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Runx3 and T-box proteins cooperate to establish the transcriptional program of effector CTLs.

Cruz-Guilloty F, Pipkin ME, Djuretic IM, Levanon D, Lotem J, Lichtenheld MG, Groner Y, Rao A - J. Exp. Med. (2009)

Bottom Line: We show that effector CTL differentiation occurs in two sequential phases in vitro, characterized by early induction of T-bet and late induction of Eomesodermin (Eomes), T-box transcription factors that regulate the early and late phases of interferon (IFN) gamma expression, respectively.Runx3 regulates Eomes expression as well as expression of three cardinal markers of the effector CTL program: IFN-gamma, perforin, and granzyme B.Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs.

View Article: PubMed Central - PubMed

Affiliation: Harvard Medical School and the Immune Disease Institute, Boston, MA 02115, USA.

ABSTRACT
Activation of naive CD8(+) T cells with antigen induces their differentiation into effector cytolytic T lymphocytes (CTLs). CTLs lyse infected or aberrant target cells by exocytosis of lytic granules containing the pore-forming protein perforin and a family of proteases termed granzymes. We show that effector CTL differentiation occurs in two sequential phases in vitro, characterized by early induction of T-bet and late induction of Eomesodermin (Eomes), T-box transcription factors that regulate the early and late phases of interferon (IFN) gamma expression, respectively. In addition, we demonstrate a critical role for the transcription factor Runx3 in CTL differentiation. Runx3 regulates Eomes expression as well as expression of three cardinal markers of the effector CTL program: IFN-gamma, perforin, and granzyme B. Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs.

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Kinetics of gene expression during CD8+ T cell differentiation. (A) Kinetics of Prf1, Gzmb, Tbx21 (T-bet), and Eomes mRNA expression in differentiating P14 CD8+ T cells analyzed by Northern blotting. RNA from day 7 Th1 cells was used as a control. Sizes of mRNA transcripts are indicated. (B) Quantification of relative mRNA amounts by phosphorimager analysis. (C) Kinetics of protein expression in differentiating P14 CD8+ T cells analyzed by immunoblotting. Sizes of protein bands are indicated. (D) Relative protein amounts quantified from the Western blots. (E) Intracellular staining for granzyme B, IFN-γ, and TNF. Granzyme B staining was specific relative to an isotype control (not depicted). Cells were restimulated with PMA and ionomycin for 4 h. (F) FACS-based assay to measure cytolytic activity of P14 CD8+ T cells against EL4 targets loaded with 0 (−) or 1 (+) μM Gp33 peptide (effector-to-target ratio = 5:1). Percentage of Annexin V+ (apoptotic) target cells in the CD8-negative EL4 target population (dot plots) was determined (histograms). Cytolytic activity was blocked by incubation with 2 mM EGTA (not depicted), confirming involvement of the granule exocytosis (perforin–granzyme B) pathway. Data are representative of at least five (A–E) or three (F) independent experiments.
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fig1: Kinetics of gene expression during CD8+ T cell differentiation. (A) Kinetics of Prf1, Gzmb, Tbx21 (T-bet), and Eomes mRNA expression in differentiating P14 CD8+ T cells analyzed by Northern blotting. RNA from day 7 Th1 cells was used as a control. Sizes of mRNA transcripts are indicated. (B) Quantification of relative mRNA amounts by phosphorimager analysis. (C) Kinetics of protein expression in differentiating P14 CD8+ T cells analyzed by immunoblotting. Sizes of protein bands are indicated. (D) Relative protein amounts quantified from the Western blots. (E) Intracellular staining for granzyme B, IFN-γ, and TNF. Granzyme B staining was specific relative to an isotype control (not depicted). Cells were restimulated with PMA and ionomycin for 4 h. (F) FACS-based assay to measure cytolytic activity of P14 CD8+ T cells against EL4 targets loaded with 0 (−) or 1 (+) μM Gp33 peptide (effector-to-target ratio = 5:1). Percentage of Annexin V+ (apoptotic) target cells in the CD8-negative EL4 target population (dot plots) was determined (histograms). Cytolytic activity was blocked by incubation with 2 mM EGTA (not depicted), confirming involvement of the granule exocytosis (perforin–granzyme B) pathway. Data are representative of at least five (A–E) or three (F) independent experiments.

Mentions: Our experiments revealed clear differences in the kinetics of perforin, granzyme B, and cytokine expression during CD8+ T cell activation (Fig. 1). Naive T cells showed detectable expression of perforin mRNA as well as perforin protein (Fig. 1, A–D). Relative to its expression in naive T cells, perforin (Prf1) mRNA expression did not increase appreciably at day 2 but showed a reproducible decrease at day 4, followed by robust reexpression between days 4 and 8 (Fig. 1, A–D). In contrast, granzyme B (Gzmb) mRNA was low or undetectable in naive T cells but was strongly up-regulated by day 2 after stimulation and increased progressively until day 6 (Fig. 1, A and B); similarly, granzyme B protein was expressed by day 4 and remained high until day 6 (Fig. 1 E). As expected, a small fraction of naive T cells expressed the cytokines IFN-γ and TNF in response to stimulation, and this capacity increased significantly in differentiated cells (Fig. 1 E; see also Fig. 2 A).


