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You are here: Home / Teams / Walzer T - LYACTS / Team presentation and main achievements

Team presentation and main achievements

GENERAL INTERESTS

Our main interests in immunology are

  • The molecular pathways that underlie differentiation, migration and activation of immune effectors and
  • How genetic or acquired modifications in these pathways may lead to immune deficits or to autoimmune reactions.

 

Our main cellular model is the Natural Killer (NK) cell, but we also study B/T cells or monocytes in some contexts. We use a wide range of experimental models, often-transgenic mice that we create by inserting new epitopes or mutations. We also apply systems biology methods, taking advantage of the full range of omics-approaches. Finally, we perform translational research, whose secondary goal is to find novel ways to manipulate immune function in various diseases.

 

Lab organization

The common interest of the four subgroups is lymphocyte activation and signaling, studied under different but complementary angles. The groups 1 and 2 study how NK cells develop, migrate and are activated during immune responses, and in particular during anti-tumor or antiviral responses. Group 2 has a special interest for immunometabolism. Group 3 studies how mutations in genes involved in lymphocyte activation may cause inflammation or autoimmunity. Group 4 is delocalized at the Lyon-Sud hospital and performs immune monitoring in patients in the context of different team projects, and with the help from the SIRIC Lyrican (collaboration with the CRCL). Although the questions addressed may appear different, these four topics benefit prominently from the complementarity in models, expertises, perspectives and methodologies within the team. The efficiency of our strategy is evidenced by the important scientific production outlined below

 

RECENT ACHIEVEMENTS

  • Group 1 (TW): S1P receptors in NK and T cells (TW)
S1P5 is one of the five members of the family of G-protein coupled receptors for sphingosine-1 phosphate (S1P). S1P is a secreted lysophospholipid bound to plasma proteins that influences cell lymphocyte trafficking, proliferation, adherence and morphogenesis. Coordinated activities of biosynthetic and biodegradative enzymes maintain S1P gradients in vivo, with high S1P concentrations in extracellular fluids and low S1P concentrations in tissues1. We previously found that S1P5 was essential for mouse NK cell egress from the bone marrow (their site of development) to the blood circulation. Mechanistically, S1P5 can sense the S1P gradient and acts as a chemotactic receptor promoting movement towards high S1P compartments.
More recently, we decided to test the relevance of our findings on S1P5 in human. This led us to start two important collaborations 1) with local clinicians who provided us with pediatric tonsils or adult lymph nodes and 2) with Biogen, who provided us with newly designed S1P5 antagonists. We found that similar to mouse, human NK cells acquire S1P5 expression upon maturation and that this receptor is essential for their responsiveness to S1P, and presumably their exit from lymphoid organs2. In the course of these experiments, we also made the original observation that human memory T cells are repulsed by S1P, which contradicted current views on the role of S1P in T cell trafficking. This repulsion is mediated by S1P2, a receptor whose role in the immune system hasn’t been fully elucidated3 (Figure 1).
 
Figure 1: S1P receptor roles in T/NK cell migration. (A) S1PR1 promotes tonsil naive T cell migration to S1P-rich sites, whereas S1PR2 inhibits memory T cell migration (Drouillard et al, J Immunol, 2018). (B) Unlike naive T cells, mature NK cells require S1PR5 to exit lymphoid organs (Mayol et al, Blood, 2011 ; Drouillard et al, J Allergy Clin Immunol, 2018).
 
  • Group 1 (TW): Sequential actions of Eomes and T-bet promote stepwise maturation of NK cells

Eomes and T-bet are T-box transcription factors (TFs) essential for type-1 cellular immunity. The homology of their DNA binding domain suggest redundancy in their transcriptional activity. However, we found that Eomes and T-bet have complementary roles in NK cells with sequential actions promoting stepwise maturation through the regulation of largely distinct gene sets (Figure 2). Eomes acted more often as an activator of transcription inducing proliferation and the expression of many NK cell signature genes at the immature stage, while T-bet acted more frequently as a repressor decreasing the expression of many lineage genes at the mature stage, the latter being linked to its ability to induce transcriptional repressors such as Zeb2 or Blimp1. We generated novel transgenic mouse strains facilitating T-bet and Eomes chromatin immunoprecipitation. Analysis of genomic binding in NK cells revealed a strong overlap between Eomes and T-bet and pointed to Runx3 as cofactor of transcriptional regulation. Yet, each TF specifically induced key target genes endowing NK cells with specific functions such as cytotoxicity for Eomes and responsiveness to IL-12 for T-bet. Moreover, analyses of chromatin revealed that the accessibility of NK cell signature genes and maturation-acquired properties are largely associated with Eomes and T-bet binding.  Finally, our integrated genomic and transcriptomic analysis points to many novel genes and pathways that deserve further investigation in NK cells. Submitted to Nat. Immunol.

