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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

Notch is required for distal TCR signaling events. Splenocytes from C57BL/6J mice were pretreated with DMSO or GSI and stimulated with plate-bound anti-CD3ε and anti-CD28 for the indicated times. Cells were harvested and analyzed by flow cytometry after gating on CD4+ T cells. Mean fluorescent intensity (MFI) values were plotted for (A) N1IC, (C) CD25, and (D) CD69. (E) IL-2 and (F) IFN-γ ELISA from supernatants of cells stimulated as described. (B) Western Blot for phosphorylated and Total Zap70. Splenocytes from C57BL/6J mice were pretreated with DMSO or GSI and stimulated with anti-CD3ε and anti-CD28 for the indicated time points. Whole cell lysates were made at for each time point and analyzed by Western Blotting. Data are representative of three independent experiments. Data represent the mean ± SEM n = 3. *p < 0.05, **p < 0.005, and ***p < 0.001.
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Figure 1: Notch is required for distal TCR signaling events. Splenocytes from C57BL/6J mice were pretreated with DMSO or GSI and stimulated with plate-bound anti-CD3ε and anti-CD28 for the indicated times. Cells were harvested and analyzed by flow cytometry after gating on CD4+ T cells. Mean fluorescent intensity (MFI) values were plotted for (A) N1IC, (C) CD25, and (D) CD69. (E) IL-2 and (F) IFN-γ ELISA from supernatants of cells stimulated as described. (B) Western Blot for phosphorylated and Total Zap70. Splenocytes from C57BL/6J mice were pretreated with DMSO or GSI and stimulated with anti-CD3ε and anti-CD28 for the indicated time points. Whole cell lysates were made at for each time point and analyzed by Western Blotting. Data are representative of three independent experiments. Data represent the mean ± SEM n = 3. *p < 0.05, **p < 0.005, and ***p < 0.001.

Mentions: Activation of T cells via the TCR accompanied by co-stimulation leads to the production of the active, intra-cellular domain of Notch1 (N1IC) and its inhibition via γ-secretase inhibitors (GSI), decreases activation, and proliferation of T cells (15, 16). While Notch has been demonstrated to influence T cell activation, precisely where Notch exerts its influence downstream of the TCR is obscure. Furthermore, whether Notch affects signaling events proximal or distal to the TCR is unclear. To address these questions, we determined the kinetics of Notch activation over time and asked how inhibition of Notch activation via GSI treatment influences downstream TCR signaling events at early and late time points after stimulation. We detected N1IC in CD4+ T cells activated with plate-bound anti-CD3ε and anti-CD28 4 h after activation and the amount of N1IC increased over time (Figure 1A). This increase was abrogated after GSI treatment (Figure 1A). Inhibition of Notch activation did not alter proximal signaling events as evidenced by intact phosphorylation of Zap 70 even in GSI treated cells (Figure 1B). On the contrary, GSI treatment significantly decreased distal TCR signaling events such as the expression of activation markers CD25, CD69, IL-2, and IFN-γ (Figures 1C–F). This decrease was most prominent close to 48 h after TCR stimulation suggesting that Notch activation is critical for signaling events distal to the TCR, but could be dispensable for proximal events. Since we observed that activating cells via the TCR also triggered the activation of Notch, we determined whether CD4+ T cells themselves express Notch ligands. We observed that surface expression of DLL1 and Jagged1 is minimal upto 6 h after activation and peaks at distal time points (Figures S1A,B in Supplementary Material). Based on this observation, we determined whether stimulating T cells in the presence of recombinant Notch ligands alters the generation of N1IC downstream of the TCR. Activation in the presence of recombinant DLL1 or Jagged1 did not alter the generation of N1IC nor did it impact T cell activation (Figures S1C–H in Supplementary Material). Finally, stimulating T cells via the TCR in the presence of DLL1 or Jagged1 did not significantly influence the acquisition of helper T cell fate, although DLL1 enhanced IFN-γ production under pre-existing TH1 conditions (Figures S1I,K in Supplementary Material). Collectively, these data show that N1IC is generated in CD4+ T cells after stimulating via the TCR and influences distal TCR signaling events.


