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Non-Canonical Notch Signaling Drives Activation and Differentiation of Peripheral CD4(+) T Cells.

Dongre A, Surampudi L, Lawlor RG, Fauq AH, Miele L, Golde TE, Minter LM, Osborne BA - Front Immunol (2014)

Bottom Line: Furthermore, differentiation to TH1 and iTreg lineages although Notch dependent, is RBP-Jκ independent.Our striking observations demonstrate that many of the cell-intrinsic functions of Notch occur independently of RBP-Jκ.This reveals a previously unknown, novel role of non-canonical Notch signaling in regulating peripheral T cell responses.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular and Cellular Biology, University of Massachusetts Amherst , Amherst, MA , USA ; Department of Veterinary and Animal Sciences, University of Massachusetts Amherst , Amherst, MA , USA.

ABSTRACT
Cleavage of the Notch receptor via a γ-secretase, results in the release of the active intra-cellular domain of Notch that migrates to the nucleus and interacts with RBP-Jκ, resulting in the activation of downstream target genes. This canonical Notch signaling pathway has been documented to influence T cell development and function. However, the mechanistic details underlying this process remain obscure. In addition to RBP-Jκ, the intra-cellular domain of Notch also interacts with other proteins in the cytoplasm and nucleus, giving rise to the possibility of an alternate, RBP-Jκ independent Notch pathway. However, the contribution of such RBP-Jκ independent, "non-canonical" Notch signaling in regulating peripheral T cell responses is unknown. In this report, we specifically demonstrate the requirement of Notch1 for regulating signal strength and signaling events distal to the T cell receptor in peripheral CD4(+) T cells. By using mice with a conditional deletion in Notch1 or RBP-Jκ, we show that Notch1 regulates activation and proliferation of CD4(+) T cells independently of RBP-Jκ. Furthermore, differentiation to TH1 and iTreg lineages although Notch dependent, is RBP-Jκ independent. Our striking observations demonstrate that many of the cell-intrinsic functions of Notch occur independently of RBP-Jκ. Such non-canonical regulation of these processes likely occurs through NF-κ B. This reveals a previously unknown, novel role of non-canonical Notch signaling in regulating peripheral T cell responses.

No MeSH data available.


Related in: MedlinePlus

Notch1 is required for TH1 differentiation and production of iTregs in vitro. CD4+ T cells from control and cN1KO mice were differentiated under TH1, TH2, or iTreg inducing conditions for 3 days followed by re-stimulation with plate-bound anti-CD3ε. Cells were analyzed by flow cytometry. Supernatants were used for ELISA. (A) Dot plots obtained from flow cytometry showing intra-cellular staining for IFN-γ and IL-4. Numbers in each quadrant represent percentage of cells. Data represent three to five independent experiments. (B) Percentage and absolute cell numbers of IFN-γ or IL-4 positive cells determined by flow cytometry n = 3–5. (C) IFN-γ and IL-4 production under TH1 and TH2 conditions, respectively, determined by an ELISA. Each data point represents one animal. (D,G) Dot plots showing intra-cellular staining for (D) T-bet and N1IC under TH1 conditions (G) or GATA3 and N1IC under TH2 conditions. Numbers in each quadrant represent percentage of cells. Data represent three to five independent experiments. (E,H) Percentage and absolute numbers of double positive cells as determined by flow cytometry n = 3–5. (F) Histograms for T-bet and (I) GATA3 expression under TH1 and TH2 conditions, respectively. Data represent three to five independent experiments. (J) Dot plots for CD25+ and FoxP3+ cells. Numbers in each quadrant represent percentage of cells. Data represent three independent experiments. (K) Percentage and absolute cell numbers of double positive cells determined by flow cytometry n = 3. Data represent mean ± SEM. *p < 0.05, **p < 0.005, and ***p < 0.001. ns, not significant.
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Figure 4: Notch1 is required for TH1 differentiation and production of iTregs in vitro. CD4+ T cells from control and cN1KO mice were differentiated under TH1, TH2, or iTreg inducing conditions for 3 days followed by re-stimulation with plate-bound anti-CD3ε. Cells were analyzed by flow cytometry. Supernatants were used for ELISA. (A) Dot plots obtained from flow cytometry showing intra-cellular staining for IFN-γ and IL-4. Numbers in each quadrant represent percentage of cells. Data represent three to five independent experiments. (B) Percentage and absolute cell numbers of IFN-γ or IL-4 positive cells determined by flow cytometry n = 3–5. (C) IFN-γ and IL-4 production under TH1 and TH2 conditions, respectively, determined by an ELISA. Each data point represents one animal. (D,G) Dot plots showing intra-cellular staining for (D) T-bet and N1IC under TH1 conditions (G) or GATA3 and N1IC under TH2 conditions. Numbers in each quadrant represent percentage of cells. Data represent three to five independent experiments. (E,H) Percentage and absolute numbers of double positive cells as determined by flow cytometry n = 3–5. (F) Histograms for T-bet and (I) GATA3 expression under TH1 and TH2 conditions, respectively. Data represent three to five independent experiments. (J) Dot plots for CD25+ and FoxP3+ cells. Numbers in each quadrant represent percentage of cells. Data represent three independent experiments. (K) Percentage and absolute cell numbers of double positive cells determined by flow cytometry n = 3. Data represent mean ± SEM. *p < 0.05, **p < 0.005, and ***p < 0.001. ns, not significant.

