rethinking treg

the two controversies that matter

In the theoretical and opinion papers published in Immunology & Cell Biology and Clinical & Experimental Immunology by the Ono group, traditional views on thymus-derived regulatory T cells (Tregs) are challenged (Ono & Tanaka, 2016; Bending & Ono, 2018).

Two Core Controversies

The Non-Reproducible Evidence for Thymus-Derived Treg

For years, the immunology community has recognized Tregs as a distinct, genetically programmed T-cell lineage crucial for immunosuppression. However, given the plasticity and dynamic regulation of Foxp3, the ‘lineage’ transcription factor for Treg, it is more straightforward to understand the temporal dynamics of Foxp3 expression over time, rather than assuming the stability of a unique T cell ‘lineage’.

Why do we still consider that Treg are distinct and unique?

For those who are TCR-seq ‘fans’ — the answer isn’t TCR. Since all T cells, including Tregs, exhibit uniqueness through their T-cell receptors (TCRs), it is misleading to single out Tregs as uniquely distinct based on TCRs alone. All T cell subsets exhibit this form of uniqueness; thus, apart from their ‘self-reactivity’, Tregs are not uniquely unique.

Furthermore, ‘self-reactivity’ isn’t exclusive to Tregs. I will delve deeper into this topic in an upcoming article.

The answer is here – the belief is primarily based on evidence that CD25+ Tregs migrate to the neonatal spleen later than their counterparts and that early thymectomy in mice leads to Treg depletion and autoimmune diseases (Asano et al, 1996).

Many esteemed immunologists appreciated this finding and accordingly established the current field of Treg research. However, Ono and Tanaka reviewed the literature, identifying contradicting evidence and noting that such foundational evidence has never been effectively reproduced, with key studies demonstrating the presence of Tregs in neonates.

Thus, simply speaking, there is no such thing as “a distinct, genetically programmed T-cell lineage crucial for immunosuppression.” There is only a status of T-cells in which Foxp3 is expressed and operating.

The article by Ono and Tanaka proposes a theoretical model: viewing Tregs not as a separate lineage but as an integral feedback mechanism for T-cell activation within the T-cell system. This conceptual shift was established as the foundation for Ono to further advance our understanding of T-cell regulation by investigating Foxp3 dynamics using the Foxp3-Tocky model.

Read the full paper here (Ono & Tanaka, 2016).

Lessons from the TGN1412 Clinical Trial —the World’s First Attempt at Treg Therapy

In a recent study from our lab, the dynamics of Foxp3 transcription were examined as key to understanding Treg-mediated immune regulation (Bending et al., 2018). This study highlights the importance of viewing Tregs not just as a distinct lineage but as part of a dynamically regulated immune system.

The catastrophic clinical trial of TGN1412 in 2006, which resulted in cytokine storm and multiorgan dysfunction in volunteers, serves as a stark reminder of what can go wrong when this dynamic is not fully understood. TGN1412, initially developed to suppress autoimmune reactions by targeting Tregs specifically, inadvertently activated a broad range of T cell responses following CD28 stimulation.

T-cell activation and CD28 costimulation.

The investigation into this incident concluded that a major factor was the ‘unexpected difference between species’ concerning the use of CD28. However, this raises a critical question: should it have been anticipated that CD28 could stimulate a wide array of T cells?

This case highlights the critical need for a deeper understanding of Treg plasticity and the dynamic nature of T cell differentiation and activation.

Read the full paper on TGN1412 and Treg dynamics here (Bending & Ono, 2018).

Implications for Future Research

The core message of these publications highlights the importance of understanding the dynamic molecular processes within T cells. Scientifically, viewing Treg as a status of activated T cells and analysing Foxp3 to investigate their function could lead to more precise interventions in autoimmune diseases, enhanced immunotherapy approaches, and a deeper understanding of T-cell dynamics.

Beyond the scientific challenges, a fundamental issue remains: the need for greater research integrity and transparency. In a field often influenced by closed circles of a limited number of ‘established scientists’, who financially benefit from ‘advances’ in immunotherapy, we must ask ourselves: are we truly prepared to embrace this necessary openness?

The journey is far from over. I am eager to continue this research and look forward to sharing further insights.

