Evaluating the potential of RANK ligand to restore effective thymic functions after bone marrow transplantation and during aging – RANKLthym

This project relied on the generation of a unique murine model with a selective ablation of the RANK receptor at the surface of thymic endothelial cells, as well as the use of multiple young and aged mouse models.

The role of the RANKL protein was analyzed in vivo in mice using a neutralizing anti-RANKL antibody as well as through administration of soluble RANKL protein.

We used a wide range of techniques to analyze thymic function during aging and following RANKL treatment, including multiparametric flow cytometry, confocal imaging, and single-cell RNAsequencing.

The benefit of thymic regeneration in aged individuals was evaluated in a tumor context using a melanoma model, as well as in a vaccination model.

We also investigated the effects of our treatment on bone using histomorphometric approaches.

Finally, we provided proof of concept that RANKL treatment stimulates thymic epithelial and endothelial cells using human thymic organ cultures.

Work Packages (WP) 2, 3, and 4 were recently published: “RANKL treatment restores thymic function and improves T cell–mediated immune responses in aged mice,” Science Translational Medicine, 2024, Santamaria, Chevallier, Dutour, Picart, Kergaravat, Cieslak, Amrane, Vincentelli, Puthier, Clave, Sergé, Cohen-Solal, Toubert, Irla. doi: 10.1126/scitranslmed.adp3171.

Our study was the subject of a commentary entitled “Rejuvenating the immune system,” Molecular Oncology, 2025, Evangelou and Gorgoulis, doi: 10.1002/1878-0261.13802.

It was also highlighted in the article “Giving the thymus a second youth for revitalized immunity!” published in La Gazette du Laboratoire, November 2025, issue no. 324.

The article was promoted by INSERM on its Instagram account as well as in issue no. 64 of its magazine (“A rejuvenation cure for the immune system”) in March 2025.

The study was also featured on the Fierce Biotech website and on X (formerly Twitter).

Age-related thymic involution, leading to reduced production of T lymphocytes, is one of the major causes of immunosenescence. It results in increased susceptibility to cancers, infections, and autoimmune diseases, as well as reduced vaccine efficacy.

We identified that the RANK/RANKL axis is altered in the thymus during aging. Using a conditional transgenic mouse model, we demonstrated that endothelial cells depend on RANK signaling for their maintenance and functional maturation. Decreased RANKL availability with age leads to reduced cellularity and function of both endothelial cells and thymic epithelial cells, thereby contributing to thymic involution.

We subsequently showed that, whereas RANKL neutralization in young mice mimics thymic involution, exogenous RANKL treatment in aged mice restores thymic architecture as well as the abundance and functional properties of endothelial and epithelial cells. Consequently, RANKL improves the homing of T cell progenitors to the thymus and enhances T cell production.

This cascade of events leads to renewal of peripheral T cells and to effective antitumor and vaccine responses in aged mice.

Finally, we conducted a proof-of-concept study demonstrating that RANKL stimulates endothelial and epithelial cells in human thymic organ cultures.

Altogether, our results suggest that targeting the RANK–RANKL axis through exogenous RANKL administration could represent a therapeutic strategy to rejuvenate thymic function and improve T cell immunity during aging.

WP1, which aims to evaluate immune responses, particularly following influenza infection, after RANKL-induced thymic regeneration in the context of bone marrow transplantation, is still ongoing.

Recent studies (Givony et al., Nature, 2023; Michelson et al., Cell, 2022; Huisman et al., Immunity, 2025) demonstrate that thymic epithelial cells are highly heterogeneous and contain subsets that mimic many peripheral tissues. However, during aging, the thymic stroma undergoes profound changes in its cellular subsets, which remain poorly described. To study thymic stromal aging and renewal following RANKL treatment, as well as precise interactions with developing T lymphocytes, we recently employed single-cell transcriptomics and spatial transcriptomics approaches. These results are the subject of a manuscript currently in preparation.

Age-related thymic involution results in the accumulation of dysfunctional T cells. This decline leads to impaired surveillance of senescent cells. Such defective surveillance promotes age-associated inflammation and progressive loss of organ integrity and function, contributing to systemic pathologies such as neurodegenerative, cardiovascular, metabolic, and musculoskeletal syndromes. Thymic regeneration represents a promising approach to restore functional T lymphocytes capable of reestablishing senescence surveillance within organs. Our preliminary results reveal that these newly produced T lymphocytes appear to preserve organs from age-related degeneration. This ongoing study opens promising perspectives for promoting healthy aging.

CONTEXT: T lymphocytes are critical cellular actors in the host response to various pathogens i.e. viruses, bacteria and fungi and in the control of malignant cell transformation. Importantly, the thymus ensures a continuous production of naïve T cells with a diverse TCR repertoire capable to mount effective immune responses. However, this organ undergoes severe changes upon aging in its mass and architecture, accompanied by a progressive decrease in T cell production, a process referred to as “thymic involution”. Furthermore, the thymus is highly sensitive to myeloablative treatments used to cure hematological disorders such as genetic disorders or cancer by hematopoietic stem cell transplantation. Both aging and myeloablative treatments have deleterious effects on thymic epithelial cells, leading to a drastic reduction in T cell production and consequently to increased susceptibility to opportunistic infections, autoimmunity and cancer. Nevertheless, the thymus shows a high plasticity and is prone to regenerative therapies. Although in mice, certain growth factors or cytokines protect or regenerate thymic epithelial cells and improve thymopoiesis, their efficacy in humans in preclinical trials either failed or remains to be determined. Identifying effective therapeutic molecules to regenerate thymic functions thus still constitutes an unmet clinical need. In this context, we recently showed in mice that the administration of the epithelial growth and differentiation cytokine, RANK ligand (RANKL), ameliorates the regeneration of the thymus and T-cell reconstitution upon myeloablative treatments and bone marrow transplantation (BMT) (Lopes et al. EMBO Mol Med 2017 et brevet WO/2018/154122).

OBJECTIVES: The objectives of this project are to determine:
(1) whether T-cell production improved by RANKL enhances protection against lethal opportunistic infections in a pre-clinical BMT mouse model,
(2) to what extent RANKL is beneficial to reverse age-related thymic involution,
(3) the impact of RANKL on the prevention of bone loss by bisphosphonates upon BMT and thymic involution,
(4) the translational potential of using RANKL to regenerate human thymic functions.
In summary, the ambition of this project is to bring the proof of concept that RANKL could be clinically pertinent to improve the recovery of T-cell functions without inducing bone loss in pathophysiological conditions in which the thymus has been severely damaged.

CONSORTIUM: This project is a close collaboration between three French research teams. It brings together a fully complementary expertise on the biology of thymic function in mice (Team #1) and humans (Team #2) as well as on bone physiology (Team #3).

BIOMEDICAL RELEVANCE: This project is expected to improve the immune system by acting on the production of T lymphocytes by regenerating the thymic function. We are convinced that the expected results could have a significant impact on public health, given the incidence of 20 to 30% of mortality related to infections after HSC transplantation and the demographic increase in the number of elderly people in our societies, a phenomenon also associated with a high rate of infections. This project has the potential to improve the health of many patients who incur morbidity and mortality due to T cell deficiency and reduce the associated medical costs.