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You are here: Home / Teams / Walzer T - LYACTS / PROJECTS AND GRANTS

PROJECTS AND GRANTS

  • Covid-19: Investigating immunological causes of disease severity and immune mechanisms of protection against infection and re-infection - collaborative study with other CIRI teams (B Lina, J Marvel, O Thaunat, FL Cosset, S Paul, M Dreux, P Vanhems) and with HCL hospital (services de réanimation, infectiologie, medecine du travail, DRCI)

SARS-CoV2 is a global health emergency and can cause fatal acute respiratory distress syndrome. We recently showed that a fraction of patients with severe disease displayed very weak type-I interferon (IFN-I) responses (Trouillet-Assant et al JACI 2020). We continue to investigate the mechanisms underlying this failed response, and in particular the role of IFN-I autoantibodies in this process. Moreover, we try to understand how local (respiratory tract) and systemic IFN-I responses regulate adaptive antiviral immunity.

Moreover, as the virus persists in the population, convalescent patients are likely re-exposed to the virus. In most cases, SARS-CoV2 specific memory should lead to protective immunity preventing the onset of symptoms. However, it becomes clear that the titer of neutralizing antibodies drops relatively rapidly in convalescent patients, especially those that were affected by mild Covid-19 disease. Whether memory B and T cells, that seem to be maintained in the long-term are sufficient to protect from re-infection is not firmly established as no studies have correlated the quality of immune memory and the re-infection rate. Moreover, new viral variants emerge with alternative surface glycoprotein structure and it is unclear if such variants may escape at least in part immune memory directed against the primary viral strain. We use various patients cohorts and associated biobanks as well as different immunological assays to address these questions. 

  • Epigenetic landscape and transcriptional regulators of NK cell maturation (TW, ANR Gambler)
As a follow up of our RNAseq/CHIPseq/ATACseq study of T-bet/Eomes roles in NK cell differentiation, we want :
1) To identify potential T-bet and Eomes interactors. For this we will take advantage of the two mouse models in which a V5-HA tag was inserted in the C-terminus region of each factor that we previously generated and used for Chipseq. Proteomics analyses will be performed after immunoprecipitation of T-bet or Eomes complexes (collaboration proteomics facility, Toulouse)
2) Using HA-V5 mice, we will try to downscale the quantity of cells needed to perform Chipseq experiments, so as to be able to compare T-bet and Eomes binding in different NK cell subsets (immature and mature). We will test the hypothesis of a “functional switch” between Eomes and T-bet upon terminal NK cell maturation, that would explain how factors with similar DNA binding capacities induce different outcomes.
3) Using a novel “procode” Crispr-Cas9 medium throughput system, we will study the role of 30 TFs in NK cell differentiation that we found to be induced specifically by T-bet or Eomes and that represent potential secondary effectors of T-box TFs. Of note many of these TFs have unknown function in the immune system.
 
  • S1P2 and S1P5 in tissue retention vs blood circulation (TW, grant Inserm-Transfert  CoPoc)

Our previous study has pointed to S1P5 as the major chemotactic receptor for S1P in NK cells, promoting their egress from the bone marrow. We then found that S1P2 was a natural inhibitor of S1P-induced migration that could promote tissue retention of NK cells. Thus, S1P2 and S1P5 may have opposite activities in the regulation of NK cell tissue infiltration. We want to study further this phenomenon in the context of anti-tumor responses. Indeed, it is well known that a major obstacle of many cell therapies of cancer is the weak ability of anti-tumor effectors (such activated NK cells or CAR-T cells) to infiltrate the tumor bed. In this context, we recently observed that S1P5 deficient mice, that have decreased numbers of circulating NK cells, are paradoxically more resistant than control mice to in vivo growth of MHC-I deficient tumor cells (RMA-KR). This could be explained by a role for S1P5 not only in NK cell egress from the BM, but also in blood retention of mature NK cells, preventing them from infiltrating tissues. As a supporting evidence of this model, we found that S1P5 cannot be internalized by high concentrations of S1P, unlike other S1P receptors, suggesting that high S1P concentrations in the blood would not desensitize S1P5 in NK cells. In this model, S1P2 would have a reciprocal effect to promote tissue infiltration. To test this model, we will inject RMA-KR cells in S1P5 KO and S1P2 KO mice (both available in the lab), and study tumor growth with respect to NK cell infiltration. S1P5 and S1P2 KO NK cells will be also compared in cell therapy protocols of established tumors in C57BL/6 mice. This project could lead to novel strategies to improve CAR-T or CAR-NK cells efficacy in patients with solid tumors.

