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Linear ubiquitin chain assembly complex coordinates late thymic T-cell differentiation and regulatory T-cell homeostasis

View Article: PubMed Central - PubMed

ABSTRACT

The linear ubiquitin chain assembly complex (LUBAC) is essential for innate immunity in mice and humans, yet its role in adaptive immunity is unclear. Here we show that the LUBAC components HOIP, HOIL-1 and SHARPIN have essential roles in late thymocyte differentiation, FOXP3+ regulatory T (Treg)-cell development and Treg cell homeostasis. LUBAC activity is not required to prevent TNF-induced apoptosis or necroptosis but is necessary for the transcriptional programme of the penultimate stage of thymocyte differentiation. Treg cell-specific ablation of HOIP causes severe Treg cell deficiency and lethal immune pathology, revealing an ongoing requirement of LUBAC activity for Treg cell homeostasis. These data reveal stage-specific requirements for LUBAC in coordinating the signals required for T-cell differentiation.

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Related in: MedlinePlus

SP thymocyte differentiation and survival in LUBAC-deficient mice.(a) Schematic representation of CD4+ T-cell development in the thymus. Earliest thymic progenitor (ETP) cells undergo progressive differentiation from DN to DP to single-positive (CD4 or CD8) cell. The different stages of thymocyte development are also accompanied by changes in CD24, CD62L, CCR7, CD69 and MHC I (H2-Kb) surface marker expression on the differentiating thymocyte. (b) Flow cytometric analysis of the surface expression of MHC I (H2-Kb) versus CD69 on whole thymocytes from 7- to 15-week-old control, HoipΔCd4, HoilΔCd4 and Sharpincpdm mice. (c) Quantification of total cell numbers and percentages of Fraction 4 (MHC Ihigh CD69high) and Fraction 5 (MHC Ihigh CD69low) populations for control, HoipΔCd4, HoilΔCd4 and Sharpincpdm mice. Surface expression of CD24 versus CD62L (d), CD24 versus CCR7 (e), Helios versus CCR7 (f) gated on CD4SP from 7- to 15-week-old control, HoipΔCd4, HoilΔCd4 and Sharpincpdm mice. Right, cell numbers of the mature CD24low CD62Lhigh, CD24low CCR7high and HELIOShigh CCR7high from CD4SP cells. (g) Contribution to different thymic T cell subsets in 50:50 mixed bone marrow chimeras 8 weeks after reconstitution. Columns show percentage of WT CD45.1+ (white bar) and CD45.2+ control, HoipΔCd4, HoilΔCd4 and Sharpincpdm (black bar) cells in individual chimeric mice. DN; CD4−CD8−, DP; CD4+CD8+ SP3; CD4+CD24lowCCR7high, W1; CD4+CCR7low,HELIOS+, W2; CD4+CCR7high,HELIOS+, Treg; CD4+FOXP3+. Each bar represents an individual mouse. (h) Surface expression of CD4 and CD8 on whole thymocytes (upper panels) and B220 and TCRβ on splenocytes (lower panels) from control, BaxΔCd4Bak−/−, HoipΔCd4 and HoipΔCd4BaxΔCd4Bak−/− mice. For c,d,e and f, each symbol represents an individual mouse; small horizontal lines indicate mean±s.d.; *P<0.05, **P<0.01, ***P<0.005 and ****P<0.001, respectively. One-way analysis of variance with a Tukey's post-hoc test for multiple comparisons was used for statistical analysis. Shpncpdm refers to Sharpincpdm mice. Data are pooled from six independent experiments with two to six mice per group (b–f) or representative of two independent experiments with four to six mice per group (g), or representative of two independent experiments with one to five mice per group (h).
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f3: SP thymocyte differentiation and survival in LUBAC-deficient mice.(a) Schematic representation of CD4+ T-cell development in the thymus. Earliest thymic progenitor (ETP) cells undergo progressive differentiation from DN to DP to single-positive (CD4 or CD8) cell. The different stages of thymocyte development are also accompanied by changes in CD24, CD62L, CCR7, CD69 and MHC I (H2-Kb) surface marker expression on the differentiating thymocyte. (b) Flow cytometric analysis of the surface expression of MHC I (H2-Kb) versus CD69 on whole thymocytes from 7- to 15-week-old control, HoipΔCd4, HoilΔCd4 and Sharpincpdm mice. (c) Quantification of total cell numbers and percentages of Fraction 4 (MHC Ihigh CD69high) and Fraction 5 (MHC Ihigh CD69low) populations for control, HoipΔCd4, HoilΔCd4 and Sharpincpdm mice. Surface expression of CD24 versus CD62L (d), CD24 versus CCR7 (e), Helios versus CCR7 (f) gated on CD4SP from 7- to 15-week-old control, HoipΔCd4, HoilΔCd4 and Sharpincpdm mice. Right, cell numbers of the mature CD24low CD62Lhigh, CD24low CCR7high and HELIOShigh CCR7high from CD4SP cells. (g) Contribution to different thymic T cell subsets in 50:50 mixed bone marrow chimeras 8 weeks after reconstitution. Columns show percentage of WT CD45.1+ (white bar) and CD45.2+ control, HoipΔCd4, HoilΔCd4 and Sharpincpdm (black bar) cells in individual chimeric mice. DN; CD4−CD8−, DP; CD4+CD8+ SP3; CD4+CD24lowCCR7high, W1; CD4+CCR7low,HELIOS+, W2; CD4+CCR7high,HELIOS+, Treg; CD4+FOXP3+. Each bar represents an individual mouse. (h) Surface expression of CD4 and CD8 on whole thymocytes (upper panels) and B220 and TCRβ on splenocytes (lower panels) from control, BaxΔCd4Bak−/−, HoipΔCd4 and HoipΔCd4BaxΔCd4Bak−/− mice. For c,d,e and f, each symbol represents an individual mouse; small horizontal lines indicate mean±s.d.; *P<0.05, **P<0.01, ***P<0.005 and ****P<0.001, respectively. One-way analysis of variance with a Tukey's post-hoc test for multiple comparisons was used for statistical analysis. Shpncpdm refers to Sharpincpdm mice. Data are pooled from six independent experiments with two to six mice per group (b–f) or representative of two independent experiments with four to six mice per group (g), or representative of two independent experiments with one to five mice per group (h).

