Innate Lymphoid Cells Populations Research Articles

Abstract A063: Multidimensional cytometric analysis of colorectal cancer reveals novel and diverse mediators of antitumor immunity

Abstract Checkpoint blockade has revived the potential of immunotherapy for cancer treatment. For optimal application and development of cancer immunotherapies, a comprehensive understanding of the antitumor immune response is required. We unraveled local and systemic immune profiles of colorectal cancer by multidimensional mass cytometric analysis of 36 immune cell markers at the single-cell level in tumor tissues, tumor-associated lymph nodes, adjacent normal mucosa, and peripheral blood samples from CRC patients. We identified 218 phenotypically distinct immune cell clusters, including a previously neglected innate lymphoid cell (CD7+CD3-CD127-CD45RO+CD56+) population with cytotoxic potential. This subset demonstrated a tissue-resident (CD69+, CD103+) phenotype, and was most abundant in the immunogenic mismatch repair deficient (MMRd) cancers. Furthermore, tumor-resident immune cell populations were identified across the adaptive (CD4+ and CD8+) and innate (gammadelta) T-cell compartments showing a highly similar activated (HLA-DR+, CD38+, PD-1+) phenotype. PD-1 intermediate and PD-1 high CD8+ T-cell subsets represented distinct states of T-cell activation that further discriminated immunogenic from non-immunogenic colorectal cancers. Remarkably, activated gammadelta T-cells were specific for MMRd cancers, and their potential role in the response to PD-1 checkpoint blockade requires further clarification. The nonactivated counterparts of the tumor-resident CD103+PD-1+ cytotoxic and gammadelta T-cells were present in both tumor and healthy colorectal tissues. We did not detect any of the aforementioned tumor-resident immune cell populations in lymph node samples, with the exception of a tumor-positive lymph node. This indicates that the critical immune cell populations with antitumor activity reside in the colorectal mucosa, and that the role of lymph nodes in the antitumor immune response should be revisited. Finally, by applying imaging mass cytometry we demonstrated that the cytotoxic anti-tumor response in colorectal cancer is highly diverse and not restricted to cytotoxic T-cells, which opens new avenues for the management of this disease.The findings presented here advance the paradigm of antitumor immunity in colorectal cancer and provide a blueprint for the detailed characterization of the involved immune cell subsets. The coordinated action of innate and adaptive immune cell populations suggests a multitargeted exploitation of their antitumor properties in a therapeutic setting. Citation Format: Noel F. de Miranda, Natasja L. de Vries, Vincent van Unen, Tamim Abdelaal, Marieke E. Ijsselsteijn, Ruud van der Breggen, Arantza Farina-Sarasqueta, Koen C.M.J. Peeters, Thomas Höllt, Boudewijn P.F. Lelieveldt, Frits Koning. Multidimensional cytometric analysis of colorectal cancer reveals novel and diverse mediators of antitumor immunity [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A063.

DIFFERENTIATION OF NK CELLS. A LOOK THROUGH THE PRISM OF TRANSCRIPTION FACTORS AND INTRACELLULAR MESSENGERS

All lymphoid cells are referred to as an innate or adaptive immunity unit in terms of the mechanisms of performing immune reactions. The functional activity of natural killer (NK) cells is not associated with pre-activation processes resulting from contact with antigen, rearrangement of antigen-recognition receptor genes, and clonal proliferation. In this regard, NK cells are traditionally referred to as cells of innate immunity. Previously, it was believed that NK cells represent the only population of innate immunity lymphoid cells, but, more recently, there has been increasing evidence in the literature concerning existence of different populations of these cells, thus serving a basis for isolating a common cluster called Innate Lymphoid Cells (ILC). According to the ILC classification, NK cells are classified as the first group of innate lymphoid cells according to their overall functional characteristics, as well as contribution of the T-bet transcription factor to their differentiation. Complexity, multistage and partially nonlinear character of NK cell differentiation are associated with influence of the cellular microenvironment, consistent expression of transcription factors and activation of various intracellular signaling pathways in NK cells. The review considers positioning of NK cells in the ILC classification, the main transcription factors involved in NK cell differentiation. The authors are seeking for generalization of the major routes of intracellular signal transmission in NK cells depending on their activation by cytokines located in the cellular microenvironment and affecting NK cells. The decidual NK cells during pregnancy represent a special object of NK cell differentiation. Stromal cells, trophoblast cells and macrophages are present in the decidua, in addition to NK cells. The review concerns a special case of microenvironmental effects upon expression of transcription factors and activation of NK intracellular messengers, while considering trophoblast cells an example of such influences. The recently discovered variety of NK cells, induced by the microenvironment in the course of their differentiation, requires further study.

Assessment of Gene Function of Mouse Innate Lymphoid Cells for In Vivo Analysis Using Retroviral Transduction.

