Multilayered Immunity by Tissue-Resident Lymphocytes in Cancer.

Lymphocytes spanning the entire innate-adaptive spectrum can stably reside in tissues and constitute an integral component of the local defense network against immunological challenges. In tight interactions with the epithelium and endothelium, tissue-resident lymphocytes sense antigens and alarmins elicited by infectious microbes and abiotic stresses at barrier sites and mount effector responses to restore tissue homeostasis. Of note, such a host cell-directed immune defense system has been recently demonstrated to surveil epithelial cell transformation and carcinoma development, as well as cancer cell metastasis at selected distant organs, and thus represents a primordial cancer immune defense module. Here we review how distinct lineages of tissue-resident innate lymphoid cells, innate-like T cells, and adaptive T cells participate in a form of multilayered cancer immunity in murine models and patients, and how their convergent effector programs may be targeted through both shared and private regulatory pathways for cancer immunotherapy.

TGF-ß Regulation of T Cells.

Transforming growth factor ß (TGF-ß) is a key cytokine regulating the development, activation, proliferation, differentiation, and death of T cells. In CD4+ T cells, TGF-ß maintains the quiescence and controls the activation of naive T cells. While inhibiting the differentiation and function of Th1 and Th2 cells, TGF-ß promotes the differentiation of Th17 and Th9 cells. TGF-ß is required for the induction of Foxp3 in naive T cells and the development of regulatory T cells. TGF-ß is crucial in the differentiation of tissue-resident memory CD8+ T cells and their retention in the tissue, whereas it suppresses effector T cell function. In addition, TGF-ß also regulates the generation or function of natural killer T cells, γδ T cells, innate lymphoid cells, and gut intraepithelial lymphocytes. Here I highlight the major findings and recent advances in our understanding of TGF-ß regulation of T cells and provide a personal perspective of the field.

Gene Regulatory Circuits in Innate and Adaptive Immune Cells.

Cell identity and function largely rely on the programming of transcriptomes during development and differentiation. Signature gene expression programs are orchestrated by regulatory circuits consisting of cis-acting promoters and enhancers, which respond to a plethora of cues via the action of transcription factors. In turn, transcription factors direct epigenetic modifications to revise chromatin landscapes, and drive contacts between distal promoter-enhancer combinations. In immune cells, regulatory circuits for effector genes are especially complex and flexible, utilizing distinct sets of transcription factors and enhancers, depending on the cues each cell type receives during an infection, after sensing cellular damage, or upon encountering a tumor. Here, we review major players in the coordination of gene regulatory programs within innate and adaptive immune cells, as well as integrative omics approaches that can be leveraged to decipher their underlying circuitry.

Group 2 Innate Lymphoid Cells: Team Players in Regulating Asthma.

Type 2 immunity helps protect the host from infection, but it also plays key roles in tissue homeostasis, metabolism, and repair. Unfortunately, inappropriate type 2 immune reactions may lead to allergy and asthma. Group 2 innate lymphoid cells (ILC2s) in the lungs respond rapidly to local environmental cues, such as the release of epithelium-derived type 2 initiator cytokines/alarmins, producing type 2 effector cytokines such as IL-4, IL-5, and IL-13 in response to tissue damage and infection. ILC2s are associated with the severity of allergic asthma, and experimental models of lung inflammation have shown how they act as playmakers, receiving signals variously from stromal and immune cells as well as the nervous system and then distributing cytokine cues to elicit type 2 immune effector functions and potentiate CD4+ T helper cell activation, both of which characterize the pathology of allergic asthma. Recent breakthroughs identifying stromal- and neuronal-derived microenvironmental cues that regulate ILC2s, along with studies recognizing the potential plasticity of ILC2s, have improved our understanding of the immunoregulation of asthma and opened new avenues for drug discovery.

New Molecular Insights into Immune Cell Development.

