Innate Lymphoid Cells (ILCs) are guardians of mucosal immunity, yet the transcriptional networks that support their function remain poorly understood. We employed inducible combinatorial deletion of key transcription factors (TFs) required for ILC development (RORγt, RORα and T-bet) to determine their necessity in maintaining ILC3 identity and function. Both RORγt and RORα were required to preserve optimum effector functions, however RORα was sufficient to support robust IL-22 production among the LTi-like ILC3 subset, but not NCR + ILC3s. LTi-like ILC3s persisted with only selective loss of phenotype and effector functions even after the loss of both TFs. In contrast, continued RORγt expression was essential to restrain transcriptional networks associated with type 1 immunity within NCR + ILC3s, which co-express T-bet. Full differentiation to an ILC1-like population required the additional loss of RORα. Together, these data demonstrate how TF networks integrate within mature ILCs post-development to sustain effector functions, imprint phenotype and restrict alternative differentiation programs.

BackgroundMultiple genes have been implicated by association studies in altering inflammatory bowel disease (IBD) predisposition. Paediatric patients often manifest more extensive disease and a particularly severe disease course. It is likely that genetic predisposition plays a more substantial role in this group.ObjectiveTo identify the spectrum of rare and novel variation in known IBD susceptibility genes using exome sequencing analysis in eight individual cases of childhood onset severe disease.DesignDNA samples from the eight patients underwent targeted exome capture and sequencing. Data were processed through an analytical pipeline to align sequence reads, conduct quality checks, and identify and annotate variants where patient sequence differed from the reference sequence. For each patient, the entire complement of rare variation within strongly associated candidate genes was catalogued.ResultsAcross the panel of 169 known IBD susceptibility genes, approximately 300 variants in 104 genes were found. Excluding splicing and HLA-class variants, 58 variants across 39 of these genes were classified as rare, with an alternative allele frequency of <5%, of which 17 were novel. Only two patients with early onset Crohn's disease exhibited rare deleterious variations within NOD2: the previously described R702W variant was the sole NOD2 variant in one patient, while the second patient also carried the L1007 frameshift insertion. Both patients harboured other potentially damaging mutations in the GSDMB, ERAP2 and SEC16A genes. The two patients severely affected with ulcerative colitis exhibited a distinct profile: both carried potentially detrimental variation in the BACH2 and IL10 genes not seen in other patients.ConclusionFor each of the eight individuals studied, all non-synonymous, truncating and frameshift mutations across all known IBD genes were identified. A unique profile of rare and potentially damaging variants was evident for each patient with this complex disease.

Tissue residency is considered a defining feature of the innate lymphoid cell (ILC) populations located within mucosal and adipose tissues. ILCs are also present within all lymphoid tissues, but whether ILCs migrate between lymphoid and nonlymphoid sites and in what context is poorly understood. To determine whether migratory ILCs exist within peripheral lymph nodes (LNs), we labeled all cells within the brachial LN (bLN) of transgenic mice expressing a photoconvertible fluorescent protein by direct exposure to light. Tracking of cellular changes in the labeled LN revealed the gradual migration of new ILCs into the tissue, balanced by egress of ILCs dependent on sphingosine-1-phosphate receptors. Most of the migratory ILCs were ILC1s, entering LNs directly from the circulation in a CD62L- and CCR7-dependent manner and thus behaving like conventional natural killer (cNK) cells. Upon egress, both ILC1s and cNK cells were found to recirculate through peripheral LNs. A distinct population of migratory ILC2s were detected in the LN, but most of the ILC3s were tissue resident. Functionally, both migratory and resident ILC1s within LNs were able to rapidly produce IFN-γ to support the generation of robust TH1 T cell responses after immunization. Thus, migratory and resident ILC populations exist within peripheral LNs, with ILC1s, akin to cNK cells, able to traffic into these tissues where they can contribute to the initiation of adaptive immunity.

Members of the innate lymphoid cell (ILC) family have been implicated in the development of thymic microenvironments and the recovery of this architecture after damage. However, a detailed characterization of this family in the thymus is lacking. To better understand the thymic ILC compartment, we have utilized multiple in vivo models including the fate mapping of inhibitor of DNA binding‐2 (Id2) expression and the use of Id2 reporter mice. Our data demonstrate that ILCs are more prominent immediately after birth, but were rapidly diluted as the T‐cell development program increased. As observed in the embryonic thymus, CCR6+NKp46− lymphoid tissue inducer (LTi) cells were the main ILC3 population present, but numbers of these cells swiftly declined in the neonate and ILC3 were barely detectable in adult thymus. This loss of ILC3 means ILC2 are the dominant ILC population in the thymus. Thymic ILC2 were able to produce IL‐5 and IL‐13, were located within the medulla, and did not result from ILC3 plasticity. Furthermore, in WT mice, thymic ILC2 express little RANKL (receptor activator of nuclear factor kappa‐B ligand) arguing that functionally, these cells provide different signals to LTi cells in the thymus. Collectively, these data reveal a dynamic switch in the ILC populations of the thymus during neonatal development.

Improving the efficacy of immune checkpoint therapies will require a better understanding of how immune cells are recruited and sustained in tumors. Here, we used the photoconversion of the tumor immune cell compartment to identify newly entering lymphocytes, determine how they change over time, and investigate their egress from the tumor. Combining single-cell transcriptomics and flow cytometry, we found that while a diverse mix of CD8 T cell subsets enter the tumor, all CD8 T cells retained within this environment for more than 72 h developed an exhausted phenotype, revealing the rapid establishment of this program. Rather than forming tumor-resident populations, non-effector subsets, which express TCF-1 and include memory and stem-like cells, were continuously recruited into the tumor, but this recruitment was balanced by concurrent egress to the tumor-draining lymph node. Thus, the TCF-1+ CD8 T cell niche in tumors is highly dynamic, with the circulation of cells between the tumor and peripheral lymphoid tissue to bridge systemic and intratumoral responses.

