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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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Themis expression(a) RT-PCR expression analysis using Themis exon 3 forward and exon 4 reverse primers on sorted T cell populations. Expression was calculated using the ΔΔCT method with β actin as the standard. Data are shown as the mean fold expression over B cell values for 4-12 biological replicates per population. (b) Rabbit polyclonal anti-Themis immunoblot on lysates from unfractionated homogenized tissues of a wild-type B6 and a Themis(Y489X) thymus, as well as other tissues from wild-type B6 mice. Data for thymus, LN and spleen are representative of ≥ 3 independent experiments. (c) Confocal imaging of unfixed HEK293 cells transiently transfected overnight with Themis-EGFP (green) and labeled with Hoechst 33342 nuclear dye (blue). Original magnification x63. Data are representative of 4 independent experiments. (d) Anti-Grb2 immunoblot showing specific co-immunoprecipitation with Themis-EGFP in both Jurkat and HEK293 cell lines that had been transiently transfected overnight with Themis-EGFP or EGFP control constructs. Data are representative of 3 independent experiments.
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Figure 5: Themis expression(a) RT-PCR expression analysis using Themis exon 3 forward and exon 4 reverse primers on sorted T cell populations. Expression was calculated using the ΔΔCT method with β actin as the standard. Data are shown as the mean fold expression over B cell values for 4-12 biological replicates per population. (b) Rabbit polyclonal anti-Themis immunoblot on lysates from unfractionated homogenized tissues of a wild-type B6 and a Themis(Y489X) thymus, as well as other tissues from wild-type B6 mice. Data for thymus, LN and spleen are representative of ≥ 3 independent experiments. (c) Confocal imaging of unfixed HEK293 cells transiently transfected overnight with Themis-EGFP (green) and labeled with Hoechst 33342 nuclear dye (blue). Original magnification x63. Data are representative of 4 independent experiments. (d) Anti-Grb2 immunoblot showing specific co-immunoprecipitation with Themis-EGFP in both Jurkat and HEK293 cell lines that had been transiently transfected overnight with Themis-EGFP or EGFP control constructs. Data are representative of 3 independent experiments.

Mentions: To characterize Themis expression, we conducted an RT PCR analysis on amplified cDNA generated from FACS-sorted lymphocyte populations of B6 mice. Themis expression was barely detectable in uncommitted DN1 thymocytes, but was highly expressed in the succeeding DN2 to DN4 populations. Expression peaked in the DP population before decreasing more than 10-fold in CD4 and CD8 SP cells in both the thymus and periphery (Fig. 5a). Published microarray studies show that Themis expression is down-regulated even further in CD4+ regulatory T cells18-20. Immunoblotting with Themis-specific rabbit polyclonal antibodies (Supplementary Fig. 7) detected a single protein species near the predicted size of 73 kDa in thymus, LN and spleen samples from wild-type mice that was absent in Themis(Y489X) thymocytes and in non-lymphoid tissues (Fig. 5b). Relative to wild-type cells, Themis(Y489X) thymocytes had a 3-fold decrease in Themis mRNA, and only a faint band near the size of the predicted truncated protein was observed in mutant thymocyte lysates (data not shown). Thus, the mutant message and protein appear to be unstable.


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)

Themis expression(a) RT-PCR expression analysis using Themis exon 3 forward and exon 4 reverse primers on sorted T cell populations. Expression was calculated using the ΔΔCT method with β actin as the standard. Data are shown as the mean fold expression over B cell values for 4-12 biological replicates per population. (b) Rabbit polyclonal anti-Themis immunoblot on lysates from unfractionated homogenized tissues of a wild-type B6 and a Themis(Y489X) thymus, as well as other tissues from wild-type B6 mice. Data for thymus, LN and spleen are representative of ≥ 3 independent experiments. (c) Confocal imaging of unfixed HEK293 cells transiently transfected overnight with Themis-EGFP (green) and labeled with Hoechst 33342 nuclear dye (blue). Original magnification x63. Data are representative of 4 independent experiments. (d) Anti-Grb2 immunoblot showing specific co-immunoprecipitation with Themis-EGFP in both Jurkat and HEK293 cell lines that had been transiently transfected overnight with Themis-EGFP or EGFP control constructs. Data are representative of 3 independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908989&req=5

Figure 5: Themis expression(a) RT-PCR expression analysis using Themis exon 3 forward and exon 4 reverse primers on sorted T cell populations. Expression was calculated using the ΔΔCT method with β actin as the standard. Data are shown as the mean fold expression over B cell values for 4-12 biological replicates per population. (b) Rabbit polyclonal anti-Themis immunoblot on lysates from unfractionated homogenized tissues of a wild-type B6 and a Themis(Y489X) thymus, as well as other tissues from wild-type B6 mice. Data for thymus, LN and spleen are representative of ≥ 3 independent experiments. (c) Confocal imaging of unfixed HEK293 cells transiently transfected overnight with Themis-EGFP (green) and labeled with Hoechst 33342 nuclear dye (blue). Original magnification x63. Data are representative of 4 independent experiments. (d) Anti-Grb2 immunoblot showing specific co-immunoprecipitation with Themis-EGFP in both Jurkat and HEK293 cell lines that had been transiently transfected overnight with Themis-EGFP or EGFP control constructs. Data are representative of 3 independent experiments.
Mentions: To characterize Themis expression, we conducted an RT PCR analysis on amplified cDNA generated from FACS-sorted lymphocyte populations of B6 mice. Themis expression was barely detectable in uncommitted DN1 thymocytes, but was highly expressed in the succeeding DN2 to DN4 populations. Expression peaked in the DP population before decreasing more than 10-fold in CD4 and CD8 SP cells in both the thymus and periphery (Fig. 5a). Published microarray studies show that Themis expression is down-regulated even further in CD4+ regulatory T cells18-20. Immunoblotting with Themis-specific rabbit polyclonal antibodies (Supplementary Fig. 7) detected a single protein species near the predicted size of 73 kDa in thymus, LN and spleen samples from wild-type mice that was absent in Themis(Y489X) thymocytes and in non-lymphoid tissues (Fig. 5b). Relative to wild-type cells, Themis(Y489X) thymocytes had a 3-fold decrease in Themis mRNA, and only a faint band near the size of the predicted truncated protein was observed in mutant thymocyte lysates (data not shown). Thus, the mutant message and protein appear to be unstable.

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