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)

Notch is required for distal TCR signaling events. Splenocytes from C57BL/6J mice were pretreated with DMSO or GSI and stimulated with plate-bound anti-CD3ε and anti-CD28 for the indicated times. Cells were harvested and analyzed by flow cytometry after gating on CD4+ T cells. Mean fluorescent intensity (MFI) values were plotted for (A) N1IC, (C) CD25, and (D) CD69. (E) IL-2 and (F) IFN-γ ELISA from supernatants of cells stimulated as described. (B) Western Blot for phosphorylated and Total Zap70. Splenocytes from C57BL/6J mice were pretreated with DMSO or GSI and stimulated with anti-CD3ε and anti-CD28 for the indicated time points. Whole cell lysates were made at for each time point and analyzed by Western Blotting. Data are representative of three independent experiments. Data represent the mean ± SEM n = 3. *p < 0.05, **p < 0.005, and ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921607&req=5

Figure 1: Notch is required for distal TCR signaling events. Splenocytes from C57BL/6J mice were pretreated with DMSO or GSI and stimulated with plate-bound anti-CD3ε and anti-CD28 for the indicated times. Cells were harvested and analyzed by flow cytometry after gating on CD4+ T cells. Mean fluorescent intensity (MFI) values were plotted for (A) N1IC, (C) CD25, and (D) CD69. (E) IL-2 and (F) IFN-γ ELISA from supernatants of cells stimulated as described. (B) Western Blot for phosphorylated and Total Zap70. Splenocytes from C57BL/6J mice were pretreated with DMSO or GSI and stimulated with anti-CD3ε and anti-CD28 for the indicated time points. Whole cell lysates were made at for each time point and analyzed by Western Blotting. Data are representative of three independent experiments. Data represent the mean ± SEM n = 3. *p < 0.05, **p < 0.005, and ***p < 0.001.
Mentions: Activation of T cells via the TCR accompanied by co-stimulation leads to the production of the active, intra-cellular domain of Notch1 (N1IC) and its inhibition via γ-secretase inhibitors (GSI), decreases activation, and proliferation of T cells (15, 16). While Notch has been demonstrated to influence T cell activation, precisely where Notch exerts its influence downstream of the TCR is obscure. Furthermore, whether Notch affects signaling events proximal or distal to the TCR is unclear. To address these questions, we determined the kinetics of Notch activation over time and asked how inhibition of Notch activation via GSI treatment influences downstream TCR signaling events at early and late time points after stimulation. We detected N1IC in CD4+ T cells activated with plate-bound anti-CD3ε and anti-CD28 4 h after activation and the amount of N1IC increased over time (Figure 1A). This increase was abrogated after GSI treatment (Figure 1A). Inhibition of Notch activation did not alter proximal signaling events as evidenced by intact phosphorylation of Zap 70 even in GSI treated cells (Figure 1B). On the contrary, GSI treatment significantly decreased distal TCR signaling events such as the expression of activation markers CD25, CD69, IL-2, and IFN-γ (Figures 1C–F). This decrease was most prominent close to 48 h after TCR stimulation suggesting that Notch activation is critical for signaling events distal to the TCR, but could be dispensable for proximal events. Since we observed that activating cells via the TCR also triggered the activation of Notch, we determined whether CD4+ T cells themselves express Notch ligands. We observed that surface expression of DLL1 and Jagged1 is minimal upto 6 h after activation and peaks at distal time points (Figures S1A,B in Supplementary Material). Based on this observation, we determined whether stimulating T cells in the presence of recombinant Notch ligands alters the generation of N1IC downstream of the TCR. Activation in the presence of recombinant DLL1 or Jagged1 did not alter the generation of N1IC nor did it impact T cell activation (Figures S1C–H in Supplementary Material). Finally, stimulating T cells via the TCR in the presence of DLL1 or Jagged1 did not significantly influence the acquisition of helper T cell fate, although DLL1 enhanced IFN-γ production under pre-existing TH1 conditions (Figures S1I,K in Supplementary Material). Collectively, these data show that N1IC is generated in CD4+ T cells after stimulating via the TCR and influences distal TCR signaling events.

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