During development innate lymphoid cells and specialized lymphocyte subsets colonize peripheral tissues, where they contribute to organogenesis and later constitute the first line of protection while maintaining tissue homeostasis. A few of these subsets are produced only during embryonic development and remain in the tissues throughout life. They are generated through a unique developmental program initiated in lympho-myeloid-primed progenitors, which lose myeloid and B cell potential. They either differentiate into innate lymphoid cells or migrate to the thymus to give rise to embryonic T cell receptor-invariant T cells. At later developmental stages, adaptive T lymphocytes are derived from lympho-myeloid progenitors that colonize the thymus, while lymphoid progenitors become specialized in the production of B cells. This sequence of events highlights the requirement for stratification in the establishment of immune functions that determine efficient seeding of peripheral tissues by a limited number of cells.

Mucosal Ecological Network of Epithelium and Immune Cells for Gut Homeostasis and Tissue Healing.

The intestinal epithelial barrier includes columnar epithelial, Paneth, goblet, enteroendocrine, and tuft cells as well as other cell populations, all of which contribute properties essential for gastrointestinal homeostasis. The intestinal mucosa is covered by mucin, which contains antimicrobial peptides and secretory IgA and prevents luminal bacteria, fungi, and viruses from stimulating intestinal immune responses. Conversely, the transport of luminal microorganisms-mediated by M, dendritic, and goblet cells-into intestinal tissues facilitates the harmonization of active and quiescent mucosal immune responses. The bacterial population within gut-associated lymphoid tissues creates the intratissue cohabitations for harmonized mucosal immunity. Intermolecular and intercellular communication among epithelial, immune, and mesenchymal cells creates an environment conducive for epithelial regeneration and mucosal healing. This review summarizes the so-called intestinal mucosal ecological network-the complex but vital molecular and cellular interactions of epithelial mesenchymal cells, immune cells, and commensal microbiota that achieve intestinal homeostasis, regeneration, and healing.

Therapeutic application of human type 2 innate lymphoid cells via induction of granzyme B-mediated tumor cell death.

The therapeutic potential for human type 2 innate lymphoid cells (ILC2s) has been underexplored. Although not observed in mouse ILC2s, we found that human ILC2s secrete granzyme B (GZMB) and directly lyse tumor cells by inducing pyroptosis and/or apoptosis, which is governed by a DNAM-1-CD112/CD155 interaction that inactivates the negative regulator FOXO1. Over time, the high surface density expression of CD155 in acute myeloid leukemia cells impairs the expression of DNAM-1 and GZMB, thus allowing for immune evasion. We describe a reliable platform capable of up to 2,000-fold expansion of human ILC2s within 4 weeks, whose molecular and cellular ILC2 profiles were validated by single-cell RNA sequencing. In both leukemia and solid tumor models, exogenously administered expanded human ILC2s show significant antitumor effects in vivo. Collectively, we demonstrate previously unreported properties of human ILC2s and identify this innate immune cell subset as a member of the cytolytic immune effector cell family.

Immunity to helminths: resistance, regulation, and susceptibility to gastrointestinal nematodes.

Helminth parasites are a highly successful group of pathogens that challenge the immune system in a manner distinct from rapidly replicating infectious agents. Of this group, roundworms (nematodes) that dwell in the intestines of humans and other animals are prevalent worldwide. Currently, more than one billion people are infected by at least one species, often for extended periods of time. Thus, host-protective immunity is rarely complete. The reasons for this are complex, but laboratory investigation of tractable model systems in which protective immunity is effective has provided a mechanistic understanding of resistance that is characterized almost universally by a type 2/T helper 2 response. Greater understanding of the mechanisms of susceptibility has also provided the basis for defining host immunoregulation and parasite-evasion strategies, helping place in context the changing patterns of immunological disease observed worldwide.

Dysregulation of ILC3s unleashes progression and immunotherapy resistance in colon cancer.

