Innate lymphoid cells (ILCs) are tissue-resident effector cells with roles in tissue homeostasis, protective immunity, and inflammatory disease. Group 3 ILCs (ILC3s) are classically defined by the master transcription factor RORγt. However, ILC3 can be further subdivided into subsets that share type 3 effector modules that exhibit significant ontological, transcriptional, phenotypic, and functional heterogeneity. Notably lymphoid tissue inducer (LTi)-like ILC3s mediate effector functions not typically associated with other RORγt-expressing lymphocytes, suggesting that additional transcription factors contribute to dictate ILC3 subset phenotypes. Here, we identify Bcl6 as a subset-defining transcription factor of LTi-like ILC3s in mice and humans. Deletion of Bcl6 results in dysregulation of the LTi-like ILC3 transcriptional program and markedly enhances expression of interleukin-17A (IL-17A) and IL-17F in LTi-like ILC3s in a manner in part dependent upon the commensal microbiota-and associated with worsened inflammation in a model of colitis. Together, these findings redefine our understanding of ILC3 subset biology.

The role of dendritic cells (DCs) in intestinal immune homeostasis remains incompletely defined. Here we show that mice lacking IRF8 transcription-factor-dependent DCs had reduced numbers of T cells in the small intestine (SI), but not large intestine (LI), including an almost complete absence of SI CD8αβ(+) and CD4(+)CD8αα(+) T cells; the latter requiring β8 integrin expression by migratory IRF8 dependent CD103(+)CD11b(-) DCs. SI homing receptor induction was impaired during T cell priming in mesenteric lymph nodes (MLN), which correlated with a reduction in aldehyde dehydrogenase activity by SI-derived MLN DCs, and inefficient T cell localization to the SI. These mice also lacked intestinal T helper 1 (Th1) cells, and failed to support Th1 cell differentiation in MLN and mount Th1 cell responses to Trichuris muris infection. Collectively these results highlight multiple non-redundant roles for IRF8 dependent DCs in the maintenance of intestinal T cell homeostasis.

Intestinal immune homeostasis is dependent upon tightly regulated and dynamic host interactions with the commensal microbiota. Immunoglobulin A (IgA) produced by mucosal B cells dictates the composition of commensal bacteria residing within the intestine. While emerging evidence suggests the majority of IgA is produced innately and may be polyreactive, mucosal-dwelling species can also elicit IgA via T cell-dependent mechanisms. However, the mechanisms that modulate the magnitude and quality of T cell-dependent IgA responses remain incompletely understood. Here we demonstrate that group 3 innate lymphoid cells (ILC3) regulate steady state interactions between T follicular helper cells (TfH) and B cells to limit mucosal IgA responses. ILC3 used conserved migratory cues to establish residence within the interfollicular regions of the intestinal draining lymph nodes, where they act to limit TfH responses and B cell class switching through antigen presentation. The absence of ILC3-intrinsic antigen presentation resulted in increased and selective IgA coating of bacteria residing within the colonic mucosa. Together these findings implicate lymph node resident, antigen-presenting ILC3 as a critical regulatory checkpoint in the generation of T cell-dependent colonic IgA and suggest ILC3 act to maintain tissue homeostasis and mutualism with the mucosal-dwelling commensal microbiota.

Group 3 innate lymphoid cells (ILC3s) sense environmental signals and are critical for tissue integrity in the intestine. Yet, which signals are sensed and what receptors control ILC3 function remain poorly understood. Here, we show that ILC3s with a lymphoid-tissue-inducer (LTi) phenotype expressed G-protein-coupled receptor 183 (GPR183) and migrated to its oxysterol ligand 7α,25-hydroxycholesterol (7α,25-OHC). In mice lacking Gpr183 or 7α,25-OHC, ILC3s failed to localize to cryptopatches (CPs) and isolated lymphoid follicles (ILFs). Gpr183 deficiency in ILC3s caused a defect in CP and ILF formation in the colon, but not in the small intestine. Localized oxysterol production by fibroblastic stromal cells provided an essential signal for colonic lymphoid tissue development, and inflammation-induced increased oxysterol production caused colitis through GPR183-mediated cell recruitment. Our findings show that GPR183 promotes lymphoid organ development and indicate that oxysterol-GPR183-dependent positioning within tissues controls ILC3 activity and intestinal homeostasis.

