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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 deficiency causes intrinsic CD4+ effector T-cell expansion.(a–g) Mixed foetal liver chimeras using CD45.2 WT, Ndfip1−/−, Ndfip2−/− or Ndfip1/Ndfip2 DKO foetal liver were analysed 6 weeks following reconstitution. (a) Representative CD44 and CD62L staining of splenic CD4+ T cells from Ndfip1−/− mixed chimera (top) and DKO mixed chimera (bottom), previously gated on live, singlet CD3+CD4+CD45.1 or CD45.2+ cells. (b,c) Flow cytometry analysis of CD45.1+ and CD45.2+ splenic T cells from chimeras showing (b) percentages of CD44+ cells and (c) percentages of CD44+ that are Ki67+. (d–f) Ex vivo stimulated splenocytes were stained for IL-4. (d) Representative flow plots of IL-4+ CD4+ T cells, (e) combined data from d, (f) percentages of CD44+ CD4 T cells that are IL-4+. (g) Percentages of CD45.2 cells among various T-cell subsets, normalized for reconstitution, as determined by the ratio for CD45.2:CD45.1 IgM+B220+ B cells in the bone marrow. Compartments analysed are as follows: A=IgM+B cells in bone marrow, B=double positive thymocytes, C=single positive CD4+ thymocytes, D=CD44+ CD4+ T cells in spleen, E=CD44+ CD4+ T cells in lung. Data shown in b and e were pooled from two experiments, 7–8 chimeras per group; (c,f,g) have 4–5 chimeras per group. Quantifications are average ±s.e.m. P values calculated by two-way ANOVA (b,c,e,f) or repeated measures one-way ANOVA (g), with Holm–Sidak test for multiple comparisons: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
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f3: Ndfip deficiency causes intrinsic CD4+ effector T-cell expansion.(a–g) Mixed foetal liver chimeras using CD45.2 WT, Ndfip1−/−, Ndfip2−/− or Ndfip1/Ndfip2 DKO foetal liver were analysed 6 weeks following reconstitution. (a) Representative CD44 and CD62L staining of splenic CD4+ T cells from Ndfip1−/− mixed chimera (top) and DKO mixed chimera (bottom), previously gated on live, singlet CD3+CD4+CD45.1 or CD45.2+ cells. (b,c) Flow cytometry analysis of CD45.1+ and CD45.2+ splenic T cells from chimeras showing (b) percentages of CD44+ cells and (c) percentages of CD44+ that are Ki67+. (d–f) Ex vivo stimulated splenocytes were stained for IL-4. (d) Representative flow plots of IL-4+ CD4+ T cells, (e) combined data from d, (f) percentages of CD44+ CD4 T cells that are IL-4+. (g) Percentages of CD45.2 cells among various T-cell subsets, normalized for reconstitution, as determined by the ratio for CD45.2:CD45.1 IgM+B220+ B cells in the bone marrow. Compartments analysed are as follows: A=IgM+B cells in bone marrow, B=double positive thymocytes, C=single positive CD4+ thymocytes, D=CD44+ CD4+ T cells in spleen, E=CD44+ CD4+ T cells in lung. Data shown in b and e were pooled from two experiments, 7–8 chimeras per group; (c,f,g) have 4–5 chimeras per group. Quantifications are average ±s.e.m. P values calculated by two-way ANOVA (b,c,e,f) or repeated measures one-way ANOVA (g), with Holm–Sidak test for multiple comparisons: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Mentions: In these chimeras, DKO CD4+ T cells were more likely to be CD44+ compared with WT CD4+ T cells within the same host. The likelihood of being CD44+ was significantly higher for DKO CD4+ T cells compared with WT cells in the same host, but only trended to be higher for Ndfip1−/− CD4+ T cells relative to their WT counterparts (Fig. 3a,b). CD44+ DKO CD4+ T cells were significantly more proliferative, as indicated by Ki67 staining (Fig. 3c). This was also true for Ndfip1−/− CD4+ T cells, though to a lesser extent. As previously published, loss of Ndfip1 was sufficient to drive IL-4 production15; further loss of Ndfip2 resulted in an increased proportion of CD4+ T cells producing IL-4, even among CD44+ cells, relative to Ndfip1-deficient cells (Fig. 3d–f). Thus, T cells lacking both Ndfip1 and Ndfip2 are much more likely than either their WT counterparts or T cells lacking only Ndfip1 to be activated in vivo, and, once activated, are more proliferative and more likely to produce cytokine.


