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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.

No MeSH data available.


Transcriptional impact of LUBAC deficiency on T-cell differentiation.(a) Venn diagrams of the numbers of genes upregulated (red) or downregulated (blue) in comparisons of CD69+ MHC Ilow or CD69+ MHC Ihigh thymocytes from HoilΔCd4 versus WT (green) and Sharpincpdm versus WT (orange) at a 5% false discovery rate (FDR) cutoff. (b) Heatmaps of individual log-expression values. Left plot shows the 25 most upregulated genes and 50 most downregulated genes for HoilΔCd4 versus WT in CD69+ MHC Ilow thymocytes. Right plot show the same for CD69+ MHC Ihigh. Genes are ordered by P-value. Red indicates relatively higher expression and blue indicates relatively lower expression. Genes highlighted in green are involved in NF-κB signalling, those in yellow are involved in thymocyte/Treg cell differentiation. Shpncpdm refers to Sharpincpdm mice. (c,d) Genes that are differentially expressed in HoilΔCd4 versus WT but show no change or opposite change in Sharpincpdm. Results for CD69+ MHC Ilow thymocytes are shown in c and CD69+ MHC Ihigh thymocytes in d. The plot shows the limma t-statistics for each gene for assessing differential expression; the dotted line indicates the 5% FDR cutoffs of t=3.25 for c and t=3.94 for d.
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f6: Transcriptional impact of LUBAC deficiency on T-cell differentiation.(a) Venn diagrams of the numbers of genes upregulated (red) or downregulated (blue) in comparisons of CD69+ MHC Ilow or CD69+ MHC Ihigh thymocytes from HoilΔCd4 versus WT (green) and Sharpincpdm versus WT (orange) at a 5% false discovery rate (FDR) cutoff. (b) Heatmaps of individual log-expression values. Left plot shows the 25 most upregulated genes and 50 most downregulated genes for HoilΔCd4 versus WT in CD69+ MHC Ilow thymocytes. Right plot show the same for CD69+ MHC Ihigh. Genes are ordered by P-value. Red indicates relatively higher expression and blue indicates relatively lower expression. Genes highlighted in green are involved in NF-κB signalling, those in yellow are involved in thymocyte/Treg cell differentiation. Shpncpdm refers to Sharpincpdm mice. (c,d) Genes that are differentially expressed in HoilΔCd4 versus WT but show no change or opposite change in Sharpincpdm. Results for CD69+ MHC Ilow thymocytes are shown in c and CD69+ MHC Ihigh thymocytes in d. The plot shows the limma t-statistics for each gene for assessing differential expression; the dotted line indicates the 5% FDR cutoffs of t=3.25 for c and t=3.94 for d.

Mentions: HOIL deficiency altered the transcriptome immediately following positive selection (124 differentially expressed genes in CD69+ MHC Ilow thymocytes) and this effect was amplified in CD69+ MHC Ihigh thymocytes (724 differentially expressed genes; Fig. 6a). Surprisingly, thymocytes from Sharpincpdm mice had more substantial alteration of the transcriptome, perhaps reflecting the consequences of moderate LUBAC defects throughout T-cell differentiation (compared with the conditional deletion of HOIL-1 at the DP stage) and minor differences in the genetic background (still largely C57BL/Ka versus WT C57BL/6). To analyse the transcriptional changes associated with the block in SP thymocyte differentiation, we performed a heat-map analysis of the most significantly upregulated (25) or downregulated (50) genes in CD69+ MHC Ilow and CD69+ MHC Ihigh thymocytes from HoilΔCd4 compared with WT mice (Fig. 6b). Reduced expression of many core NF-κB target genes was a common feature of thymocytes from HoilΔCd4 and Sharpincpdm mice. These included genes involved in negative feedback (for example, Nfkbia (or IκBα), Nfkbie (or IκBɛ), Birc3 (or cIAP2) and Tnfaip3 (or A20)) and T-cell differentiation (particularly of FOXP3+ Treg cells; for example, Gadd45b, Il2ra, Tnfrsf18, Tnfrsf4 (or OX40) and Relb; Fig. 6b). To determine whether there was a defect in the induction of NF-κB target genes in the LUBAC-deficient strains during the differentiation from CD69+ MHC Ilow into CD69+ MHC Ihigh thymocytes, we first identified 154 NF-κB target genes that were significantly up- or downregulated during this transition in WT cells. Barcode enrichment plots show that, despite the dampened transcription of NF-κB target genes, the magnitude and direction of changes induced in these transcripts during the post-positive selection stages was maintained in HOIL- and SHARPIN-deficient cells (Supplementary Fig. 4). These findings support our earlier data showing that, although LUBAC-deficient thymocytes have impaired NF-κB signalling, this defect does not by itself explain the block in thymocyte differentiation observed in HoilΔCd4 and HoipΔCd4 mice.


