Interleukin-33 Induces the Enzyme Tryptophan Hydroxylase 1 to Promote Inflammatory Group 2 Innate Lymphoid Cell-Mediated Immunity.

Group 2 innate lymphoid cells (ILC2s) regulate immunity, inflammation, and tissue homeostasis. Two distinct subsets of ILC2s have been described: steady-state natural ILC2s and inflammatory ILC2s, which are elicited following helminth infection. However, how tissue-specific cues regulate these two subsets of ILC2s and their effector functions remains elusive. Here, we report that interleukin-33 (IL-33) promotes the generation of inflammatory ILC2s (ILC2INFLAM) via induction of the enzyme tryptophan hydroxylase 1 (Tph1). Tph1 expression was upregulated in ILC2s upon activation with IL-33 or following helminth infection in an IL-33-dependent manner. Conditional deletion of Tph1 in lymphocytes resulted in selective impairment of ILC2INFLAM responses and increased susceptibility to helminth infection. Further, RNA sequencing analysis revealed altered gene expression in Tph1 deficient ILC2s including inducible T cell co-stimulator (Icos). Collectively, these data reveal a previously unrecognized function for IL-33, Tph1, and ICOS in promoting inflammatory ILC2 responses and type 2 immunity at mucosal barriers.

Differentiation of type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages.

Innate lymphoid cells (ILCs) are a recently recognized group of lymphocytes that have important functions in protecting epithelial barriers against infections and in maintaining organ homeostasis. ILCs have been categorized into three distinct groups, transcriptional circuitry and effector functions of which strikingly resemble the various T helper cell subsets. Here, we identify a common, Id2-expressing progenitor to all interleukin 7 receptor-expressing, "helper-like" ILC lineages, the CHILP. Interestingly, the CHILP differentiated into ILC2 and ILC3 lineages, but not into conventional natural killer (cNK) cells that have been considered an ILC1 subset. Instead, the CHILP gave rise to a peculiar NKp46(+) IL-7Rα(+) ILC lineage that required T-bet for specification and was distinct of cNK cells or other ILC lineages. Such ILC1s coproduced high levels of IFN-γ and TNF and protected against infections with the intracellular parasite Toxoplasma gondii. Our data significantly advance our understanding of ILC differentiation and presents evidence for a new ILC lineage that protects barrier surfaces against intracellular infections.

Innate lymphoid cells as regulators of immunity, inflammation and tissue homeostasis.

Research over the last 7 years has led to the formal identification of innate lymphoid cells (ILCs), increased the understanding of their tissue distribution and has established essential functions of ILCs in diverse physiological processes. These include resistance to pathogens, the regulation of autoimmune inflammation, tissue remodeling, cancer and metabolic homeostasis. Notably, many ILC functions appear to be regulated by mechanisms distinct from those of other innate and adaptive immune cells. In this Review, we focus on how group 2 ILC (ILC2) and group 3 ILC (ILC3) responses are regulated and how these cells interact with other immune and non-immune cells to mediate their functions. We highlight experimental evidence from mouse models and patient-based studies that have elucidated the effects of ILCs on the maintenance of tissue homeostasis and the consequences for health and disease.

Neuropeptide regulation of non-redundant ILC2 responses at barrier surfaces.

Emerging studies indicate that cooperation between neurons and immune cells regulates antimicrobial immunity, inflammation and tissue homeostasis. For example, a neuronal rheostat provides excitatory or inhibitory signals that control the functions of tissue-resident group 2 innate lymphoid cells (ILC2s) at mucosal barrier surfaces1-4. ILC2s express NMUR1, a receptor for neuromedin U (NMU), which is a prominent cholinergic neuropeptide that promotes ILC2 responses5-7. However, many functions of ILC2s are shared with adaptive lymphocytes, including the production of type 2 cytokines8,9 and the release of tissue-protective amphiregulin (AREG)10-12. Consequently, there is controversy regarding whether innate lymphoid cells and adaptive lymphocytes perform redundant or non-redundant functions13-15. Here we generate a new genetic tool to target ILC2s for depletion or gene deletion in the presence of an intact adaptive immune system. Transgenic expression of iCre recombinase under the control of the mouse Nmur1 promoter enabled ILC2-specific deletion of AREG. This revealed that ILC2-derived AREG promotes non-redundant functions in the context of antiparasite immunity and tissue protection following intestinal damage and inflammation. Notably, NMU expression levels increased in inflamed intestinal tissues from both mice and humans, and NMU induced AREG production in mouse and human ILC2s. These results indicate that neuropeptide-mediated regulation of non-redundant functions of ILC2s is an evolutionarily conserved mechanism that integrates immunity and tissue protection.

