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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

Jak1 degradation is dependent on Ndfip1/Ndfip2.(a–j) Immunoblotting of restimulated WT and cDKO CD4+ T cells. (a) Cycloheximide was added 2 h after CD3/CD28 stimulation; cells were then incubated for an additional 4 h. (b) Level of Jak1 was normalized to GAPDH. The stability of Jak1 was determined by normalizing the percent Jak1 remaining in cDKO or cKO cells to the percent remaining in experiment-matched control cells. Data shown are average ±s.e.m. from three to five biologic replicates in more than three experiments. (c,d) p-STAT5 was quantified relative to tubulin. Relative levels of p-STAT5 in cDKO and cKO CD4+ T cells were normalized to experiment-matched control cells at 2 and 6 h of restimulation. Data shown is average ±s.e.m. for two biologic replicates. (e,f) Immunoblotting of total STAT5 in restimulated WT and cDKO CD4+ T cells. Cells were stimulated for 2 h, and cycloheximide was added for an additional 2 h of stimulation. (f) Level of STAT5 was normalized to GAPDH. The stability of STAT5 was determined by normalizing the relative STAT5 remaining after stimulation to the amount of STAT5 at 2 h. Data shown are average ±s.e.m. from three biologic replicates. (g–j) Levels of Jak1, normalized to GAPDH, at various timepoints following restimulation of WT and cDKO CD4+ T cells relative to Jak1 in IL-2-rested cells at time 0. (h) Rate of Jak1 degradation in relative units per hour over 6 h of stimulation. (i,j) Levels of Jak1, normalized to GAPDH, at various timepoints following rest in the absence of IL-2/TCR, of WT and cDKO CD4+ T cells relative to Jak1 in IL-2-rested cells at time 0. (j) As in g and i, levels of Jak1, normalized to GAPDH, 24 h ±TCR stimulation of WT and cDKO CD4+ T cells relative to Jak1 in IL-2-rested cells at time 0. Data shown in g–j average ±s.e.m. from two to four biologic replicates in more than three experiments. P value calculated by two sample, unpaired t-test *P<0.05.
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f7: Jak1 degradation is dependent on Ndfip1/Ndfip2.(a–j) Immunoblotting of restimulated WT and cDKO CD4+ T cells. (a) Cycloheximide was added 2 h after CD3/CD28 stimulation; cells were then incubated for an additional 4 h. (b) Level of Jak1 was normalized to GAPDH. The stability of Jak1 was determined by normalizing the percent Jak1 remaining in cDKO or cKO cells to the percent remaining in experiment-matched control cells. Data shown are average ±s.e.m. from three to five biologic replicates in more than three experiments. (c,d) p-STAT5 was quantified relative to tubulin. Relative levels of p-STAT5 in cDKO and cKO CD4+ T cells were normalized to experiment-matched control cells at 2 and 6 h of restimulation. Data shown is average ±s.e.m. for two biologic replicates. (e,f) Immunoblotting of total STAT5 in restimulated WT and cDKO CD4+ T cells. Cells were stimulated for 2 h, and cycloheximide was added for an additional 2 h of stimulation. (f) Level of STAT5 was normalized to GAPDH. The stability of STAT5 was determined by normalizing the relative STAT5 remaining after stimulation to the amount of STAT5 at 2 h. Data shown are average ±s.e.m. from three biologic replicates. (g–j) Levels of Jak1, normalized to GAPDH, at various timepoints following restimulation of WT and cDKO CD4+ T cells relative to Jak1 in IL-2-rested cells at time 0. (h) Rate of Jak1 degradation in relative units per hour over 6 h of stimulation. (i,j) Levels of Jak1, normalized to GAPDH, at various timepoints following rest in the absence of IL-2/TCR, of WT and cDKO CD4+ T cells relative to Jak1 in IL-2-rested cells at time 0. (j) As in g and i, levels of Jak1, normalized to GAPDH, 24 h ±TCR stimulation of WT and cDKO CD4+ T cells relative to Jak1 in IL-2-rested cells at time 0. Data shown in g–j average ±s.e.m. from two to four biologic replicates in more than three experiments. P value calculated by two sample, unpaired t-test *P<0.05.

Mentions: Jak1 was robustly degraded in TCR-stimulated WT cells treated with cycloheximide, a translation inhibitor (Fig. 7a). Ndfip-deficient CD4+ T cells showed significantly less degradation (Fig. 7a,b). We also observed increased phosphorylation of STAT5a/b after stimulation of Ndfip-deficient cells compared with WT cells (Fig. 7c,d). We did not observe any change in Jak1 stability or p-STAT5a/b signal in CD4+ T cells from Ndfip1 cKO mice, suggesting that loss of Ndfip1 alone is not sufficient to affect Jak1 stability and STAT5 activity (Fig. 7a–d). Levels of total STAT5 were not significantly increased in cDKO CD4+ T cells, and STAT5 did not show cycloheximide sensitivity (Fig. 7e,f), consistent with published data37. Thus increased levels of p-STAT5 are indicative of increased Jak1 activity in Ndfip-deficient cells.


