Régulation du développement des cellules lymphoïdes innées par les facteurs de transcription : approche centrée sur de nouveaux modèles murins multi-rapporteurs

Les cellules lymphoïdes innées (ILC) forment une famille de rares cellules hématopoïétiques à laquelle appartiennes les cellules cytotoxiques « natural killer » (NK) et les ILC auxiliaires. Chez l’adulte, les ILC sont générés dans la moelle osseuse, et dépendent de l’expression séquentielle de plusieurs facteurs de transcription afin de se développer complétement. Parmi ces facteurs de transcription, le facteur répresseur ID2 a rapidement été démontré comme crucial pour permettre aux progéniteurs communs lymphoïdes de se différencier en ILC et non en lymphocyte T ou B. Dans un premier temps, nous avons mis au point une nouvelle souche de souris rapportrice Id2RFP, afin de mieux caractériser les précurseurs ILC (ILCP) Id2+ de la moelle osseuse. Nous avons remarqué que les ILCP Id2+ formaient un groupe hétérogène de cellules, capable de générer aussi bien des cellules NK que des ILC auxiliaires, in vitro comme in vivo. Nous avons également mis au point un nouveau modèle murin multi-rapporteur adapté au développement des ILC, en multiplexant notre rapporteur Id2RFP avec d’autres rapporteurs de facteurs de transcription (Zbtb16 et Bcl11b). Nous avons mis en évidence que les ILCP Id2+Zbtb16+ maintenaient leur capacité à générer des cellules NK, tandis que le ILCP Id2+Zbtb16+/-Bcl11b+ n’étaient plus capables que de générer des ILC du deuxième groupe. Dans un second temps, nous avons tiré avantage de cette technique et obtenu des souris quadruple-rapportrice, combinant des rapporteurs pour Id2, Gata3, Nfil3 et Bcl11b. En utilisant ce modèle en combinaison avec du séquençage ARN en cellule unique, nous avons identifié des progéniteurs Id2+ ayant des profils transcriptomiques compatibles avec ceux de précurseurs unipotents. L’acquisition de ces profils transcriptomiques nous est apparue comme graduelle et continue, par contraste avec les modèles de différentiation généralement admis « en escalier ». Nous avons également mis en évidence que Gata3 et Bcl11b étaient de bons prédicteurs de précurseurs biaisés vers une différentiation en ILC du deuxième groupe, tandis que de faibles niveaux de Gata3 et de hauts niveaux de Nfil3 étaient davantage prédicteurs d’un biais vers des ILC de groupe 1 ou 3. En conclusion, l’ensemble de ces résultats appellent à reconsidérer et précisent le modèle de différentiation des ILC murins.Innate lymphoid cells (ILC) form a family of rare hematopoietic cells that comprises cytotoxic natural killer (NK) cells and “helper” ILCs. In adult, ILCs are generated in the bone marrow and rely on the sequential expression of several transcription factors to get fully differentiated. Among these transcription factors, transcriptional repressor ID2 has early been shown to counteract T or B cell fate in common lymphoid progenitors and to promote ILC lineage commitment. First, we engineered a novel Id2RFP reporter mouse model to better characterize bone marrow Id2+ ILC progenitors (ILCP). Id2+ ILCP were highly heterogeneous and gave rise to both NK cells and “helper” ILCs in vitro and in vivo. To further investigate precursors potential, we generated a new multi-TF reporter mouse model for ILCP development, by multiplexing Id2RFP with other transcription factor reporters (Zbtb16 and Bcl11b). We found that the Id2+Zbtb16+ ILCP compartment surprisingly retains significant NK cell potential, while Id2+Zbtb16+/-Bcl11b+ ILCP precursors lacking expression of ICOS or CD25 were already restricted to a group 2 ILC fate. Second, we took advantage of this technique and obtained a quadruple TF-reporter model, combining reporters for Id2, Gata3, Nfil3 and Bcl11b. Using this model in combination with single-cell RNA sequencing, we identified Id2+ progenitors that had transcriptomic profiles compatible with uni-lineage progenitors. Interestingly, acquisition of these profiles was gradual and continuous, in contrast with commonly used stepwise models of differentiation. We also discovered that Gata3 and Bcl11b were predictors of an ILC2 fate, while low Gata3 and high Nfil3 levels were characteristic of group 1 and 3 ILCs progenitors. Taken together, these results challenge and redefine the current model of murine ILC development

