THEMIS, the new kid on the block for T-cell development

Understanding T-cell development is a major goal of the immunology field and is crucial for the elucidation of the mechanisms behind self-tolerance and the occurrence of autoimmune disorders. T-cell development is a complex process that begins in the bone marrow or fetal liver, where lymphoid progenitors arise. Then, these progenitor cells migrate to the thymus to start the commitment into the T-cell lineage.^{1, 2} Thymocyte development has been described as a series of well-documented differentiation steps characterized according to the cell-surface expression of CD4 and CD8 co-receptors.¹ In the initial step, thymocytes are double negative (DN; CD4⁻ CD8⁻). In the second step, they become double positive (DP; CD4⁺ CD8⁺) before finally maturing into single-positive T cells (SP; CD4 or CD8).²

Thymocyte selection is essential for lineage commitment. This phenomenon occurs at the DP stage. The T-cell receptor (TCR) interacts with self-peptide–MHC ligands on the surface of cortical thymic epithelial cells.¹ Then, thymocytes are selected according to the nature of TCR signaling induced by the self-peptide–MHC ligands. While it is thought that signaling derived from weak TCR/MHC-peptide interactions leads to delayed apoptosis (death by neglect), strong TCR signaling promotes acute apoptosis (negative selection). An optimal intermediate activation of TCR signaling leads to positive selection and continues the maturation process to the CD4SP or CD8 stage.¹

The thymocyte-expressed molecule involved in selection (Themis) is an important regulator of thymocyte positive selection,³ and mice lacking Themis display a defect in the selection process mediated by the positive selection checkpoints, resulting in reduced numbers of SP cells and mature peripheral T cells.⁴

Themis is expressed exclusively in the T-cell lineage, with the highest expression levels found in DP thymocytes.^{4, 5, 6} Themis is a member of a small gene family conserved throughout vertebrate evolution and is tyrosine-phosphorylated by Lck (and possibly ZAP70) immediately after TCR crosslinking.^{5, 7, 8} Themis constitutively binds to GRB2^{6, 7, 9} by means of a polyproline region that interacts with the C-terminal SH3 domain of GRB2.³ Furthermore, Themis also co-immunoprecipitates with other molecules of the LAT/SLP76 signaling module.⁷

Themis contains two novel cysteine-based CABIT (cysteine-containing, all beta in Themis) domains,¹⁰ a bipartite type nuclear localization sequence and a proline-rich sequence.¹⁰ The CABIT domain is a recently designated domain structure conserved among metazoans, which has been predicted by means of multiple-sequence alignments to adopt an all-beta-strand structure with at least 12 strands,¹¹ suggesting either an extended beta-sandwich-like fold or a dyad of six-stranded beta-barrel units.¹⁰ In mammals, the CABIT domains are conserved among three Themis family proteins (Themis/Themis1, ICB1/Themis2 and 9130404H23Rik/Themis3), harboring two tandemly repeated CABIT domains (CABIT1 and CABIT2) and two Fam59 proteins (Fam59a and b) containing one CABIT domain.¹⁰ Although several proteins containing CABIT domains have been identified, their function is still unknown. The identification of the biological function of the CABIT domain is extremely important to understand the contribution of Themis in the positive selection process during T-cell development.

In a recent issue of Nature Immunology, Choi et al.¹² demonstrated a critical function of Themis during T-cell development and identified a biological function for the CABIT domain during positive selection and T-cell development. They showed that Themis, through CABIT, negatively regulates the activity of the PTP SHP-1 in DP thymocytes and enhances TCR signaling in response to low-affinity self-pMHC. As a result, positive selection is promoted (Figure 1). The authors demonstrated that the CABIT domain modulates Themis by interacting directly with the PTP domain of SHP-1, using cell-free in vitro protein-binding assays. This observation is consistent with previous findings made by Paster et al.,¹³ who showed that Themis constitutively associates with the phosphatases SHP-1 and SHP-2.¹³ In addition, Choi et al. demonstrated that Themis directly regulates the catalytic activity of SHP-1 through the CABIT domain, which binds to the phosphatase domain, promoting or stabilizing oxidation of the catalytic cysteine residue of SHP-1, leading to inhibition of the tyrosine phosphatase activity of SHP-1.

Figure 1.

Model of the function of Themis in positive selection. (a) In Themis^+/+ DP thymocytes: (1) binding of the TCR on these cells to the self-peptide and MHC complexes causes phosphorylation of ITAM domains on the CD3 co-receptor by Lck; (2) ZAP70 is recruited and phosphorylated; (3) LAT is phosphorylated by ZAP70; (4) LAT recruits GRB2, Themis and SHP-1; and (5) downstream signaling pathways are activated that are sufficient to support positive selection. (b) In thymocytes without a functional Themis: (1) binding of the TCR to the self-peptide and MHC complexes causes phosphorylation of ITAM domains of CD3 by Lck kinase; (2) ZAP70 is recruited and phosphorylated; and (3) ZAP70 and Lck are blocked by the action of SHP-1, which results in the failure of positive selection and death by non-selection.

Choi et al. also identified a key role for ROS in thymocyte selection, which agrees with data previously published by Moon et al.¹⁴ and Jin et al.¹⁵ suggesting that ROS might play a role during positive selection and the maturation of SP thymocytes. Here, Jin et al.¹⁵ published data suggesting that the transition from pre- to post-selected DP stages is accompanied by an increase in ROS and transient expression of a variety of redox regulators, such as the thioredoxin (Trx)1/thioredoxin reductase (TrxR)1 system.¹⁵

Importantly, Choi et al. determined the importance of Themis in vivo by demonstrating that the deletion of SHP-1 in the Themis^−/− Ptpn6^fl/fl Cd4-Cre mice alleviated the developmental block in Themis^−/− thymocytes. Furthermore, these results, together with the profound block in the positive selection exhibited by Themis^−/− thymocytes, suggested that this protein might be responsible for the selective sensitivity of DP thymocytes to TCR engagement. These findings are in agreement with a previous report by Lesourne et al.⁴ showing that although DP thymocytes highly express Themis, expression is downregulated as thymocytes transition to the SP stage and become less responsive to low-affinity self ligands. Such a property is thought necessary for the prevention of autoimmunity.¹⁶ That activity, coupled with the stage-specific regulation of Themis during T-cell development, provides an explanation for the unusual sensitivity of DP thymocytes to TCR stimulation, a feature that is essential for positive selection. Previous independent studies had already identified the same gene, Themis, whose elimination has a profound effect on T-cell development. Collectively, these authors had provided a comprehensive investigation into the function of Themis but without defining the mechanism of action. The critical finding from the work of Choi et al. was the identification of one of the possible mechanisms of action of Themis in the process of positive selection. However, there are several questions that remain to be addressed. Finally, the findings made by Choi et al. contribute to the understanding of the process of tolerance of T cells to self and to defining some of the causes of the development of autoimmune diseases.