Project description:Subtypes of innate lymphoid cells (ILC), defined by effector function and transcription factor expression, have recently been identified. In the adult, ILC derive from common lymphoid progenitors in bone marrow, although transcriptional regulation of the developmental pathways involved remains poorly defined. TOX is required for development of lymphoid tissue inducer cells, a type of ILC3 required for lymph node organogenesis, and NK cells, a type of ILC1. We show here that production of multiple ILC lineages requires TOX, as a result of TOX-dependent development of common ILC progenitors. Comparative transcriptome analysis demonstrated failure to induce various aspects of the ILC gene program in the absence of TOX, implicating this nuclear factor as a key early determinant of ILC lineage specification. TOX KO vs. wild tyype
Project description:Subtypes of innate lymphoid cells (ILC), defined by effector function and transcription factor expression, have recently been identified. In the adult, ILC derive from common lymphoid progenitors in bone marrow, although transcriptional regulation of the developmental pathways involved remains poorly defined. TOX is required for development of lymphoid tissue inducer cells, a type of ILC3 required for lymph node organogenesis, and NK cells, a type of ILC1. We show here that production of multiple ILC lineages requires TOX, as a result of TOX-dependent development of common ILC progenitors. Comparative transcriptome analysis demonstrated failure to induce various aspects of the ILC gene program in the absence of TOX, implicating this nuclear factor as a key early determinant of ILC lineage specification.
Project description:Innate lymphoid cells (ILC) in the small intestine govern immune homeostasis and protect the host against gut pathogens. While distinct cell-intrinsic signals have been identified that determine ILC development and differentiation, it has remained unclear which cell population regulates ILC sustenance. Using unbiased single cell RNA transcriptomic analysis of intestinal fibroblasts, we have identified a specialized Ccl19-expressing fibroblastic reticular cell (FRC) population that underpins solitary intestinal lymphoid tissue (SILT) structures including cryptopatches and isolated lymphoid follicles. Conditional ablation of lymphotoxin-β receptor (LTβR) signalling in SILT FRC impeded the maturation of isolated lymphoid follicles and blocked ILC maintenance through the downregulation of IL-7, consequently resulting in the elevated susceptibility to bacterial infection. Moreover, specific Ltbr ablation in FRC during adulthood revealed that constant LTβR-dependent FRC-ILC interaction is required to maintain SILT structures and ILC populations. Taken together, our study unveils a critical intestinal FRC niche that secures protective gut immunity.
Project description:In order to determine the dependency of mature tissue-resident ILCs on combinations of synergistic and antagonising Transcription Factors (TFs), we generated mice with conditional deletions of RORc, RORa and Tbx21 driven from the Id2 allele, utilizing a tamoxifen inducible Cre-recombinase in combination with a tdRFP allele to report Cre-excision (Id2creERT2 x ROSAtdRFP). RFP+ small intestinal ILCs were sorted from the lamina propria digests (gated as CD45+, Lineage negative, CD90+ CD127+, tdRFP+ - see manuscript for full methodological details), from control mice or mice containing loxP flanked alleles for RORc, RORc and RORa or RORc and Tbx21. Four samples of sort-purified ILCs were then subjected to 10X single cell sequencing; - Id2creERT2 - Id2idRORc - Id2idRORc/RORa - Id2idRORc/Tbx21
Project description:Innate lymphoid cells (ILCs) develop from common lymphoid progenitors (CLP), which further differentiate into the common ILC progenitor (CILP) that can give rise to both ILCs and NK cells. Murine ILC intermediates have recently been characterized, but the human counterparts and their developmental trajectories have not yet been identified, largely due to the lack of homologous surface receptors in both organisms. Here, we show that human CILPs (CD34+CD117+α4β7+Lin-) acquire CD48 and CD52, which define NK progenitors (NKPs) and innate lymphoid cell precursors (ILCPs). Two distinct NK cell subsets were generated in vitro from CD34+CD117+α4β7+Lin-CD48-CD52+ and CD34+CD117+α4β7+Lin-CD48+CD52+ NKPs, respectively. Independent of NKPs, ILCPs exist in the CD34+CD117+α4β7+Lin-CD48+CD52+ subset and give rise to ILC1s, ILC2s and NCR+ ILC3s, whereas CD34+CD117+α4β7+Lin-CD48+CD52- ILCPs give rise to a distinct subset of ILC3s that have lymphoid tissue inducer (LTi)-like properties. Additionally, CD48 expressing CD34+CD117+α4β7+Lin- precursors give rise to tissue-associated ILCs in vivo. We also observed that the interaction of 2B4 with CD48 induced differentiation of ILC2s, and together these findings show that expression of CD48 by human ILCPs modulates ILC differentiation.
Project description:Innate lymphoid cells (ILC) represent innate versions of T helper and cytotoxic T cells that differentiate from committed ILC precursors (ILCP). Still, how ILCP relate to mature tissue-resident ILCs remains unclear. We observed that a population of CD117+ ILC from peripheral blood (PB) of healthy donors does not represent any conical ILC subset, but expressed marker (CD117) commonly expressed by hemato-lymphoid progenitors. We therefore hypothesized PB CD117+ ILC might include uncommitted lymphoid precursors. In order to further understand the identity of PB CD117+ ILC, we profiled the transcriptome of highly purified circulating CD117+ ILC compared to CD34+ HSC, the latter representing immature hematopoietic progenitors with multi-lineage potential. Clear differences in gene expression profiles emerged, with a large cluster of 1540 genes expressed at substantially higher levels in CD117+ ILC. In contrast, CD34+ HSC cells highly expressed genes involved in the broad development of diverse hematopoietic lineages. Compared to HSC, CD117+ ILC express high levels of TF that have been shown to be essential for murine ILC development and we did not detect transcripts characteristic of T and B cells development. Transcriptomic analysis suggested that CD117+ ILC represent lymphoid-biased progenitors carrying a TF expression profile resembling a multi-potent ILC precursor (ILCP).
Project description:Innate lymphoid cells (ILC) in the small intestine govern immune homeostasis and protect the host against gut pathogens. While distinct cell-intrinsic signals have been identified that determine ILC development and differentiation, it has remained unclear which cell population regulates ILC sustenance. Using unbiased single cell RNA transcriptomic analysis of intestinal fibroblasts, we have identified a specialized Ccl19-expressing fibroblastic reticular cell (FRC) population that underpins solitary intestinal lymphoid tissue (SILT) structures including cryptopatches and isolated lymphoid follicles. Conditional ablation of lymphotoxin-β receptor (LTβR) signalling in SILT FRC impeded the maturation of isolated lymphoid follicles and blocked ILC maintenance through the downregulation of IL-7, consequently resulting in the elevated susceptibility to bacterial infection. Moreover, specific Ltbr ablation in FRC during adulthood revealed that constant LTβR-dependent FRC-ILC interaction is required to maintain SILT structures and ILC populations. Taken together, our study unveils a critical intestinal FRC niche that secures protective gut immunity.
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. Transcriptome analysis of CD56+CD127hi tonsil ILC, Ex vivo and after stimulation with aNKp44, IL-1/IL-7/IL-23 or aNKp44/IL-1/IL-7/IL-23 for 3.5h. RNA was extracted and pooled from 2 donors each, Amplified and labeled according to manufacturer´s instructions (GeneChipU133plus2® , Affymetrix). The analysis was performed in triplicates.