Group 3 innate lymphoid cells (ILC3s) regulate immunity and inflammation, yet their role in cancer remains elusive. Here, we identify that colorectal cancer (CRC) manifests with altered ILC3s that are characterized by reduced frequencies, increased plasticity, and an imbalance with T cells. We evaluated the consequences of these changes in mice and determined that a dialog between ILC3s and T cells via major histocompatibility complex class II (MHCII) is necessary to support colonization with microbiota that subsequently induce type-1 immunity in the intestine and tumor microenvironment. As a result, mice lacking ILC3-specific MHCII develop invasive CRC and resistance to anti-PD-1 immunotherapy. Finally, humans with dysregulated intestinal ILC3s harbor microbiota that fail to induce type-1 immunity and immunotherapy responsiveness when transferred to mice. Collectively, these data define a protective role for ILC3s in cancer and indicate that their inherent disruption in CRC drives dysfunctional adaptive immunity, tumor progression, and immunotherapy resistance.

The microbiota coordinates diurnal rhythms in innate immunity with the circadian clock.

Environmental light cycles entrain circadian feeding behaviors in animals that produce rhythms in exposure to foodborne bacteria. Here, we show that the intestinal microbiota generates diurnal rhythms in innate immunity that synchronize with feeding rhythms to anticipate microbial exposure. Rhythmic expression of antimicrobial proteins was driven by daily rhythms in epithelial attachment by segmented filamentous bacteria (SFB), members of the mouse intestinal microbiota. Rhythmic SFB attachment was driven by the circadian clock through control of feeding rhythms. Mechanistically, rhythmic SFB attachment activated an immunological circuit involving group 3 innate lymphoid cells. This circuit triggered oscillations in epithelial STAT3 expression and activation that produced rhythmic antimicrobial protein expression and caused resistance to Salmonella Typhimurium infection to vary across the day-night cycle. Thus, host feeding rhythms synchronize with the microbiota to promote rhythms in intestinal innate immunity that anticipate exogenous microbial exposure.

Opposing Functions of Interferon Coordinate Adaptive and Innate Immune Responses to Cancer Immune Checkpoint Blockade.

Interferon-gamma (IFNG) augments immune function yet promotes T cell exhaustion through PDL1. How these opposing effects are integrated to impact immune checkpoint blockade (ICB) is unclear. We show that while inhibiting tumor IFNG signaling decreases interferon-stimulated genes (ISGs) in cancer cells, it increases ISGs in immune cells by enhancing IFNG produced by exhausted T cells (TEX). In tumors with favorable antigenicity, these TEX mediate rejection. In tumors with neoantigen or MHC-I loss, TEX instead utilize IFNG to drive maturation of innate immune cells, including a PD1+TRAIL+ ILC1 population. By disabling an inhibitory circuit impacting PD1 and TRAIL, blocking tumor IFNG signaling promotes innate immune killing. Thus, interferon signaling in cancer cells and immune cells oppose each other to establish a regulatory relationship that limits both adaptive and innate immune killing. In melanoma and lung cancer patients, perturbation of this relationship is associated with ICB response independent of tumor mutational burden.

Metabolic regulator LKB1 controls adipose tissue ILC2 PD-1 expression and mitochondrial homeostasis to prevent insulin resistance.

Adipose tissue group 2 innate lymphoid cells (ILC2s) help maintain metabolic homeostasis by sustaining type 2 immunity and promoting adipose beiging. Although impairment of the ILC2 compartment contributes to obesity-associated insulin resistance, the underlying mechanisms have not been elucidated. Here, we found that ILC2s in obese mice and humans exhibited impaired liver kinase B1 (LKB1) activation. Genetic ablation of LKB1 disrupted ILC2 mitochondrial metabolism and suppressed ILC2 responses, resulting in exacerbated insulin resistance. Mechanistically, LKB1 deficiency induced aberrant PD-1 expression through activation of NFAT, which in turn enhanced mitophagy by suppressing Bcl-xL expression. Blockade of PD-1 restored the normal functions of ILC2s and reversed obesity-induced insulin resistance in mice. Collectively, these data present the LKB1-PD-1 axis as a promising therapeutic target for the treatment of metabolic disease.

Intestinal CXCR6+ ILC3s migrate to the kidney and exacerbate renal fibrosis via IL-23 receptor signaling enhanced by PD-1 expression.

