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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

Activation and Proliferation of CD4+ T cells is RBP-Jκ independent but Notch and NF-κB dependent. CD4+ T cells were isolated from cRBP-Jκ-KO mice, pretreated with DMSO, GSI, or NS-GSI (A–E,L,M) or DHMEQ (F–J) and stimulated with plate-bound anti-CD3ε and anti-CD28 for 24 h. Cells were surface stained for CD4, CD25, and CD69 and intra-cellular stained for N1IC and analyzed by flow cytometry. Supernatants were used to detect IL-2 and IFN-γ by an ELISA mean fluorescent intensity (MFI) values were plotted for (A,F) N1IC, (B,G) CD25, (C,H) CD69 n = 3–5. Histograms to the right of (A–C) show expression of N1IC, CD25, and CD69. Data represent three independent experiments. (D,I) IL-2 and (E,J) IFN-γ as determined by an ELISA. (K) Counts per minute (CPM) obtained after 3H-thymidine uptake in CD4+ T cells from cRBP-Jκ-KO mice treated with DMSO or DHMEQ and stimulated for 48 h. (L,M) Histograms representing a CFSE Proliferation Assay. CD4+ T cells from cRBP-Jκ-KO mice pretreated with DMSO, GSI, or NS-GSI were labeled with CFSE and activated with plate-bound anti-CD3ε and anti-CD28 for 48 h followed by flow cytometry analysis. Data represent three independent experiments. (M) Bar graph showing the percentage of CFSE negative cells obtained by flow cytometry. Data represent three independent experiments. Data represent mean ± SEM. *p < 0.05, **p < 0.005, and ***p < 0.001. ns, not significant.
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Figure 6: Activation and Proliferation of CD4+ T cells is RBP-Jκ independent but Notch and NF-κB dependent. CD4+ T cells were isolated from cRBP-Jκ-KO mice, pretreated with DMSO, GSI, or NS-GSI (A–E,L,M) or DHMEQ (F–J) and stimulated with plate-bound anti-CD3ε and anti-CD28 for 24 h. Cells were surface stained for CD4, CD25, and CD69 and intra-cellular stained for N1IC and analyzed by flow cytometry. Supernatants were used to detect IL-2 and IFN-γ by an ELISA mean fluorescent intensity (MFI) values were plotted for (A,F) N1IC, (B,G) CD25, (C,H) CD69 n = 3–5. Histograms to the right of (A–C) show expression of N1IC, CD25, and CD69. Data represent three independent experiments. (D,I) IL-2 and (E,J) IFN-γ as determined by an ELISA. (K) Counts per minute (CPM) obtained after 3H-thymidine uptake in CD4+ T cells from cRBP-Jκ-KO mice treated with DMSO or DHMEQ and stimulated for 48 h. (L,M) Histograms representing a CFSE Proliferation Assay. CD4+ T cells from cRBP-Jκ-KO mice pretreated with DMSO, GSI, or NS-GSI were labeled with CFSE and activated with plate-bound anti-CD3ε and anti-CD28 for 48 h followed by flow cytometry analysis. Data represent three independent experiments. (M) Bar graph showing the percentage of CFSE negative cells obtained by flow cytometry. Data represent three independent experiments. Data represent mean ± SEM. *p < 0.05, **p < 0.005, and ***p < 0.001. ns, not significant.