Mentions: To parse out thymocyte differentiation following positive selection, we employed a staging scheme validated by Mingueneau et al.1 (schematically represented in Fig. 3a), comparing expression of the early activation marker CD69 and the mature SP marker MHC class I (MHC I, H2-Kb). The proportions of pre-selection (CD69− MHC Ilow) and early selection thymocytes (CD69low/high MHC Ilow) were comparable among all strains (Fig. 3b), with normal numbers of CD4+CD8low/intermediate cells27 progressing through differentiation (Supplementary Fig. 3). However, both HoipΔCd4 and HoilΔCd4 mice exhibited specific loss of the late selection/mature subsets (CD69high MHC Ihigh and CD69low MHC Ihigh, termed Fractions 4 and 5, ref. 1), Fig. 3b and Supplementary Fig. 3). This defect corresponded to the loss of ‘mature' CD24low CD62Lhigh CCR7+/− CD4SP (Fig. 3c,d and Supplementary Fig. 3). By contrast, thymocytes from Sharpincpdm mice exhibited largely normal progression through these differentiation stages (Fig. 3b–e). These data establish that, in the absence of HOIL-1 or HOIP, early positive and negative selection events occur normally, but that SP thymocyte maturation is almost completely blocked.


Linear ubiquitin chain assembly complex coordinates late thymic T-cell differentiation and regulatory T-cell homeostasis
SP thymocyte differentiation and survival in LUBAC-deficient mice.(a) Schematic representation of CD4+ T-cell development in the thymus. Earliest thymic progenitor (ETP) cells undergo progressive differentiation from DN to DP to single-positive (CD4 or CD8) cell. The different stages of thymocyte development are also accompanied by changes in CD24, CD62L, CCR7, CD69 and MHC I (H2-Kb) surface marker expression on the differentiating thymocyte. (b) Flow cytometric analysis of the surface expression of MHC I (H2-Kb) versus CD69 on whole thymocytes from 7- to 15-week-old control, HoipΔCd4, HoilΔCd4 and Sharpincpdm mice. (c) Quantification of total cell numbers and percentages of Fraction 4 (MHC Ihigh CD69high) and Fraction 5 (MHC Ihigh CD69low) populations for control, HoipΔCd4, HoilΔCd4 and Sharpincpdm mice. Surface expression of CD24 versus CD62L (d), CD24 versus CCR7 (e), Helios versus CCR7 (f) gated on CD4SP from 7- to 15-week-old control, HoipΔCd4, HoilΔCd4 and Sharpincpdm mice. Right, cell numbers of the mature CD24low CD62Lhigh, CD24low CCR7high and HELIOShigh CCR7high from CD4SP cells. (g) Contribution to different thymic T cell subsets in 50:50 mixed bone marrow chimeras 8 weeks after reconstitution. Columns show percentage of WT CD45.1+ (white bar) and CD45.2+ control, HoipΔCd4, HoilΔCd4 and Sharpincpdm (black bar) cells in individual chimeric mice. DN; CD4−CD8−, DP; CD4+CD8+ SP3; CD4+CD24lowCCR7high, W1; CD4+CCR7low,HELIOS+, W2; CD4+CCR7high,HELIOS+, Treg; CD4+FOXP3+. Each bar represents an individual mouse. (h) Surface expression of CD4 and CD8 on whole thymocytes (upper panels) and B220 and TCRβ on splenocytes (lower panels) from control, BaxΔCd4Bak−/−, HoipΔCd4 and HoipΔCd4BaxΔCd4Bak−/− mice. For c,d,e and f, each symbol represents an individual mouse; small horizontal lines indicate mean±s.d.; *P<0.05, **P<0.01, ***P<0.005 and ****P<0.001, respectively. One-way analysis of variance with a Tukey's post-hoc test for multiple comparisons was used for statistical analysis. Shpncpdm refers to Sharpincpdm mice. Data are pooled from six independent experiments with two to six mice per group (b–f) or representative of two independent experiments with four to six mice per group (g), or representative of two independent experiments with one to five mice per group (h).
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Related In: Results  -  Collection