Group 3 innate lymphoid cells (ILC3s) regulate inflammation and tissue repair at mucosal sites, but whether these functions pertain to other tissues-like the kidneys-remains unclear. Here, we observed that renal fibrosis in humans was associated with increased ILC3s in the kidneys and blood. In mice, we showed that CXCR6+ ILC3s rapidly migrated from the intestinal mucosa and accumulated in the kidney via CXCL16 released from the injured tubules. Within the fibrotic kidney, ILC3s increased the expression of programmed cell death-1 (PD-1) and subsequent IL-17A production to directly activate myofibroblasts and fibrotic niche formation. ILC3 expression of PD-1 inhibited IL-23R endocytosis and consequently amplified the JAK2/STAT3/RORγt/IL-17A pathway that was essential for the pro-fibrogenic effect of ILC3s. Thus, we reveal a hitherto unrecognized migration pathway of ILC3s from the intestine to the kidney and the PD-1-dependent function of ILC3s in promoting renal fibrosis.

Circulating NK cells establish tissue residency upon acute infection of skin and mediate accelerated effector responses to secondary infection.

Natural killer (NK) cells are present in the circulation and can also be found residing in tissues, and these populations exhibit distinct developmental requirements and are thought to differ in terms of ontogeny. Here, we investigate whether circulating conventional NK (cNK) cells can develop into long-lived tissue-resident NK (trNK) cells following acute infections. We found that viral and bacterial infections of the skin triggered the recruitment of cNK cells and their differentiation into Tcf1hiCD69hi trNK cells that share transcriptional similarity with CD56brightTCF1hi NK cells in human tissues. Skin trNK cells arose from interferon (IFN)-γ-producing effector cells and required restricted expression of the transcriptional regulator Blimp1 to optimize Tcf1-dependent trNK cell formation. Upon secondary infection, trNK cells rapidly gained effector function and mediated an accelerated NK cell response. Thus, cNK cells redistribute and permanently position at sites of previous infection via a mechanism promoting tissue residency that is distinct from Hobit-dependent developmental paths of NK cells and ILC1 seeding tissues during ontogeny.

Innate Lymphoid Cells: 10 Years On.

Innate lymphoid cells (ILCs) are lymphocytes that do not express the type of diversified antigen receptors expressed on T cells and B cells. ILCs are largely tissue-resident cells and are deeply integrated into the fabric of tissues. The discovery and investigation of ILCs over the past decade has changed our perception of immune regulation and how the immune system contributes to the maintenance of tissue homeostasis. We now know that cytokine-producing ILCs contribute to multiple immune pathways by, for example, sustaining appropriate immune responses to commensals and pathogens at mucosal barriers, potentiating adaptive immunity, and regulating tissue inflammation. Critically, the biology of ILCs also extends beyond classical immunology to metabolic homeostasis, tissue remodeling, and dialog with the nervous system. The last 10 years have also contributed to our greater understanding of the transcriptional networks that regulate lymphocyte commitment and delineation. This, in conjunction with the recent advances in our understanding of the influence of local tissue microenvironments on the plasticity and function of ILCs, has led to a re-evaluation of their existing categorization. In this review, we distill the advances in ILC biology over the past decade to refine the nomenclature of ILCs and highlight the importance of ILCs in tissue homeostasis, morphogenesis, metabolism, repair, and regeneration.

Natural Killer Cells Control Tumor Growth by Sensing a Growth Factor.

Many tumors produce platelet-derived growth factor (PDGF)-DD, which promotes cellular proliferation, epithelial-mesenchymal transition, stromal reaction, and angiogenesis through autocrine and paracrine PDGFRß signaling. By screening a secretome library, we found that the human immunoreceptor NKp44, encoded by NCR2 and expressed on natural killer (NK) cells and innate lymphoid cells, recognizes PDGF-DD. PDGF-DD engagement of NKp44 triggered NK cell secretion of interferon gamma (IFN)-γ and tumor necrosis factor alpha (TNF-α) that induced tumor cell growth arrest. A distinctive transcriptional signature of PDGF-DD-induced cytokines and the downregulation of tumor cell-cycle genes correlated with NCR2 expression and greater survival in glioblastoma. NKp44 expression in mouse NK cells controlled the dissemination of tumors expressing PDGF-DD more effectively than control mice, an effect enhanced by blockade of the inhibitory receptor CD96 or CpG-oligonucleotide treatment. Thus, while cancer cell production of PDGF-DD supports tumor growth and stromal reaction, it concomitantly activates innate immune responses to tumor expansion.