Non-redundant functions of group 2 innate lymphoid cells.

Protective immunity relies on the interplay of innate and adaptive immune cells with complementary and redundant functions. Innate lymphoid cells (ILCs) have recently emerged as tissue-resident, innate mirror images of the T cell system, with which they share lineage-specifying transcription factors and effector machinery1. Located at barrier surfaces, ILCs are among the first responders against invading pathogens and thus could potentially determine the outcome of the immune response2. However, so far it has not been possible to dissect the unique contributions of ILCs to protective immunity owing to limitations in specific targeting of ILC subsets. Thus, all of the available data have been generated either in mice lacking the adaptive immune system or with tools that also affect other immune cell subsets. In addition, it has been proposed that ILCs might be dispensable for a proper immune response because other immune cells could compensate for their absence3-7. Here we report the generation of a mouse model based on the neuromedin U receptor 1 (Nmur1) promoter as a driver for simultaneous expression of Cre recombinase and green fluorescent protein, which enables gene targeting in group 2 ILCs (ILC2s) without affecting other innate and adaptive immune cells. Using Cre-mediated gene deletion of Id2 and Gata3 in Nmur1-expressing cells, we generated mice with a selective and specific deficiency in ILC2s. ILC2-deficient mice have decreased eosinophil counts at steady state and are unable to recruit eosinophils to the airways in models of allergic asthma. Further, ILC2-deficient mice do not mount an appropriate immune and epithelial type 2 response, resulting in a profound defect in worm expulsion and a non-protective type 3 immune response. In total, our data establish non-redundant functions for ILC2s in the presence of adaptive immune cells at steady state and during disease and argue for a multilayered organization of the immune system on the basis of a spatiotemporal division of labour.

IL-33 controls IL-22-dependent antibacterial defense by modulating the microbiota.

IL-22 plays a critical role in defending against mucosal infections, but how IL-22 production is regulated is incompletely understood. Here, we show that mice lacking IL-33 or its receptor ST2 (IL-1RL1) were more resistant to Streptococcus pneumoniae lung infection than wild-type animals and that single-nucleotide polymorphisms in IL33 and IL1RL1 were associated with pneumococcal pneumonia in humans. The effect of IL-33 on S. pneumoniae infection was mediated by negative regulation of IL-22 production in innate lymphoid cells (ILCs) but independent of ILC2s as well as IL-4 and IL-13 signaling. Moreover, IL-33's influence on IL-22-dependent antibacterial defense was dependent on housing conditions of the mice and mediated by IL-33's modulatory effect on the gut microbiota. Collectively, we provide insight into the bidirectional crosstalk between the innate immune system and the microbiota. We conclude that both genetic and environmental factors influence the gut microbiota, thereby impacting the efficacy of antibacterial immune defense and susceptibility to pneumonia.

Interleukin-22 protects intestinal stem cells against genotoxic stress.

Environmental genotoxic factors pose a challenge to the genomic integrity of epithelial cells at barrier surfaces that separate host organisms from the environment. They can induce mutations that, if they occur in epithelial stem cells, contribute to malignant transformation and cancer development1-3. Genome integrity in epithelial stem cells is maintained by an evolutionarily conserved cellular response pathway, the DNA damage response (DDR). The DDR culminates in either transient cell-cycle arrest and DNA repair or elimination of damaged cells by apoptosis4,5. Here we show that the cytokine interleukin-22 (IL-22), produced by group 3 innate lymphoid cells (ILC3) and γδ T cells, is an important regulator of the DDR machinery in intestinal epithelial stem cells. Using a new mouse model that enables sporadic inactivation of the IL-22 receptor in colon epithelial stem cells, we demonstrate that IL-22 is required for effective initiation of the DDR following DNA damage. Stem cells deprived of IL-22 signals and exposed to carcinogens escaped DDR-controlled apoptosis, contained more mutations and were more likely to give rise to colon cancer. We identified metabolites of glucosinolates, a group of phytochemicals contained in cruciferous vegetables, to be a widespread source of genotoxic stress in intestinal epithelial cells. These metabolites are ligands of the aryl hydrocarbon receptor (AhR)6, and AhR-mediated signalling in ILC3 and γδ T cells controlled their production of IL-22. Mice fed with diets depleted of glucosinolates produced only very low levels of IL-22 and, consequently, the DDR in epithelial cells of mice on a glucosinolate-free diet was impaired. This work identifies a homeostatic network protecting stem cells against challenge to their genome integrity by AhR-mediated 'sensing' of genotoxic compounds from the diet. AhR signalling, in turn, ensures on-demand production of IL-22 by innate lymphocytes directly regulating components of the DDR in epithelial stem cells.

