Nature Communications publishes findings from a Humanitas study aimed at understanding the characteristics, functions and relationships of regulatory T cells, key components of the immune system that, in tumors, help dampen the anti-tumor response. The findings pave the way for the development of new therapeutic strategies aimed at enhancing responses to immunotherapy.

Regulatory T cells, or Tregs, play a key role in maintaining immune system balance and immune tolerance, namely the ability to avoid attacking the body’s healthy tissues. This is also an area of research recognised by the 2025 Nobel Prize in Physiology or Medicine. In tumors, these cells can be recruited and end up “helping the enemy.” This contributes to limiting the anti-tumor immune response, thereby promoting disease progression and resistance to treatment.

Understanding which Tregs are present in tumors, what their characteristics are and how they are related to one another is therefore important for developing therapeutic strategies that can selectively target immunosuppressive populations while preserving the physiological functions of Tregs.

These are the key questions addressed by a new study published in Nature Communications, coordinated by Enrico Lugli – Director of the Immunology Program and Head of the Translational Immunology Laboratory and Flow Cytometry Facility at Humanitas Research Hospital. Julian Swatler and Simone Puccio, from Lugli’s laboratory, are co-first authors.

The study also involved Matteo Simonelli – Head of Early Development of New Drugs and Therapeutic Strategies for Solid Tumors and of Neuro-Oncology at Humanitas Research Hospital and Professor at Humanitas University; Giuseppe Marulli – Head of Thoracic Surgery and Professor at Humanitas University; Agnese Losurdo – Medical Oncologist and Valentina Errico – thoracic surgeon at Humanitas Research Hospital.

A common signature of Tregs across tumors

To more precisely define the characteristics of Tregs present in tumors, the researchers integrated several approaches, including single-cell RNA sequencing, spectral flow cytometry and T-cell receptor (TCR) sequencing. Combining these methods made it possible to analyse the cells from complementary perspectives, characterising both their molecular profiles and the relationships between different cell populations. In particular, collaboration with the Flow Cytometry Facility and bioinformatics experts made it possible to link the molecular characteristics of the cells to their identity and distribution.

Applying this approach to nine different solid tumors – breast, colorectal, lung, kidney, ovarian, hepatocellular, endometrial and esophageal cancers, as well as cholangiocarcinoma — the researchers identified a common molecular signature comprising 88 genes. This signature was characteristic of tumor-infiltrating Tregs and consistently more highly expressed than in other T-cell populations.

However, the analysis also revealed significant heterogeneity behind this common signature. Intratumoral Tregs are not a uniform population: the researchers identified four subpopulations with distinct molecular and functional characteristics, showing different distributions within tumor tissues and different suppressive capacities.

“Our study shows just how complex and dynamic the Treg compartment is within tumors,” says Dr. Enrico Lugli. “The presence of distinct populations, characterised by specific states of activity and differentiation, suggests that these cells may perform different functions within the tumor microenvironment. Defining their characteristics and relationships could therefore help us better understand the immune response and develop increasingly selective therapeutic strategies.”

From an identity card to a family tree: CCR8+ Tregs and their origins

Tregs have been at the center of important discoveries on immune tolerance, an area of research also recognised by the 2025 Nobel Prize in Physiology or Medicine, awarded to Mary E. Brunkow, Fred Ramsdell and Shimon Sakaguchi for their discoveries concerning peripheral immune tolerance. Today, one of the challenges is to better understand how these cells differentiate and adapt in different contexts, including tumors.

Among the different Treg populations identified, CCR8+ effector Tregs have attracted particular attention because of their high level of activation and their immunosuppressive properties. But how do these cells arise, and what is their relationship with the other Tregs present in tumors? This question guided the next phase of the study.

To reconstruct the possible relationships between the different cell populations, the researchers analysed the T-cell receptor (TCR) at the single-cell level. By using the TCR as a kind of “molecular surname,” the researchers were able to identify cells belonging to the same clone and reconstruct their relationships. The analysis showed that CCR8+ Tregs do not simply derive from the other Tregs already present in the tumor: their history appears to be more complex. CCR8+ Tregs are closely related to cells found in the tumor-draining lymph nodes, a common site of tumor metastasis.

A potential therapeutic target for a translational trial

Characterising CCR8+ Tregs also has important translational implications. Identifying specific features of tumor-associated Tregs could make it possible to develop strategies that selectively target these cells, while avoiding indiscriminate depletion of Tregs in healthy tissues.

In this context, CCR8 represents a promising therapeutic target. The hypothesis is that reducing this immunosuppressive component of the tumor microenvironment could enhance the immune response against the tumor. This is the basis for a new translational trial being conducted at Humanitas, with the aim of translating these biological findings into a potential new therapeutic strategy.

“The trial, – explains Prof. Matteo Simonelli, who will coordinate the study – will involve the use of an innovative anti-CCR8 monoclonal antibody that is still under investigation, in combination with standard immunotherapy, in patients with advanced solid tumors. The study, which will be partially supported by Fondazione Humanitas per la Ricerca, will primarily aim to identify biomarkers of response and mechanisms of resistance to treatment.”

In-depth molecular analyses will be performed on samples collected from patients, with the aim of understanding how the treatment modifies the tumor microenvironment and identifying biological features associated with response.

The transition from characterising cells in the laboratory to evaluating a therapeutic strategy in patients represents a concrete example of translational research: from understanding the mechanisms that regulate the tumor microenvironment to potentially turning this knowledge into new treatment strategies.

Overall, the study adds a new piece to our understanding of Treg heterogeneity in cancer and of their relationships with other T-cell populations. The findings outline a more complex and dynamic picture of regulatory cells within tumors and further support the development of increasingly targeted immunotherapy strategies.