Mentions: Although Notch has been implicated in influencing differentiation of T cells, the precise role of Notch1 in favoring TH1 versus TH2 lineage decisions is unclear. While some studies have shown that inhibiting Notch using GSIs diminishes the ability of CD4+ T cells to adopt a TH1 fate, other studies using different strategies to inhibit Notch signaling have reported conflicting observations (21, 22, 33). To determine the precise role of Notch1 in helper T cell differentiation, CD4+ T cells from control or cN1KO mice were polarized in vitro to TH1, TH2, or iTreg lineages. Absence of Notch1 impaired TH1 differentiation in vitro. CD4+ T cells from cN1KO mice had significantly fewer cells that stained positive for intra-cellular IFN-γ (Figures 4A,B). Secreted IFN-γ was also reduced significantly in the absence of Notch1 (Figure 4C). This decrease was accompanied by a reduction in the amount of the master TH1 transcription factor T-bet (Figures 4D–F). In contrast, no marked effect was observed in TH2 differentiation. Although, the amount of GATA3 was reduced in the absence of Notch1 (Figures 4G–I), both control as well as cN1KO mice had similar percentages and absolute number of CD4+ T cells that were positive for intra-cellular IL-4 (Figures 4A,B) and expressed comparable levels of secreted IL-4 (Figure 4C). Whether CD4+ T cells from cN1KO mice proliferated differently under different polarizing conditions, was determined by 3H-thymidine uptake under non-polarized (NP), TH1 and TH2 conditions. Proliferative capability of CD4+ T cells from cN1KO mice was the same under different polarizing conditions (Figure S3C in Supplementary Material), despite of differences in cytokine secretion. In addition to TH1, Notch1 deficiency significantly reduced induced T-reg populations as observed by a significant decrease in the frequency of CD25+FoxP3+ cells in cN1KO animals (Figures 4J,K). These results show that Notch1 is required for TH1 and iTreg differentiation but is dispensable for TH2 cell fate acquisition in vitro. Furthermore, these data demonstrate an intrinsic role of Notch1 in regulating helper T cell differentiation.


Non-Canonical Notch Signaling Drives Activation and Differentiation of Peripheral CD4(+) T Cells.

Dongre A, Surampudi L, Lawlor RG, Fauq AH, Miele L, Golde TE, Minter LM, Osborne BA - Front Immunol (2014)