Microscopic image of CD4+ T cells. Green represents CD4 protein; red indicates Foxp3 protein; blue stains DNA. (Image credit: Ono)

References

2018

From stability to dynamics: understanding molecular mechanisms of regulatory T cells through Foxp3 transcriptional dynamics

David Bending , and Masahiro Ono

Clinical and Experimental Immunology, Sep 2018

Abstract Publication

Studies on regulatory T cells (Treg) have focused on thymic Treg as a stable lineage of immunosuppressive T cells, the differentiation of which is controlled by the transcription factor forkhead box protein 3 (Foxp3). This lineage perspective, however, may constrain hypotheses regarding the role of Foxp3 and Treg in vivo, particularly in clinical settings and immunotherapy development. In this review, we synthesize a new perspective on the role of Foxp3 as a dynamically expressed gene, and thereby revisit the molecular mechanisms for the transcriptional regulation of Foxp3. In particular, we introduce a recent advancement in the study of Foxp3-mediated T cell regulation through the development of the Timer of cell kinetics and activity (Tocky) system, and show that the investigation of Foxp3 transcriptional dynamics can reveal temporal changes in the differentiation and function of Treg in vivo. We highlight the role of Foxp3 as a gene downstream of T cell receptor (TCR) signalling and show that temporally persistent TCR signals initiate Foxp3 transcription in self-reactive thymocytes. In addition, we feature the autoregulatory transcriptional circuit for the Foxp3 gene as a mechanism for consolidating Treg differentiation and activating their suppressive functions. Furthermore, we explore the potential mechanisms behind the dynamic regulation of epigenetic modifications and chromatin architecture for Foxp3 transcription. Lastly, we discuss the clinical relevance of temporal changes in the differentiation and activation of Treg.
A temporally dynamic Foxp3 autoregulatory transcriptional circuit controls the effector Treg programme

David Bending , Alina Paduraru , Catherine B Ducker , Paz Prieto Martı́n , Tessa Crompton , and Masahiro Ono

The EMBO journal, Sep 2018

The second Tocky paper from the Ono lab uncovers the temporally dynamic regulation of Foxp3 transcription, offering new insights into T cell regulation.

Abstract Publication

Regulatory T cells (Treg) are negative regulators of the immune response; however, it is poorly understood whether and how Foxp3 transcription is induced and regulated in the periphery during T‐cell responses. Using Foxp3‐Timer of cell kinetics and activity (Tocky) mice, which report real‐time Foxp3 expression, we show that the flux of new Foxp3 expressors and the rate of Foxp3 transcription are increased during inflammation. These persistent dynamics of Foxp3 transcription determine the effector Treg programme and are dependent on a Foxp3 autoregulatory transcriptional circuit. Persistent Foxp3 transcriptional activity controls the expression of coinhibitory molecules, including CTLA‐4 and effector Treg signature genes. Using RNA‐seq, we identify two groups of surface proteins based on their relationship to the temporal dynamics of Foxp3 transcription, and we show proof of principle for the manipulation of Foxp3 dynamics by immunotherapy: new Foxp3 flux is promoted by anti‐TNFRII antibody, and high‐frequency Foxp3 expressors are targeted by anti‐OX40 antibody. Collectively, our study dissects time‐dependent mechanisms behind Foxp3‐driven T‐cell regulation and establishes the Foxp3‐Tocky system as a tool to investigate the mechanisms behind T‐cell immunotherapies.

2016

Controversies concerning thymus‐derived regulatory T cells: fundamental issues and a new perspective

Masahiro Ono, and Reiko J Tanaka

Immunology and cell biology, Sep 2016

A landmark opinion piece challenging the reproducibility of a foundational Treg experiment, while introducing a groundbreaking dynamic view on Foxp3-mediated T cell regulation.

Abstract Publication

Thymus-derived regulatory T cells (Tregs) are considered to be a distinct T-cell lineage that is genetically programmed and specialised for immunosuppression. This perspective is based on the key evidence that CD25+ Tregs emigrate to neonatal spleen a few days later than other T cells and that thymectomy of 3-day-old mice depletes Tregs only, causing autoimmune diseases. Although widely believed, the evidence has never been reproduced as originally reported, and some studies indicate that Tregs exist in neonates. Thus we examine the consequences of the controversial evidence, revisit the fundamental issues of Tregs and thereby reveal the overlooked relationship of T-cell activation and Foxp3-mediated control of the T-cell system. Here we provide a new model of Tregs and Foxp3, a feedback control perspective, which views Tregs as a component of the system that controls T-cell activation, rather than as a distinct genetically programmed lineage. This perspective provides new insights into the roles of self-reactivity, T cell–antigen-presenting cell interaction and T-cell activation in Foxp3-mediated immune regulation.