Multipotent hematopoietic stem cells originating in the bone marrow (BM) enter the thymus and become committed to the T cell lineage. Immature CD4-CD8- double negative (DN) T cells that successfully rearrange and express a TCR beta chain, proliferate and upregulate both co-receptors to become CD4+CD8+ double positive (DP). Positive selection, negative selection, and lineage commitment decisions are then based in large part on the duration and strength of TCR signaling, which is heavily influenced by the extracellular milieu and nature of the peptide:MHC complexes presented by thymic stromal cells. Only cells with intermediate activation of TCR signaling pathways are positively selected, while cells that are activated with too strong or too weak a signal die by negative selection or neglect, respectively. Lineage commitment to the mature single positive (SP) stages is also thought to depend on signal strength and duration, as stronger, sustained signaling leads to CD4 SP commitment while weaker and shorter signals result in the development of CD8 SP cells. In the present work we looked at an ENU-induced mutation whose peripheral blood T cells proliferated poorly to stimulation with anti-CD3 and anti-CD28 antibodies. This was caused by an 8 fold decrease in the number of naive CD4+ T cells. Naive CD8+ T cells were also decreased, but not as dramatically. The numbers of CD44hi memory cells of both types were normal. The immunological defect appeared to arise during T cell development at the level of positive selection. DP, CD69+ thymocytes are generated, but as the cells move into the single positive stage, the CD4 T cells decrease by 6-10 fold compared to WT thymocytes and the CD8 SPs decrease by 2-3 fold. CD25+ Treg cells are also diminished, but NKT cell numbers are normal. Negative selection by superantigens also appeared normal. Radiation chimera experiments with mixed bone marrow suggested that the defect is T cell intrinsic. The mutation is a stop codon at the C-terminal end of a previously uncharacterized gene (now called Themis) that eliminates any detectable protein. The gene expression is T cell-specific and first turns on at the DN2 stage of development. Expression peaks at the DP stage and then declines about 10 fold as WT thymocytes mature. The protein is a member of a small family found in all vertebrates and each has a duplicated conserved domain structure with an invariant cysteine motif. Each member is expressed in a different tissue. The cysteine motif, which we call the CABIT domain, can also be followed in evolution to define a metazoan superfamily going back to Cnidarians. Each member has one or two CABIT domains and a separate region containing a protein interaction sequence such as a SAM domain or a proline rich region for interaction with an SH3 domain. Purified naive CD4+ T cells from the mutant proliferate normally to anti-CD3 plus anti-CD28 stimulation. They also make comparable amounts of IL-2 and express the activation markers CD69 and CD25 normally. DP thymocytes also showed normal CD69 expression and a comparable calcium response when stimulated with various doses of anti-CD3, as well as normal Erk and general tyrosine phosphorylation following anti-TCR and anti-CD4 crosslinking. The only abnormality in TCR signaling we detected was a slight increase in I kappa B alpha degradation in mutant thymocytes, but no downstream consequences of this were noted. CD69 expression was normal even in response to weak peptide agonists stimulating mutant TCR transgenic thymocytes. We therefore turned to a microarray analysis on DP thymocytes, both CD69+ and CD69- subsets, as well as on early CD4+ SP thymocytes (CD24hi, H-2Klo) to look for differences between the mutant and WT cells at the mRNA level. This analysis revealed that Themis-deficient thymocytes fail to maintain normal expression of cell cycle and survival genes and to appropriately regulate metabolic pathways. As a consequence of this deficiency we think there is premature death and a failure to complete positive selection. During the past year we have focused more heavily on analyzing the genes uncovered in the microarray study. We first repeated the analysis in mixed bone marrow chimeric mice to be certain that the gene expression differences we detected were intrinsic to the DP thymocytes and not secondary to the impact of a few genes on the general developmental environment in the thymus. Purified mutant CD69 negative DP thymocytes showed a similar down regulation (relative to purified WT cells from the same chimeric thymus) of about 90% of the cell cycle genes observed in the original experiment, giving us confidence that the genes we planned to study further were intrinsically altered in their expression in the thymocytes. Next we did a preliminary microarray analysis on mutant vs WT double negative (DN) thymocytes. This revealed ony 4 genes, which had been identified in the original screen, that still manifested significant differential expression. These genes are currently being examined for which stages of DN thymocyte development their expression is being altered by the themis mutation, and what impact they might be having on T cell development. The latter is being studied by over-expressing each gene using transgenic mice and/or by knocking down the gene expression using shRNAs.
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