Mentions: To confirm that RBP-Jκ-independent activation and proliferation was in fact Notch dependent but RBP-Jκ-independent, we used the following strategies. We first inhibited activation of Notch in CD4+ T cells from cRBP-Jκ-KO by treating these cells with GSI. To control for the off-target effects of GSIs, we also treated cells with a Notch sparing GSI (NS-GSI) that inhibited all GSI substrates except Notch. GSI treatment of CD4+ T cells from cRBP-Jκ-KO mice inhibited intra-cellular Notch (Figure 6A) and significantly reduced the expression of the activation markers CD25 and CD69 (Figures 6B,C). This was accompanied by a significant decrease in the cytokines IL-2 and IFN-γ (Figures 6D,E). Importantly, NS-GSI treatment rescued Notch activation as well as CD25, CD69, and IL-2 (Figures 6A–D). A partial rescue was observed with IFN-γ (Figure 6E). Furthermore, a decrease in proliferation of CD4+ T cells from cRBP-Jκ-KO mice after GSI treatment was also rescued by the NS-GSI (Figures 6L,M). These data suggest that while canonical Notch signaling is dispensable for the activation and proliferation of peripheral CD4+ T cells, these processes require intra-cellular Notch. The role of Notch and NF-κB in regulating T cell activation and differentiation processes has been well documented (3, 15, 36, 37). We have shown that the intra-cellular domain of Notch1 binds to and exerts some of its effects via NF-κB suggestive of cross talk between the two pathways (3, 15). Hence, we asked whether these RBP-Jκ-independent processes, were also dependent on NF-κB. This was determined by examining activation markers after inhibiting NF-κB in CD4+ T cells lacking RBP-Jκ using a pharmacological inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) (38). DHMEQ has been used to block NF-κB activity in different types of solid tumors, malignant cells, and T cells (39, 40). DHMEQ treatment effectively inhibited nuclear translocation of NF-κB (Figures S6A,B in Supplementary Material). Although DHMEQ treatment did not alter the levels of N1IC (Figure 6F), DHMEQ treated CD4+ T cells from cRBP-Jκ-KO mice showed a significant reduction in the amounts of CD25, CD69, IL-2, and IFN-γ (Figures 6G–J). Furthermore, DHMEQ treatment of CD4+ T cells from cRBP-Jκ-KO animals significantly impaired proliferation (Figure 6K). These data show that activation and proliferation of CD4+ T cells is an RBP-Jκ-independent but Notch dependent process. Furthermore, our data suggest that non-canonical Notch signaling may control these processes, at least in part through NF-κB.


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)

