Functional characterization of noncoding sequences is crucial for understanding the human genome and learning how genetic variation contributes to disease. 3' untranslated regions (UTRs) are an important class of noncoding sequences, but their functions remain largely uncharacterized. We developed a method for massively parallel functional annotation of sequences from 3' UTRs (fast-UTR) and used this approach to measure the effects of a total of >450 kilobases of 3' UTR sequences from >2,000 human genes on steady-state mRNA abundance, mRNA stability and protein production. We found widespread regulatory effects on mRNA that were coupled to effects on mRNA stability and protein production. Furthermore, we discovered 87 novel cis-regulatory elements and measured the effects of genetic variation within known and novel 3' UTR motifs. This work shows how massively parallel approaches can improve the functional annotation of noncoding sequences, advance our understanding of cis-regulatory mechanisms and quantify the effects of human genetic variation.

Group 2 innate lymphoid cells (ILC2s) and CD4⁺ type 2 helper T cells (T_H2 cells) are defined by their similar effector cytokines, which together mediate the features of allergic immunity. We found that tissue ILC2s and T_H2 cells differentiated independently but shared overlapping effector function programs that were mediated by exposure to the tissue-derived cytokines interleukin 25 (IL-25), IL-33 and thymic stromal lymphopoietin (TSLP). Loss of these three tissue signals did not affect lymph node priming, but abrogated the terminal differentiation of effector T_H2 cells and adaptive lung inflammation in a T cell-intrinsic manner. Our findings suggest a mechanism by which diverse perturbations can activate type 2 immunity and reveal a shared local-tissue-elicited checkpoint that can be exploited to control both innate and adaptive allergic inflammation.

The tumor immune microenvironment (TIME) is commonly infiltrated by diverse collections of myeloid cells. Yet, the complexity of myeloid-cell identity and plasticity has challenged efforts to define bona fide populations and determine their connections to T-cell function and their relationship to patient outcome. Here, we have leveraged single-cell RNA-sequencing analysis of several mouse and human tumors and found that monocyte-macrophage diversity is characterized by a combination of conserved lineage states as well as transcriptional programs accessed along the differentiation trajectory. We also found in mouse models that tumor monocyte-to-macrophage progression was profoundly tied to regulatory T cell (Treg) abundance. In human kidney cancer, heterogeneity in macrophage accumulation and myeloid composition corresponded to variance in, not only Treg density, but also the quality of infiltrating CD8+ T cells. In this way, holistic analysis of monocyte-to-macrophage differentiation creates a framework for critically different immune states.

Background

Asthma is characterized by increased airway narrowing in response to nonspecific stimuli. The disorder is influenced by both environmental and genetic factors. Exosomes are nanosized vesicles of endosomal origin released from inflammatory and epithelial cells that have been implicated in asthma. In this study we characterized the microRNA (miRNA) content of exosomes in healthy control subjects and patients with mild intermittent asthma both at unprovoked baseline and in response to environmental challenge.

Objective

To investigate alterations in bronchoalveolar lavage fluid (BALF) exosomal miRNA profiles due to asthma, and following subway air exposure.

Methods

Exosomes were isolated from BALF from healthy control subjects (n = 10) and patients with mild intermittent asthma (n = 10) after subway and control exposures. Exosomal RNA was analyzed by using microarrays containing probes for 894 human miRNAs, and selected findings were validated with quantitative RT-PCR. Results were analyzed by using multivariate modeling.

Results

The presence of miRNAs was confirmed in exosomes from BALF of both asthmatic patients and healthy control subjects. Significant differences in BALF exosomal miRNA was detected for 24 miRNAs with a subset of 16 miRNAs, including members of the let-7 and miRNA-200 families, providing robust classification of patients with mild nonsymptomatic asthma from healthy subjects with 72% cross-validated predictive power (Q(2) = 0.72). In contrast, subway exposure did not cause any significant alterations in miRNA profiles.

Conclusion

These studies demonstrate substantial differences in exosomal miRNA profiles between healthy subjects and patients with unprovoked, mild, stable asthma. These changes might be important in the inflammatory response leading to bronchial hyperresponsiveness and asthma.

Differentiation of proinflammatory CD4⁺ conventional T cells (T_conv) is critical for productive antitumor responses yet their elicitation remains poorly understood. We comprehensively characterized myeloid cells in tumor draining lymph nodes (tdLN) of mice and identified two subsets of conventional type-2 dendritic cells (cDC2) that traffic from tumor to tdLN and present tumor-derived antigens to CD4⁺ T_conv, but then fail to support antitumor CD4⁺ T_conv differentiation. Regulatory T cell (T_reg) depletion enhanced their capacity to elicit strong CD4⁺ T_conv responses and ensuing antitumor protection. Analogous cDC2 populations were identified in patients, and as in mice, their abundance relative to T_reg predicts protective ICOS⁺ PD-1^lo CD4⁺ T_conv phenotypes and survival. Further, in melanoma patients with low T_reg abundance, intratumoral cDC2 density alone correlates with abundant CD4⁺ T_conv and with responsiveness to anti-PD-1 therapy. Together, this highlights a pathway that restrains cDC2 and whose reversal enhances CD4⁺ T_conv abundance and controls tumor growth.