 
Figure 2 : Eomes and T-bet are sequentially required for NK cell maturation. (A) Image stream X (ISX) analysis of T-bet and Eomes expression in gated NK cells. Representative images are shown for the three NK cell subsets. CD122 staining was used to visualize the membrane. (B) Eomes and T-bet control different transitions during NK cell maturation. They also have complementary roles in the control of gene expression upon maturation as T-bet and Eomes regulate mostly distinct gene sets and at different maturation stages both transcriptionally and epigenetically.
 
  • Group 2 (AM): Regulation of mTOR in NK cells (AM, TW)

IL-15 has long been known to be essential for NK cell development and homeostasis6. Moreover, high concentrations of this cytokine enhance NK cell proliferation and cytotoxicity7. The molecular basis of this dual activity remained obscure until we found that low IL-15 concentrations were sufficient to induce STAT5 phosphorylation while high IL-15 concentrations are required to induce mTOR phosphorylation and activation. mTOR is a S/T kinase that regulates translation and metabolic activity in response to a wide variety of signals. Increasing evidence show that the control of mTOR activity is key to regulate effector functions of a wide variety of immune cells8. Using a specific deletion of mTOR in mouse NK cells, we showed that mTOR was required for proper NK cell development and also for their activation in vitro and in vivo in antiviral settings. Mechanistically, mTOR regulates NK cell metabolic activity (glycolysis and mitochondrial function) to sustain proliferation, and also increases IL-15 receptor levels in a positive feedback-signaling loop. mTOR is also essential for up regulation of granzyme B and the cytolytic machinery. Treatment of mice with rapamycin inhibits NK cell capacity to kill MHC-I deficient cells in vivo. As this drug is currently used in several clinical conditions, our work has important implications (see “projects”). More recently, we also found that mTOR activity is tightly regulated by inhibitory and activating receptors during NK cell “education”. High mTOR activity is favored by chronic engagement of inhibitory receptors and is required to maintain NK cell responsiveness. Upon stimulation, the mTOR/Akt pathway amplifies signaling through activating NK cell receptors by enhancing calcium flux and LFA-1 integrin activation9.

  • Group 2 (AM): Long-term treatment with rapamycin impairs NK cell maturation and function in women with metastatic breast cancer (AM, TW)

Building up on our discovery that mTOR is a central rheostat of NK cell reactivity we sought to study its role in human NK cells. We collaborated with oncologists from the Centre Léon Bérard, in the context of the “Lyrican” network, to monitor NK cell phenotype and function in women treated with everolimus (rapamycin) as monotherapy for metastatic breast cancer. We monitored peripheral NK cell activity at T0, 1 month, 3 months and 9 months after the start of treatment. We found that rapamycin profoundly impaired NK cell response to IL-15, which led to a progressive block in their maturation and a decrease in their effector functions (especially IFNg secretion) in response to ex vivo stimulation with tumor or antibody-coated cells. These results highlight the essential role of mTOR in human NK cell homeostasis and function and also suggest that long-term treatment with mTOR inhibitors impair anti-tumor immune function.

  • Group 2 (AM): Peripheral Natural Killer cells from chronic hepatitis B patients display molecular hallmarks of T cell exhaustion (UH, AM, TW)

A significant proportion of individuals infected by HBV develops chronic infection. Antiviral effectors such as Natural Killer (NK) cells have impaired functions in these patients, but the molecular mechanism responsible for this dysfunction remains poorly characterized. Here, we show that peripheral NK cells from chronic hepatitis B (CHB) patients have a defective capacity to produce IFN-γ, MIP1-β and TNF-α but retain an intact killing capacity. This functional phenotype was associated with a decrease in the expression of NKp30 and CD16, combined with defects in IL-15 stimulation of the mTOR pathway. Transcriptome analysis of NK cells in CHB patients further revealed a strong enrichment for transcripts typically expressed in exhausted T cells suggesting that NK cell dysfunction and T cell exhaustion rely on common molecular mechanisms (Figure 3). In particular, the transcription factors thymocyte selection-associated HMG box protein (TOX) and several of its targets, including immune checkpoints, were over-expressed in NK cells of CHB patients. This T cell exhaustion signature was predicted to be dependent on the calcium (Ca2+)-associated transcription factor NFAT. In line with this, when stimulating the Ca2+-dependent pathway in isolation, we recapitulated the dysfunctional phenotype. Thus, deregulated Ca2+ signalling could be a central event in both T cell exhaustion and NK cell dysfunction that occur during chronic infections.  Published in eLife (see publications)


Figure 3: Volcano plot showing differential gene expression between NK cells from chronic HBV carriers and healthy donors, as measured by RNAseq. A few genes involved in T cell exhaustion are highlighted.  
 

