Project description:RORγt+ innate lymphoid cells (ILC) are crucial players of innate immune responses and represent a major source of IL-22, which has an important role in mucosal homeostasis. The signals required by RORγt+ ILC to express IL-22 and other cytokines, including TNF, have only partially been elucidated. Here we show that RORγt+ ILC can directly sense the environment by the engagement of the activating receptor NKp44. NKp44 triggering in RORγt+ ILC selectively activates a coordinated pro-inflammatory program, including TNF, while cytokine stimulation induces preferentially IL-22 expression. However, combined engagement of NKp44 and cytokine receptors results in a strong synergistic effect. These data support the concept that NKp44+ RORγt+ ILC can be activated without cytokines and are able to switch between IL-22 or TNF production, depending on the triggering stimulus.
Project description:Autoimmune regulator (Aire) plays an indispensable role in the induction of central immune tolerance. We identify a novel subset of Rorγt-dependent innate lymphoid cell-type in secondary lymphoid organs, which presents endogenously generated antigens and contributes to peripheral T cell tolerance. In this experiment, transcriptomes of these Aire-ILC3 cells are characterized in wild-type Balb/C and Aire-/- Balb/C mice [Ramsey, C. et al. (2002) Hum Mol Genet 11:397-409] using RNA-seq method. To obtain the cells, peripheral lymph nodes were minced, enzymatically digested, and Aire-ILC3 cells were FACS-sorted as Lin-, MHCII+, CD80+, IL7Ra+ cells.
Project description:The aim of this study was to analyze the global transcriptional profiles of small intestine (SI) Innate Lymphoid Cells (ILCs) expressing the NK cell marker NKp46. Based on differential expression of the RORgt transcription factor SI NKp46+ ILCs can be divided in NKp46+RORgt- and NKp46+RORgt+ cells. While NKp46+RORgt- cells produce IFN-g, like conventional Natural Killer (NK) cells, NKp46+RORgt+ cells secrete IL-22, like Lymphoid Tissue inducer (LTi) cells. We compared the global transcriptional profiles of both NKp46+RORgt- and NKp46+RORgt+ cells to conventional splenic NK cells and to SI NKp46-RORgt+ cells, which contain adult LTi cells. By following this approach, we showed that SI NKp46+RORγt- ILCs correspond to SI NK cells. We also identified a transcriptional program conserved in adult SI NKp46+RORγt+, NKp46-RORγt+ ILCs and fetal LTi. The various ILC cell populations analyzed in this study were isolated from C57BL/6 RORc(gt)+/GFP reporter mice. SI NKp46+RORγt- (NKp46+GFP-) cells, SI NKp46+RORγt+ cells (NKp46+GFPlow and NKp46+GFPhigh cells) and NKp46-RORγt+ ILCs, including adult LTi cells , were sorted by flow cytometry from CD3- lamina propria cells of small intestine (SI) of RORc(γt)+/GFP reporter mice . Splenic NKp46+RORγt- (NKp46+GFP-) cells were also sorted as the reference for conventional NK cells. Two replicates of each populations were produced and analyzed.
Project description:Group 3 innate lymphoid cells (ILC3) are defined by the expression of RORγt, which is selectively required for their development. The lineage-specified progenitor cells of human ILC3 and their developmental site after birth remain undefined. Here we identified a novel population of human CD34+ hematopoietic progenitor cells (HPC) expressing RORγt and sharing with ILC3 a distinct transcriptional signature. RORγt+ CD34+ HPC were located in tonsils and intestinal lamina propria (LP) and selectively differentiated towards ILC3. Conversely, RORγt- CD34+ HPC displayed commitment potential for both ILC3 and NK cells and the differentiation fate towards these two cell lineages was determined by cytokine and aryl hydrocarbon receptor (AhR) signaling. Thus, we propose that RORγt+ CD34+ cells represent human lineage-specified progenitors of IL-22+ ILC3 and that tonsils as well as intestinal LP might be preferential sites of their differentiation.
Project description:Lymphoid tissue inducer (LTi) cells are regarded as a subset of innate lymphoid cells (ILCs). However, these cells are not derived from the ILC common progenitor, which generates other ILC subsets and is defined by the expression of the transcription factor PLZF. Here we examined transcription factor(s) determining the fate of LTi progenitor versus non-LTi ILC progenitor. Conditional deletion of Gata3 resulted in the loss of PLZF+ non-LTi progenitors but not the LTi progenitors that expressed the transcription factor RORγt. Consistently, PLZF+ non-LTi progenitors expressed high amounts of GATA3 whereas GATA3 expression was low in RORγt+ LTi progenitors. The generation of both progenitors required the transcriptional regulator Id2, which defines the common helper-like innate lymphoid progenitor, but not cytokine signaling. Nevertheless, low GATA3 expression was necessary for the generation of functionally mature LTi cells. Thus, differential expression of GATA3 determines the fates and functions of distinct ILC progenitors.