Activation and Proliferation of CD4+ T cells is RBP-Jκ independent but Notch and NF-κB dependent. CD4+ T cells were isolated from cRBP-Jκ-KO mice, pretreated with DMSO, GSI, or NS-GSI (A–E,L,M) or DHMEQ (F–J) and stimulated with plate-bound anti-CD3ε and anti-CD28 for 24 h. Cells were surface stained for CD4, CD25, and CD69 and intra-cellular stained for N1IC and analyzed by flow cytometry. Supernatants were used to detect IL-2 and IFN-γ by an ELISA mean fluorescent intensity (MFI) values were plotted for (A,F) N1IC, (B,G) CD25, (C,H) CD69 n = 3–5. Histograms to the right of (A–C) show expression of N1IC, CD25, and CD69. Data represent three independent experiments. (D,I) IL-2 and (E,J) IFN-γ as determined by an ELISA. (K) Counts per minute (CPM) obtained after 3H-thymidine uptake in CD4+ T cells from cRBP-Jκ-KO mice treated with DMSO or DHMEQ and stimulated for 48 h. (L,M) Histograms representing a CFSE Proliferation Assay. CD4+ T cells from cRBP-Jκ-KO mice pretreated with DMSO, GSI, or NS-GSI were labeled with CFSE and activated with plate-bound anti-CD3ε and anti-CD28 for 48 h followed by flow cytometry analysis. Data represent three independent experiments. (M) Bar graph showing the percentage of CFSE negative cells obtained by flow cytometry. Data represent three independent experiments. Data represent mean ± SEM. *p < 0.05, **p < 0.005, and ***p < 0.001. ns, not significant.
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Figure 6: Activation and Proliferation of CD4+ T cells is RBP-Jκ independent but Notch and NF-κB dependent. CD4+ T cells were isolated from cRBP-Jκ-KO mice, pretreated with DMSO, GSI, or NS-GSI (A–E,L,M) or DHMEQ (F–J) and stimulated with plate-bound anti-CD3ε and anti-CD28 for 24 h. Cells were surface stained for CD4, CD25, and CD69 and intra-cellular stained for N1IC and analyzed by flow cytometry. Supernatants were used to detect IL-2 and IFN-γ by an ELISA mean fluorescent intensity (MFI) values were plotted for (A,F) N1IC, (B,G) CD25, (C,H) CD69 n = 3–5. Histograms to the right of (A–C) show expression of N1IC, CD25, and CD69. Data represent three independent experiments. (D,I) IL-2 and (E,J) IFN-γ as determined by an ELISA. (K) Counts per minute (CPM) obtained after 3H-thymidine uptake in CD4+ T cells from cRBP-Jκ-KO mice treated with DMSO or DHMEQ and stimulated for 48 h. (L,M) Histograms representing a CFSE Proliferation Assay. CD4+ T cells from cRBP-Jκ-KO mice pretreated with DMSO, GSI, or NS-GSI were labeled with CFSE and activated with plate-bound anti-CD3ε and anti-CD28 for 48 h followed by flow cytometry analysis. Data represent three independent experiments. (M) Bar graph showing the percentage of CFSE negative cells obtained by flow cytometry. Data represent three independent experiments. Data represent mean ± SEM. *p < 0.05, **p < 0.005, and ***p < 0.001. ns, not significant.
Mentions: To confirm that RBP-Jκ-independent activation and proliferation was in fact Notch dependent but RBP-Jκ-independent, we used the following strategies. We first inhibited activation of Notch in CD4+ T cells from cRBP-Jκ-KO by treating these cells with GSI. To control for the off-target effects of GSIs, we also treated cells with a Notch sparing GSI (NS-GSI) that inhibited all GSI substrates except Notch. GSI treatment of CD4+ T cells from cRBP-Jκ-KO mice inhibited intra-cellular Notch (Figure 6A) and significantly reduced the expression of the activation markers CD25 and CD69 (Figures 6B,C). This was accompanied by a significant decrease in the cytokines IL-2 and IFN-γ (Figures 6D,E). Importantly, NS-GSI treatment rescued Notch activation as well as CD25, CD69, and IL-2 (Figures 6A–D). A partial rescue was observed with IFN-γ (Figure 6E). Furthermore, a decrease in proliferation of CD4+ T cells from cRBP-Jκ-KO mice after GSI treatment was also rescued by the NS-GSI (Figures 6L,M). These data suggest that while canonical Notch signaling is dispensable for the activation and proliferation of peripheral CD4+ T cells, these processes require intra-cellular Notch. The role of Notch and NF-κB in regulating T cell activation and differentiation processes has been well documented (3, 15, 36, 37). We have shown that the intra-cellular domain of Notch1 binds to and exerts some of its effects via NF-κB suggestive of cross talk between the two pathways (3, 15). Hence, we asked whether these RBP-Jκ-independent processes, were also dependent on NF-κB. This was determined by examining activation markers after inhibiting NF-κB in CD4+ T cells lacking RBP-Jκ using a pharmacological inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) (38). DHMEQ has been used to block NF-κB activity in different types of solid tumors, malignant cells, and T cells (39, 40). DHMEQ treatment effectively inhibited nuclear translocation of NF-κB (Figures S6A,B in Supplementary Material). Although DHMEQ treatment did not alter the levels of N1IC (Figure 6F), DHMEQ treated CD4+ T cells from cRBP-Jκ-KO mice showed a significant reduction in the amounts of CD25, CD69, IL-2, and IFN-γ (Figures 6G–J). Furthermore, DHMEQ treatment of CD4+ T cells from cRBP-Jκ-KO animals significantly impaired proliferation (Figure 6K). These data show that activation and proliferation of CD4+ T cells is an RBP-Jκ-independent but Notch dependent process. Furthermore, our data suggest that non-canonical Notch signaling may control these processes, at least in part through NF-κB.

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