Helminths, allergens, and certain protists induce type 2 immune responses, but the underlying mechanisms of immune activation remain poorly understood. In the small intestine, chemosensing by epithelial tuft cells results in the activation of group 2 innate lymphoid cells (ILC2s), which subsequently drive increased tuft cell frequency. This feedforward circuit is essential for intestinal remodeling and helminth clearance. ILC2 activation requires tuft-cellderived interleukin-25 (IL-25), but whether additional signals regulate the circuit is unclear. Here, we show that tuft cells secrete cysteinyl leukotrienes (cysLTs) to rapidly activate type 2 immunity following chemosensing of helminth infection. CysLTs cooperate with IL-25 to activate ILC2s, and tuft-cell-specific ablation of leukotriene synthesis attenuates type 2 immunity and delays helminth clearance. Conversely, cysLTs are dispensable for the tuft cell response induced by intestinal protists. Our findings identify an additional tuft cell effector function and suggest context-specific regulation of tuft-ILC2 circuits within the small intestine.

Tuft cells were first discovered in epithelial barriers decades ago, but their function remained unclear until recently. In the last 2 years, a series of studies has provided important advances that link tuft cells to infectious diseases and the host immune responses. Broadly, a model has emerged in which tuft cells use chemosensing to monitor their surroundings and translate environmental signals into effector functions that regulate immune responses in the underlying tissue. In this article, we review the current understanding of tuft cell immune function in the intestines, airways, and thymus. In particular, we discuss the role of tuft cells in type 2 immunity, norovirus infection, and thymocyte development. Despite recent advances, many fundamental questions about the function of tuft cells in immunity remain to be answered.

SummaryRegulation of respiratory mucosal immunity by microbial-derived metabolites has been a proposed mechanism that may provide airway protection. Here we examine the effect of oral Lactobacillus johnsonii-supplementation on metabolic and immune response dynamics during respiratory syncytial virus (RSV) infection. L. johnsonii-supplementation reduced airway Th2 cytokines, dendritic cell function, increased T-regulatory cells, and was associated with a reprogrammed circulating metabolic environment, including docosahexanoic acid (DHA) enrichment. RSV-infected bone-marrow derived dendritic cells (BMDC) from L. johnsonii-supplemented mice had altered cytokine secretion, reduced expression of co-stimulatory molecules, and modified CD4+ T cell cytokines. This was replicated upon co-incubation of wild-type BMDCs with either plasma from L. johnsonii-supplemented mice, or DHA. Finally, airway transfer of BMDCs from L. johnsonii-supplemented mice, or with wild-type derived BMDCs pre-treated with plasma from L. johnsonii-supplemented mice, reduced airway pathologic responses to infection in recipient animals. Thus, L. johnsonii-supplementation mediates airway mucosal protection via immunomodulatory metabolites and altered immune function.

The generation of regulatory T (Treg) cells is driven by Foxp3 and is responsible for dampening inflammation and reducing autoimmunity. In this study the epigenetic regulation of iTreg cells was examined and identified a H3K4 histone methyltransferase, SMYD3, which regulates expression of Foxp3 by a TGFβ1/Smad3 dependent mechanism. Using ChIP assays, SMYD3 depletion led to reduction in H3K4me3 in the promoter region and CNS-1 of the foxp3 locus. SMYD3 abrogation affected iTreg cell formation while allowing dysregulated IL-17 production. In a mouse model of respiratory syncytial virus infection (RSV), a model where iTreg cells play a critical role in regulating lung pathogenesis, SMYD3−/− mice demonstrated exacerbation of RSV-induced disease related to enhanced proinflammatory responses and worsened pathogenesis within the lung. Our data highlight a novel activation role for the TGFβ-inducible SMYD3 in regulating iTreg cell formation leading to increased severity of virus-related disease.

Notch ligand Delta-like ligand 4 (DLL4) has been shown to regulate CD4 T-cell differentiation, including regulatory T cells (T). Epigenetic alterations, which include histone modifications, are critical in cell differentiation decisions. Recent genome-wide studies demonstrated that T have increased trimethylation on histone H3 at lysine 4 (H3K4me3) around the T master transcription factor, Foxp3 loci. Here we report that DLL4 dynamically increased H3K4 methylation around the Foxp3 locus that was dependent upon upregulated SET and MYDN domain containing protein 3 (SMYD3). DLL4 promoted Smyd3 through the canonical Notch pathway in iT differentiation. DLL4 inhibition during pulmonary respiratory syncytial virus (RSV) infection decreased Smyd3 expression and Foxp3 expression in T leading to increased Il17a. On the other hand, DLL4 supported Il10 expression in vitro and in vivo, which was also partially dependent upon SMYD3. Using genome-wide unbiased mRNA sequencing, novel sets of DLL4- and Smyd3-dependent differentially expressed genes were discovered, including lymphocyte-activation gene 3 (Lag3), a checkpoint inhibitor that has been identified for modulating Th cell activation. Together, our data demonstrate a novel mechanism of DLL4/Notch-induced Smyd3 epigenetic pathways that maintain regulatory CD4 T cells in viral infections.

