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

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.

Figure 1. Decreased CD4⁺ and CD8⁺ T cell production in 5AT161 mutant mice

Representative (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. TCRβ^hi SP thymocyte subsets were gated as follows: 24⁺MHC-I^lo = CD4⁺CD8^loCD24⁺MHC-I^lo; immature (Imm) = MHC-I^hiCD24⁺; and mature (Mat) = MHC-I^hiCD24^lo. (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 TCR^hi DP population.

Figure 2. Impaired positive selection and normal negative selection

Representative (1 of 5 experiments for 5C.C7 and 2 experiments for H-Y) flow cytometry profiles of CD4 and CD8 expression (left) and cellularity (right, shown as mean with standard error) of thymocytes from 3 month-old 5C.C7 TCR transgenic mice or female H-Y TCR transgenic mice. * P<0.01. (b) The percentage of V_β5⁺ cells within the CD4⁺ or CD8⁺ T cell populations from the thymus and spleen of 2-4 month old mutant (Y489X) or wild-type (WT) mice on B6 (BB: non-deleting) and B10.BR (KK: deleting) backgrounds is shown (n ≥ 5 mice per group).

Figure 3. Induced premature stop codon in the Themis gene (E430004N04Rik) in 5AT161 mutant mice

(a) The critical mapping interval of the 5AT161 mutation with the location of key SNPs and microsatellite markers between B6 and 129 or CBA strains. B6 homozygotes (black squares) and heterozygotes (white squares) are shown for affected and unaffected F2 mice along with the number of recombinants (Rec) observed in affected mice for each genetic marker. (b) Sequence trace histograms of the Tyr-489 mutated codon in wild-type and 5AT161 mice. (c) Amino acid sequence alignment of the region surrounding the conserved cysteine residue in the CABIT domain(s) of multiple Themis protein family members. Highly conserved hydrophobic residues (yellow), conserved cysteine residue(s) (red), and putative coiled-coil motif (underlined) are highlighted. In the case of the two domain proteins like Themis itself, the two CABIT domains are indicated by the prefix 1 or 2. The organism abbreviations are: Ccap : Capitella capitata; Dmel : Drosophila melanogaster; Drer : Danio rerio; Hsap : Homo sapiens; Mmus : Mus musculus; Olat : Oryzias latipes.

Figure 4. Domain architectures and evolution of the CABIT domain

A domain schematic of Themis showing two globular CABIT domains (purple), a proline-rich region (blue), a conserved helical segment (green), conserved cysteine motifs (orange bars), the site of the mutation (black bar), and the location of key amino acid residues is shown in the lower left corner of the figure. In the rest of the figure, similar domain architectures for all of the CABIT domain proteins have been superimposed on the animal phylogenetic tree. The major animal groups are indicated to the right of the figure. Proteins show the CABIT domain (C), the SAM domain (S), and the low complexity proline-rich stretch (P). The conserved helical segments preceding the proline-rich region are shown in green as distinct elements and extensions unique to particular proteins are shown as grey elements. In gnathostome vertebrates and insects, the names of the orthologs are also indicated. The location of the conserved cysteine in the CABIT domain is shown as an orange bar; those representatives lacking it are presumed to be inactive versions of the domain.

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 ΔΔC_T 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.

Figure 6. Normal TCR-driven upregulation of activation markers and decreased expression of development markers in Themis mutant DP thymocytes

(a) Mean fluorescence intensity (MFI) of CD5 expression on Themis(Y489X) (black with solid line) and wild-type (gray with dashed line) DP thymocytes after overnight stimulation with varying doses of plate-bound anti-CD3ε. The data points to the left of the break are for unstimulated samples. Live DP cells were gated based on forward scatter and the absence of 7AAD staining. (b) Percentage of CD69⁺ cells after overnight stimulation of Themis(Y489X) (red) or wild-type (blue) AND thymocytes cultered 2:1 with P13.9 cells and varying doses of the agonist (PCC_88-104), the weak agonist (K99P), or the very weak agonist (K99Q, insert) peptide ligands. Live DP cells were gated as in (a). Data are representative of 3 independent experiments for both (a) and (b). (c) MFI expression of the indicated cell surface proteins on TCRβ^lo/mid DP thymocytes from non-transgenic (B6) and the indicated TCR transgenic mice. Values were normalized to the maximum MFI within each experiment. Horizontal bars are the mean for each genotype. Data are representative of 2 independent experiments for the H-Y mice and ≥5 experiments for all others.

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.