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 deficiency causes intrinsic CD4+ effector T-cell expansion.(a–g) Mixed foetal liver chimeras using CD45.2 WT, Ndfip1−/−, Ndfip2−/− or Ndfip1/Ndfip2 DKO foetal liver were analysed 6 weeks following reconstitution. (a) Representative CD44 and CD62L staining of splenic CD4+ T cells from Ndfip1−/− mixed chimera (top) and DKO mixed chimera (bottom), previously gated on live, singlet CD3+CD4+CD45.1 or CD45.2+ cells. (b,c) Flow cytometry analysis of CD45.1+ and CD45.2+ splenic T cells from chimeras showing (b) percentages of CD44+ cells and (c) percentages of CD44+ that are Ki67+. (d–f) Ex vivo stimulated splenocytes were stained for IL-4. (d) Representative flow plots of IL-4+ CD4+ T cells, (e) combined data from d, (f) percentages of CD44+ CD4 T cells that are IL-4+. (g) Percentages of CD45.2 cells among various T-cell subsets, normalized for reconstitution, as determined by the ratio for CD45.2:CD45.1 IgM+B220+ B cells in the bone marrow. Compartments analysed are as follows: A=IgM+B cells in bone marrow, B=double positive thymocytes, C=single positive CD4+ thymocytes, D=CD44+ CD4+ T cells in spleen, E=CD44+ CD4+ T cells in lung. Data shown in b and e were pooled from two experiments, 7–8 chimeras per group; (c,f,g) have 4–5 chimeras per group. Quantifications are average ±s.e.m. P values calculated by two-way ANOVA (b,c,e,f) or repeated measures one-way ANOVA (g), with Holm–Sidak test for multiple comparisons: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
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Related In: Results  -  Collection

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f3: Ndfip deficiency causes intrinsic CD4+ effector T-cell expansion.(a–g) Mixed foetal liver chimeras using CD45.2 WT, Ndfip1−/−, Ndfip2−/− or Ndfip1/Ndfip2 DKO foetal liver were analysed 6 weeks following reconstitution. (a) Representative CD44 and CD62L staining of splenic CD4+ T cells from Ndfip1−/− mixed chimera (top) and DKO mixed chimera (bottom), previously gated on live, singlet CD3+CD4+CD45.1 or CD45.2+ cells. (b,c) Flow cytometry analysis of CD45.1+ and CD45.2+ splenic T cells from chimeras showing (b) percentages of CD44+ cells and (c) percentages of CD44+ that are Ki67+. (d–f) Ex vivo stimulated splenocytes were stained for IL-4. (d) Representative flow plots of IL-4+ CD4+ T cells, (e) combined data from d, (f) percentages of CD44+ CD4 T cells that are IL-4+. (g) Percentages of CD45.2 cells among various T-cell subsets, normalized for reconstitution, as determined by the ratio for CD45.2:CD45.1 IgM+B220+ B cells in the bone marrow. Compartments analysed are as follows: A=IgM+B cells in bone marrow, B=double positive thymocytes, C=single positive CD4+ thymocytes, D=CD44+ CD4+ T cells in spleen, E=CD44+ CD4+ T cells in lung. Data shown in b and e were pooled from two experiments, 7–8 chimeras per group; (c,f,g) have 4–5 chimeras per group. Quantifications are average ±s.e.m. P values calculated by two-way ANOVA (b,c,e,f) or repeated measures one-way ANOVA (g), with Holm–Sidak test for multiple comparisons: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Mentions: In these chimeras, DKO CD4+ T cells were more likely to be CD44+ compared with WT CD4+ T cells within the same host. The likelihood of being CD44+ was significantly higher for DKO CD4+ T cells compared with WT cells in the same host, but only trended to be higher for Ndfip1−/− CD4+ T cells relative to their WT counterparts (Fig. 3a,b). CD44+ DKO CD4+ T cells were significantly more proliferative, as indicated by Ki67 staining (Fig. 3c). This was also true for Ndfip1−/− CD4+ T cells, though to a lesser extent. As previously published, loss of Ndfip1 was sufficient to drive IL-4 production15; further loss of Ndfip2 resulted in an increased proportion of CD4+ T cells producing IL-4, even among CD44+ cells, relative to Ndfip1-deficient cells (Fig. 3d–f). Thus, T cells lacking both Ndfip1 and Ndfip2 are much more likely than either their WT counterparts or T cells lacking only Ndfip1 to be activated in vivo, and, once activated, are more proliferative and more likely to produce cytokine.

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