When dormant naïve T cells first become activated by antigenpresenting cells, they express the autocrine growth factor IL-2 which transforms them into rapidly dividing effector T cells. During this process, hundreds of genes undergo epigenetic reprogramming for efficient activation, and also for potential reactivation after they return to quiescence as memory T cells. However, the relative contributions of IL-2 and T cell receptor signaling to this process are unknown. Here, we show that IL-2 signaling is required to maintain open chromatin at hundreds of gene regulatory elements, many of which control subsequent stimulus-dependent alternative pathways of T cell differentiation. We demonstrate that IL-2 activates binding of AP-1 and STAT5 at sites that can subsequently bind lineage-determining transcription factors, depending upon what other external factors exist in the local T cell environment. Once established, priming can also be maintained by the stroma-derived homeostatic cytokine IL-7, and priming diminishes if Il7r is subsequently deleted in vivo. Hence, IL-2 is not just a growth factor; it lays the foundation for T cell differentiation and immunological memory.

Angeborene lymphoide Zellen (ILCs, engl. für Innate Lymphoid Cells) bilden eine kürzlich beschriebene Familie von Effektorzellen des angeborenen Immunsystems, die in verschiedenste immunologische Prozesse involviert sind. ILCs fehlen somatisch rekombinierte Antigenrezeptoren und können aufgrund ihrer Master-Transkriptionsfaktoren und Effektorfunktionen in drei Hauptgruppen eingeteilt werden. In der Embryonalentwicklung spielt ein spezifisches Mitglied der Typ 3 ILCs, sogenannte LTi (engl. für Lymphoid Tissue inducer) Zellen, eine zentrale Rolle in der Entwicklung von Lymphknoten. Wie die gesamte ILC3 Familie, sind auch LTi Zellen abhängig von dem Master-Transkriptionsfaktor ROR t, was sich in ROR t-defizienten Mäuse nicht nur durch die Abwesenheit aller ILC3, sondern makroskopisch auch durch fehlende Lymphknoten äußert. ILC3 persistieren bis ins Erwachsenenalter und befinden sich hauptsächlich im Darmgewebe und den assoziierten mesenterialen Lymphknoten, wo sie die Homöostase der Barrierefunktionen, Immunüberwachung, sowie die Typ-3-Gewebeimmunität unterstützen. Während postnatale Ko-expression der Transkriptionsfaktoren T-bet und ROR t in spezifischen ILC3-Subpopulationen und deren Bedeutung für Differenzierung, Phänotyp und Funktionen fest etabliert sind, ist der Einfluss von T-bet in fötalen ILC3 und auf die Generation von Lymphknoten noch unbekannt. Um diese Mechanismen genau zu untersuchen, wurden fötale ILCs mittels Einzelzell-RNA-Sequenzierung detailliert charakterisiert, wodurch eine unerwartete Heterogenität innerhalb der ILC3 mit T-bet-exprimierenden Zellen aufgedeckt wurde. Außerdem wurden PLZF + ILC-Vorläufer (ILCP, engl. für Innate Lymphoid Cell Precursor) im sich entwickelnden Darm nachgewiesen, was darauf hindeutet, dass der embryonale Darm eine aktive Differenzierungsnische für ILCs während der frühen Entwicklung darstellt. Weiterhin, bes ätigen verschiedene Mausmodelle eine Schlüsselrolle für T-bet in der Regulation der ILC-Differenzierung und der Entstehung von Lymphknoten. Im Detail konnte gezeigt werden, dass die zusätzliche genetische Ablation von T-bet in ROR tdefizienten Mäusen Differenzierungsentscheidungen in fötalen ILCP zentral beeinflusst. Die Abwesenheit von T-bet in ILCP ermöglichte die Akkumulation von ILCP mit LTi-Aktivität, wodurch die Organogenese von Lymphknoten, unabhängig von ROR t wiederhergestellt wurde. PLZF + ILCP von ROR t/T-bet-Doppeldefizienten Mäusen bestanden bis ins Erwachsenenalter, wo diese Zellen die Darmbarrierefunktionen durch Produktion von IL-22 wiederherstellten. Darüber hinaus erwies sich ROR als entscheidend für die Entwicklung von PLZF + ILCP und die damit verbundene Bildung von Lymphknoten. Insgesamt enthüllen diese Ergebnisse eine neue Rolle für T-bet und ROR in der embryonalen ILC-Differenzierung und der Lymphknoten-Organogenese, und decken die antagonistische Funktion von ROR t innerhalb der differenzierenden ILCP auf, T-bet entgegenzuwirken. Altogether, these data unveil a novel role for T-bet and ROR in embryonic ILC differentiation and LN organogene...

The OX40-OX40L pathway provides crucial co-stimulatory signals for CD4 T cell responses, however the precise cellular interactions critical for OX40L provision in vivo and when these occur, remains unclear. Here, we demonstrate that provision of OX40L by dendritic cells (DCs), but not T cells, B cells nor group 3 innate lymphoid cells (ILC3s), is critical specifically for the effector Th1 response to an acute systemic infection with Listeria monocytogenes (Lm). OX40L expression by DCs is regulated by cross-talk with NK cells, with IFNγ signalling to the DC to enhance OX40L in a mechanism conserved in both mouse and human DCs. Strikingly, DC expression of OX40L is redundant in a chronic intestinal Th1 response and expression by ILC3s is necessary. Collectively these data reveal tissue specific compartmentalisation of the cellular provision of OX40L and define a mechanism controlling DC expression of OX40L in vivo.