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f3: SP thymocyte differentiation and survival in LUBAC-deficient mice.(a) Schematic representation of CD4+ T-cell development in the thymus. Earliest thymic progenitor (ETP) cells undergo progressive differentiation from DN to DP to single-positive (CD4 or CD8) cell. The different stages of thymocyte development are also accompanied by changes in CD24, CD62L, CCR7, CD69 and MHC I (H2-Kb) surface marker expression on the differentiating thymocyte. (b) Flow cytometric analysis of the surface expression of MHC I (H2-Kb) versus CD69 on whole thymocytes from 7- to 15-week-old control, HoipΔCd4, HoilΔCd4 and Sharpincpdm mice. (c) Quantification of total cell numbers and percentages of Fraction 4 (MHC Ihigh CD69high) and Fraction 5 (MHC Ihigh CD69low) populations for control, HoipΔCd4, HoilΔCd4 and Sharpincpdm mice. Surface expression of CD24 versus CD62L (d), CD24 versus CCR7 (e), Helios versus CCR7 (f) gated on CD4SP from 7- to 15-week-old control, HoipΔCd4, HoilΔCd4 and Sharpincpdm mice. Right, cell numbers of the mature CD24low CD62Lhigh, CD24low CCR7high and HELIOShigh CCR7high from CD4SP cells. (g) Contribution to different thymic T cell subsets in 50:50 mixed bone marrow chimeras 8 weeks after reconstitution. Columns show percentage of WT CD45.1+ (white bar) and CD45.2+ control, HoipΔCd4, HoilΔCd4 and Sharpincpdm (black bar) cells in individual chimeric mice. DN; CD4−CD8−, DP; CD4+CD8+ SP3; CD4+CD24lowCCR7high, W1; CD4+CCR7low,HELIOS+, W2; CD4+CCR7high,HELIOS+, Treg; CD4+FOXP3+. Each bar represents an individual mouse. (h) Surface expression of CD4 and CD8 on whole thymocytes (upper panels) and B220 and TCRβ on splenocytes (lower panels) from control, BaxΔCd4Bak−/−, HoipΔCd4 and HoipΔCd4BaxΔCd4Bak−/− mice. For c,d,e and f, each symbol represents an individual mouse; small horizontal lines indicate mean±s.d.; *P<0.05, **P<0.01, ***P<0.005 and ****P<0.001, respectively. One-way analysis of variance with a Tukey's post-hoc test for multiple comparisons was used for statistical analysis. Shpncpdm refers to Sharpincpdm mice. Data are pooled from six independent experiments with two to six mice per group (b–f) or representative of two independent experiments with four to six mice per group (g), or representative of two independent experiments with one to five mice per group (h).
Mentions: To parse out thymocyte differentiation following positive selection, we employed a staging scheme validated by Mingueneau et al.1 (schematically represented in Fig. 3a), comparing expression of the early activation marker CD69 and the mature SP marker MHC class I (MHC I, H2-Kb). The proportions of pre-selection (CD69− MHC Ilow) and early selection thymocytes (CD69low/high MHC Ilow) were comparable among all strains (Fig. 3b), with normal numbers of CD4+CD8low/intermediate cells27 progressing through differentiation (Supplementary Fig. 3). However, both HoipΔCd4 and HoilΔCd4 mice exhibited specific loss of the late selection/mature subsets (CD69high MHC Ihigh and CD69low MHC Ihigh, termed Fractions 4 and 5, ref. 1), Fig. 3b and Supplementary Fig. 3). This defect corresponded to the loss of ‘mature' CD24low CD62Lhigh CCR7+/− CD4SP (Fig. 3c,d and Supplementary Fig. 3). By contrast, thymocytes from Sharpincpdm mice exhibited largely normal progression through these differentiation stages (Fig. 3b–e). These data establish that, in the absence of HOIL-1 or HOIP, early positive and negative selection events occur normally, but that SP thymocyte maturation is almost completely blocked.

View Article: PubMed Central - PubMed

ABSTRACT

The linear ubiquitin chain assembly complex (LUBAC) is essential for innate immunity in mice and humans, yet its role in adaptive immunity is unclear. Here we show that the LUBAC components HOIP, HOIL-1 and SHARPIN have essential roles in late thymocyte differentiation, FOXP3+ regulatory T (Treg)-cell development and Treg cell homeostasis. LUBAC activity is not required to prevent TNF-induced apoptosis or necroptosis but is necessary for the transcriptional programme of the penultimate stage of thymocyte differentiation. Treg cell-specific ablation of HOIP causes severe Treg cell deficiency and lethal immune pathology, revealing an ongoing requirement of LUBAC activity for Treg cell homeostasis. These data reveal stage-specific requirements for LUBAC in coordinating the signals required for T-cell differentiation.

No MeSH data available.


Related in: MedlinePlus