The transcription factor T-bet is induced by IL-15 and thymic agonist selection and controls CD8αα(+) intraepithelial lymphocyte development.

CD8αα(+) intraepithelial lymphocytes (IELs) are instrumental in maintaining the epithelial barrier in the intestine. Similar to natural killer cells and other innate lymphoid cells, CD8αα(+) IELs constitutively express the T-box transcription factor T-bet. However, the precise role of T-bet for the differentiation or function of IELs is unknown. Here we show that mice genetically deficient for T-bet lacked both TCRαß(+) and TCRγδ(+) CD8αα(+) IELs and thus are more susceptible to chemically induced colitis. Although T-bet was induced in thymic IEL precursors (IELPs) as a result of agonist selection and interleukin-15 (IL-15) receptor signaling, it was dispensable for the generation of IELPs. Subsequently, T-bet was required for the IL-15-dependent activation, differentiation, and expansion of IELPs in the periphery. Our study reveals a function of T-bet as a central transcriptional regulator linking agonist selection and IL-15 signaling with the emergence of CD8αα(+) IELs.

The neuropeptide neuromedin U stimulates innate lymphoid cells and type 2 inflammation.

The type 2 cytokines interleukin (IL)-4, IL-5, IL-9 and IL-13 have important roles in stimulating innate and adaptive immune responses that are required for resistance to helminth infection, promotion of allergic inflammation, metabolic homeostasis and tissue repair. Group 2 innate lymphoid cells (ILC2s) produce type 2 cytokines, and although advances have been made in understanding the cytokine milieu that promotes ILC2 responses, how ILC2 responses are regulated by other stimuli remains poorly understood. Here we demonstrate that ILC2s in the mouse gastrointestinal tract co-localize with cholinergic neurons that express the neuropeptide neuromedin U (NMU). In contrast to other haematopoietic cells, ILC2s selectively express the NMU receptor 1 (NMUR1). In vitro stimulation of ILC2s with NMU induced rapid cell activation, proliferation, and secretion of the type 2 cytokines IL-5, IL-9 and IL-13 that was dependent on cell-intrinsic expression of NMUR1 and Gαq protein. In vivo administration of NMU triggered potent type 2 cytokine responses characterized by ILC2 activation, proliferation and eosinophil recruitment that was associated with accelerated expulsion of the gastrointestinal nematode Nippostrongylus brasiliensis or induction of lung inflammation. Conversely, worm burden was higher in Nmur1-/- mice than in control mice. Furthermore, use of gene-deficient mice and adoptive cell transfer experiments revealed that ILC2s were necessary and sufficient to mount NMU-elicited type 2 cytokine responses. Together, these data indicate that the NMU-NMUR1 neuronal signalling circuit provides a selective mechanism through which the enteric nervous system and innate immune system integrate to promote rapid type 2 cytokine responses that can induce anti-microbial, inflammatory and tissue-protective type 2 responses at mucosal sites.

The neuropeptide NMU amplifies ILC2-driven allergic lung inflammation.

