Decoding Foxp3 – From Treg Dogma to T-cell Dynamics

The second Tocky publication from the Ono Lab

Foxp3 - a Lineage Factor or a Marker for Regulatory Function?

It is widely considered that Foxp3-expressing regulatory T cells (Treg) are a separate type of T-cells specialised for suppressing immune cell activities. Okay, this may be true if all Treg are functioning as suppressor T cells. However, in reality, it is difficult to identify which Treg cells are functioning and which ones are not. Further, the majority of Foxp3-expressing T-cells are not functional Treg and can contribute to the induction of T-cell response in humans (Fujii et al., 2016; Miyara et al., 2009; Fujii et al., 2011).

Importantly, the belief that Tregs constitute a unique lineage of cells originates from, and is supported by, the findings of Asano et al. Should this evidence no longer be valid (Ono & Tanaka, 2016), how can we then investigate Foxp3 and Treg?

Our second Tocky publication (Bending et al., 2018) aimed to answer this question by demonstrating the power of Foxp3-Tocky – the Tocky mouse strain for monitoring temporal dynamics of Foxp3 transcription (Fujii et al., 2016; Bending et al., 2018). Our results support that Foxp3 acts as a switch for the suppressive mechanism that is dynamically induced during homeostasis and upon stimulation.

Experimental Validation of Foxp3-Tocky

First, to better understand Foxp3 transcription dynamics, we experimentally determined the half-life of Fluorescent Timer protein. Using degradation assays, the half-life of blue fluorescence and red fluorescence was 4 h and 120 h, respectively. These are improved measurements, as previous reports relied on estimation through modelling.

Immunological Significance of Foxp3 Transcirptional Dynamics Revealed by Tocky

New Foxp3 Transcription

By analysing homeostasis and inflammation, we showed that Foxp3 expression is newly induced in a small yet significant percentage of CD4+ T-cells and that this is increased during self-containing inflammation. These results support more dynamic regulation of Foxp3 than previously imagined.

Persistent Foxp3 Transcription

Intriguingly, we found that temporally sustained Foxp3 transcription (Persistent transcription) is a hallmark of activated Treg with enhanced immunoregulatory phenotype (effector Treg).

Autoregulatory Transcriptional Loop for Foxp3 Revealed by Tocky

The utilization of a triple transgenic system, combining Foxp3-Tocky, Foxp3-EGFP knock-in, and Foxp3 scurfy hypomorphic mutant loci, allowed for an unprecedented analysis of Foxp3 transcriptional regulation in the presence and absence of functional Foxp3 protein. This approach unveiled a significant finding: the Foxp3 protein itself amplifies the transcription of the Foxp3 gene, indicating an autoregulatory loop that enhances the regulatory functions of Foxp3.

Immunotherapy Application of Tocky

Understanding of temporally dynamic features of Foxp3 expressors enabled a new immunotherapy targeting Foxp3 dynamics. We demonstrated this using anti-OX40 antibody, which accelerated apoptosis of Foxp3 Persistent expressors.

References

2018

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, May 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.
A timer for analyzing temporally dynamic changes in transcription during differentiation in vivo

David Bending , Paz Prieto Martı́n , Alina Paduraru , Catherine Ducker , Erik Marzaganov , Marie Laviron , Satsuki Kitano , Hitoshi Miyachi , Tessa Crompton , and Masahiro Ono

Journal of Cell Biology, May 2018

The foundational publication introducing Tocky technology by the Ono lab, marking a breakthrough in T cell and B cell studies.

Abstract Publication

Understanding the mechanisms of cellular differentiation is challenging because differentiation is initiated by signaling pathways that drive temporally dynamic processes, which are difficult to analyze in vivo. We establish a new tool, Timer of cell kinetics and activity (Tocky; or toki [time in Japanese]). Tocky uses the fluorescent Timer protein, which spontaneously shifts its emission spectrum from blue to red, in combination with computer algorithms to reveal the dynamics of differentiation in vivo. Using a transcriptional target of T cell receptor (TCR) signaling, we establish Nr4a3-Tocky to follow downstream effects of TCR signaling. Nr4a3-Tocky reveals the temporal sequence of events during regulatory T cell (Treg) differentiation and shows that persistent TCR signals occur during Treg generation. Remarkably, antigen-specific T cells at the site of autoimmune inflammation also show persistent TCR signaling. In addition, by generating Foxp3-Tocky, we reveal the in vivo dynamics of demethylation of the Foxp3 gene. Thus, Tocky is a tool for cell biologists to address previously inaccessible questions by directly revealing dynamic processes in vivo.

2016

Regulatory T cells in melanoma revisited by a computational clustering of FOXP3+ T cell subpopulations

Hiroko Fujii , Julie Josse , Miki Tanioka , Yoshiki Miyachi , François Husson , and Masahiro Ono

The Journal of Immunology, May 2016

The pioneering study ntroduces a data-driven methodology to explore Treg subpopulations, employing Principal Component Analysis (PCA) for the first time.

