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Themis is a member of a new metazoan gene family and is required for the completion of thymocyte positive selection.

Johnson AL, Aravind L, Shulzhenko N, Morgun A, Choi SY, Crockford TL, Lambe T, Domaschenz H, Kucharska EM, Zheng L, Vinuesa CG, Lenardo MJ, Goodnow CC, Cornall RJ, Schwartz RH - Nat. Immunol. (2009)

Bottom Line: T cell antigen receptor (TCR) signaling in CD4(+)CD8(+) double-positive thymocytes determines cell survival and lineage commitment, but the genetic and molecular basis of this process is poorly defined.Themis contains a tandem repeat of a unique globular domain (called 'CABIT' here) that includes a cysteine motif that defines a family of five uncharacterized vertebrate proteins with orthologs in most animal species.Themis-deficient thymocytes showed no substantial impairment in early TCR signaling but did show altered expression of genes involved in the cell cycle and survival before and during positive selection.

View Article: PubMed Central - PubMed

Affiliation: Nuffield Department of Clinical Medicine, Oxford University, UK.

ABSTRACT
T cell antigen receptor (TCR) signaling in CD4(+)CD8(+) double-positive thymocytes determines cell survival and lineage commitment, but the genetic and molecular basis of this process is poorly defined. To address this issue, we used ethylnitrosourea mutagenesis to identify a previously unknown T lineage-specific gene, Themis, which is critical for the completion of positive selection. Themis contains a tandem repeat of a unique globular domain (called 'CABIT' here) that includes a cysteine motif that defines a family of five uncharacterized vertebrate proteins with orthologs in most animal species. Themis-deficient thymocytes showed no substantial impairment in early TCR signaling but did show altered expression of genes involved in the cell cycle and survival before and during positive selection. Our data suggest a unique function for Themis in sustaining positive selection.

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Altered expression of survival, cell cycle and lipid metabolism genes in Themis(Y489X) thymocytes(a) Log2 expression of Erk and NF-AT target genes in wild-type (gray circles with mean as dotted line) and Themis(Y489X) (black circles with mean as solid line) thymocyte populations. Data taken from gene-specific probes are relative to a reference of mixed wild-type and mutant samples from all populations. (b) Microarray gene expression analysis identified 325 differentially expressed transcripts between Themis(Y489X) and wild-type thymocytes that were separated into 5 clusters based on differential expression between mutant and wild-type cells as well as expression changes between populations in wild-type samples (see Supplementary Table 1). Gene Ontology categories enriched in each cluster (*P<0.001, **P<10-5, and P<0.01 for all others), and median relative expression of genes in each cluster in mutant (black with solid line) and wild-type (gray with dotted line) samples are shown. The fold-change between averaged mutant and wild-type expression in each population (n = 8 or 9) is displayed in the heatmap for each transcript.
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Figure 7: Altered expression of survival, cell cycle and lipid metabolism genes in Themis(Y489X) thymocytes(a) Log2 expression of Erk and NF-AT target genes in wild-type (gray circles with mean as dotted line) and Themis(Y489X) (black circles with mean as solid line) thymocyte populations. Data taken from gene-specific probes are relative to a reference of mixed wild-type and mutant samples from all populations. (b) Microarray gene expression analysis identified 325 differentially expressed transcripts between Themis(Y489X) and wild-type thymocytes that were separated into 5 clusters based on differential expression between mutant and wild-type cells as well as expression changes between populations in wild-type samples (see Supplementary Table 1). Gene Ontology categories enriched in each cluster (*P<0.001, **P<10-5, and P<0.01 for all others), and median relative expression of genes in each cluster in mutant (black with solid line) and wild-type (gray with dotted line) samples are shown. The fold-change between averaged mutant and wild-type expression in each population (n = 8 or 9) is displayed in the heatmap for each transcript.

Mentions: Despite the lack of any major TCR-dependent signaling abnormalities and the presence of normal absolute numbers of cells, freshly isolated Themis(Y489X) DP thymocytes expressed slightly lower amounts of the developmental markers CD2, CD27, and CD5 (Fig. 6c). Given that Themis is first expressed at the DN1 stage of thymocyte development, these observations suggest that the loss of Themis(Y489X) DP thymocytes following positive selection might be due to a functional impairment established at a stage prior to positive selection. To search for alternative pathways affected by Themis deficiency at all stages of development, we compared gene expression profiles in pre-selection DP cells (CD5loCD69lo), post-selection DP cells (CD5hiCD69+) and uncommitted CD24+MHC-Ilo cells (CD4+CD8loTCR╬▓hiCD24+MHC-Ilo) sorted from wild-type and Themis(Y489X) mice by flow cytometry. We identified 325 genes that were either differentially expressed in at least one of the three populations in mutant mice or had altered expression changes during development of these thymocyte populations when compared to wild-type expression patterns (false discovery rate of 10%; Supplementary Table 1). Importantly, the transcription pattern did not demonstrate any changes in downstream gene targets of the two principal pathways known to be involved in positive selection, those involving Erk and NF-AT (Fig. 7a)25. The normal induction of these target genes bolsters the conclusion that initial TCR signaling in DP thymocytes is unaffected by the Themis mutation.


