The many roles of TOX in the immune system

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TOX is a member of an evolutionarily conserved DNA-binding protein family and is expressed in several immune-relevant cell subsets. Here, we review the key role of TOX in regulating development of CD4 T cells, natural killer cells and lymphoid tissue inducer cells, the latter responsible for the generation of lymph nodes. Although the exact molecular mechanism of action of TOX remains to be elucidated, the role of TOX in establishment of gene programs in the thymus and the potential of TOX as a regulator of E protein activity are discussed.

Highlights

► The TOX protein is a member of a small subfamily of HMG-box DNA binding proteins. ► The TOX protein is predicted to interact with DNA in a sequence-independent fashion. ► TOX is required for CD4 T cell lineage development in the thymus. ► TOX regulates NK cell development in the bone marrow and lymph node organogenesis.

Introduction

Development of mature cells requires coordinated expression of gene regulatory networks that promote differentiation of precursors, while simultaneously inhibiting alternate cell fates. Many nuclear factors, including transcription factors, cofactors, and chromatin modifiers, have been described to play essential roles in the development of the immune system. In this review, we focus on the specific role of nuclear factor TOX (Thymocyte selection-associated HMG bOX protein) in the immune system. TOX was originally identified by microarray as a thymic transcript that was highly upregulated in CD4+CD8+ double positive (DP) thymocytes activated with pharmacological agents phorbol ester and ionomycin ex vivo, as a mimic of the TCR signaling that initiates thymic positive selection [1]. Subsequent analysis at the level of gene and protein expression confirmed that TOX was expressed in the thymus in a stage-specific regulated manner. However, TOX expression is not limited to the thymus and is detected in other tissues, including liver, where it likely plays additional functions in regulating metabolic processes (J. Kaye, unpublished data). TOX is not only a part of the larger HMG-box superfamily of proteins, but also defines a small subfamily of proteins including TOX2, TOX3 and TOX4, all of which are highly conserved in vertebrate species (Figure 1) [2]. While the in vivo functions of TOX have been best characterized, TOX2 may play a role in reproductive organs [3], TOX3 has been implicated in regulation of neuron cell survival [4] and breast cancer [5, 6], and TOX4 is known to interact with a phosphatase complex involved in the control of chromatin structure and cell cycle progression [7, 8].

The sequence of the HMG-box domain of the TOX family of proteins places these factors in the sequence-independent but structure-dependent family of DNA-binding proteins [2]. Like other HMG-box proteins, the DNA binding domain of TOX comprises three helices that fold into an L-shaped structure (Protein Data Bank; http://www.pdb.org/pdb/explore/explore.do?structureId=2co9), and can interact with distorted DNA structures including cisplatinated DNA and double stranded circular DNA (J. Kaye, unpublished data). Unlike many HMG-box proteins, however, the TOX DNA binding domain cannot induce bending of DNA due to lack of a key hydrophobic wedge residue (J. Kaye, unpublished data). Among TOX family members, the DNA binding domain is near identical (and with shared genomic organization), the N-terminal domain is fairly conserved and has transactivation activity, and the C-terminal domain is family-member specific. Using genetically modified mice we have revealed the first in vivo function for a TOX-family member, in this case as a key regulator of immune system development.

Section snippets

Role of TOX in T cell development

TOX is transiently upregulated during β-selection and positive selection of developing thymocytes [1]. In terms of the latter, upregulation of TOX by DP cells is mediated by TCR-mediated calcineurin signaling [9]. We have taken both transgenic and knockout approaches to identify the role of TOX in the thymus. At the CD4CD8 (double negative, DN) stage, enforced expression of TOX was sufficient to induce upregulation of both CD4 and CD8αβ, although not the cell proliferation normally associated

Role of TOX in development of lymph nodes and Peyer's patches

We also observed that TKO mice lacked lymph nodes and had a significant decrease in the frequency and size of Peyer's patches [25••]. This was shown to be independent of the T cell developmental defect in these animals [25••]. Lymphoid tissue inducer (LTi) cells are key regulators of lymph node organogenesis [26, 27, 28]. These cells are of hematopoietic origin, are found in fetal liver during embryogenesis, and initiate formation of lymph node and Peyer's patch anlagen through interactions

Role of TOX in formation of NK cells

TOX is also expressed in the NK cell lineage, with highest expression in immature NK (iNK, LinIL-15Rα+NK1.1+DX5) and mature NK (mNK, LinIL-15Rα+NK1.1+DX5+) cells in the bone marrow [25••]. In TKO mice, NK cells were significantly reduced in both the bone marrow and spleen, with a block in the transition of NK progenitor cells (LinIL15Rα+NK1.1DX5) to the iNK stage. The loss of NK cells was also reflected in reduction in NK cell-mediated cytotoxicity in vivo in TKO mice [25••].

Development

Conclusions

TOX has multiple roles in the generation of the immune system, including development of CD4 T cells and NK cells, and lymph node organogenesis, the latter via regulation of LTi cell development. It is not clear if there are overlapping or distinct roles for TOX in each of these cell lineages. The one commonality may be upregulation of Id2, which is associated with each developmental process (Figure 3). Indeed, loss of TOX was associated with decreases in Id2 expression in both thymocytes and NK

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

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