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 regulateemetabolic pathways. As a consequence of this deficiency we think there is premature death and a failure to complete positive selection. The past year was spent trying to make retrogenic bone marrow chimeric mice in order to reconstitute the Themis mutation and to study the importance of the CABIT domain cysteine residues in the function of the molecule. This turned out to be technially very difficult as retroviral expression of Themis in bone marrow cells was not robust. Nonetheless, after optimization of expression and viral titers at every step we were able to reconstitute normal CD4 SP thymocyte development in GFP-expressing cells (compared to GFP negative cells in the same thymus)in the Themis mutant, demonstrating that the mutation was responsible for the defect in thymic developmental. We then looked at themis constructs in which we mutated the cysteines in the CABIT domain to alanines. Chimeric Themis mutant mice reconstituted with retroviral infected bone marrow expressing these Cys to Ala substitutions showed only a partial reconstitution of the normal CD4 SP thymocyte phenotype. Double substitution of alanine at both CABIT domains led to more impairment in CD4 SP development, but did not completely prevent it. Protein expression in 293 cells of all 3 of these Cys/Ala mutations showed that the molecules could still bind Grb2 normally, suggesting that their three dimensional structure had not been significantly altered by the amino acid substitutions. Overall, these observations suggest that the cysteine residues do play a role in Themis function in vivo.
|Johnson, Andy L; Aravind, L; Shulzhenko, Natalia et al. (2009) Themis is a member of a new metazoan gene family and is required for the completion of thymocyte positive selection. Nat Immunol 10:831-9|