Retroviral transduction is commonly used to modulate gene expression and is a powerful approach to understand the role of a gene using gain- or loss-of-function strategies. Retroviral vectors can stably integrate non-viral genes into host genomes, providing long-term modulation of gene expression in infected cells and their progeny. Here we describe the generation of retroviral supernatants and the steps to efficiently transduce genes in innate lymphoid cell (ILC) progenitors for subsequent analysis of ILC populations in vivo.

Dynamic changes in intrathymic ILC populations during murine neonatal development.

Members of the innate lymphoid cell (ILC) family have been implicated in the development of thymic microenvironments and the recovery of this architecture after damage. However, a detailed characterization of this family in the thymus is lacking. To better understand the thymic ILC compartment, we have utilized multiple in vivo models including the fate mapping of inhibitor of DNA binding‐2 (Id2) expression and the use of Id2 reporter mice. Our data demonstrate that ILCs are more prominent immediately after birth, but were rapidly diluted as the T‐cell development program increased. As observed in the embryonic thymus, CCR6+NKp46− lymphoid tissue inducer (LTi) cells were the main ILC3 population present, but numbers of these cells swiftly declined in the neonate and ILC3 were barely detectable in adult thymus. This loss of ILC3 means ILC2 are the dominant ILC population in the thymus. Thymic ILC2 were able to produce IL‐5 and IL‐13, were located within the medulla, and did not result from ILC3 plasticity. Furthermore, in WT mice, thymic ILC2 express little RANKL (receptor activator of nuclear factor kappa‐B ligand) arguing that functionally, these cells provide different signals to LTi cells in the thymus. Collectively, these data reveal a dynamic switch in the ILC populations of the thymus during neonatal development.

Enrichment of Innate Lymphoid Cell Populations in Gingival Tissue

Innate lymphoid cells (ILCs) are a population of lymphocytes that act as the first line of immunologic defense at mucosal surfaces. The ILC family in the skin, lungs, and gastrointestinal tissues has been investigated, and there are reports of individual subsets of ILCs in the oral tissues. We sought to investigate the whole ILC population (group 1, 2, and 3 subsets) in the murine gingivae and the lymph nodes draining the oral cavity. We show that ILCs made up a greater proportion of the whole CD45+ lymphocyte population in the murine gingivae (0.356% ± 0.039%) as compared with the proportion of ILCs in the draining lymph nodes (0.158% ± 0.005%). Cytokine profiling of the ILC populations demonstrated different proportions of ILC subsets in the murine gingivae versus the regional lymph nodes. The majority of ILCs in the draining lymph nodes expressed IL-5, whereas there were equal proportions of IFN-γ- and IL-5 expressing ILCs in the oral mucosa. The percentage of IL-17+ ILCs was comparable between the murine gingivae and the oral draining lymph nodes. These data suggest an enrichment of ILCs in the murine gingivae, and these ILCs reflect a cytokine profile discrepant to that of the local draining lymph nodes. These studies indicate diversity and enrichment of ILCs at the oral mucosal surface. The function of ILCs in the oral cavity remains to be determined; here, we provide a premise of ILC populations that merits future consideration in investigations of mouse models and human tissues.

Immune cell census in murine atherosclerosis: cytometry by time of flight illuminates vascular myeloid cell diversity.

AimsAtherosclerosis is characterized by the abundant infiltration of myeloid cells starting at early stages of disease. Myeloid cells are key players in vascular immunity during atherogenesis. However, the subsets of vascular myeloid cells have eluded resolution due to shared marker expression and atypical heterogeneity in vascular tissues. We applied the high-dimensionality of mass cytometry to the study of myeloid cell subsets in atherosclerosis.Methods and resultsApolipoprotein E-deficient (ApoE−/−) mice were fed a chow or a high fat (western) diet for 12 weeks. Single-cell aortic preparations were probed with a panel of 35 metal-conjugated antibodies using cytometry by time of flight (CyTOF). Clustering of marker expression on live CD45+ cells from the aortas of ApoE−/− mice identified 13 broad populations of leucocytes. Monocyte, macrophage, type 1 and type 2 conventional dendritic cell (cDC1 and cDC2), plasmacytoid dendritic cell (pDC), neutrophil, eosinophil, B cell, CD4+ and CD8+ T cell, γδ T cell, natural killer (NK) cell, and innate lymphoid cell (ILC) populations accounted for approximately 95% of the live CD45+ aortic cells. Automated clustering algorithms applied to the Lin-CD11blo-hi cells revealed 20 clusters of myeloid cells. Comparison between chow and high fat fed animals revealed increases in monocytes (both Ly6C+ and Ly6C−), pDC, and a CD11c+ macrophage subset with high fat feeding. Concomitantly, the proportions of CD206+ CD169+ subsets of macrophages were significantly reduced as were cDC2.ConclusionsA CyTOF-based comprehensive mapping of the immune cell subsets within atherosclerotic aortas from ApoE−/− mice offers tools for myeloid cell discrimination within the vascular compartment and it reveals that high fat feeding skews the myeloid cell repertoire toward inflammatory monocyte-macrophage populations rather than resident macrophage phenotypes and cDC2 during atherogenesis.

