR fragments from pHIV-CAT and pΔ-κB-HIV-CAT (AIDS Research and Reference Reagent Program, NIAID/NIH) into the multiple cloning site of pGL4-luc2.
T254 45210-45462 Sentence denotes HIV-1 wt LTR and HIV-1 Δ-κB LTR luciferase reporter vectors were constructed by cloning the XhoI/HindIII LTR fragments from pHIV-CAT and pΔ-κB-HIV-CAT (AIDS Research and Reference Reagent Program, NIAID/NIH) into the multiple cloning site of pGL4-luc2.
T255 45463-45577 Sentence denotes NF-κB, HTLV-I LTR, and CREB luciferase reporter and pCMV-p300-HA expression vectors were generously provided by B.
T19224 45463-45626 Sentence denotes NF-κB, HTLV-I LTR, and CREB luciferase reporter and pCMV-p300-HA expression vectors were generously provided by B. Wigdahl (Drexel University College of Medicine).
T256 45578-45626 Sentence denotes Wigdahl (Drexel University College of Medicine).
T19225 45627-45999 Sentence denotes HTLV-I pACH infectious molecular clone has been described previously [30]. pFR-luc, pFA-CMV, pFA2-CREB-1, and pFA2-c-Jun were purchased from Stratagene. pFA-Tax (encoding a fusion protein consisting of the Gal4 DNA-binding domain fused in-frame to HTLV-I Tax) was constructed by PCR amplification of HTLV-I Tax using pCMV4-Tax as a template and BamHI/BglII-tagged primers.
T257 45627-45999 Sentence denotes HTLV-I pACH infectious molecular clone has been described previously [30]. pFR-luc, pFA-CMV, pFA2-CREB-1, and pFA2-c-Jun were purchased from Stratagene. pFA-Tax (encoding a fusion protein consisting of the Gal4 DNA-binding domain fused in-frame to HTLV-I Tax) was constructed by PCR amplification of HTLV-I Tax using pCMV4-Tax as a template and BamHI/BglII-tagged primers.
T19226 46000-46084 Sentence denotes The amplified insert was digested and ligated into the BamHI/BglII sites of pFA-CMV.
T258 46000-46084 Sentence denotes The amplified insert was digested and ligated into the BamHI/BglII sites of pFA-CMV.
T19227 46085-46135 Sentence denotes Plasmid contents were confirmed by DNA sequencing.
T259 46085-46135 Sentence denotes Plasmid contents were confirmed by DNA sequencing.
T19707 46137-46177 Sentence denotes Isolation of primary human CD4+ T cells.
T260 46137-46177 Sentence denotes Isolation of primary human CD4+ T cells.
T19708 46178-46390 Sentence denotes CD4+ T cells were isolated from cryopreserved healthy donor PBMCs by negative selection with the CD4+ T Cell Isolation Kit II (Miltenyi Biotech, Bergisch Gladbach, Germany) according to manufacturer's guidelines.
T261 46178-46390 Sentence denotes CD4+ T cells were isolated from cryopreserved healthy donor PBMCs by negative selection with the CD4+ T Cell Isolation Kit II (Miltenyi Biotech, Bergisch Gladbach, Germany) according to manufacturer's guidelines.
T19709 46391-46499 Sentence denotes Purity of negatively selected CD4+ T cells was consistently higher than 96% as determined by flow cytometry.
T262 46391-46499 Sentence denotes Purity of negatively selected CD4+ T cells was consistently higher than 96% as determined by flow cytometry.
T19893 46501-46544 Sentence denotes Transient expression and luciferase assays.
T263 46501-46544 Sentence denotes Transient expression and luciferase assays.
T19894 46545-46826 Sentence denotes HEK 293T cells were plated at a density of 5 × 105 cells/well in six-well culture plates (BD Biosciences, San Diego, California, United States) 1 d prior to transfection with the appropriate plasmid DNA (~2 μg total) using FuGene 6 transfection reagent (Roche, Basel, Switzerland).
T264 46545-46826 Sentence denotes HEK 293T cells were plated at a density of 5 × 105 cells/well in six-well culture plates (BD Biosciences, San Diego, California, United States) 1 d prior to transfection with the appropriate plasmid DNA (~2 μg total) using FuGene 6 transfection reagent (Roche, Basel, Switzerland).
T19895 46827-47010 Sentence denotes Jurkat T cells were plated at 1 × 106 cells/well in six-well culture plates the day of transfection with the appropriate plasmid DNA (~2 μg total) using FuGene 6 transfection reagent.
