Periodic Reporting for period 1 - Kidney-Treg (Characterisation and impact of kidney-resident Tregs in kidney physiology and pathologies)
Berichtszeitraum: 2020-04-01 bis 2022-03-31
Regulatory T cells (Tregs) are a subtype of T cells exerting immunosuppressive functions that are vital to prevent autoimmunity. Recently, tissue-resident Tregs have been proposed to be distinct from the generic Tregs in circulation. Such Treg subtypes have already been identified in the gut, muscle and adipose tissue. Beyond the classical immunosuppressive function of Tregs, tissue-resident Tregs also modulate non-immunological process in the tissue. For example, muscle Tregs promote tissue repair and adipose tissue Tregs contribute to maintain insulin sensitivity. Apart from these notable exceptions, our knowledge on tissue-resident Tregs is severely limited. The aims of this project are to characterize Tregs located in the selected tissues and to expand up these tissue resident Treg populations to evaluate their specific impact in the physiology and pathology of these tissues.
Previous studies strongly support a role of Tregs for protection from injury and promotion of tissue repair in kidney diseases and highlight the exciting potential of therapies harnessing Tregs in these pathologies. However, a key limitation of these studies, is the systemic nature of the Treg manipulation. Previous works looking at Treg function in this organ are based on a systemic depletion, which cause systemic inflammation, or on systemic supplementation in Tregs isolated from blood or lymphoid organs. Without experiments that distinguish between the function of kidney-resident Tregs and systemic Tregs it remains unknown as to whether the key function resides in peripheral impacts, e.g. reducing priming in the draining lymph nodes, or in tissue-based impacts. Formal analysis of function of Tregs in the kidney requires a new approach, where kidney-Tregs are specifically targeted without impacting the peripheral population.
The recent Covid 19 pandemic has highlighted the need for a deep understanding of lung immunity. The lung is in contact daily with many opportunistic pathogens. Upon pathogen invasion, the lung elicits an immune response which leads to the clearance of the pathogens. However, in some individuals, an exacerbated production of proinflammatory cytokines, chemokines, and reactive oxygen species can occur, damaging the lung tissue and affecting breathing. Animal studies demonstrated that more Tregs positively correlates with reduced severity of lung inflammation and that therapeutic transfer of Tregs can revert airway inflammation and tissue damages. These studies provide compelling evidence of Tregs involvement in lung immunity that could be harnessed to potentiate tissue repair. In this study, we investigate the potential of local Tregs expansion in the lung to dampen local inflammation and promote tissue repair following acute viral infection, without affecting the systemic immune responses established in the draining lymph nodes.
Previous studies strongly support a role of Tregs for protection from injury and promotion of tissue repair in kidney diseases and highlight the exciting potential of therapies harnessing Tregs in these pathologies. However, a key limitation of these studies, is the systemic nature of the Treg manipulation. Previous works looking at Treg function in this organ are based on a systemic depletion, which cause systemic inflammation, or on systemic supplementation in Tregs isolated from blood or lymphoid organs. Without experiments that distinguish between the function of kidney-resident Tregs and systemic Tregs it remains unknown as to whether the key function resides in peripheral impacts, e.g. reducing priming in the draining lymph nodes, or in tissue-based impacts. Formal analysis of function of Tregs in the kidney requires a new approach, where kidney-Tregs are specifically targeted without impacting the peripheral population.
The recent Covid 19 pandemic has highlighted the need for a deep understanding of lung immunity. The lung is in contact daily with many opportunistic pathogens. Upon pathogen invasion, the lung elicits an immune response which leads to the clearance of the pathogens. However, in some individuals, an exacerbated production of proinflammatory cytokines, chemokines, and reactive oxygen species can occur, damaging the lung tissue and affecting breathing. Animal studies demonstrated that more Tregs positively correlates with reduced severity of lung inflammation and that therapeutic transfer of Tregs can revert airway inflammation and tissue damages. These studies provide compelling evidence of Tregs involvement in lung immunity that could be harnessed to potentiate tissue repair. In this study, we investigate the potential of local Tregs expansion in the lung to dampen local inflammation and promote tissue repair following acute viral infection, without affecting the systemic immune responses established in the draining lymph nodes.