Abstract Publication

CD4+ T cells that express the transcription factor FOXP3 (FOXP3+ T cells) are commonly regarded as immunosuppressive regulatory T cells (Tregs). FOXP3+ T cells are reported to be increased in tumor-bearing patients or animals and are considered to suppress antitumor immunity, but the evidence is often contradictory. In addition, accumulating evidence indicates that FOXP3 is induced by antigenic stimulation and that some non-Treg FOXP3+ T cells, especially memory-phenotype FOXP3low cells, produce proinflammatory cytokines. Accordingly, the subclassification of FOXP3+ T cells is fundamental for revealing the significance of FOXP3+ T cells in tumor immunity, but the arbitrariness and complexity of manual gating have complicated the issue. In this article, we report a computational method to automatically identify and classify FOXP3+ T cells into subsets using clustering algorithms. By analyzing flow cytometric data of melanoma patients, the proposed method showed that the FOXP3+ subpopulation that had relatively high FOXP3, CD45RO, and CD25 expressions was increased in melanoma patients, whereas manual gating did not produce significant results on the FOXP3+ subpopulations. Interestingly, the computationally identified FOXP3+ subpopulation included not only classical FOXP3high Tregs, but also memory-phenotype FOXP3low cells by manual gating. Furthermore, the proposed method successfully analyzed an independent data set, showing that the same FOXP3+ subpopulation was increased in melanoma patients, validating the method. Collectively, the proposed method successfully captured an important feature of melanoma without relying on the existing criteria of FOXP3+ T cells, revealing a hidden association between the T cell profile and melanoma, and providing new insights into FOXP3+ T cells and Tregs.
Controversies concerning thymus‐derived regulatory T cells: fundamental issues and a new perspective

Masahiro Ono, and Reiko J Tanaka

Immunology and cell biology, May 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.

2011

Perturbations of both nonregulatory and regulatory FOXP3+ T cells in patients with malignant melanoma

Hiroko Fujii , A Arakawa , A Kitoh , M Miyara , M Kato , S Kore‐Eda , S Sakaguchi , Y Miyachi , M Tanioka , and M Ono

British Journal of Dermatology, May 2011

Abstract Publication

Background: ’FOXP3+ regulatory T cells’ (Tregs) are reported to be increased in tumour-bearing hosts including patients with melanoma, leading to tumour immune suppression. However, this idea is challenged by recent evidence that the ’FOXP3+ Treg’ fraction in fact contains activated ’nonregulatory’ T cells. Also, FOXP3+ T cells are reported to have functionally and kinetically distinct subsets. Objectives: To investigate whether either or both of regulatory and ’nonregulatory’ FOXP3+ T cells are perturbed in patients with melanoma. Methods: FOXP3+ T cells were classified into three subsets, namely CD45RO+FOXP3(low) nonregulatory T cells, CD45RO+FOXP3(high) effector Tregs, and CD45RO-FOXP3(low) naïve Tregs, according to their expression levels of FOXP3 and CD45RO. The percentage and cytokine production of these FOXP3+ T-cell subsets were assessed by flow cytometry. Results: Both regulatory and nonregulatory T cells were increased in patients with melanoma. Moreover, we found three unexpected perturbations in FOXP3+ T-cell subsets: (i) patients with melanoma showed higher frequencies of FOXP3(low) nonregulatory T cells, which decreased and normalized after tumour removal; (ii) FOXP3(low) naïve Tregs containing higher frequencies of interferon-γ+ cells increased with tumour progression; and (iii) CD45RO+FOXP3(high) effector Tregs were pronouncedly infiltrated around tumour tissues. Conclusions: These findings demonstrate that patients with melanoma have distinct and differential perturbation of both regulatory and nonregulatory FOXP3+ T cells. The degree of perturbation is associated with tumour burden and progression, suggesting that the perturbation reflects fundamental pathophysiological processes in patients with melanoma. The presented analysis provides a practical approach to investigate the immunological environment of cancer patients.

2009

Functional Delineation and Differentiation Dynamics of Human CD4⁺ T Cells Expressing the FoxP3 Transcription Factor

Makoto Miyara , Yumiko Yoshioka , Akihiko Kitoh , Tomoko Shima , Kajsa Wing , Akira Niwa , Christophe Parizot , Cécile Taflin , Toshio Heike , Dominique Valeyre , Alexis Mathian , Tatsutoshi Nakahata , Tomoyuki Yamaguchi , Takashi Nomura , Masahiro Ono, Zahir Amoura , Guy Gorochov , and Shimon Sakaguchi

Immunity, May 2009

Abstract Publication

FoxP3 is a key transcription factor for the development and function of natural CD4+ regulatory T cells (Treg cells). Here we show that human FoxP3+CD4+ T cells were composed of three phenotypically and functionally distinct subpopulations: CD45RA+FoxP3lo resting Treg cells (rTreg cells) and CD45RA?FoxP3hi activated Treg cells (aTreg cells), both of which were suppressive in vitro, and cytokine-secreting CD45RA?FoxP3lo nonsuppressive T cells. The proportion of the three subpopulations differed between cord blood, aged individuals, and patients with immunological diseases. Terminally differentiated aTreg cells rapidly died whereas rTreg cells proliferated and converted into aTreg cells in vitro and in vivo. This was shown by the transfer of rTreg cells into NOD-scid-common ?-chain-deficient mice and by TCR sequence-based T cell clonotype tracing in peripheral blood in a normal individual. Taken together, the dissection of FoxP3+ cells into subsets enables one to analyze Treg cell differentiation dynamics and interactions in normal and disease states, and to control immune responses through manipulating particular FoxP3+ subpopulations.