T265 46827-47010 Sentence denotes Jurkat T cells were plated at 1 × 106 cells/well in six-well culture plates the day of transfection with the appropriate plasmid DNA (~2 μg total) using FuGene 6 transfection reagent.
T19896 47011-47198 Sentence denotes Primary human CD4+ T cells (2 × 106) were nucleofected with the specified plasmid DNA (5 μg total) using the Human T Cell Nucleofection Kit (Amaxa, Gaithersburg, Maryland, United States).
T266 47011-47198 Sentence denotes Primary human CD4+ T cells (2 × 106) were nucleofected with the specified plasmid DNA (5 μg total) using the Human T Cell Nucleofection Kit (Amaxa, Gaithersburg, Maryland, United States).
T19897 47199-47319 Sentence denotes Forskolin (10 μM; Calbiochem, San Diego, California, United States) was added in some experiments 20 h posttransfection.
T267 47199-47319 Sentence denotes Forskolin (10 μM; Calbiochem, San Diego, California, United States) was added in some experiments 20 h posttransfection.
T19898 47320-47693 Sentence denotes Cells were harvested 24 h posttransfection and luciferase activity was analyzed using the Dual-luciferase Reporter Assay System (Promega) and a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, California, United States) according to manufacturer's guidelines. pGL4-TKhRluc2 was used as an internal control to normalize for transfection efficiency.
T268 47320-47693 Sentence denotes Cells were harvested 24 h posttransfection and luciferase activity was analyzed using the Dual-luciferase Reporter Assay System (Promega) and a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, California, United States) according to manufacturer's guidelines. pGL4-TKhRluc2 was used as an internal control to normalize for transfection efficiency.
T19899 47694-47820 Sentence denotes Nucleofected CD4+ T cells were also monitored for transfection efficiency and cell viability 24 h posttransfection as follows.
T269 47694-47820 Sentence denotes Nucleofected CD4+ T cells were also monitored for transfection efficiency and cell viability 24 h posttransfection as follows.
T19900 47821-47925 Sentence denotes Transfection efficiency was routinely ~30% as determined by flow cytometric analysis of EGFP expression.
T270 47821-47925 Sentence denotes Transfection efficiency was routinely ~30% as determined by flow cytometric analysis of EGFP expression.
T19901 47926-48036 Sentence denotes Cell viability, determined by staining with 7-amino-actinomycin D (7-AAD; BD Biosciences), was routinely ~70%.
T271 47926-48036 Sentence denotes Cell viability, determined by staining with 7-amino-actinomycin D (7-AAD; BD Biosciences), was routinely ~70%.
T19902 48037-48151 Sentence denotes Both transfection efficiency and cell viability in nucleofected CD4+ T cells was independent of the plasmids used.
T272 48037-48151 Sentence denotes Both transfection efficiency and cell viability in nucleofected CD4+ T cells was independent of the plasmids used.
T20630 48153-48214 Sentence denotes Foxp3 and HTLV-I Tax expression analysis by real-time RT-PCR.
T273 48153-48214 Sentence denotes Foxp3 and HTLV-I Tax expression analysis by real-time RT-PCR.
T20631 48215-48321 Sentence denotes Real-time RT-PCR analysis of Foxp3 and HTLV-I Tax expression was performed as previously described [8,47].
T274 48215-48321 Sentence denotes Real-time RT-PCR analysis of Foxp3 and HTLV-I Tax expression was performed as previously described [8,47].
T20632 48322-48636 Sentence denotes Briefly, total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, California, United States) according to manufacturer's guidelines, and cDNA was synthesized by reverse transcription using TaqMan Gold RT-PCR Kit using random hexamer primers (Applied Biosystems, Foster City, California, United States).
T275 48322-48636 Sentence denotes Briefly, total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, California, United States) according to manufacturer's guidelines, and cDNA was synthesized by reverse transcription using TaqMan Gold RT-PCR Kit using random hexamer primers (Applied Biosystems, Foster City, California, United States).
T20633 48637-48774 Sentence denotes Foxp3 and HTLV-I Tax mRNA expression was quantified by real-time PCR using ABI PRISM 7700 Sequence Detection System (Applied Biosystems).
T276 48637-48774 Sentence denotes Foxp3 and HTLV-I Tax mRNA expression was quantified by real-time PCR using ABI PRISM 7700 Sequence Detection System (Applied Biosystems).
