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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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Decreased CD4+ and CD8+ T cell production in 5AT161 mutant miceRepresentative (1 of 4 independent experiments) flow cytometry profiles of CD4 and CD8 expression (left) and cellularity (right, shown as mean with standard error) of (a) LN T cells and (b) thymocyte subsets from 7 week-old mice. * P<0.02. TCRβhi SP thymocyte subsets were gated as follows: 24+MHC-Ilo = CD4+CD8loCD24+MHC-Ilo; immature (Imm) = MHC-IhiCD24+; and mature (Mat) = MHC-IhiCD24lo. (c) Percentage of 5AT161 mutant-derived CD45.1- cells in lymphocyte populations analyzed 8 weeks after CD45.1+ wild-type mice were lethally irradiated and injected i.v. with an equal mix of CD45.1- 5AT161 mutant and CD45.1+ wild-type BM. Data show geometric mean with 95% confidence interval error bars (n=5). * indicates P<0.02 compared to the TCRhi DP population.
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Figure 1: Decreased CD4+ and CD8+ T cell production in 5AT161 mutant miceRepresentative (1 of 4 independent experiments) flow cytometry profiles of CD4 and CD8 expression (left) and cellularity (right, shown as mean with standard error) of (a) LN T cells and (b) thymocyte subsets from 7 week-old mice. * P<0.02. TCRβhi SP thymocyte subsets were gated as follows: 24+MHC-Ilo = CD4+CD8loCD24+MHC-Ilo; immature (Imm) = MHC-IhiCD24+; and mature (Mat) = MHC-IhiCD24lo. (c) Percentage of 5AT161 mutant-derived CD45.1- cells in lymphocyte populations analyzed 8 weeks after CD45.1+ wild-type mice were lethally irradiated and injected i.v. with an equal mix of CD45.1- 5AT161 mutant and CD45.1+ wild-type BM. Data show geometric mean with 95% confidence interval error bars (n=5). * indicates P<0.02 compared to the TCRhi DP population.

Mentions: By immunological screening of a library of pedigrees segregating thousands of ENU-induced nucleotide substitutions10, we identified one, 5AT161, with an abnormal response to immunization, anti-nuclear autoantibodies, low anti-CD3ε+CD28-induced proliferation and a decreased percentage of naive CD44low CD4+ T cells in the blood (Supplementary Fig. 1a and data not shown). The latter two phenotypes proved to be inherited as a fully penetrant, recessive trait that was readily scored and fixed in subsequent generations. In homozygous 5AT161 mutants, flow cytometry of blood and lymph nodes (LN) showed a reversal of the normal CD4:CD8 ratio and an 8-fold decrease in the absolute number of naïve CD4+ cells and a 3-fold decrease in the naïve CD8+ cells (Fig. 1a). By contrast, the number of antigen-experienced CD44hi cells in both subsets was normal (Fig. 1a). Peripheral naïve CD44lo cells sorted from 5AT161 mice proliferated and upregulated CD25 equivalently to control counterparts following stimulation with anti-CD3ε and anti-CD28 (Supplementary Fig. 1b), confirming that the decreased proliferation in the original screen was due to decreased cellularity. Absolute numbers of other non-T cell hematopoietic lineages (B cells, natural killer (NK) cells, dendritic cells (DCs), and granulocytes) were similar in the central and peripheral lymphoid organs of 5AT161 and control mice, indicating a selective defect in T cell development (Supplementary Fig. 2 and data not shown).


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)

Decreased CD4+ and CD8+ T cell production in 5AT161 mutant miceRepresentative (1 of 4 independent experiments) flow cytometry profiles of CD4 and CD8 expression (left) and cellularity (right, shown as mean with standard error) of (a) LN T cells and (b) thymocyte subsets from 7 week-old mice. * P<0.02. TCRβhi SP thymocyte subsets were gated as follows: 24+MHC-Ilo = CD4+CD8loCD24+MHC-Ilo; immature (Imm) = MHC-IhiCD24+; and mature (Mat) = MHC-IhiCD24lo. (c) Percentage of 5AT161 mutant-derived CD45.1- cells in lymphocyte populations analyzed 8 weeks after CD45.1+ wild-type mice were lethally irradiated and injected i.v. with an equal mix of CD45.1- 5AT161 mutant and CD45.1+ wild-type BM. Data show geometric mean with 95% confidence interval error bars (n=5). * indicates P<0.02 compared to the TCRhi DP population.
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Related In: Results  -  Collection

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Figure 1: Decreased CD4+ and CD8+ T cell production in 5AT161 mutant miceRepresentative (1 of 4 independent experiments) flow cytometry profiles of CD4 and CD8 expression (left) and cellularity (right, shown as mean with standard error) of (a) LN T cells and (b) thymocyte subsets from 7 week-old mice. * P<0.02. TCRβhi SP thymocyte subsets were gated as follows: 24+MHC-Ilo = CD4+CD8loCD24+MHC-Ilo; immature (Imm) = MHC-IhiCD24+; and mature (Mat) = MHC-IhiCD24lo. (c) Percentage of 5AT161 mutant-derived CD45.1- cells in lymphocyte populations analyzed 8 weeks after CD45.1+ wild-type mice were lethally irradiated and injected i.v. with an equal mix of CD45.1- 5AT161 mutant and CD45.1+ wild-type BM. Data show geometric mean with 95% confidence interval error bars (n=5). * indicates P<0.02 compared to the TCRhi DP population.
Mentions: By immunological screening of a library of pedigrees segregating thousands of ENU-induced nucleotide substitutions10, we identified one, 5AT161, with an abnormal response to immunization, anti-nuclear autoantibodies, low anti-CD3ε+CD28-induced proliferation and a decreased percentage of naive CD44low CD4+ T cells in the blood (Supplementary Fig. 1a and data not shown). The latter two phenotypes proved to be inherited as a fully penetrant, recessive trait that was readily scored and fixed in subsequent generations. In homozygous 5AT161 mutants, flow cytometry of blood and lymph nodes (LN) showed a reversal of the normal CD4:CD8 ratio and an 8-fold decrease in the absolute number of naïve CD4+ cells and a 3-fold decrease in the naïve CD8+ cells (Fig. 1a). By contrast, the number of antigen-experienced CD44hi cells in both subsets was normal (Fig. 1a). Peripheral naïve CD44lo cells sorted from 5AT161 mice proliferated and upregulated CD25 equivalently to control counterparts following stimulation with anti-CD3ε and anti-CD28 (Supplementary Fig. 1b), confirming that the decreased proliferation in the original screen was due to decreased cellularity. Absolute numbers of other non-T cell hematopoietic lineages (B cells, natural killer (NK) cells, dendritic cells (DCs), and granulocytes) were similar in the central and peripheral lymphoid organs of 5AT161 and control mice, indicating a selective defect in T cell development (Supplementary Fig. 2 and data not shown).

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