Role of Sting in lymphocyte development and function – LYMPHO-STING

We propose in aim 1 to perform STING GOF bone marrow reconstitution of lethally irradiated WT mice. To confirm developmental block in B and T cell lineages, we will perform bone marrow cultures with stromal cells in vitro. Using this model and/or ex vivo progenitors, we will analyze the implication of different biological pathways. Results will be confirmed with rescue experiments in vitro and in vivo. Finally, a high-throughput sequencing strategy by RNAseq on purified progenitors from STING GOF or WT control mice will be done in parallel.
As mentioned above, the anti- proliferative effect of STING GOF on mature T cells is partially reversed by the deficit of IFNAR, therefore implicating both IFN-dependent and independent pathways. To decipher the mechanisms of mature lymphocyte defects in our mouse model, we will use the same strategies as in aim 1, to study the role of key pathways in STING GOF mice on IFNAR KO or WT background. In addition, a high-throughput sequencing strategy by RNAseq in purified mature splenic B and T cells from STING GOF or control mice will be done in parallel.

The analysis of hematopoietic development in the bone marrow and thymus of STING GOF mice and control mice allowed us to identify more precisely the stages of development impacted by Sting GOF.
In addition, RNAseq analysis of the transcriptome of peripheral T lymphocytes revealed, in addition to the gain in apoptosis already described by us and other teams, the activation of other biological pathways that may explain the phenotype of these cells. These preliminary results are currently being confirmed.

The rest of the project will be devoted to the confirmation of the results obtained, and to prepare them for publication. In particular, the analysis of the RNAseq of progenitors will allow us to better identify the mechanisms of developmental blockade due to Sting gain of function, which may help explain the lymphopenia in patients with SAVI. In addition, confirmation of the biological pathways involved in the phenotype of peripheral Sting GOF T lymphocyte will give us new fundamental data on the role of Sting in lymphocyte function, but also on the impact of Sting targeting in therapy.

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Stimulator of Interferon Genes (STING), encoded by TMEM173, belongs to cytosolic DNA-sensing pathways in innate immunity, and has emerged as a central player in antiviral and antibacterial immunity, autoinflammation and cancer. STING is an endoplasmic reticulum (ER)-associated membrane protein, expressed in a wide variety of tissues including endothelial, epithelial and hematopoietic cell types. When activated by cyclic dinucleotides produced by cGAS enzyme, or directly by microbes, STING translocates from the ER to perinuclear vesicles where it activates TANK-binding kinase 1 (TBK1), which then phosphorylates interferon regulatory factor 3 (IRF3) and induces the expression of the type I interferons (IFNs). Type I IFNs trigger the expression of a set of interferon-stimulated genes (ISGs), upon activation of the type I IFNs receptor (IFNAR). The NF?B pathway will also be engaged, in a TBK1 dependent or independent manner, driving inflammatory response.
Gain of function (GOF) heterozygous mutations in TMEM173 (leading to constitutive activation of STING), have been described in patients with an autoinflammatory phenotype designated STING Associated Vasculopathy with onset in Infancy (SAVI), which is classified as a type I interferonopathy, with increased expression of ISGs. We describe a heterozygous STING GOF mutation (V155M mutation), in the context of a previous collaborative ANR project (ANR Lumugene: 2014-2018; ANR-14-CE14-0026-04). SAVI patients exhibit variable signs of systemic inflammation, with growth delay and reduced survival. The skin pathology in particular, is improved after treatment with JAK1-JAK2 inhibitors (as ruxolitinib), targeting IFNAR and other cytokine receptors’ pathways. SAVI patients frequently present CD4 and CD8 T lymphopenia and NK lymphopenia. B lymphopenia is also detectable in a significative subset of patients. Significant infections are observed in 25% of the patients. Today, the exact molecular mechanisms leading to these various features remain uncertain, which led us to generate a knock-in murine model carrying a heterozygous point mutation (V154M) in STING corresponding to the most frequent mutation (V155M) identified in SAVI patients (STING V154M/WT (wild-type) mice, thereafter called STING GOF mice), recently published.
We have shown that STING GOF mouse model exhibits a severe combined immunodeficiency (SCID) phenotype with T, B and NK lymphopenia. In addition, peripheral T and B cells show an abnormal phenotype in terms of activation, proliferation and apoptosis. These data emphasize a new role of STING in lymphocyte development and function. Surprisingly, the SCID phenotype of this STING GOF mouse model and others is globally independent of type I IFN.
This project proposes to decipher the molecular mechanisms responsible for this new role of STING in lymphoid development and functions, with two main objectives: 1) understand the mechanisms driving the early lymphoid development defects linked to STING GOF and 2) understand the mechanisms underlying the peripheral T and B cell abnormalities linked to STING GOF, using this new murine model of STING GOF, in vitro cultures, and human samples from SAVI patients. In the two aims, we will combine hypothesis-driven strategies and omics analysis.
This project will contribute to understand the different biological pathways induced by STING GOF in immature and mature lymphocytes leading to lymphopenia and abnormal phenotype of peripheral T and B cells. This project will give, in a fundamental point of view, new insights into the role of STING in adaptive immunity. In addition, a better comprehension of B and T cell development and function in STING GOF mutants will contribute to devise new therapeutic strategies for lymphopenic SAVI patients.