Innate Lymphoid Cell Development: A T Cell Perspective

International audienceInnate lymphoid cells (ILCs) and natural killer (NK) cells have garnered considerable interest due to their unique functional properties in immune defense and tissue homeostasis. Our current understanding of how these cells develop has been greatly facilitated by knowledge of T cell biology. Models of T cell differentiation provided the basis for a conceptual classification of these innate effectors and inspired a scheme of their activation and regulation. In this review, we discuss NK cell and ILC development from a "T cell standpoint" in an attempt to extend the analogy between adaptive T cells and their innate ILC and NK cell counterparts

An Id2RFP-Reporter Mouse Redefines Innate Lymphoid Cell Precursor Potentials

International audienceInnate lymphoid cell (ILC) development proposes that ILC precursors (ILCPs) segregate along natural killer (NK) cell versus helper cell (ILC1, ILC2, ILC3) pathways, the latter depending on expression of Id2, Zbtb16, and Gata3. We have developed an Id2-reporter strain expressing red fluorescent protein (RFP) in the context of normal Id2 expression to re-examine ILCP phenotype and function. We show that bone-marrow ILCPs were heterogeneous and harbored extensive NK-cell potential in vivo and in vitro. By multiplexing Id2RFP with Zbtb16CreGFP and Bcl11btdTomato strains, we made a single-cell dissection of the ILCP compartment. In contrast with the current model, we have demonstrated that Id2+Zbtb16+ ILCPs included multi-potent ILCPs that retained NK-cell potential. Late-stage ILC2P and ILC3P compartments could be defined by differential Zbtb16 and Bcl11b expression. We suggest a revised model for ILC differentiation that redefines the cell-fate potential of helper-ILC-restricted Zbtb16+ ILCPs

Zeb1 represses TCR signaling, promotes the proliferation of T cell progenitors and is essential for NK1.1+ T cell development

International audienceT cell development proceeds under the influence of a network of transcription factors (TFs). The precise role of Zeb1, a member of this network, remains unclear. Here, we report that Zeb1 expression is induced early during T cell development in CD4 − CD8 − double-negative (DN) stage 2 (DN2). Zeb1 expression was further increased in the CD4 + CD8 + double-positive (DP) stage before decreasing in more mature T cell subsets. We performed an exhaustive characterization of T cells in Cellophane mice that bear Zeb1 hypomorphic mutations. The Zeb1 mutation profoundly affected all thymic subsets, especially DN2 and DP cells. Zeb1 promoted the survival and proliferation of both cell populations in a cell-intrinsic manner. In the periphery of Cellophane mice, the number of conventional T cells was near normal, but invariant NKT cells, NK1.1 + γδ T cells and Ly49 + CD8 T cells were virtually absent. This suggested that Zeb1 regulates the development of unconventional T cell types from DP progenitors. A transcriptomic analysis of WT and Cellophane DP cells revealed that Zeb1 regulated the expression of multiple genes involved in the cell cycle and TCR signaling, which possibly occurred in cooperation with Tcf1 and Heb. Indeed, Cellophane DP cells displayed stronger signaling than WT DP cells upon TCR engagement in terms of the calcium response, phosphorylation events, and expression of early genes. Thus, Zeb1 is a key regulator of the cell cycle and TCR signaling during thymic T cell development. We propose that thymocyte selection is perturbed in Zeb1-mutated mice in a way that does not allow the survival of unconventional T cell subsets

Reproducibility of fluorescent expression from engineered biological constructs in E. coli

We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices.Peer reviewe