Cross-talk between different components of the intestinal barrier and the immune system may be important in maintaining gut homeostasis. A crucial part of the gut barrier is the mucus layer, a cross-linked gel on top of the intestinal epithelium that consists predominantly of the mucin glycoprotein MUC2. However, whether the mucin layer actively regulates intestinal immune cell responses is not clear. Because recent evidence suggests that intestinal dendritic cells (DCs) may be regulated by the mucus layer, we purified intestinal mucin, incubated it with human DCs, and determined the functional effects. Here we show that expression of the chemokine IL-8 and co-stimulatory DC markers CD86 and CD83 are significantly up-regulated on human DCs in the presence of intestinal mucins. Additionally, mucin-exposed DCs promoted neutrophil migration in an IL-8-dependent manner. The stimulatory effects of mucins on DCs were not due to mucin sample contaminants such as lipopolysaccharide, DNA, or contaminant proteins. Instead, mucin glycans are important for the pro-inflammatory effects on DCs. Thus, intestinal mucins are capable of inducing important pro-inflammatory functions in DCs, which could be important in driving inflammatory responses upon intestinal barrier damage.

Group 3 innate lymphoid cells (ILC3) are potent effector cells with critical roles in enforcing immunity, barrier integrity and tissue homeostasis along the gastrointestinal tract. ILC3 are considered primarily tissue-resident cells, seeding the gastrointestinal tract during embryonic stages and early life. However, the mechanisms through which mature ILC3 are maintained within adult tissues are poorly understood. Here, we report that lymphoid tissue-inducer-like (LTi-like) ILC3 exhibit minimal turnover in the healthy adult intestinal tract, persist for extended periods of time, and display a quiescent phenotype. Strikingly, during enteric bacterial infection LTi-like ILC3 also exhibit negligible hematopoietic replenishment and remain non-proliferative, despite robustly producing cytokines. Survival of LTi-like ILC3 was found to be dependent upon the balance between the metabolic activity required to drive effector function and anti-apoptotic programs. Notably, the pro-survival protein B-cell lymphoma-2 (Bcl-2) was required for the survival of LTi-like ILC3 ex vivo but was rendered partially dispensable if mitochondrial respiration was inhibited. Together we demonstrate LTi-like ILC3 are a tissue-resident, quiescent population that persist independently of hematopoietic replenishment to survive within the intestinal microenvironment.

Interactions between the mammalian host and commensal microbiota are enforced through a range of immune responses that confer metabolic benefits and promote tissue health and homeostasis. Immunoglobulin A (IgA) responses directly determine the composition of commensal species that colonize the intestinal tract but require substantial metabolic resources to fuel antibody production by tissue-resident plasma cells. Here, we demonstrate that IgA responses are subject to diurnal regulation over the course of a circadian day. Specifically, the magnitude of IgA secretion, as well as the transcriptome of intestinal IgA+ plasma cells, was found to exhibit rhythmicity. Oscillatory IgA responses were found to be entrained by time of feeding and were also found to be in part coordinated by the plasma cell-intrinsic circadian clock via deletion of the master clock gene Arntl. Moreover, reciprocal interactions between the host and microbiota dictated oscillatory dynamics among the commensal microbial community and its associated transcriptional and metabolic activity in an IgA-dependent manner. Together, our findings suggest that circadian networks comprising intestinal IgA, diet, and the microbiota converge to align circadian biology in the intestinal tract and to ensure host-microbial mutualism.