Parabiosis experiment performed by the host lab revealed the overall kinetics of Tregs residency. However, the potential pre-determination to residency in a given tissue and the fate of these cells when leaving this tissue are unknown. These Tregs could constitute a set of cells dedicated to this tissue only, migrating in and out of this specific organ. Alternatively, they could circulate across multiple organs. Finally, tissue-residency could be the terminal fate of these Tregs, with new waves of cells constantly renewing the pool of tissue-Tregs. To fate-map tissue-Tregs, we decided to set up a new mouse model based on the synthetic Notch system (SynNotch). This system allows a cell-contact dependent activation of gene transcription. The interaction of a SynNotch receptor expressed by Tregs, with a SynNotch ligand expressed by cells from a given tissue will induces the permanent expression of a reporter by Tregs; allowing the detection of these cells even after leaving the tissue. Foxp3-SynNotch receptor, SynNotch ligand and SynNotch reporter transgenic mouse strains has been generated. We validated the Treg specific expression of the SynNotch receptor and that the excision of a stop cassette by a Cre-recombinase induces expression of the SynNotch ligand. These strains are currently crossed together in the host laboratory and with tissue-specific Cre lines, to generate a fully functional SynNotch mouse to determine the fate of kidney and lung-Tregs.
We used single cell RNA sequencing to identify and compare Tregs isolated from the blood, spleen and a set of non-lymphoid tissues. We highlighted that numerous Tregs harboring the same T cell receptor (TCR) can be found in several non-lymphoid tissue. However, the overlap of the TCR repertoire of Tregs from non-lymphoid tissues and from blood or spleen is much more restricted. This observation seems to support the existence of a population of tissue-Tregs distinct from the circulating pool.
To expand Tregs locally, we used a strategy developed in the host laboratory that relies on IL2. IL2 is a proven growth factor for Tregs. Therefore, increased level of IL2 can drive Treg expansion, either systemically or locally. To expand Tregs located in the kidney, we designed AAVs driving the expression of IL2 under the control of kidney specific promoters described in the literature. We observed that intravenous injection of these AAVs in mice induces an up to ten times expansion of Tregs in the kidney. However, it also induces a massive expansion of Tregs and other immune cell types in the liver. Future work in the host laboratory will investigate other route of administrations, targeting more specifically the kidney, e.g. retro-ureteral route or intra-renal pelvis injection, to increase specificity to the kidney.
We applied the AAV approaches to Treg expansion in the lung and designed several AAVs for expression of IL2 specifically in the lung. We succeed to observe a significant increase of Tregs percentage among CD4 cells in the lung without significant difference in other immune cell types or in the other organs tested, like liver or spleen. This demonstrates the feasibility of restricted and specific Tregs expansion in the lung thanks to local IL2 delivery. Evaluation of the impact of lung-Treg expansion on the lung immune response to viral infections and tissue repair is pursued in the host laboratory. A publication and a patent about this work are under preparation.
We used single cell RNA sequencing to identify and compare Tregs isolated from the blood, spleen and a set of non-lymphoid tissues. We highlighted that numerous Tregs harboring the same T cell receptor (TCR) can be found in several non-lymphoid tissue. However, the overlap of the TCR repertoire of Tregs from non-lymphoid tissues and from blood or spleen is much more restricted. This observation seems to support the existence of a population of tissue-Tregs distinct from the circulating pool.
To expand Tregs locally, we used a strategy developed in the host laboratory that relies on IL2. IL2 is a proven growth factor for Tregs. Therefore, increased level of IL2 can drive Treg expansion, either systemically or locally. To expand Tregs located in the kidney, we designed AAVs driving the expression of IL2 under the control of kidney specific promoters described in the literature. We observed that intravenous injection of these AAVs in mice induces an up to ten times expansion of Tregs in the kidney. However, it also induces a massive expansion of Tregs and other immune cell types in the liver. Future work in the host laboratory will investigate other route of administrations, targeting more specifically the kidney, e.g. retro-ureteral route or intra-renal pelvis injection, to increase specificity to the kidney.
We applied the AAV approaches to Treg expansion in the lung and designed several AAVs for expression of IL2 specifically in the lung. We succeed to observe a significant increase of Tregs percentage among CD4 cells in the lung without significant difference in other immune cell types or in the other organs tested, like liver or spleen. This demonstrates the feasibility of restricted and specific Tregs expansion in the lung thanks to local IL2 delivery. Evaluation of the impact of lung-Treg expansion on the lung immune response to viral infections and tissue repair is pursued in the host laboratory. A publication and a patent about this work are under preparation.
This work allowed the generation of several new tools to study tissue-Tregs. In addition to the potential impact of the better knowledge about these cells on the scientific community, we also developed new strategy to expand tissue-Tregs. While adoptive transfer of in vitro expanded Tregs is effective in pre-clinical models, the feasibility and suitability of large-scale expansion of the systemic Treg population in patients would be challenging and likely to create unwanted side-effects (i.e. systemic immunosuppression). Specific targeting of a tissue-resident population is a promising and feasible alternative. For instance, the recent Covid 19 caused extensive lung damage in elderly patients, resulting in COVID-19-induced fibrotic lung disease. Expanding Tregs in lungs could be used in clinic to reverse or prevent lung damage.