  • Group 3 (AB): Identification of novel genetic causes of early-onset autoimmunity

The contribution of genetic mutations in the development of autoimmune or autoinflammatory diseases is believed to be more important for pediatric-onset than for adult-onset cases. Based on this, Alexandre Belot constituted a cohort of pediatric cases, in the context of a newly created national reference center (centre de reference des rhumatismes inflammatoires et maladies autoimmunes de l’enfant, RAISE, plan national maladies rares, >400 patients) that he coordinates. Exome sequencing is performed on all familial cases, so as to identify potential genetic causes and mouse models are sometimes produced to test the causality of the gene variants identified. This work led to the discovery of several novel monogenic or oligogenic causes of autoimmune or autoinflammatory diseases (mutations in Prkdc10, Socs1, Def6, Lyn). Moreover, we contributed to collaborative efforts for a better diagnosis of autoinflammatory disorders (Interferon signature, with Yanick Crow for lupus patients, inflammasome activation for FMF patients, with Thomas Henry, CIRI) and for personalized treatments of patients (collaboration Imagine Institute, Paris, patients with mutations in Tmem173 (STING), treated with Ruxolitinib). Recently, we generated a mouse model of PKCd deficiency that recapitulates lupus features found in patients. We perform biochemical studies to try to understand how PKCd regulates B cell proliferation and activation.

  • Group 3 (AB): LACC1 deficiency defines a novel form of inherited juvenile arthritis associated with impaired autophagy in macrophages (AB, TW)

Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease in children and its aetiology remains poorly understood. We explored three families with early-onset arthritis carrying homozygous loss-of-expression mutations in LACC1. To understand the link between LACC1 and inflammation, we performed a functional study of LACC1 in human immune cells. We showed that LACC1 was primarily expressed in macrophages upon mTOR-signaling. We found that LACC1 deficiency had no obvious impact on inflammasome activation, the type-I interferon response or NF-κB regulation. We used bimolecular fluorescence complementation to identify novel LACC1 interactants. Biochemical experiments in primary macrophages confirmed that autophagy-inducing proteins, RACK1 and AMPK interacted with LACC1. Autophagy blockade in macrophages was also associated with LACC1 cleavage and degradation. LACC1 deficiency dramatically impaired autophagy in primary macrophages, while LACC1 overexpression induced autophagy in cell lines (Figure 4). This was associated with a defect in the accumulation of lipid droplets and mitochondrial respiration, suggesting that LACC1-dependent autophagy fuels macrophage bioenergetic metabolism. Altogether, LACC1 deficiency defines a novel form of genetically inherited juvenile arthritis associated with impaired autophagy in macrophages


 Figure 4: Overexpression of LACC1 in HeLa cells induces autophagy. LC3-GFP HeLa cells were transfected with a Flag-Lacc1 expressing or a control vector, as indicated. Cells were then stained for Lacc1 and nucleus (DAPI) and fluorescence (including GFP) was recorded by confocal microscopy. Representative images are shown.
 
  • Group 3-4: (SV, AB) Type I IFN immunoprofiling in COVID-19 patients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which causes coronavirus disease 2019 (COVID-19), is characterized by a wide spectrum of disease encompassing asymptomatic carriage, mild to severe upper respiratory tract illness that can evolve into respiratory failure, or rapidly progressing severe viral pneumonia with acute respiratory distress syndrome. We assessed the kinetics of plasma IFN-I in a cohort of 26 patients with COVID from the intensive care unit at Hospices Civils de Lyon. Our data demonstrate a heterogeneous pattern of IFN-α response in patients with COVID-19, with IFN-I response being impaired in about 20% of critically ill patients. Patients with an impaired IFN-I response had on average a poorer outcome, suggesting that defective innate immunity could contribute to disease exacerbation11.

 

 

 

SPONSORS

Inserm, erc, university, anr, league, finovi