Notch1 is required for TH1 differentiation and production of iTregs in vitro. CD4+ T cells from control and cN1KO mice were differentiated under TH1, TH2, or iTreg inducing conditions for 3 days followed by re-stimulation with plate-bound anti-CD3ε. Cells were analyzed by flow cytometry. Supernatants were used for ELISA. (A) Dot plots obtained from flow cytometry showing intra-cellular staining for IFN-γ and IL-4. Numbers in each quadrant represent percentage of cells. Data represent three to five independent experiments. (B) Percentage and absolute cell numbers of IFN-γ or IL-4 positive cells determined by flow cytometry n = 3–5. (C) IFN-γ and IL-4 production under TH1 and TH2 conditions, respectively, determined by an ELISA. Each data point represents one animal. (D,G) Dot plots showing intra-cellular staining for (D) T-bet and N1IC under TH1 conditions (G) or GATA3 and N1IC under TH2 conditions. Numbers in each quadrant represent percentage of cells. Data represent three to five independent experiments. (E,H) Percentage and absolute numbers of double positive cells as determined by flow cytometry n = 3–5. (F) Histograms for T-bet and (I) GATA3 expression under TH1 and TH2 conditions, respectively. Data represent three to five independent experiments. (J) Dot plots for CD25+ and FoxP3+ cells. Numbers in each quadrant represent percentage of cells. Data represent three independent experiments. (K) Percentage and absolute cell numbers of double positive cells determined by flow cytometry n = 3. Data represent mean ± SEM. *p < 0.05, **p < 0.005, and ***p < 0.001. ns, not significant.
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Figure 4: Notch1 is required for TH1 differentiation and production of iTregs in vitro. CD4+ T cells from control and cN1KO mice were differentiated under TH1, TH2, or iTreg inducing conditions for 3 days followed by re-stimulation with plate-bound anti-CD3ε. Cells were analyzed by flow cytometry. Supernatants were used for ELISA. (A) Dot plots obtained from flow cytometry showing intra-cellular staining for IFN-γ and IL-4. Numbers in each quadrant represent percentage of cells. Data represent three to five independent experiments. (B) Percentage and absolute cell numbers of IFN-γ or IL-4 positive cells determined by flow cytometry n = 3–5. (C) IFN-γ and IL-4 production under TH1 and TH2 conditions, respectively, determined by an ELISA. Each data point represents one animal. (D,G) Dot plots showing intra-cellular staining for (D) T-bet and N1IC under TH1 conditions (G) or GATA3 and N1IC under TH2 conditions. Numbers in each quadrant represent percentage of cells. Data represent three to five independent experiments. (E,H) Percentage and absolute numbers of double positive cells as determined by flow cytometry n = 3–5. (F) Histograms for T-bet and (I) GATA3 expression under TH1 and TH2 conditions, respectively. Data represent three to five independent experiments. (J) Dot plots for CD25+ and FoxP3+ cells. Numbers in each quadrant represent percentage of cells. Data represent three independent experiments. (K) Percentage and absolute cell numbers of double positive cells determined by flow cytometry n = 3. Data represent mean ± SEM. *p < 0.05, **p < 0.005, and ***p < 0.001. ns, not significant.
Mentions: Although Notch has been implicated in influencing differentiation of T cells, the precise role of Notch1 in favoring TH1 versus TH2 lineage decisions is unclear. While some studies have shown that inhibiting Notch using GSIs diminishes the ability of CD4+ T cells to adopt a TH1 fate, other studies using different strategies to inhibit Notch signaling have reported conflicting observations (21, 22, 33). To determine the precise role of Notch1 in helper T cell differentiation, CD4+ T cells from control or cN1KO mice were polarized in vitro to TH1, TH2, or iTreg lineages. Absence of Notch1 impaired TH1 differentiation in vitro. CD4+ T cells from cN1KO mice had significantly fewer cells that stained positive for intra-cellular IFN-γ (Figures 4A,B). Secreted IFN-γ was also reduced significantly in the absence of Notch1 (Figure 4C). This decrease was accompanied by a reduction in the amount of the master TH1 transcription factor T-bet (Figures 4D–F). In contrast, no marked effect was observed in TH2 differentiation. Although, the amount of GATA3 was reduced in the absence of Notch1 (Figures 4G–I), both control as well as cN1KO mice had similar percentages and absolute number of CD4+ T cells that were positive for intra-cellular IL-4 (Figures 4A,B) and expressed comparable levels of secreted IL-4 (Figure 4C). Whether CD4+ T cells from cN1KO mice proliferated differently under different polarizing conditions, was determined by 3H-thymidine uptake under non-polarized (NP), TH1 and TH2 conditions. Proliferative capability of CD4+ T cells from cN1KO mice was the same under different polarizing conditions (Figure S3C in Supplementary Material), despite of differences in cytokine secretion. In addition to TH1, Notch1 deficiency significantly reduced induced T-reg populations as observed by a significant decrease in the frequency of CD25+FoxP3+ cells in cN1KO animals (Figures 4J,K). These results show that Notch1 is required for TH1 and iTreg differentiation but is dispensable for TH2 cell fate acquisition in vitro. Furthermore, these data demonstrate an intrinsic role of Notch1 in regulating helper T cell differentiation.

Bottom Line: Furthermore, differentiation to TH1 and iTreg lineages although Notch dependent, is RBP-Jκ independent.Our striking observations demonstrate that many of the cell-intrinsic functions of Notch occur independently of RBP-Jκ.This reveals a previously unknown, novel role of non-canonical Notch signaling in regulating peripheral T cell responses.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular and Cellular Biology, University of Massachusetts Amherst , Amherst, MA , USA ; Department of Veterinary and Animal Sciences, University of Massachusetts Amherst , Amherst, MA , USA.

ABSTRACT
Cleavage of the Notch receptor via a γ-secretase, results in the release of the active intra-cellular domain of Notch that migrates to the nucleus and interacts with RBP-Jκ, resulting in the activation of downstream target genes. This canonical Notch signaling pathway has been documented to influence T cell development and function. However, the mechanistic details underlying this process remain obscure. In addition to RBP-Jκ, the intra-cellular domain of Notch also interacts with other proteins in the cytoplasm and nucleus, giving rise to the possibility of an alternate, RBP-Jκ independent Notch pathway. However, the contribution of such RBP-Jκ independent, "non-canonical" Notch signaling in regulating peripheral T cell responses is unknown. In this report, we specifically demonstrate the requirement of Notch1 for regulating signal strength and signaling events distal to the T cell receptor in peripheral CD4(+) T cells. By using mice with a conditional deletion in Notch1 or RBP-Jκ, we show that Notch1 regulates activation and proliferation of CD4(+) T cells independently of RBP-Jκ. Furthermore, differentiation to TH1 and iTreg lineages although Notch dependent, is RBP-Jκ independent. Our striking observations demonstrate that many of the cell-intrinsic functions of Notch occur independently of RBP-Jκ. Such non-canonical regulation of these processes likely occurs through NF-κ B. This reveals a previously unknown, novel role of non-canonical Notch signaling in regulating peripheral T cell responses.

No MeSH data available.


Related in: MedlinePlus