Project description:The intestinal immune system maintains tolerance to harmless food proteins and gut microbiota through peripherally-derived RORγt+Tregs (pTregs), which prevent food intolerance and inflammatory bowel disease. Recent studies suggested that RORγt+ antigen-presenting cells (APCs), which encompass rare dendritic cell (DC) subsets and type 3 innate lymphoid cells (ILC3s), are key to pTregs induction. Here, we developed a mouse with reduced RORγt+APCs by deleting a specific cis-regulatory element of Rorc encoding RORγt. Single-cell RNA-sequencing and flow cytometry analyses confirmed the depletion of a RORγt+DC subset and ILC3s. These mice showed a secondary reduction in pTregs, impaired tolerance to oral antigens and increase in Th2 cells. Conversely, newly and previously generated ILC3-deficient mice showed no pTregs or Th2 cells abnormalities. Lineage tracing revealed that RORγt+DCs share a lymphoid origin with ILC3s, consistent with their similar phenotypic traits. These findings highlight a unique role of lymphoid RORγt+DCs in maintaining intestinal immune balance and preventing conditions like food allergies.
Project description:The intestinal immune system maintains tolerance to harmless food proteins and gut microbiota through peripherally-derived RORγt+Tregs (pTregs), which prevent food intolerance and inflammatory bowel disease. Recent studies suggested that RORγt+ antigen-presenting cells (APCs), which encompass rare dendritic cell (DC) subsets and type 3 innate lymphoid cells (ILC3s), are key to pTregs induction. Here, we developed a mouse with reduced RORγt+APCs by deleting a specific cis-regulatory element of Rorc encoding RORγt. Single-cell RNA-sequencing and flow cytometry analyses confirmed the depletion of a RORγt+DC subset and ILC3s. These mice showed a secondary reduction in pTregs, impaired tolerance to oral antigens and increase in Th2 cells. Conversely, newly and previously generated ILC3-deficient mice showed no pTregs or Th2 cells abnormalities. Lineage tracing revealed that RORγt+DCs share a lymphoid origin with ILC3s, consistent with their similar phenotypic traits. These findings highlight a unique role of lymphoid RORγt+DCs in maintaining intestinal immune balance and preventing conditions like food allergies.
Project description:The intestinal immune system maintains tolerance to harmless food proteins and gut microbiota through peripherally-derived RORγt+Tregs (pTregs), which prevent food intolerance and inflammatory bowel disease. Recent studies suggested that RORγt+ antigen-presenting cells (APCs), which encompass rare dendritic cell (DC) subsets and type 3 innate lymphoid cells (ILC3s), are key to pTregs induction. Here, we developed a mouse with reduced RORγt+APCs by deleting a specific cis-regulatory element of Rorc encoding RORγt. Single-cell RNA-sequencing and flow cytometry analyses confirmed the depletion of a RORγt+DC subset and ILC3s. These mice showed a secondary reduction in pTregs, impaired tolerance to oral antigens and increase in Th2 cells. Conversely, newly and previously generated ILC3-deficient mice showed no pTregs or Th2 cells abnormalities. Lineage tracing revealed that RORγt+DCs share a lymphoid origin with ILC3s, consistent with their similar phenotypic traits. These findings highlight a unique role of lymphoid RORγt+DCs in maintaining intestinal immune balance and preventing conditions like food allergies.
Project description:We analyzed the total proteome of group 2 innate lymphoid cells (ILC2s) after different stimulation with interleukin-33 (IL-33), a cytokine playing a critical role in human asthma, and TL1A, a TNF-family cytokine also known to activate ILC2s. Upon combined stimulation with IL-33 plus TL1A, we show that lung ILC2s produce high amounts of IL-9 and acquire a transient ‘ILC9’ phenotype. This phenotype is characterized by simultaneous production of large amounts of type 2 cytokines (IL-5, IL-13 and IL-9), induction of the IL-2 receptor CD25 (Il2ra), and of the transcription factors IRF4, JunB and BATF, that form immune-specific complexes known to induce IL-9 expression.