Shared Transcriptional Control of Innate Lymphoid Cell and Dendritic Cell Development.

Innate immunity and adaptive immunity consist of highly specialized immune lineages that depend on transcription factors for both function and development. In this review, we dissect the similarities between two innate lineages, innate lymphoid cells (ILCs) and dendritic cells (DCs), and an adaptive immune lineage, T cells. ILCs, DCs, and T cells make up four functional immune modules and interact in concert to produce a specified immune response. These three immune lineages also share transcriptional networks governing the development of each lineage, and we discuss the similarities between ILCs and DCs in this review.

Dopamine inhibits group 2 innate lymphoid cell-driven allergic lung inflammation by dampening mitochondrial activity.

Neuronal signals have emerged as pivotal regulators of group 2 innate lymphoid cells (ILC2s) that regulate tissue homeostasis and allergic inflammation. The molecular pathways underlying the neuronal regulation of ILC2 responses in lungs remain to be fully elucidated. Here, we found that the abundance of neurotransmitter dopamine was negatively correlated with circulating ILC2 numbers and positively associated with pulmonary function in humans. Dopamine potently suppressed lung ILC2 responses in a DRD1-receptor-dependent manner. Genetic deletion of Drd1 or local ablation of dopaminergic neurons augmented ILC2 responses and allergic lung inflammation. Transcriptome and metabolic analyses revealed that dopamine impaired the mitochondrial oxidative phosphorylation (OXPHOS) pathway in ILC2s. Augmentation of OXPHOS activity with oltipraz antagonized the inhibitory effect of dopamine. Local administration of dopamine alleviated allergen-induced ILC2 responses and airway inflammation. These findings demonstrate that dopamine represents an inhibitory regulator of ILC2 responses in allergic airway inflammation.

Circumvention of luteolysis reveals parturition pathways in mice dependent upon innate type 2 immunity.

Although mice normally enter labor when their ovaries stop producing progesterone (luteolysis), parturition can also be triggered in this species through uterus-intrinsic pathways potentially analogous to the ones that trigger parturition in humans. Such pathways, however, remain largely undefined in both species. Here, we report that mice deficient in innate type 2 immunity experienced profound parturition delays when manipulated endocrinologically to circumvent luteolysis, thus obliging them to enter labor through uterus-intrinsic pathways. We found that these pathways were in part driven by the alarmin IL-33 produced by uterine interstitial fibroblasts. We also implicated important roles for uterine group 2 innate lymphoid cells, which demonstrated IL-33-dependent activation prior to labor onset, and eosinophils, which displayed evidence of elevated turnover in the prepartum uterus. These findings reveal a role for innate type 2 immunity in controlling the timing of labor onset through a cascade potentially relevant to human parturition.

Retinoid X receptor gamma dictates the activation threshold of group 2 innate lymphoid cells and limits type 2 inflammation in the small intestine.

Group 2 innate lymphoid cells (ILC2s) are crucial in promoting type 2 inflammation that contributes to both anti-parasite immunity and allergic diseases. However, the molecular checkpoints in ILC2s that determine whether to immediately launch a proinflammatory response are unknown. Here, we found that retinoid X receptor gamma (Rxrg) was highly expressed in small intestinal ILC2s and rapidly suppressed by alarmin cytokines. Genetic deletion of Rxrg did not impact ILC2 development but facilitated ILC2 responses and the tissue inflammation induced by alarmins. Mechanistically, RXRγ maintained the expression of its target genes that support intracellular cholesterol efflux, which in turn reduce ILC2 proliferation. Furthermore, RXRγ expression prevented ILC2 response to mild stimulations, including low doses of alarmin cytokine and mechanical skin injury. Together, we propose that RXRγ expression and its mediated lipid metabolic states function as a cell-intrinsic checkpoint that confers the threshold of ILC2 activation in the small intestine.