Themis is a member of a new metazoan gene family and is required for the completion of thymocyte positive selection.

Johnson AL, Aravind L, Shulzhenko N, Morgun A, Choi SY, Crockford TL, Lambe T, Domaschenz H, Kucharska EM, Zheng L, Vinuesa CG, Lenardo MJ, Goodnow CC, Cornall RJ, Schwartz RH - Nat. Immunol. (2009)

Altered expression of survival, cell cycle and lipid metabolism genes in Themis(Y489X) thymocytes(a) Log2 expression of Erk and NF-AT target genes in wild-type (gray circles with mean as dotted line) and Themis(Y489X) (black circles with mean as solid line) thymocyte populations. Data taken from gene-specific probes are relative to a reference of mixed wild-type and mutant samples from all populations. (b) Microarray gene expression analysis identified 325 differentially expressed transcripts between Themis(Y489X) and wild-type thymocytes that were separated into 5 clusters based on differential expression between mutant and wild-type cells as well as expression changes between populations in wild-type samples (see Supplementary Table 1). Gene Ontology categories enriched in each cluster (*P<0.001, **P<10-5, and P<0.01 for all others), and median relative expression of genes in each cluster in mutant (black with solid line) and wild-type (gray with dotted line) samples are shown. The fold-change between averaged mutant and wild-type expression in each population (n = 8 or 9) is displayed in the heatmap for each transcript.
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Figure 7: Altered expression of survival, cell cycle and lipid metabolism genes in Themis(Y489X) thymocytes(a) Log2 expression of Erk and NF-AT target genes in wild-type (gray circles with mean as dotted line) and Themis(Y489X) (black circles with mean as solid line) thymocyte populations. Data taken from gene-specific probes are relative to a reference of mixed wild-type and mutant samples from all populations. (b) Microarray gene expression analysis identified 325 differentially expressed transcripts between Themis(Y489X) and wild-type thymocytes that were separated into 5 clusters based on differential expression between mutant and wild-type cells as well as expression changes between populations in wild-type samples (see Supplementary Table 1). Gene Ontology categories enriched in each cluster (*P<0.001, **P<10-5, and P<0.01 for all others), and median relative expression of genes in each cluster in mutant (black with solid line) and wild-type (gray with dotted line) samples are shown. The fold-change between averaged mutant and wild-type expression in each population (n = 8 or 9) is displayed in the heatmap for each transcript.
Mentions: Despite the lack of any major TCR-dependent signaling abnormalities and the presence of normal absolute numbers of cells, freshly isolated Themis(Y489X) DP thymocytes expressed slightly lower amounts of the developmental markers CD2, CD27, and CD5 (Fig. 6c). Given that Themis is first expressed at the DN1 stage of thymocyte development, these observations suggest that the loss of Themis(Y489X) DP thymocytes following positive selection might be due to a functional impairment established at a stage prior to positive selection. To search for alternative pathways affected by Themis deficiency at all stages of development, we compared gene expression profiles in pre-selection DP cells (CD5loCD69lo), post-selection DP cells (CD5hiCD69+) and uncommitted CD24+MHC-Ilo cells (CD4+CD8loTCR╬▓hiCD24+MHC-Ilo) sorted from wild-type and Themis(Y489X) mice by flow cytometry. We identified 325 genes that were either differentially expressed in at least one of the three populations in mutant mice or had altered expression changes during development of these thymocyte populations when compared to wild-type expression patterns (false discovery rate of 10%; Supplementary Table 1). Importantly, the transcription pattern did not demonstrate any changes in downstream gene targets of the two principal pathways known to be involved in positive selection, those involving Erk and NF-AT (Fig. 7a)25. The normal induction of these target genes bolsters the conclusion that initial TCR signaling in DP thymocytes is unaffected by the Themis mutation.

Bottom Line: T cell antigen receptor (TCR) signaling in CD4(+)CD8(+) double-positive thymocytes determines cell survival and lineage commitment, but the genetic and molecular basis of this process is poorly defined.Themis contains a tandem repeat of a unique globular domain (called 'CABIT' here) that includes a cysteine motif that defines a family of five uncharacterized vertebrate proteins with orthologs in most animal species.Themis-deficient thymocytes showed no substantial impairment in early TCR signaling but did show altered expression of genes involved in the cell cycle and survival before and during positive selection.

View Article: PubMed Central - PubMed

Affiliation: Nuffield Department of Clinical Medicine, Oxford University, UK.

ABSTRACT
T cell antigen receptor (TCR) signaling in CD4(+)CD8(+) double-positive thymocytes determines cell survival and lineage commitment, but the genetic and molecular basis of this process is poorly defined. To address this issue, we used ethylnitrosourea mutagenesis to identify a previously unknown T lineage-specific gene, Themis, which is critical for the completion of positive selection. Themis contains a tandem repeat of a unique globular domain (called 'CABIT' here) that includes a cysteine motif that defines a family of five uncharacterized vertebrate proteins with orthologs in most animal species. Themis-deficient thymocytes showed no substantial impairment in early TCR signaling but did show altered expression of genes involved in the cell cycle and survival before and during positive selection. Our data suggest a unique function for Themis in sustaining positive selection.

Show MeSH