BackgroundChimeric antigen receptor (CAR) T-cell therapies have demonstrated transformational outcomes in the treatment of B-cell malignancies, but their widespread use is hindered by technical and logistical challenges associated with ex vivo cell manufacturing. To overcome these challenges, we developed VivoVec, a lentiviral vector-based platform for in vivo engineering of T cells. UB-VV100, a VivoVec clinical candidate for the treatment of B-cell malignancies, displays an anti-CD3 single-chain variable fragment (scFv) on the surface and delivers a genetic payload that encodes a second-generation CD19-targeted CAR along with a rapamycin-activated cytokine receptor (RACR) system designed to overcome the need for lymphodepleting chemotherapy in supporting successful CAR T-cell expansion and persistence. In the presence of exogenous rapamycin, non-transduced immune cells are suppressed, while the RACR system in transduced cells converts rapamycin binding to an interleukin (IL)-2/IL-15 signal to promote proliferation.MethodsUB-VV100 was administered to peripheral blood mononuclear cells (PBMCs) from healthy donors and from patients with B-cell malignancy without additional stimulation. Cultures were assessed for CAR T-cell transduction and function. Biodistribution was evaluated in CD34-humanized mice and in canines. In vivo efficacy was evaluated against normal B cells in CD34-humanized mice and against systemic tumor xenografts in PBMC-humanized mice.ResultsIn vitro, administration of UB-VV100 resulted in dose-dependent and anti-CD3 scFv-dependent T-cell activation and CAR T-cell transduction. The resulting CAR T cells exhibited selective expansion in rapamycin and antigen-dependent activity against malignant B-cell targets. In humanized mouse and canine studies, UB-VV100 demonstrated a favorable biodistribution profile, with transduction events limited to the immune compartment after intranodal or intraperitoneal administration. Administration of UB-VV100 to humanized mice engrafted with B-cell tumors resulted in CAR T-cell transduction, expansion, and elimination of systemic malignancy.ConclusionsThese findings demonstrate that UB-VV100 generates functional CAR T cells in vivo, which could expand patient access to CAR T technology in both hematological and solid tumors without the need for ex vivo cell manufacturing.

E-protein transcription factors limit group 2 innate lymphoid cell (ILC2) development while promoting T cell differentiation from common lymphoid progenitors. Inhibitors of DNA binding (ID) proteins block E-protein DNA binding in common lymphoid progenitors to allow ILC2 development. However, whether E-proteins influence ILC2 function upon maturity and activation remains unclear. Mice that overexpress ID1 under control of the thymus-restricted proximal Lck promoter (ID1 tg/WT ) have a large pool of primarily thymus-derived ILC2s in the periphery that develop in the absence of E-protein activity. We used these mice to investigate how the absence of E-protein activity affects ILC2 function and the genomic landscape in response to house dust mite (HDM) allergens. ID1 tg/WT mice had increased KLRG1 2 ILC2s in the lung compared with wild-type (WT; ID1 WT/WT ) mice in response to HDM, but ID1 tg/WT ILC2s had an impaired capacity to produce type 2 cytokines. Analysis of WT ILC2 accessible chromatin suggested that AP-1 and C/EBP transcription factors but not E-proteins were associated with ILC2 inflammatory gene programs. Instead, E-protein binding sites were enriched at functional genes in ILC2s during development that were later dynamically regulated in allergic lung inflammation, including genes that control ILC2 response to cytokines and interactions with T cells. Finally, ILC2s from ID1 tg/WT compared with WT mice had fewer regions of open chromatin near functional genes that were enriched for AP-1 factor binding sites following HDM treatment. These data show that E-proteins shape the chromatin landscape during ILC2 development to dictate the functional capacity of mature ILC2s during allergic inflammation in the lung.

Notch signaling facilitates inducible Foxp3+ regulatory T cells (iTreg) differentiation. Epigenetic modifications, which include histone modifications, are critical in cell differentiation. Recent genome-wide studies showed that iTreg have increased trimethylation on histone H3 at lysine 4 (H3K4me3) around Foxp3 loci, but the role of Notch signaling and its underlying epigenetic mechanisms remain elusive. Here we report that Delta-like ligand 4 (Dll4) but not Jagged1 can activate Notch signaling, facilitate Foxp3+ iTreg differentiation, and increased H3K4me3 around Foxp3 promoter and conserved noncoding sequences (CNS). We performed epigenetic enzyme array analysis and found one histone methyltransferase—SET and MYND domain containing protein 3 (Smyd3) that is abundant in iTreg. Dll4/Notch signaling amplified and accelerated Smyd3 expression in vitro during iTreg cell differentiation via an RBPJ-mediated transcription activation. Inhibition of Notch signaling decreased Smyd3 expression. When Smyd3 was genetically knocked out in CD4 T cells, the Dll4-enhanced H3K4me3 around the Foxp3 loci and Foxp3+ Tregs were decreased in vitro and in vivo. These results demonstrate that Notch ligand Dll4 can epigenetically regulate Foxp3+ Tregs through Smyd3.