T20634 48775-48949 Sentence denotes The normalized values in each sample were calculated as the relative quantity of Foxp3 or HTLV-I Tax mRNA expression divided by the relative quantity of HPRT mRNA expression.
T277 48775-48949 Sentence denotes The normalized values in each sample were calculated as the relative quantity of Foxp3 or HTLV-I Tax mRNA expression divided by the relative quantity of HPRT mRNA expression.
T20635 48950-49099 Sentence denotes The values were calculated by the following formula: normalized Foxp3 or HTLV-I Tax expression = 2Ct value of HPRT – Ct value of Foxp3 or HTLV-I Tax.
T278 48950-49099 Sentence denotes The values were calculated by the following formula: normalized Foxp3 or HTLV-I Tax expression = 2Ct value of HPRT – Ct value of Foxp3 or HTLV-I Tax.
T21163 49101-49115 Sentence denotes Real-time PCR.
T279 49101-49115 Sentence denotes Real-time PCR.
T21164 49116-49215 Sentence denotes Real-time PCR analysis of HTLV-I (Tax) proviral load was performed as previously described [47,50].
T280 49116-49215 Sentence denotes Real-time PCR analysis of HTLV-I (Tax) proviral load was performed as previously described [47,50].
T21165 49216-49401 Sentence denotes DNA was extracted from 1 × 106 cells using Puregene DNA Isolation Kit (Gentra, Minneapolis, Minnesota, United States), and 100 ng of the sample DNA solution was analyzed by this system.
T281 49216-49401 Sentence denotes DNA was extracted from 1 × 106 cells using Puregene DNA Isolation Kit (Gentra, Minneapolis, Minnesota, United States), and 100 ng of the sample DNA solution was analyzed by this system.
T21166 49402-49568 Sentence denotes The HTLV-I proviral DNA load was calculated by the following formula: copy number of HTLV-I (pX) per 100 cells = (copy number of pX)/(copy number of β-actin/2) × 100.
T282 49402-49568 Sentence denotes The HTLV-I proviral DNA load was calculated by the following formula: copy number of HTLV-I (pX) per 100 cells = (copy number of pX)/(copy number of β-actin/2) × 100.
T21405 49570-49592 Sentence denotes Western blot analysis.
T283 49570-49592 Sentence denotes Western blot analysis.
T21406 49593-49815 Sentence denotes HEK 293T cells were plated at a density of 5 × 105 cells/well in six-well culture plates (BD Biosciences) 1 d prior to transfection with the appropriate plasmid DNA (2 μg total) using FuGene 6 transfection reagent (Roche).
T284 49593-49815 Sentence denotes HEK 293T cells were plated at a density of 5 × 105 cells/well in six-well culture plates (BD Biosciences) 1 d prior to transfection with the appropriate plasmid DNA (2 μg total) using FuGene 6 transfection reagent (Roche).
T21407 49816-50062 Sentence denotes Cells were harvested 24 h posttransfection for whole-cell lysates in RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Igepal (NP-40), 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 1× Complete Mini Protease Inhibitor [Roche]).
T285 49816-50062 Sentence denotes Cells were harvested 24 h posttransfection for whole-cell lysates in RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Igepal (NP-40), 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 1× Complete Mini Protease Inhibitor [Roche]).
T21408 50063-50319 Sentence denotes Protein concentration was determined by Lowry assay (Bio-Rad, Hercules, California, United States) and colorimetric reactions were read using a VersaMax microplate reader (Molecular Devices, Sunnyvale, California, United States) at an absorbance of 750 nm.
T286 50063-50319 Sentence denotes Protein concentration was determined by Lowry assay (Bio-Rad, Hercules, California, United States) and colorimetric reactions were read using a VersaMax microplate reader (Molecular Devices, Sunnyvale, California, United States) at an absorbance of 750 nm.
T21409 50320-50524 Sentence denotes Size fractionation was performed on 20 μg of protein/sample by SDS-PAGE, and the protein was transferred to nitrocellulose or PVDF membranes and subjected to immunoblotting using the indicated antibodies.
T287 50320-50524 Sentence denotes Size fractionation was performed on 20 μg of protein/sample by SDS-PAGE, and the protein was transferred to nitrocellulose or PVDF membranes and subjected to immunoblotting using the indicated antibodies.
T21410 50525-50758 Sentence denotes Foxp3 was detected using rabbit anti-human Foxp3 polyclonal antibody (ab4728 or ab10563; Abcam, Cambridge, United Kingdom) and anti-rabbit IgG-HRP secondary antibody (Cell Signaling Technology, Beverly, Massachusetts, United States).