  • Regulation of mTOR and immunometabolism of NK cells (AM, TW, Labellisation ligue, LVMH)
We recently found that mTOR is a major immunological checkpoint that controls the reactivity and effector functions of NK cells. Our results raise many questions:
1) How is mTOR activated in NK cells in relation to protein partners, nutrients availability and subcellular localization,
2) what are the relevant metabolic and signalling pathways involved in NK cell activation by mTOR, and
3) what are the negative feedback mechanisms.
To address these questions, we will use a multi-scale approach combining functional screens of metabolites and kinase inhibitors (collaboration PO Vidalain, CIRI), proteomics analyses of mTOR complexes using a novel and unique tool (mTOR-OST mice expressing an endogenously tagged mTOR, in collaboration with B Malissen, CIML Marseille), analyses of metabolic pathways (Seahorse) and translation (proteomics) in NK cells. Moreover, we will investigate consequences of constitutive PI3K activation using novel KI mice (Pik3cd-CA, ongoing gene-editing) on NK cell function (we generated these mice, they are already available). Indeed, several articles previously reported that constitutive PI3K activation induced T and NK cell exhaustion, but the mechanisms involved are mostly unknown. Moreover, we observed persistent mTOR activity in NK cells from the mouse tumour model described above. We will compare NK cells from those different mice with the various models of mTOR up- or down-regulation that we already have in the lab (in which NK cells are deficient for TSC1, AMPK, Raptor or Rictor,), for a number of functional readouts and intracellular events.
 
  • NK cell dysfunction in patients with multiple myeloma (SV, AM, TW, INCA “Reclaim”)

Multiple myeloma (MM) is a yet incurable disease characterized by the expansion of tumoral plasma B cells. NK cells can recognize and kill MM cells in vitro. This observation suggests that harnessing NK cell activity could be a valid therapeutic strategy to improve current anti-MM treatments, especially new MM-targeting anti-CD38 antibodies (Daratumab), whose efficacy may rely on NK cells. Whether NK cell activity correlates with MM patients’ response to standard treatments, relapse and overall survival is however unknown, and this clearly limits the development of NK cell-based immune therapies in MM. To address these points, we will take advantage of a unique resource of bone marrow (BM) samples for about 600 patients at diagnosis and after treatment and associated clinical data (IFN 2009 trial coordinated by our collaborators (L Martinet, H Avet-Loiseau, Toulouse). NK cell activity will be monitored in details in these samples. Moreover, transcriptomic data for 350 patients are already available for MM cells in this cohort, and will also be used to correlate NK cell activity and MM phenotype and genetic alterations, with the goal to find molecular pathways associated with NK cell function or dysfunction that could be targeted in patients. NK cell dysfunction will also be studied in a mouse model of lymphoma that we generated in the lab (RMA-KR, see above) and that allows the generation of large numbers of exhausted NK cells. We will use multiple techniques (RNAseq, proteomics, metabolic assays) to characterize exhausted NK cells in order to understand the molecular causes of effector dysfunction.

  • Genetic anomalies of cytokine/BCR signaling in lupus (TW, AB)

We recently identified a series of mutations in familial cases of lupus in BCR signaling molecules, including inactivating mutations in PKCd, Lyn, Socs1  and Def6. Many of these proteins regulate MAPK activation downstream BCR or cytokine signaling, suggesting that defective ERK activation could define a novel category of monogenic lupus. We generated KI mice bearing the identified human mutations in Prkcd or Lyn using Crispr/Cas9 mediated genome editing. We already validated that these mice develop lupus-like symptoms. A similar strategy will be used to generate mouse models of Socs1 and def6 mediated autoimmunity. In all these models, we will characterize the autoimmune phenotype (lymphoproliferation, autoantibodies, kidney histology and proteinuria, interferon signature etc), BCR signaling and activation in details (phospho-flow analyses), the development of immune subsets, in particular B cells. We will also explore possible therapies (BCR signaling inhibitors, B cell depletion etc), in collaboration with the team of L Genestier / G Salles (CRCL Lyon) that uses these therapies for B cell lymphomas (drug repurposing).