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Ndfip-mediated degradation of Jak1 tunes cytokine signalling to limit expansion of CD4+ effector T cells.

O'Leary CE, Riling CR, Spruce LA, Ding H, Kumar S, Deng G, Liu Y, Seeholzer SH, Oliver PM - Nat Commun (2016)

Bottom Line: Nedd4 family E3 ubiquitin ligases have been shown to restrict T-cell function and impact T-cell differentiation.We show here that Ndfip1 and Ndfip2, activators of Nedd4 family ligases, together limit accumulation and function of effector CD4+ T cells.In wild-type cells, Jak1 degradation lessens CD4+ cell sensitivity to cytokines during TCR stimulation, while in Ndfip-deficient cells cytokine responsiveness persists, promoting increased expansion and survival of pathogenic effector T cells.

View Article: PubMed Central - PubMed

Affiliation: Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Nedd4 family E3 ubiquitin ligases have been shown to restrict T-cell function and impact T-cell differentiation. We show here that Ndfip1 and Ndfip2, activators of Nedd4 family ligases, together limit accumulation and function of effector CD4+ T cells. Using a three-part proteomics approach in primary T cells, we identify stabilization of Jak1 in Ndfip1/2-deficient T cells stimulated through the TCR. Jak1 degradation is aborted in activated T cells that lack Ndfips. In wild-type cells, Jak1 degradation lessens CD4+ cell sensitivity to cytokines during TCR stimulation, while in Ndfip-deficient cells cytokine responsiveness persists, promoting increased expansion and survival of pathogenic effector T cells. Thus, Ndfip1/Ndfip2 regulate the cross talk between the T-cell receptor and cytokine signalling pathways to limit inappropriate T-cell responses.

No MeSH data available.


Related in: MedlinePlus

Ndfip-deficient T cells show persistent cytokine signalling.(a–c) Sorted naive CD4+ T cells from WT and cDKO mice were stimulated with αCD3/CD28 in the presence of Jak inhibitor I (JAKi) and analysed on day 5 by flow cytometry. (a) Representative plots of CFSE dilution and viability staining in WT and cDKO CD4+ T cells ±Jak inhibitor (31.2 nM). (b,c) Quantification of (b) percent of cDKO and WT cells divided four or more times relative to DMSO-treated experiment-matched WT cells and (c) viability of cDKO and WT CD4+ T cells normalized to DMSO-treated experiment-matched WT cells. Data shown is average ±s.e.m. from six biologic replicates. P values calculated by multiple t-test with Holm–Sidak correction. (d,e) p-STAT5 staining in cDKO and WT CD4+ T cells rested in the absence of IL-2 overnight then treated ±αCD3/CD28 beads before addition of IL-2. (d) Representative flow cytometry histograms. (e) Quantification of p-STAT5 MFI for eight to nine biologic replicates and four independent experiments, showing average ±s.e.m. Patterned bars indicate addition of exogenous IL-2. P values calculated by two-way ANOVA with Holm–Sidak test for multiple comparisons. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
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f8: Ndfip-deficient T cells show persistent cytokine signalling.(a–c) Sorted naive CD4+ T cells from WT and cDKO mice were stimulated with αCD3/CD28 in the presence of Jak inhibitor I (JAKi) and analysed on day 5 by flow cytometry. (a) Representative plots of CFSE dilution and viability staining in WT and cDKO CD4+ T cells ±Jak inhibitor (31.2 nM). (b,c) Quantification of (b) percent of cDKO and WT cells divided four or more times relative to DMSO-treated experiment-matched WT cells and (c) viability of cDKO and WT CD4+ T cells normalized to DMSO-treated experiment-matched WT cells. Data shown is average ±s.e.m. from six biologic replicates. P values calculated by multiple t-test with Holm–Sidak correction. (d,e) p-STAT5 staining in cDKO and WT CD4+ T cells rested in the absence of IL-2 overnight then treated ±αCD3/CD28 beads before addition of IL-2. (d) Representative flow cytometry histograms. (e) Quantification of p-STAT5 MFI for eight to nine biologic replicates and four independent experiments, showing average ±s.e.m. Patterned bars indicate addition of exogenous IL-2. P values calculated by two-way ANOVA with Holm–Sidak test for multiple comparisons. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Mentions: To determine if Jak1 stabilization mediates the observed in vitro and in vivo hyperactivation and hyperviability of Ndfip-deficient CD4+ T cells, we tested whether Jak inhibition could normalize cDKO levels of proliferation/survival. cDKO CD4+ T cells stimulated in vitro in the presence of a Jak inhibitor showed reduced proliferation, down to WT levels, in a dose-dependent manner; WT cell proliferation was responsive to Jak inhibition but did not show dose dependence, suggesting complete Jak inhibition occurred at a lower dose (Fig. 8a,b). CD25 decreased in both WT and cDKO CD4+ T cells in a dose-dependent manner (Supplementary Fig. 7g). Strikingly, cDKO CD4+ T cells showed a robust decline in viability, down to WT levels, when treated with the Jak inhibitor (Fig. 8a,c).