T288 50525-50758 Sentence denotes Foxp3 was detected using rabbit anti-human Foxp3 polyclonal antibody (ab4728 or ab10563; Abcam, Cambridge, United Kingdom) and anti-rabbit IgG-HRP secondary antibody (Cell Signaling Technology, Beverly, Massachusetts, United States).
T21411 50759-50901 Sentence denotes NF-κB p65 and CREB-1 were detected using rabbit anti-human polyclonal (p65) or monoclonal antibody (CREB-1; 48H2) (Cell Signaling Technology).
T289 50759-50901 Sentence denotes NF-κB p65 and CREB-1 were detected using rabbit anti-human polyclonal (p65) or monoclonal antibody (CREB-1; 48H2) (Cell Signaling Technology).
T21412 50902-51011 Sentence denotes Beta-actin was detected using a mouse monoclonal antibody (AC-15; Sigma, St. Louis, Missouri, United States).
T290 50902-51011 Sentence denotes Beta-actin was detected using a mouse monoclonal antibody (AC-15; Sigma, St. Louis, Missouri, United States).
T21413 51012-51340 Sentence denotes For coimmunoprecipitation analysis, cell lysates were precleared with 30 μl of protein A/G plus-agarose beads (Santa Cruz Biotechnology, Santa Cruz, California, United States) and then incubated with mouse monoclonal anti-HA antibody (6E2; 1:100; Cell Signaling Technology) and 30 μl of protein A/G plus-agarose beads overnight.
T291 51012-51340 Sentence denotes For coimmunoprecipitation analysis, cell lysates were precleared with 30 μl of protein A/G plus-agarose beads (Santa Cruz Biotechnology, Santa Cruz, California, United States) and then incubated with mouse monoclonal anti-HA antibody (6E2; 1:100; Cell Signaling Technology) and 30 μl of protein A/G plus-agarose beads overnight.
T21414 51341-51469 Sentence denotes The immunoprecipitates were washed four times with RIPA buffer, resuspended in SDS sample buffer, and heated at 95 °C for 5 min.
T292 51341-51469 Sentence denotes The immunoprecipitates were washed four times with RIPA buffer, resuspended in SDS sample buffer, and heated at 95 °C for 5 min.
T21415 51470-51536 Sentence denotes Proteins were then treated as described for Western blot analysis.
T293 51470-51536 Sentence denotes Proteins were then treated as described for Western blot analysis.
T22264 51538-51591 Sentence denotes Transcription factor DNA-binding analysis (TF-ELISA).
T294 51538-51591 Sentence denotes Transcription factor DNA-binding analysis (TF-ELISA).
T22265 51592-51753 Sentence denotes CREB-1 and ATF-2 DNA-binding activity was analyzed with the TransFactor Profiling (Inflammation 1) Kit (BD Biosciences) according to the manufacturer's protocol.
T295 51592-51753 Sentence denotes CREB-1 and ATF-2 DNA-binding activity was analyzed with the TransFactor Profiling (Inflammation 1) Kit (BD Biosciences) according to the manufacturer's protocol.
T22266 51754-51994 Sentence denotes Nuclear extracts were prepared from HEK 293T cells transfected with a control vector (EGFP) or Foxp3 expression vector (1,000 ng) in the presence or absence of forskolin (10 μM for 4 h) using the TransFactor Extraction Kit (BD Biosciences).
T296 51754-51994 Sentence denotes Nuclear extracts were prepared from HEK 293T cells transfected with a control vector (EGFP) or Foxp3 expression vector (1,000 ng) in the presence or absence of forskolin (10 μM for 4 h) using the TransFactor Extraction Kit (BD Biosciences).
T22267 51995-52084 Sentence denotes Protein concentration was determined using a Biophotometer (Eppendorf, Hamburg, Germany).
T297 51995-52084 Sentence denotes Protein concentration was determined using a Biophotometer (Eppendorf, Hamburg, Germany).
T22268 52085-52262 Sentence denotes Nuclear extracts (20 μg) were incubated in preblocked wells containing plate-bound double-stranded oligonucleotides corresponding to an ATF/CREB consensus sequence (…TGACATCA…).
T298 52085-52262 Sentence denotes Nuclear extracts (20 μg) were incubated in preblocked wells containing plate-bound double-stranded oligonucleotides corresponding to an ATF/CREB consensus sequence (…TGACATCA…).