Type 2 innate lymphoid cells (ILC2s) both contribute to mucosal homeostasis and initiate pathologic inflammation in allergic asthma. However, the signals that direct ILC2s to promote homeostasis versus inflammation are unclear. To identify such molecular cues, we profiled mouse lung-resident ILCs using single-cell RNA sequencing at steady state and after in vivo stimulation with the alarmin cytokines IL-25 and IL-33. ILC2s were transcriptionally heterogeneous after activation, with subpopulations distinguished by expression of proliferative, homeostatic and effector genes. The neuropeptide receptor Nmur1 was preferentially expressed by ILC2s at steady state and after IL-25 stimulation. Neuromedin U (NMU), the ligand of NMUR1, activated ILC2s in vitro, and in vivo co-administration of NMU with IL-25 strongly amplified allergic inflammation. Loss of NMU-NMUR1 signalling reduced ILC2 frequency and effector function, and altered transcriptional programs following allergen challenge in vivo. Thus, NMUR1 signalling promotes inflammatory ILC2 responses, highlighting the importance of neuro-immune crosstalk in allergic inflammation at mucosal surfaces.

Erratum: The neuropeptide NMU amplifies ILC2-driven allergic lung inflammation.

This corrects the article DOI: 10.1038/nature24029.

Neuroimmune interactions in peripheral tissues.

Neuroimmune interactions have been revealed to be at the centre stage of tissue defence, organ homeostasis, and organismal physiology. Neuronal and immune cell subsets have been shown to colocalize in discrete tissue environments, forming neuroimmune cell units that constitute the basis for bidirectional interactions. These multitissue units drive coordinated neuroimmune responses to local and systemic signals, which represents an important challenge to our current views of mucosal physiology and immune regulation. In this review, we focus on the impact of reciprocal neuroimmune interactions, focusing on the anatomy of neuronal innervation and on the neuronal regulation of immune cells in peripheral tissues. Finally, we shed light on recent studies that explore how neuroimmune interactions maximise sensing and integration of environmental aggressions, modulating immune function in health and disease.

Eomes cannot replace its paralog T-bet during expansion and differentiation of CD8 effector T cells.

The two T-box transcription factors T-bet and Eomesodermin (Eomes) are important regulators of cytotoxic lymphocytes (CTLs), such as activated CD8 T cells, which are essential in the fight against intracellular pathogens and tumors. Both transcription factors share a great degree of homology based on sequence analysis and as a result exert partial functional redundancy during viral infection. However, the actual degree of redundancy between T-bet and Eomes remains a matter of debate and is further confounded by their distinct spatiotemporal expression pattern in activated CD8 T cells. To directly investigate the functional overlap of these transcription factors, we generated a new mouse model in which Eomes expression is under the transcriptional control of the endogenous Tbx21 (encoding for T-bet) locus. Applying this model, we demonstrate that the induction of Eomes in lieu of T-bet cannot rescue T-bet deficiency in CD8 T cells during acute lymphocytic choriomeningitis virus (LCMV) infection. We found that the expression of Eomes instead of T-bet was not sufficient for early cell expansion or effector cell differentiation. Finally, we show that imposed expression of Eomes after acute viral infection promotes some features of exhaustion but must act in concert with other factors during chronic viral infection to establish all hallmarks of exhaustion. In summary, our results clearly underline the importance of T-bet in guiding canonical CTL development during acute viral infections.

MCL1 Is Required for Maintenance of Intestinal Homeostasis and Prevention of Carcinogenesis in Mice.

BACKGROUND & AIMS: Intestinal epithelial homeostasis depends on a tightly regulated balance between intestinal epithelial cell (IEC) death and proliferation. While the disruption of several IEC death regulating factors result in intestinal inflammation, the loss of the anti-apoptotic BCL2 family members BCL2 and BCL2L1 has no effect on intestinal homeostasis in mice. We investigated the functions of the antiapoptotic protein MCL1, another member of the BCL2 family, in intestinal homeostasis in mice. METHODS: We generated mice with IEC-specific disruption of Mcl1 (Mcl1ΔIEC mice) or tamoxifen-inducible IEC-specific disruption of Mcl1 (i-Mcl1ΔIEC mice); these mice and mice with full-length Mcl1 (controls) were raised under normal or germ-free conditions. Mice were analyzed by endoscopy and for intestinal epithelial barrier permeability. Intestinal tissues were analyzed by histology, in situ hybridization, proliferation assays, and immunoblots. Levels of calprotectin, a marker of intestinal inflammation, were measured in intestinal tissues and feces. RESULTS: Mcl1ΔIEC mice spontaneously developed apoptotic enterocolopathy, characterized by increased IEC apoptosis, hyperproliferative crypts, epithelial barrier dysfunction, and chronic inflammation. Loss of MCL1 retained intestinal crypts in a hyperproliferated state and prevented the differentiation of intestinal stem cells. Proliferation of intestinal stem cells in MCL1-deficient mice required WNT signaling and was associated with DNA damage accumulation. By 1 year of age, Mcl1ΔIEC mice developed intestinal tumors with morphologic and genetic features of human adenomas and carcinomas. Germ-free housing of Mcl1ΔIEC mice reduced markers of microbiota-induced intestinal inflammation but not tumor development. CONCLUSION: The antiapoptotic protein MCL1, a member of the BCL2 family, is required for maintenance of intestinal homeostasis and prevention of carcinogenesis in mice. Loss of MCL1 results in development of intestinal carcinomas, even under germ-free conditions, and therefore does not involve microbe-induced chronic inflammation. Mcl1ΔIEC mice might be used to study apoptotic enterocolopathy and inflammatory bowel diseases.

