A Fateful Decision in the Thymus Controlled by the Transcription Factor ThPOK

Introduction

T cells that express heterodimerized α-β TCR play central roles in host defense against infections and cancers (1–4). Conventional αβT cells are divided into CD4 helper and CD8 cytotoxic T cells. CD4 T cells possess plasticity to differentiate into distinct populations of cytokine producers that subsequently modulate immune responses by diverse cell types, including macrophages, mast cells, neutrophils, and epithelial cells. This functional polarization of CD4 effector T cells is driven by the cytokine milieu established by innate immune cells that sense invasion by microbes and thus amplifies coordinated immune responses involving multiple cell types for efficient control of pathogens. A distinct subset of CD4 T cells generated from the same naive pool provides help to license professional Ag-presenting cells for efficient priming of CD8 T cells, while also supporting survival and clonal expansion of B cells for Ab affinity maturation in the germinal center. By contrast, CD8 T cells predominantly function as cytotoxic cells through their capacity for enormous clonal expansion, production of cytotoxic effector molecules, and expression of type-I cytokines, such as IFN-γ and TNF.

Although these two cell types are crucial mediators of adaptive immunity through their unique roles, the lineage bifurcation occurs at a narrow developmental window during positive selection of αβTCR precursors in the thymus (5–7). In this regard, thymocyte development is initiated by the entry of bone marrow– or fetal liver–derived multipotent or lymphoid primed progenitor cells. These progenitors generate a diverse array of CD4⁺ CD8⁺ double-positive (DP) immature T cells following rearrangements of both Tcra and Tcrb loci. DP cells are then tested for the ability of their clonal αβTCR to bind self-peptide presented in the context of MHC expressed on thymic epithelial cells. Whereas a majority of DP thymocytes die “by neglect,” clones with proper avidity for peptide–MHC complex receive TCR signaling and are positively selected. These clones are not only allowed to survive, but they also differentiate, a fate decision restricted by TCR recognition of either MHC class I (MHC-I) or class II (MHC-II) for cytotoxic or helper T cell lineages, respectively.

Helper versus cytotoxic T cell differentiation in the thymus has been studied intensively and serves as a model for binary cell fate decisions that are instructed or supported by cell extrinsic signals in multicellular organisms. Despite a tight link between TCR specificity to MHC-I versus MHC-II and the fate of selected thymocytes, there exists a long history of debates about stochastic versus instructive roles of signaling through TCR and coreceptors in this developmental process (7–15). Several general models were proposed to explain the CD4/CD8 cell fate decision process, including the strength-of-signal instructive and the stochastic selection models. The former proposed that qualitative differences existed in TCR signaling when engaged by CD4–MHC-II versus CD8–MHC-I. The differential signals would be mediated by the proximal tyrosine kinase Lck and, in turn, determine the fate of selected clones (16, 17). The latter model proposed that the differentiation of DP thymocytes to mature helper or cytotoxic T cells required MHC restriction of a TCR and continued expression of the properly matched coreceptor following stochastic downregulation of one coreceptor (18, 19). Later, the kinetic signaling model was proposed that incorporated signaling duration as a determinant for thymocyte fate decisions, with asymmetric downregulation of the coreceptors following signal initiation, regardless of MHC restriction (20). However, a major unanswered question had remained as to how these signaling events were connected to the differentiation program at the gene level. Indeed, any cell-intrinsic mechanism critical for thymocyte lineage decisions had remained elusive until the breakthrough discovery of Dietmar Kappes and colleagues, identifying ThPOK/Zbtb7b as the master transcription factor regulating helper versus cytotoxic T cell fates, as featured in this Pillars of Immunology article (21).

In the late 1990s, Kappes and colleagues serendipitously identified a peculiar phenotype in a cohort of mice in their colony, which completely lacked CD4 T cells despite normal numbers of their CD8 counterparts, naming them helper-deficient (HD) mice (22). The HD phenotype was cell intrinsic, as it was independent of CD4 or MHC-I expression, indicating that CD4 versus CD8 T cell fate decision was mechanistically distinct from initial positive selection (23). After several years of diligent effort, Kappes and colleagues completed genetic mapping and bacterial artificial chromosome complementation of the HD mutation, identifying the broad-complex, tramtrack, and bric-a-brac–poxvirus and zinc finger transcription factor, ThPOK (21). Among the major cell populations in the thymus, expression of ThPOK is specific to mature CD4⁺ CD8⁻ single-positive thymocytes but absent in DP thymocytes and CD4⁻ CD8⁺ single-positive thymocytes. ThPOK is upregulated in the CD4⁺ CD8^lo intermediate thymocytes immediately after positive selection and is substantially higher in cells undergoing MHC-II– versus MHC-I–restricted selection. Furthermore, forced expression of ThPOK directs both MHC-I– or MHC-II–restricted thymocytes into the helper lineage, indicating that it is both necessary and sufficient for differentiation of CD4 T cells. As is often the case, a breakthrough comes with accompanying independent studies by others. It is worth noting that Remy Bosselut and colleagues almost simultaneously discovered ThPOK, which they coined as cKrox, as a factor upregulated in positively selected DP thymocytes that directs them into the helper lineage (24).

These discoveries placed ThPOK at the core of a gene regulatory network governing thymocyte lineage decisions. Subsequent studies revealed that the fate decision of selected thymocytes is a progressive, multistep process, in which initial positive selection signals establish a mixed population of CD4⁺ CD8^lo intermediate cells that have already initiated differentiation toward either lineage, instructed by initial MHC-restricted TCR signals (lineage specification) (25, 26). During helper lineage differentiation, the transcription factor GATA3 is necessary for specification and promotes upregulation of ThPOK (25, 27). Whereas helper-specified intermediate cells retain plasticity and can be redirected to the cytotoxic lineage by Runx proteins (26), continued expression of ThPOK by persistent TCR signaling stabilizes the initial fate-choice instructed by MHC-II restriction through the suppression of Runx3, the primary Runx protein for the CTL lineage, and Runx target genes (26, 28–31). Thus, ThPOK reinforces thymocyte fate choices initiated by MHC-II–restricted TCR signaling during positive selection. In MHC-I–selected thymocytes, in contrast, ThPOK is barely induced by positive selection, and its subsequent upregulation is actively repressed through the activity of a silencer cis-element, which is bound by Runx and other transcription factors (32, 33). Thus, ThPOK represents the primary transcription factor in the network governing thymocyte fate decision, and its discovery was a major conceptual advance beyond immunology, showing that the lineage commitment of a differentiating cell during a binary fate decision is completed by inactivation of the alternative fate.

In summary, the identification of ThPOK moved the field of thymocyte lineage decision from phenomenon-based studies to the realm of molecular mechanisms. Now the original question has been transformed into more-specific questions: how do positive selection signals turn on ThPOK, and how does ThPOK establish helper T cell identity in CD4 T cells during their development, immune responses, and lymphomagenesis (34)?