Human macrophage signatures (gene lists)

Tumor-associated macrophages (TAM) and regulatory T cells (Treg) are major immune components of the tumor microenvironment, promoting tumor growth and limiting the efficacy of chemotherapy in almost all cancer indications. Although Tregs are well known for their immunosuppressive activity toward the adaptive immune system, less is known about their regulatory activity toward the innate compartment. In this study, we have shown that in human and mouse lung cancer, chemotherapy transiently reduced Treg number and switched the mononuclear phagocyte (MP) landscape toward not only a proinflammatory signature but also an increased TGFβ-expressing TAM accumulation over time. Preventing Treg recovery further increased the recruitment of monocytes and limited TGFβ expression upon TAM differentiation, demonstrating that Tregs dampen the proinflammatory status of the MP compartment induced by chemotherapy and promote tumor relapse. Anti-TNFR2 antibody treatment during the Treg recovery phase affected the direct interaction between Tregs and MPs, increased the proinflammatory signature of the MPs, and improved survival in the mouse model. Targeting the cross-talk between tumor-associated Tregs and the MP compartment limits the reconstitution of an anti-inflammatory environment following chemotherapy and improves therapeutic outcome.

Chemotherapy changes the myeloid landscape in mouse lung tumors

Myeloid landscape in human lung tumors

In vivo imaging of the lung showed Treg (TdTomato+, red) behaviors and interactions with tumor-associated mononuclear phagocytes (ECFP+EGFP+, cyan) 7 days after chemotherapy. White square represents Treg behavior with low velocity, and white arrow represents Treg behavior with high velocity.

TNFR2 blockade improves chemotherapy response

Regulation of cellular metabolism is a central element governing the fate and function of T cells. However, the in vivo metabolic characteristics of rare cells, such as nonlymphoid tissue T cells, are poorly understood because of experimental limitations. Most techniques measuring cell metabolism require large cell numbers. The recent SCENITH method allows for studying the metabolism of rare cells by flow cytometry. However, this technique requires cells to be isolated and cultured ex vivo , which may alter their metabolism. Here, we propose a new experimental approach, called in vivo SCENITH, to investigate the cellular metabolism of T cells in vivo at a steady state in the spleen and lungs. For this purpose, we administered the metabolic modulators directly in mice, instead of applying these reagents ex vivo , as in the classical SCENITH method. Whereas ex vivo manipulation impacted the viability and phenotype of T cells, this toxic effect was not observed in the in vivo SCENITH. We observed that conventional and regulatory T cells shared similar metabolic profiles. Importantly, whereas spleen T cells used both oxidative phosphorylation and glycolysis, the metabolism of T cells in the lungs was mainly based on oxidative phosphorylation. Finally, metabolic inhibitors that interfere with protein translation and energy availability downregulated Foxp3 expression in regulatory T cells. These results describe an expansion of SCENITH that allows to measure the metabolic profile of rare cells in vivo , revealing a high dependence on oxidative phosphorylation of lung T cells.

Foxp3+ regulatory T cells (Tregs) reside in both lymphoid and nonlymphoid organs, where they play a crucial role in immune tolerance and tissue homeostasis. In mice, Tregs begin colonizing these tissues shortly after birth, contributing to long-term immune response regulation therein. However, the kinetics of Treg generation across different tissues remains unclear. Here, we investigate Treg ontogeny from birth to adulthood in various tissues. In lymphoid organs, the adult Treg pool is continuously replenished with cells generated at different ages. In contrast, the skin retains a large fraction of Tregs that colonize the tissue during the neonatal period, with minimal turnover in adulthood. The liver, lungs, and colon exhibit intermediate Treg renewal dynamics. Notably, neonatal Tregs that persist into adulthood display a more activated phenotype and express markers associated with tissue-resident Tregs and type 2 immunity. Our findings reveal tissue-specific differences in Treg generation kinetics and highlight a major phenotypic shift between neonatal and adult-derived Tregs.

Gene lists of the DGE between treated and untreated subsets in human and mouse.

Regulation of cellular metabolism is a central element governing the fate and function of T cells. However, the in vivo metabolic characteristics of rare cells, such as nonlymphoid tissue T cells, are poorly understood because of experimental limitations. Most techniques measuring cell metabolism require large cell numbers. The recent SCENITH method allows for studying the metabolism of rare cells by flow cytometry. However, this technique requires cells to be isolated and cultured ex vivo , which may alter their metabolism. Here, we propose a new experimental approach, called in vivo SCENITH, to investigate the cellular metabolism of T cells in vivo at a steady state in the spleen and lungs. For this purpose, we administered the metabolic modulators directly in mice, instead of applying these reagents ex vivo , as in the classical SCENITH method. Whereas ex vivo manipulation impacted the viability and phenotype of T cells, this toxic effect was not observed in the in vivo SCENITH. We observed that conventional and regulatory T cells shared similar metabolic profiles. Importantly, whereas spleen T cells used both oxidative phosphorylation and glycolysis, the metabolism of T cells in the lungs was mainly based on oxidative phosphorylation. Finally, metabolic inhibitors that interfere with protein translation and energy availability downregulated Foxp3 expression in regulatory T cells. These results describe an expansion of SCENITH that allows to measure the metabolic profile of rare cells in vivo , revealing a high dependence on oxidative phosphorylation of lung T cells.