The transcription factor GATA-3 controls cell fate and maintenance of type 2 innate lymphoid cells.

Innate lymphoid cells (ILCs) reside at mucosal surfaces and control immunity to intestinal infections. Type 2 innate lymphoid cells (ILC2s) produce cytokines such as IL-5 and IL-13, are required for immune defense against helminth infections, and are involved in the pathogenesis of airway hyperreactivity. Here, we have investigated the role of the transcription factor GATA-3 for ILC2 differentiation and maintenance. We showed that ILC2s and their lineage-specified bone marrow precursors (ILC2Ps), as identified here, were characterized by continuous high expression of GATA-3. Analysis of mice with temporary deletion of GATA-3 in all ILCs showed that GATA-3 was required for the differentiation and maintenance of ILC2s but not for RORγt(+) ILCs. Thus, our data demonstrate that GATA-3 is essential for ILC2 fate decisions and reveal similarities between the transcriptional programs controlling ILC and T helper cell fates.

A T-bet gradient controls the fate and function of CCR6-RORγt+ innate lymphoid cells.

At mucosal surfaces, the immune system should not initiate inflammatory immune responses to the plethora of antigens constantly present in the environment, but should remain poised to unleash a potent assault on intestinal pathogens. The transcriptional programs and regulatory factors required for immune cells to switch from homeostatic (often tissue-protective) function to potent antimicrobial immunity are poorly defined. Mucosal retinoic-acid-receptor-related orphan receptor-γt-positive (RORγt(+)) innate lymphoid cells (ILCs) are emerging as an important innate lymphocyte population required for immunity to intestinal infections. Various subsets of RORγt(+) ILCs have been described but the transcriptional programs controlling their specification and fate remain largely unknown. Here we provide evidence that the transcription factor T-bet determines the fate of a distinct lineage of CCR6(-)RORγt(+) ILCs. Postnatally emerging CCR6(-)RORγt(+) ILCs upregulated T-bet and this was controlled by cues from the commensal microbiota and interleukin-23 (IL-23). In contrast, CCR6(+)RORγt(+) ILCs, which arise earlier during ontogeny, did not express T-bet. T-bet instructed the expression of T-bet target genes such as interferon-γ (IFN-γ) and of the natural cytotoxicity receptor NKp46. Mice genetically lacking T-bet showed normal development of CCR6(-)RORγt(+) ILCs, but they could not differentiate into NKp46-expressing RORγt(+) ILCs (that is, IL-22-producing natural killer (NK-22) cells) and failed to produce IFN-γ. The production of IFN-γ by T-bet-expressing CCR6(-)RORγt(+) ILCs was essential for the release of mucus-forming glycoproteins required to protect the epithelial barrier during Salmonella enterica infection. Salmonella infection also causes severe enterocolitis that is at least partly driven by IFN-γ. Mice deficient for T-bet or depleted of ILCs developed only mild enterocolitis. Thus, graded expression of T-bet in CCR6(-)RORγt(+) ILCs facilitates the differentiation of IFN-γ-producing CCR6(-)RORγt(+) ILCs required to protect the epithelial barrier against Salmonella infections. Co-expression of T-bet and RORγt, which is also found in subsets of IL-17-producing T-helper (T(H)17) cells, may be an evolutionarily conserved transcriptional program that originally developed as part of the innate defence against infections but that also confers an increased risk of immune-mediated pathology.

Transcription factors controlling innate lymphoid cell fate decisions.