Runx3 and T-box proteins cooperate to establish the transcriptional program of effector CTLs.

Cruz-Guilloty F, Pipkin ME, Djuretic IM, Levanon D, Lotem J, Lichtenheld MG, Groner Y, Rao A - J. Exp. Med. (2009)

Kinetics of gene expression during CD8+ T cell differentiation. (A) Kinetics of Prf1, Gzmb, Tbx21 (T-bet), and Eomes mRNA expression in differentiating P14 CD8+ T cells analyzed by Northern blotting. RNA from day 7 Th1 cells was used as a control. Sizes of mRNA transcripts are indicated. (B) Quantification of relative mRNA amounts by phosphorimager analysis. (C) Kinetics of protein expression in differentiating P14 CD8+ T cells analyzed by immunoblotting. Sizes of protein bands are indicated. (D) Relative protein amounts quantified from the Western blots. (E) Intracellular staining for granzyme B, IFN-γ, and TNF. Granzyme B staining was specific relative to an isotype control (not depicted). Cells were restimulated with PMA and ionomycin for 4 h. (F) FACS-based assay to measure cytolytic activity of P14 CD8+ T cells against EL4 targets loaded with 0 (−) or 1 (+) μM Gp33 peptide (effector-to-target ratio = 5:1). Percentage of Annexin V+ (apoptotic) target cells in the CD8-negative EL4 target population (dot plots) was determined (histograms). Cytolytic activity was blocked by incubation with 2 mM EGTA (not depicted), confirming involvement of the granule exocytosis (perforin–granzyme B) pathway. Data are representative of at least five (A–E) or three (F) independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
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fig1: Kinetics of gene expression during CD8+ T cell differentiation. (A) Kinetics of Prf1, Gzmb, Tbx21 (T-bet), and Eomes mRNA expression in differentiating P14 CD8+ T cells analyzed by Northern blotting. RNA from day 7 Th1 cells was used as a control. Sizes of mRNA transcripts are indicated. (B) Quantification of relative mRNA amounts by phosphorimager analysis. (C) Kinetics of protein expression in differentiating P14 CD8+ T cells analyzed by immunoblotting. Sizes of protein bands are indicated. (D) Relative protein amounts quantified from the Western blots. (E) Intracellular staining for granzyme B, IFN-γ, and TNF. Granzyme B staining was specific relative to an isotype control (not depicted). Cells were restimulated with PMA and ionomycin for 4 h. (F) FACS-based assay to measure cytolytic activity of P14 CD8+ T cells against EL4 targets loaded with 0 (−) or 1 (+) μM Gp33 peptide (effector-to-target ratio = 5:1). Percentage of Annexin V+ (apoptotic) target cells in the CD8-negative EL4 target population (dot plots) was determined (histograms). Cytolytic activity was blocked by incubation with 2 mM EGTA (not depicted), confirming involvement of the granule exocytosis (perforin–granzyme B) pathway. Data are representative of at least five (A–E) or three (F) independent experiments.
Mentions: Our experiments revealed clear differences in the kinetics of perforin, granzyme B, and cytokine expression during CD8+ T cell activation (Fig. 1). Naive T cells showed detectable expression of perforin mRNA as well as perforin protein (Fig. 1, A–D). Relative to its expression in naive T cells, perforin (Prf1) mRNA expression did not increase appreciably at day 2 but showed a reproducible decrease at day 4, followed by robust reexpression between days 4 and 8 (Fig. 1, A–D). In contrast, granzyme B (Gzmb) mRNA was low or undetectable in naive T cells but was strongly up-regulated by day 2 after stimulation and increased progressively until day 6 (Fig. 1, A and B); similarly, granzyme B protein was expressed by day 4 and remained high until day 6 (Fig. 1 E). As expected, a small fraction of naive T cells expressed the cytokines IFN-γ and TNF in response to stimulation, and this capacity increased significantly in differentiated cells (Fig. 1 E; see also Fig. 2 A).

Bottom Line: We show that effector CTL differentiation occurs in two sequential phases in vitro, characterized by early induction of T-bet and late induction of Eomesodermin (Eomes), T-box transcription factors that regulate the early and late phases of interferon (IFN) gamma expression, respectively.Runx3 regulates Eomes expression as well as expression of three cardinal markers of the effector CTL program: IFN-gamma, perforin, and granzyme B.Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs.

View Article: PubMed Central - PubMed

Affiliation: Harvard Medical School and the Immune Disease Institute, Boston, MA 02115, USA.

ABSTRACT
Activation of naive CD8(+) T cells with antigen induces their differentiation into effector cytolytic T lymphocytes (CTLs). CTLs lyse infected or aberrant target cells by exocytosis of lytic granules containing the pore-forming protein perforin and a family of proteases termed granzymes. We show that effector CTL differentiation occurs in two sequential phases in vitro, characterized by early induction of T-bet and late induction of Eomesodermin (Eomes), T-box transcription factors that regulate the early and late phases of interferon (IFN) gamma expression, respectively. In addition, we demonstrate a critical role for the transcription factor Runx3 in CTL differentiation. Runx3 regulates Eomes expression as well as expression of three cardinal markers of the effector CTL program: IFN-gamma, perforin, and granzyme B. Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs.

Show MeSH
Related in: MedlinePlus