Intratumoral stimulatory dendritic cells (SDCs) play an important role in stimulating cytotoxic T cells and driving immune responses against cancer. Understanding the mechanisms that regulate their abundance in the tumor microenvironment (TME) could unveil new therapeutic opportunities. We find that in human melanoma, SDC abundance is associated with intratumoral expression of the gene encoding the cytokine FLT3LG. FLT3LG is predominantly produced by lymphocytes, notably natural killer (NK) cells in mouse and human tumors. NK cells stably form conjugates with SDCs in the mouse TME, and genetic and cellular ablation of NK cells in mice demonstrates their importance in positively regulating SDC abundance in tumor through production of FLT3L. Although anti-PD-1 'checkpoint' immunotherapy for cancer largely targets T cells, we find that NK cell frequency correlates with protective SDCs in human cancers, with patient responsiveness to anti-PD-1 immunotherapy, and with increased overall survival. Our studies reveal that innate immune SDCs and NK cells cluster together as an excellent prognostic tool for T cell-directed immunotherapy and that these innate cells are necessary for enhanced T cell tumor responses, suggesting this axis as a target for new therapies.

It is well understood that antigen-presenting cells (APCs) within tumors typically do not maintain cytotoxic T cell (CTL) function, despite engaging them. Across multiple mouse tumor models and human tumor biopsies, we have delineated the intratumoral dendritic cell (DC) populations as distinct from macrophage populations. Within these, CD103(+) DCs are extremely sparse and yet remarkably capable CTL stimulators. These are uniquely dependent on IRF8, Zbtb46, and Batf3 transcription factors and are generated by GM-CSF and FLT3L cytokines. Regressing tumors have higher proportions of these cells, T-cell-dependent immune clearance relies on them, and abundance of their transcripts in human tumors correlates with clinical outcome. This cell type presents opportunities for prognostic and therapeutic approaches across multiple cancer types.

The medial ganglionic eminence (MGE) is an embryonic forebrain structure that generates the majority of cortical interneurons. MGE transplantation into specific regions of the postnatal central nervous system modifies circuit function and improves deficits in mouse models of epilepsy, Parkinson's disease, pain, and phencyclidine-induced cognitive deficits. Herein, we describe approaches to generate MGE-like progenitor cells from mouse embryonic stem (ES) cells. Using a modified embryoid body method, we provided gene expression evidence that mouse ES-derived Lhx6(+) cells closely resemble immature interneurons generated from authentic MGE-derived Lhx6(+) cells. We hypothesized that enhancers that are active in the mouse MGE would be useful tools in detecting when ES cells differentiate into MGE cells. Here we demonstrate the utility of enhancer elements [422 (DlxI12b), Lhx6, 692, 1056, and 1538] as tools to mark MGE-like cells in ES cell differentiation experiments. We found that enhancers DlxI12b, 692, and 1538 are active in Lhx6-GFP(+) cells, while enhancer 1056 is active in Olig2(+) cells. These data demonstrate unique techniques to follow and purify MGE-like derivatives from ES cells, including GABAergic cortical interneurons and oligodendrocytes, for use in stem cell-based therapeutic assays and treatments.

The thymus is responsible for generating a diverse yet self-tolerant pool of T cells¹. Although the thymic medulla consists mostly of developing and mature AIRE⁺ epithelial cells, recent evidence has suggested that there is far greater heterogeneity among medullary thymic epithelial cells than was previously thought². Here we describe in detail an epithelial subset that is remarkably similar to peripheral tuft cells that are found at mucosal barriers³. Similar to the periphery, thymic tuft cells express the canonical taste transduction pathway and IL-25. However, they are unique in their spatial association with cornified aggregates, ability to present antigens and expression of a broad diversity of taste receptors. Some thymic tuft cells pass through an Aire-expressing stage and depend on a known AIRE-binding partner, HIPK2, for their development. Notably, the taste chemosensory protein TRPM5 is required for their thymic function through which they support the development and polarization of thymic invariant natural killer T cells and act to establish a medullary microenvironment that is enriched in the type 2 cytokine, IL-4. These findings indicate that there is a compartmentalized medullary environment in which differentiation of a minor and highly specialized epithelial subset has a non-redundant role in shaping thymic function.

In the small intestine, type 2 responses are regulated by a signaling circuit that involves tuft cells and group 2 innate lymphoid cells (ILC2s). Here, we identified the microbial metabolite succinate as an activating ligand for small intestinal (SI) tuft cells. Sequencing analyses of tuft cells isolated from the small intestine, gall bladder, colon, thymus, and trachea revealed that expression of tuft cell chemosensory receptors is tissue specific. SI tuft cells expressed the succinate receptor (SUCNR1), and providing succinate in drinking water was sufficient to induce a multifaceted type 2 immune response via the tuft-ILC2 circuit. The helminth Nippostrongylus brasiliensis and a tritrichomonad protist both secreted succinate as a metabolite. In vivo sensing of the tritrichomonad required SUCNR1, whereas N. brasiliensis was SUCNR1 independent. These findings define a paradigm wherein tuft cells monitor microbial metabolites to initiate type 2 immunity and suggest the existence of other sensing pathways triggering the response to helminths.