Ndfip-mediated degradation of Jak1 tunes cytokine signalling to limit expansion of CD4+ effector T cells.

O'Leary CE, Riling CR, Spruce LA, Ding H, Kumar S, Deng G, Liu Y, Seeholzer SH, Oliver PM - Nat Commun (2016)

Ndfip-deficient T cells show persistent cytokine signalling.(a–c) Sorted naive CD4+ T cells from WT and cDKO mice were stimulated with αCD3/CD28 in the presence of Jak inhibitor I (JAKi) and analysed on day 5 by flow cytometry. (a) Representative plots of CFSE dilution and viability staining in WT and cDKO CD4+ T cells ±Jak inhibitor (31.2 nM). (b,c) Quantification of (b) percent of cDKO and WT cells divided four or more times relative to DMSO-treated experiment-matched WT cells and (c) viability of cDKO and WT CD4+ T cells normalized to DMSO-treated experiment-matched WT cells. Data shown is average ±s.e.m. from six biologic replicates. P values calculated by multiple t-test with Holm–Sidak correction. (d,e) p-STAT5 staining in cDKO and WT CD4+ T cells rested in the absence of IL-2 overnight then treated ±αCD3/CD28 beads before addition of IL-2. (d) Representative flow cytometry histograms. (e) Quantification of p-STAT5 MFI for eight to nine biologic replicates and four independent experiments, showing average ±s.e.m. Patterned bars indicate addition of exogenous IL-2. P values calculated by two-way ANOVA with Holm–Sidak test for multiple comparisons. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
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f8: Ndfip-deficient T cells show persistent cytokine signalling.(a–c) Sorted naive CD4+ T cells from WT and cDKO mice were stimulated with αCD3/CD28 in the presence of Jak inhibitor I (JAKi) and analysed on day 5 by flow cytometry. (a) Representative plots of CFSE dilution and viability staining in WT and cDKO CD4+ T cells ±Jak inhibitor (31.2 nM). (b,c) Quantification of (b) percent of cDKO and WT cells divided four or more times relative to DMSO-treated experiment-matched WT cells and (c) viability of cDKO and WT CD4+ T cells normalized to DMSO-treated experiment-matched WT cells. Data shown is average ±s.e.m. from six biologic replicates. P values calculated by multiple t-test with Holm–Sidak correction. (d,e) p-STAT5 staining in cDKO and WT CD4+ T cells rested in the absence of IL-2 overnight then treated ±αCD3/CD28 beads before addition of IL-2. (d) Representative flow cytometry histograms. (e) Quantification of p-STAT5 MFI for eight to nine biologic replicates and four independent experiments, showing average ±s.e.m. Patterned bars indicate addition of exogenous IL-2. P values calculated by two-way ANOVA with Holm–Sidak test for multiple comparisons. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Mentions: To determine if Jak1 stabilization mediates the observed in vitro and in vivo hyperactivation and hyperviability of Ndfip-deficient CD4+ T cells, we tested whether Jak inhibition could normalize cDKO levels of proliferation/survival. cDKO CD4+ T cells stimulated in vitro in the presence of a Jak inhibitor showed reduced proliferation, down to WT levels, in a dose-dependent manner; WT cell proliferation was responsive to Jak inhibition but did not show dose dependence, suggesting complete Jak inhibition occurred at a lower dose (Fig. 8a,b). CD25 decreased in both WT and cDKO CD4+ T cells in a dose-dependent manner (Supplementary Fig. 7g). Strikingly, cDKO CD4+ T cells showed a robust decline in viability, down to WT levels, when treated with the Jak inhibitor (Fig. 8a,c).

Bottom Line: Nedd4 family E3 ubiquitin ligases have been shown to restrict T-cell function and impact T-cell differentiation.We show here that Ndfip1 and Ndfip2, activators of Nedd4 family ligases, together limit accumulation and function of effector CD4+ T cells.In wild-type cells, Jak1 degradation lessens CD4+ cell sensitivity to cytokines during TCR stimulation, while in Ndfip-deficient cells cytokine responsiveness persists, promoting increased expansion and survival of pathogenic effector T cells.

View Article: PubMed Central - PubMed

Affiliation: Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Nedd4 family E3 ubiquitin ligases have been shown to restrict T-cell function and impact T-cell differentiation. We show here that Ndfip1 and Ndfip2, activators of Nedd4 family ligases, together limit accumulation and function of effector CD4+ T cells. Using a three-part proteomics approach in primary T cells, we identify stabilization of Jak1 in Ndfip1/2-deficient T cells stimulated through the TCR. Jak1 degradation is aborted in activated T cells that lack Ndfips. In wild-type cells, Jak1 degradation lessens CD4+ cell sensitivity to cytokines during TCR stimulation, while in Ndfip-deficient cells cytokine responsiveness persists, promoting increased expansion and survival of pathogenic effector T cells. Thus, Ndfip1/Ndfip2 regulate the cross talk between the T-cell receptor and cytokine signalling pathways to limit inappropriate T-cell responses.

No MeSH data available.


Related in: MedlinePlus