The mucosal epithelium is in direct contact with symbiotic and pathogenic microorganisms. Therefore, the mucosal surface is the principal portal of entry for invading pathogens and immune cells accumulated in the intestine to prevent infections. In addition to these conventional immune system functions, it has become clear that immune cells during steady-state continuously integrate microbial and nutrient-derived signals from the environment to support organ homeostasis. A major role in both processes is played by a recently discovered group of lymphocytes referred to as innate lymphoid cells (ILCs) Innate lymphoid cells (ILCs) that are specifically enriched at mucosal surfaces but are rather rare in secondary lymphoid organs. In analogy to the dichotomy between CD8 and CD4 T cells, we propose to classify ILCs into interleukin-7 receptor α-negative cytotoxic ILCs and IL-7Rα(+) helper-like ILCs. Dysregulated immune responses triggered by the various ILC subsets have been linked to inflammatory diseases such as inflammatory bowel disease, atopic dermatitis and airway hyperresponsiveness. Here, we will review recent progress in determining the transcriptional and developmental programs that control ILC fate decisions.

Cooperation of ILC2s and TH2 cells in the expulsion of intestinal helminth parasites.

Type 2 immune responses form a critical defence against enteric worm infections. In recent years, mouse models have revealed shared and unique functions for group 2 innate lymphoid cells and T helper 2 cells in type 2 immune response to intestinal helminths. Both cell types use similar innate effector functions at the site of infection, whereas each population has distinct roles during different stages of infection. In this Perspective, we review the underlying mechanisms used by group 2 innate lymphoid cells and T helper 2 cells to cooperate with each other and suggest an overarching model of the interplay between these cell types over the course of a helminth infection.

Tuft cell IL-17RB restrains IL-25 bioavailability and reveals context-dependent ILC2 hypoproliferation.

The tuft cell-ILC2 circuit orchestrates rapid type 2 responses upon detecting microbe-derived succinate and luminal helminths. Our findings delineate key mechanistic steps, involving IP3R2 engagement and Ca 2+ flux, governing IL-25 production by tuft cells triggered by succinate detection. While IL-17RB plays a pivotal intrinsic role in ILC2 activation, it exerts a regulatory function in tuft cells. Tuft cells exhibit constitutive Il25 expression, placing them in an anticipatory state that facilitates rapid production of IL-25 protein for ILC2 activation. Tuft cell IL-17RB is crucial for restraining IL-25 bioavailability, preventing excessive tonic ILC2 stimulation due to basal Il25 expression. Suboptimal ILC2 stimulation by IL-25 resulting from tuft cell Il17rb -deficiency or prolonged succinate exposure induces a state of hypoproliferation in ILC2s, also observed in chronic helminth infection. Our study offers critical insights into the regulatory dynamics of IL-25 in this circuit, highlighting the delicate tuning required for responses to diverse luminal states.

Eomes is sufficient to regulate IL-10 expression and cytotoxic effector molecules in murine CD4+ T cells.

The T-box transcription factors T-bet and Eomesodermin regulate type 1 immune responses in innate and adaptive lymphocytes. T-bet is widely expressed in the immune system but was initially identified as the lineage-specifying transcription factor of Th1 CD4+ T cells, where it governs expression of the signature cytokine IFN- γ and represses alternative cell fates like Th2 and Th17. T-bet's paralog Eomes is less abundantly expressed and Eomes+ CD4+ T cells are mostly found in the context of persistent antigen exposure, like bone marrow transplantation, chronic infection or inflammation as well as malignant disorders. However, it has remained unresolved whether Eomes executes similar transcriptional activities as T-bet in CD4+ T cells. Here we use a novel genetic approach to show that Eomes expression in CD4+ T cells drives a distinct transcriptional program that shows only partial overlap with T-bet. We found that Eomes is sufficient to induce the expression of the immunoregulatory cytokine IL-10 and, together with T-bet, promotes a cytotoxic effector profile, including Prf1, Gzmb, Gzmk, Nkg7 and Ccl5, while repressing alternative cell fates. Our results demonstrate that Eomes+ CD4+ T cells, which are often found in the context of chronic antigen stimulation, are likely to be a unique CD4+ T cell subset that limits inflammation and immunopathology as well as eliminates antigen-presenting and malignant cells.