How autoreactive thymocytes differentiate into regulatory versus effector CD4⁺ T cells after avoiding clonal deletion

Thymic selection is a crucial process ensuring self-tolerance and the generation of a functional T cell repertoire. Developing thymocytes express T cell receptors (TCRs) that are screened for their reactivity to self-antigens. Those with high affinity for self-ligands, along with co-stimulatory signals (like those mediated by CD28 and B7), receive agonist signals. These signals can trigger three main outcomes: clonal deletion (apoptosis of autoreactive thymocytes), differentiation into regulatory T cells (Tregs), or differentiation into effector T cells (Teffs). Tregs maintain self-tolerance by suppressing autoreactive Teffs, whereas Teffs contribute to immunity by producing cytokines, including IL-2, which supports T cell survival and differentiation. While the roles of these cell types are understood, the precise mechanisms directing autoreactive thymocytes towards these distinct fates remain largely unclear. The timing of these signals—whether they are sequential or simultaneous—is a key question. Previous research suggests complexities in Treg development, involving multiple signaling pathways and factors like TGF-β, whose role isn't fully elucidated. Little is known about Teff development from autoreactive thymocytes, and how the choice between Treg and Teff lineages is made. This study aims to clarify the molecular mechanisms and timing of these differentiation pathways, shedding light on how agonist signaling drives these distinct developmental outcomes and impacting our understanding of self-tolerance.

Extensive research has explored Treg development, revealing a complex interplay of molecular requirements and signaling pathways leading to Foxp3 expression and differentiation. TCR-CD28 signaling has been shown to both promote and inhibit Foxp3 expression. The origin of Tregs has also been a source of debate, with conflicting evidence regarding the existence of distinct Treg precursors or a common precursor-progeny relationship. TGF-β has been implicated as a necessary factor in thymic Treg development, but the specifics of its involvement remain unclear. In contrast, information on the differentiation of agonist-signaled thymocytes into Teffs and the underlying mechanisms driving Treg/Teff lineage decisions is scarce. Existing models often link TCR affinity to cell fate—high affinity leading to deletion, intermediate affinity to Teffs, and low affinity to Tregs—but the molecular basis for this correlation is not well understood. This lack of clarity necessitates further investigation to fully understand the intricacies of thymic selection and its implications for maintaining immune homeostasis and self-tolerance.

The study employed a comprehensive approach using various mouse models and techniques to investigate the differentiation of autoreactive thymocytes. First, they identified five sequential stages of thymic selection using CD69 and CCR7 expression, coupled with a Rag-GFP reporter to track TCR signaling. To determine the timing of clonal deletion and Treg development, they analyzed the presence of these cell types across the five stages. They used superantigens and conventional antigens to induce clonal deletion, allowing them to observe the temporal relationship between CD28 engagement and deletion. To study the role of late agonist signaling in Treg generation, they created ZAP70TBKO mice, lacking late-stage agonist signals. They investigated the expression of transcription factors cRel and Foxo1, known to influence Foxp3 expression. In vitro experiments were performed using immobilized anti-TCR/CD28 antibodies and recombinant TGF-β (rTGF-β) to manipulate agonist signaling and observe the effects on Foxp3 expression and Treg development. In vivo experiments involved intra-thymic transfer of T cell precursors into different host strains to observe their differentiation in various contexts, including IL-2-deficient and TGF-β-deficient mice, and mice with genetic modifications of Foxo proteins. Flow cytometry was used extensively to analyze cell surface markers and intracellular proteins, including transcription factors and cytokines. Confocal microscopy and immunohistochemistry techniques were used to analyze protein localization within cells and tissues. Quantitative RT-PCR was employed to measure gene expression levels. ELISA was used to measure IL-2 protein levels. Statistical analyses, including t-tests and ANOVA, were used to assess the significance of the findings.

The study's key findings center around the timing and regulation of autoreactive thymocyte differentiation. First, clonal deletion was found to occur at stage 3 of thymic selection, coinciding with CD28-B7 engagement. In contrast, Foxp3+ Treg cells appeared only at stage 5. Using Rag-GFP and Foxp3-RFP double-reporter mice, they demonstrated that clonal deletion occurs approximately 20 hours after Rag gene termination, whereas Treg generation occurs around 90 hours. They established that late-stage agonist signaling is essential for Treg development, as evidenced by the abrogation of Treg generation in ZAP70TBKO mice. The study revealed that disruption of agonist signaling, rather than its persistence, is critical for Foxp3 gene induction and Treg development. In vitro experiments demonstrated that continuous agonist signaling failed to generate Foxp3+ cells, but signaling disruption led to Foxp3 expression. The CD25+ subset of stage 4 thymocytes was identified as the Treg precursor population. TGF-β was found to be crucial for Treg development in vivo, acting by disrupting weaker agonist signals and upregulating n-Foxo1. An alternative pathway for Treg development was identified, dependent on excessive IL-2 and independent of TGF-β and Foxo proteins. This pathway was shown to be non-physiological, only occurring in situations with abnormally high IL-2 levels. The study also showed that IL-2+ Teff cells require late agonist signaling and persistent signals for their development. TGF-β signaling was shown to inhibit Teff development, promoting Treg development instead. The study demonstrated that both Tregs and Teffs develop from CD25+ precursors, with their fate being determined by whether TGF-β disrupts or allows persistent agonist signaling.

This study significantly advances our understanding of thymic selection by providing a detailed temporal and mechanistic framework for the differentiation of autoreactive thymocytes. The finding that TCR signaling disruption, mediated by TGF-β, is crucial for initiating Foxp3 expression and Treg development challenges the long-held view that IL-2 is the primary driver of Foxp3 expression. The identification of two distinct pathways for Treg development—a primary TGF-β-dependent pathway and an alternative IL-2-dependent pathway—offers a more nuanced perspective on Treg generation. The temporal relationship between clonal deletion and Treg development, with deletion preceding Treg generation, clarifies a key aspect of thymic selection. The study also illuminates the role of TGF-β in modulating Treg/Teff lineage decisions. Its ability to disrupt weaker agonist signals and promote Treg development highlights its central role in maintaining immune homeostasis. The findings reconcile existing observations regarding TCR affinity and developmental fate. The interplay between TCR affinity, the timing of agonist signaling, and the influence of TGF-β provides a comprehensive explanation for the correlation between TCR affinity and cell fate. The study opens new avenues for research into manipulating thymic selection to improve immune regulation and treat autoimmune diseases.

This research demonstrates that the developmental fates of autoreactive CD4+ thymocytes are determined by the timing and strength of agonist signals. Early agonist signals induce clonal deletion, while late signals, modulated by TGF-β, direct differentiation into either Tregs or Teffs. TGF-β disrupts weaker signals, promoting Treg development, while persistent strong signals lead to Teff differentiation. An alternative Treg pathway exists but relies on non-physiological IL-2 levels. This work provides a comprehensive model of thymic selection, revealing the intricate mechanisms controlling self-tolerance and suggesting potential therapeutic strategies for immune-related disorders. Future studies should investigate the precise molecular mechanisms of TGF-β-mediated signaling disruption and explore the therapeutic potential of manipulating these pathways.

The study primarily utilizes mouse models, limiting the direct translatability of the findings to human systems. The in vitro experiments, while valuable, may not fully recapitulate the complexity of the in vivo environment. The definition of 'excessive' IL-2 levels in the context of the alternative Treg pathway could benefit from further quantitative analysis across a wider range of physiological and pathological conditions. Further investigation is needed to fully elucidate the molecular mechanisms involved in TGF-β-mediated signaling disruption and the precise interplay between different signaling pathways.