T22269 52263-52443 Sentence denotes Wells were washed, incubated with the appropriate primary antibody, washed, incubated with secondary antibody (HRP-labeled), washed again, and finally developed with TMB substrate.
T299 52263-52443 Sentence denotes Wells were washed, incubated with the appropriate primary antibody, washed, incubated with secondary antibody (HRP-labeled), washed again, and finally developed with TMB substrate.
T22270 52444-52559 Sentence denotes Colorimetric reactions were read using a VersaMax microplate reader (Molecular Devices) at an absorbance of 655 nm.
T300 52444-52559 Sentence denotes Colorimetric reactions were read using a VersaMax microplate reader (Molecular Devices) at an absorbance of 655 nm.
T22718 52561-52614 Sentence denotes Flow cytometric analysis of Foxp3 protein expression.
T301 52561-52614 Sentence denotes Flow cytometric analysis of Foxp3 protein expression.
T22719 52615-52748 Sentence denotes Cryopreserved PBMCs from HAM/TSP patients or HTLV-I-infected ACs were thawed and washed with FACS buffer (1× PBS, 0.1% NaN3, 5% FBS).
T302 52615-52748 Sentence denotes Cryopreserved PBMCs from HAM/TSP patients or HTLV-I-infected ACs were thawed and washed with FACS buffer (1× PBS, 0.1% NaN3, 5% FBS).
T22720 52749-52834 Sentence denotes Cells (1.5 × 106) were fixed by sequential formaldehyde/methanol fixation as follows.
T303 52749-52834 Sentence denotes Cells (1.5 × 106) were fixed by sequential formaldehyde/methanol fixation as follows.
T304 52835-53076 Sentence denotes Cells were carefully resuspended in FACS buffer and fixed with 100 μl of reagent A (Fix & Perm kit; Caltag Laboratories, Burlingame, California, United States) at room temperature for 3 min followed by 2 ml of 70% methanol for 5 min at 4 °C.
T22721 52835-53318 Sentence denotes Cells were carefully resuspended in FACS buffer and fixed with 100 μl of reagent A (Fix & Perm kit; Caltag Laboratories, Burlingame, California, United States) at room temperature for 3 min followed by 2 ml of 70% methanol for 5 min at 4 °C. Cells were washed twice and permeabilized with 100 μl of reagent B (Fix & Perm kit) and stained for intracellular Foxp3 with mouse anti-human Foxp3 monoclonal antibody (0.5 μg of ab22510; Abcam) or the appropriate isotype control for 30 min.
T305 53077-53318 Sentence denotes Cells were washed twice and permeabilized with 100 μl of reagent B (Fix & Perm kit) and stained for intracellular Foxp3 with mouse anti-human Foxp3 monoclonal antibody (0.5 μg of ab22510; Abcam) or the appropriate isotype control for 30 min.
T22722 53319-53480 Sentence denotes Cells were washed twice and stained with Cy5-conjugated goat anti-mouse immunoglobulin F(ab′)2 secondary antibody (Caltag Laboratories) for an additional 30 min.
T306 53319-53480 Sentence denotes Cells were washed twice and stained with Cy5-conjugated goat anti-mouse immunoglobulin F(ab′)2 secondary antibody (Caltag Laboratories) for an additional 30 min.
T22723 53481-53627 Sentence denotes Cells were washed twice and stained for surface CD4 expression with PE-labeled anti-CD4 (BD) and CD25 expression with FITC-labeled anti-CD25 (BD).
T307 53481-53627 Sentence denotes Cells were washed twice and stained for surface CD4 expression with PE-labeled anti-CD4 (BD) and CD25 expression with FITC-labeled anti-CD25 (BD).
T22724 53628-53687 Sentence denotes Cells were washed twice and analyzed on a FACSCalibur (BD).
T308 53628-53687 Sentence denotes Cells were washed twice and analyzed on a FACSCalibur (BD).
T22725 53688-53773 Sentence denotes Data analysis was performed using FlowJo (Tree Star, Ashland, Oregon, United States).
T309 53688-53773 Sentence denotes Data analysis was performed using FlowJo (Tree Star, Ashland, Oregon, United States).
T23276 53775-53796 Sentence denotes Statistical analyses.
T310 53775-53796 Sentence denotes Statistical analyses.
T23277 53797-53887 Sentence denotes The Mann-Whitney U test was used to compare the data between patients with HAM/TSP and AC.
T311 53797-53887 Sentence denotes The Mann-Whitney U test was used to compare the data between patients with HAM/TSP and AC.