Linear ubiquitin chain assembly complex coordinates late thymic T-cell differentiation and regulatory T-cell homeostasis
Transcriptional impact of LUBAC deficiency on T-cell differentiation.(a) Venn diagrams of the numbers of genes upregulated (red) or downregulated (blue) in comparisons of CD69+ MHC Ilow or CD69+ MHC Ihigh thymocytes from HoilΔCd4 versus WT (green) and Sharpincpdm versus WT (orange) at a 5% false discovery rate (FDR) cutoff. (b) Heatmaps of individual log-expression values. Left plot shows the 25 most upregulated genes and 50 most downregulated genes for HoilΔCd4 versus WT in CD69+ MHC Ilow thymocytes. Right plot show the same for CD69+ MHC Ihigh. Genes are ordered by P-value. Red indicates relatively higher expression and blue indicates relatively lower expression. Genes highlighted in green are involved in NF-κB signalling, those in yellow are involved in thymocyte/Treg cell differentiation. Shpncpdm refers to Sharpincpdm mice. (c,d) Genes that are differentially expressed in HoilΔCd4 versus WT but show no change or opposite change in Sharpincpdm. Results for CD69+ MHC Ilow thymocytes are shown in c and CD69+ MHC Ihigh thymocytes in d. The plot shows the limma t-statistics for each gene for assessing differential expression; the dotted line indicates the 5% FDR cutoffs of t=3.25 for c and t=3.94 for d.
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f6: Transcriptional impact of LUBAC deficiency on T-cell differentiation.(a) Venn diagrams of the numbers of genes upregulated (red) or downregulated (blue) in comparisons of CD69+ MHC Ilow or CD69+ MHC Ihigh thymocytes from HoilΔCd4 versus WT (green) and Sharpincpdm versus WT (orange) at a 5% false discovery rate (FDR) cutoff. (b) Heatmaps of individual log-expression values. Left plot shows the 25 most upregulated genes and 50 most downregulated genes for HoilΔCd4 versus WT in CD69+ MHC Ilow thymocytes. Right plot show the same for CD69+ MHC Ihigh. Genes are ordered by P-value. Red indicates relatively higher expression and blue indicates relatively lower expression. Genes highlighted in green are involved in NF-κB signalling, those in yellow are involved in thymocyte/Treg cell differentiation. Shpncpdm refers to Sharpincpdm mice. (c,d) Genes that are differentially expressed in HoilΔCd4 versus WT but show no change or opposite change in Sharpincpdm. Results for CD69+ MHC Ilow thymocytes are shown in c and CD69+ MHC Ihigh thymocytes in d. The plot shows the limma t-statistics for each gene for assessing differential expression; the dotted line indicates the 5% FDR cutoffs of t=3.25 for c and t=3.94 for d.
Mentions: HOIL deficiency altered the transcriptome immediately following positive selection (124 differentially expressed genes in CD69+ MHC Ilow thymocytes) and this effect was amplified in CD69+ MHC Ihigh thymocytes (724 differentially expressed genes; Fig. 6a). Surprisingly, thymocytes from Sharpincpdm mice had more substantial alteration of the transcriptome, perhaps reflecting the consequences of moderate LUBAC defects throughout T-cell differentiation (compared with the conditional deletion of HOIL-1 at the DP stage) and minor differences in the genetic background (still largely C57BL/Ka versus WT C57BL/6). To analyse the transcriptional changes associated with the block in SP thymocyte differentiation, we performed a heat-map analysis of the most significantly upregulated (25) or downregulated (50) genes in CD69+ MHC Ilow and CD69+ MHC Ihigh thymocytes from HoilΔCd4 compared with WT mice (Fig. 6b). Reduced expression of many core NF-κB target genes was a common feature of thymocytes from HoilΔCd4 and Sharpincpdm mice. These included genes involved in negative feedback (for example, Nfkbia (or IκBα), Nfkbie (or IκBɛ), Birc3 (or cIAP2) and Tnfaip3 (or A20)) and T-cell differentiation (particularly of FOXP3+ Treg cells; for example, Gadd45b, Il2ra, Tnfrsf18, Tnfrsf4 (or OX40) and Relb; Fig. 6b). To determine whether there was a defect in the induction of NF-κB target genes in the LUBAC-deficient strains during the differentiation from CD69+ MHC Ilow into CD69+ MHC Ihigh thymocytes, we first identified 154 NF-κB target genes that were significantly up- or downregulated during this transition in WT cells. Barcode enrichment plots show that, despite the dampened transcription of NF-κB target genes, the magnitude and direction of changes induced in these transcripts during the post-positive selection stages was maintained in HOIL- and SHARPIN-deficient cells (Supplementary Fig. 4). These findings support our earlier data showing that, although LUBAC-deficient thymocytes have impaired NF-κB signalling, this defect does not by itself explain the block in thymocyte differentiation observed in HoilΔCd4 and HoipΔCd4 mice.

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.