Deconstructing Intestinal Immune Tolerance

Project Information

DeconstrucTolerance

Grant agreement ID: 101116251

DOI

10.3030/101116251

EC signature date 30 August 2023

Start date 1 January 2024

End date 31 December 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 500 000,00

EU contribution

€ 1 500 000,00

Coordinated by

FORSCHUNGSINSTITUT FUR MOLEKULARE PATHOLOGIE GESELLSCHAFT MBH
Austria

Periodic Reporting for period 1 - DeconstrucTolerance (Deconstructing Intestinal Immune Tolerance)

Reporting period: 2024-01-01 to 2026-06-30

Summary of the context and overall objectives of the project

The intestinal immune system must continuously balance protective immunity to pathogens with tolerance to self antigens, diet, and harmless commensal microbes. Failure to maintain this balance underlies chronic inflammatory diseases, food allergy, and autoimmunity, yet the regulatory principles that maintain intestinal immune homeostasis remain poorly understood. Central to this process are Foxp3-expressing regulatory T (Treg) cells, whose sustained activity is essential to suppress lethal autoimmunity and allergic inflammation. Recent work has revealed that intestinal Treg cells comprise at least two distinct subsets defined by developmental origin and transcriptional programming. How these specialized Treg cells acquire their identity and how Treg cells exert their suppressive effects within complex tissue environments remain fundamental open questions.

The proposal addresses these questions by establishing a conceptual and technological framework to “deconstruct” intestinal immune tolerance. The overarching objective is to define the molecular and cellular networks that maintain intestinal immune homeostasis, with a focus on transcriptional control, cell–cell interactions, and developmental timing. By employing acute and reversible chemical-genetic perturbations, the project aims to identify the direct gene regulatory functions of Treg cell-specific transcription factors and the subset-specific activities of intestinal Treg cell populations.

The expected impact of this work is twofold. Conceptually, it will provide fundamentally new insights into how immune tolerance is established, maintained, and disrupted in a vital barrier tissue. Methodologically, it will deliver broadly applicable genetic strategies to interrogate gene regulation and immune cell function in complex organs with unprecedented precision. Together, these advances have the potential to reshape our understanding of immune regulation and inform future approaches to treat inflammatory and autoimmune diseases of the intestine.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Since the start of the project, substantial progress has been made toward all major objectives. We have established and validated a versatile experimental toolkit that enables rapid, reversible perturbation of key transcription factors in defined immune cell populations, both in vitro and in vivo, using the auxin-inducible degron 2 (AID2 system). This platform provides the temporal and cell type–specific resolution required to disentangle the direct and indirect consequences of perturbing key regulatory proteins that define intestinal Treg cells.

Using this approach, we have made significant advances in defining the direct gene regulatory activities of the Treg cell lineage-defining transcription factor Foxp3. This work revealed that suppressive Treg cells in non-inflamed tissues can persist for extended periods in the near-complete absence of Foxp3, whereas Treg cells operating in inflammatory environments exhibit a strict, ongoing requirement for Foxp3 activity. These findings demonstrate that Foxp3 primarily functions as a transcriptional repressor whose continuous activity is selectively required under inflammatory conditions, uncovering an unexpected context-dependent function with important implications for cancer immunotherapy.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The selective role for Foxp3 in Treg cells exposed to inflammatory environments was unexpected and is a breakthrough discovery that opens many new opportunities for the field. Foxp3 has been synonymous with Treg cells for approximately 20 years, and it had been assumed that Treg cells would be continuously dependent on Foxp3. Our results surprisingly revealed that Treg cells can persist and continue to suppress autoimmune inflammation in the absence of Foxp3. However, Treg cells exposed to inflammatory environments appear to be much more sensitive to Foxp3 protein depletion. This effect is due to Foxp3’s role as a transcriptional repressor that counteracts inflammation-induced transcriptional activators. This stimulation-dependent requirement for Foxp3 can be leveraged for the treatment of cancer, as Treg cells within the tumor microenvironment are known to become activated in response to the local inflammatory milieu. Accordingly, global depletion of Foxp3 protein perturbs the functionality of tumor-infiltrating Treg cells and raises an effective anti-tumor immune response, while leaving Treg cells in healthy organs relatively unperturbed. Further clinical translation of these findings will require the development of small molecule FOXP3 degraders.