Objective: Regulatory T cells (Tregs) are essential in maintaining immune tolerance and controlling inflammation. Treg stability relies on transcriptional and post-translational mechanisms, including histone acetylation at the Foxp3 locus and FoxP3 protein acetylation. Additionally, Tregs depend on specific metabolic programs for differentiation, yet the underlying molecular mechanisms remain elusive. We aimed to investigate the role of acetyl-CoA carboxylase 1 (ACC1) in the differentiation, stability, and function of regulatory T cells (Tregs). Methods: We used either T cell-specific ACC1 knockout mice or ACC1 inhibition via a pharmacological agent to examine the effects on Treg differentiation and stability. The impact of ACC1 inhibition on Treg function was assessed in vivo through adoptive transfer models of Th1/Th17driven inflammatory diseases. Results: Inhibition or genetic deletion of ACC1 led to an increase in acetyl-CoA availability, promoting enhanced histone and protein acetylation, and sustained FoxP3 transcription even under inflammatory conditions. Mice with T cell-specific ACC1 deletion exhibited an enrichment of double positive RORgt+FoxP3+ cells. Moreover, Tregs treated with an ACC1 inhibitor demonstrated superior long-term stability and an enhanced capacity to suppress Th1/Th17-driven inflammatory diseases in adoptive transfer models. Conclusions: We identified ACC1 as a metabolic checkpoint in Treg biology. Our data demonstrate that ACC1 inhibition promotes Treg differentiation and long-term stability in vitro and in vivo. Thus, ACC1 serves as a dual metabolic and epigenetic hub, regulating immune tolerance and inflammation by balancing de novo lipid synthesis and protein acetylation. (c) 2025 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Treg depletion improves chemotherapy response

Supplementary Table and Video Legends

TNFR2 blockade changes the transcriptomic profile of myeloid cells

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, highly resistant to standard chemotherapy and immunotherapy. Regulatory T cells (Tregs) expressing tumor necrosis factor α receptor 2 (TNFR2) contribute to immunosuppression in PDAC. Treg infiltration correlates with poor survival and tumor progression in patients with PDAC. We hypothesized that TNFR2 inhibition using a blocking monoclonal antibody (mAb) could shift the Treg-effector T cell balance in PDAC, thus enhancing antitumoral responses.MethodTo support this hypothesis, we first described TNFR2 expression in a cohort of 24 patients with PDAC from publicly available single-cell analysis data. In orthotopic and immunocompetent mouse models of PDAC, we also described the immune environment of PDAC after immune cell sorting and single-cell analysis. The modifications of the immune environment before and after anti-TNFR2 mAb treatment were evaluated as well as the effect on tumor progression.ResultsPatients with PDAC exhibited elevated TNFR2 expression in Treg, myeloid cells and endothelial cells and lower level in tumor cells. By flow cytometry and single-cell RNA-seq analysis, we identified two Treg populations in orthotopic mouse models: Resting and activated Tregs. The anti-TNFR2 mAb selectively targeted activated tumor-infiltrating Tregs, reducing T cell exhaustion markers in CD8+T cells. However, anti-TNFR2 treatment alone had limited efficacy in activating CD8+T cells and only slightly reduced the tumor growth. The combination of the anti-TNFR2 mAb with agonistic anti-CD40 mAb promoted stronger T cell activation, tumor growth inhibition, and improved survival and immunological memory in PDAC-bearing mice.ConclusionOur data suggest that combining a CD40 agonist with a TNFR2 antagonist represents a promising therapeutic strategy for patients with PDAC.

Four supplementary figures and their legends.

CD137 (4-1BB)-activating receptor represents a promising cancer immunotherapeutic target. Yet, the cellular program driven by CD137 and its role in cancer immune surveillance remain unresolved. Using T cell-specific deletion and agonist antibodies, we found that CD137 modulates tumor infiltration of CD8+-exhausted T (Tex) cells expressing PD1, Lag-3, and Tim-3 inhibitory receptors. T cell-intrinsic, TCR-independent CD137 signaling stimulated the proliferation and the terminal differentiation of Tex precursor cells through a mechanism involving the RelA and cRel canonical NF-κB subunits and Tox-dependent chromatin remodeling. While Tex cell accumulation induced by prophylactic CD137 agonists favored tumor growth, anti-PD1 efficacy was improved with subsequent CD137 stimulation in pre-clinical mouse models. Better understanding of T cell exhaustion has crucial implications for the treatment of cancer and infectious diseases. Our results identify CD137 as a critical regulator of Tex cell expansion and differentiation that holds potential for broad therapeutic applications.