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)

Jak1 degradation is dependent on Ndfip1/Ndfip2.(a–j) Immunoblotting of restimulated WT and cDKO CD4+ T cells. (a) Cycloheximide was added 2 h after CD3/CD28 stimulation; cells were then incubated for an additional 4 h. (b) Level of Jak1 was normalized to GAPDH. The stability of Jak1 was determined by normalizing the percent Jak1 remaining in cDKO or cKO cells to the percent remaining in experiment-matched control cells. Data shown are average ±s.e.m. from three to five biologic replicates in more than three experiments. (c,d) p-STAT5 was quantified relative to tubulin. Relative levels of p-STAT5 in cDKO and cKO CD4+ T cells were normalized to experiment-matched control cells at 2 and 6 h of restimulation. Data shown is average ±s.e.m. for two biologic replicates. (e,f) Immunoblotting of total STAT5 in restimulated WT and cDKO CD4+ T cells. Cells were stimulated for 2 h, and cycloheximide was added for an additional 2 h of stimulation. (f) Level of STAT5 was normalized to GAPDH. The stability of STAT5 was determined by normalizing the relative STAT5 remaining after stimulation to the amount of STAT5 at 2 h. Data shown are average ±s.e.m. from three biologic replicates. (g–j) Levels of Jak1, normalized to GAPDH, at various timepoints following restimulation of WT and cDKO CD4+ T cells relative to Jak1 in IL-2-rested cells at time 0. (h) Rate of Jak1 degradation in relative units per hour over 6 h of stimulation. (i,j) Levels of Jak1, normalized to GAPDH, at various timepoints following rest in the absence of IL-2/TCR, of WT and cDKO CD4+ T cells relative to Jak1 in IL-2-rested cells at time 0. (j) As in g and i, levels of Jak1, normalized to GAPDH, 24 h ±TCR stimulation of WT and cDKO CD4+ T cells relative to Jak1 in IL-2-rested cells at time 0. Data shown in g–j average ±s.e.m. from two to four biologic replicates in more than three experiments. P value calculated by two sample, unpaired t-test *P<0.05.
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f7: Jak1 degradation is dependent on Ndfip1/Ndfip2.(a–j) Immunoblotting of restimulated WT and cDKO CD4+ T cells. (a) Cycloheximide was added 2 h after CD3/CD28 stimulation; cells were then incubated for an additional 4 h. (b) Level of Jak1 was normalized to GAPDH. The stability of Jak1 was determined by normalizing the percent Jak1 remaining in cDKO or cKO cells to the percent remaining in experiment-matched control cells. Data shown are average ±s.e.m. from three to five biologic replicates in more than three experiments. (c,d) p-STAT5 was quantified relative to tubulin. Relative levels of p-STAT5 in cDKO and cKO CD4+ T cells were normalized to experiment-matched control cells at 2 and 6 h of restimulation. Data shown is average ±s.e.m. for two biologic replicates. (e,f) Immunoblotting of total STAT5 in restimulated WT and cDKO CD4+ T cells. Cells were stimulated for 2 h, and cycloheximide was added for an additional 2 h of stimulation. (f) Level of STAT5 was normalized to GAPDH. The stability of STAT5 was determined by normalizing the relative STAT5 remaining after stimulation to the amount of STAT5 at 2 h. Data shown are average ±s.e.m. from three biologic replicates. (g–j) Levels of Jak1, normalized to GAPDH, at various timepoints following restimulation of WT and cDKO CD4+ T cells relative to Jak1 in IL-2-rested cells at time 0. (h) Rate of Jak1 degradation in relative units per hour over 6 h of stimulation. (i,j) Levels of Jak1, normalized to GAPDH, at various timepoints following rest in the absence of IL-2/TCR, of WT and cDKO CD4+ T cells relative to Jak1 in IL-2-rested cells at time 0. (j) As in g and i, levels of Jak1, normalized to GAPDH, 24 h ±TCR stimulation of WT and cDKO CD4+ T cells relative to Jak1 in IL-2-rested cells at time 0. Data shown in g–j average ±s.e.m. from two to four biologic replicates in more than three experiments. P value calculated by two sample, unpaired t-test *P<0.05.
Mentions: Jak1 was robustly degraded in TCR-stimulated WT cells treated with cycloheximide, a translation inhibitor (Fig. 7a). Ndfip-deficient CD4+ T cells showed significantly less degradation (Fig. 7a,b). We also observed increased phosphorylation of STAT5a/b after stimulation of Ndfip-deficient cells compared with WT cells (Fig. 7c,d). We did not observe any change in Jak1 stability or p-STAT5a/b signal in CD4+ T cells from Ndfip1 cKO mice, suggesting that loss of Ndfip1 alone is not sufficient to affect Jak1 stability and STAT5 activity (Fig. 7a–d). Levels of total STAT5 were not significantly increased in cDKO CD4+ T cells, and STAT5 did not show cycloheximide sensitivity (Fig. 7e,f), consistent with published data37. Thus increased levels of p-STAT5 are indicative of increased Jak1 activity in Ndfip-deficient cells.

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