Tu1270 - Altered Population of Group3 Innate Lymphoid Cells in Lymph from Indomethacin-Induced Inflamed Mucosa in Rat

Tu1270 - Altered Population of Group3 Innate Lymphoid Cells in Lymph from Indomethacin-Induced Inflamed Mucosa in Rat

Group 2 Innate Lymphoid Cells in Pulmonary Immunity and Tissue Homeostasis

Group 2 innate lymphoid cells (ILC2) represent an evolutionary rather old but only recently identified member of the family of innate lymphoid cells and have received much attention since their detailed description in 2010. They can orchestrate innate as well as adaptive immune responses as they interact with and influence several immune and non-immune cell populations. Moreover, ILC2 are able to rapidly secrete large amounts of type 2 cytokines that can contribute to protective but also detrimental host immune responses depending on timing, location, and physiological context. Interestingly, ILC2, despite their scarcity, are the dominant innate lymphoid cell population in the lung, indicating a key role as first responders and amplifiers upon immune challenge at this site. In addition, the recently described tissue residency of ILC2 further underlines the importance of their respective microenvironment. In this review, we provide an overview of lung physiology including a description of the most prominent pulmonary resident cells together with a review of known and potential ILC2 interactions within this unique environment. We will further outline recent observations regarding pulmonary ILC2 during immune challenge including respiratory infections and discuss different models and approaches to study ILC2 biology in the lung.

Dependence of innate lymphoid cell 1 development on NKp46.

NKp46, a natural killer (NK) cell–activating receptor, is involved in NK cell cytotoxicity against virus-infected cells or tumor cells. However, the role of NKp46 in other NKp46+ non-NK innate lymphoid cell (ILC) populations has not yet been characterized. Here, an NKp46 deficiency model of natural cytotoxicity receptor 1 (Ncr1)gfp/gfp and Ncr1gfp/+ mice, i.e., homozygous and heterozygous knockout (KO), was used to explore the role of NKp46 in regulating the development of the NKp46+ ILCs. Surprisingly, our studies demonstrated that homozygous NKp46 deficiency resulted in a nearly complete depletion of the ILC1 subset (ILC1) of group 1 ILCs, and heterozygote KO decreased the number of cells in the ILC1 subset. Moreover, transplantation studies confirmed that ILC1 development depends on NKp46 and that the dependency is cell intrinsic. Interestingly, however, the cell depletion specifically occurred in the ILC1 subset but not in the other ILCs, including ILC2s, ILC3s, and NK cells. Thus, our studies reveal that NKp46 selectively participates in the regulation of ILC1 development.

Activated innate lymphoid cell populations accumulate in human tumour tissues

BackgroundInnate lymphoid cells (ILC) are part of a heterogeneous family of haematopoietic effector cells which lack re-arranged antigen-specific receptors. They promote host defense and contribute to tissue and metabolic homeostasis, wound healing and immune surveillance. Their role in human cancer immunity is less defined, and therefore we aimed to identify the frequency and phenotype of distinct ILC groups in various types of cancer.MethodsTissue samples and peripheral blood were collected from patients undergoing surgical resection of gastrointestinal and breast tumours. Single cell suspension of tumour tissue was immediately obtained following surgery using tumour dissociation.ResultsWe observed significantly higher frequencies of ILC2 (p value: 0.04) in malignant breast cancer tissue and significantly higher frequencies of group 1 ILC (p value: 0.001) in malignant gastrointestinal tumours. Tumour infiltrating ILC were found to show an activated phenotype with higher expression of MHC-II, KLRG1, early activation marker CD69 and CD44.ConclusionsActivated innate lymphoid cells infiltrate tumours dependent on tumour type and location.

Characterisation of innate lymphoid cell populations at different sites in mice with defective T cell immunity.

Background: Innate lymphoid cells (ILCs) have now been identified within most tissues of the body and current evidence indicates that this family of cells play a fundamental role in maintaining tissue homeostasis. However, few studies have compared the ILC populations between several tissues. Methods: We sought to generate a comprehensive characterisation of the ILC populations in different tissues of C57BL/6 WT and genetically modified mice targeting costimulatory pathways, using transcription factor expression to define specific groups. Results: Consistent with studies individually describing the ILC composition in different tissues, our analysis revealed different ILC groups dominate the ILC population in different tissues. Additionally, we observed a population of IL-7Rα +Id2 + cells lacking expression of lineage markers but also lacking expression of GATA-3, RORgt or T-bet. This population was most evident in ear skin where it outnumbered the defined ILC groups, however, further experiments demonstrated that detection of these cells was influenced by how the tissue was digested, raising concerns as to its real nature. Since both ILC2 and ILC3 express ICOS, we then investigated the requirement for ICOS:ICOSL interactions in the homeostasis of ILC populations at these sites. Surprisingly, no significant differences were detected in the number of ILC1, ILC2 or ILC3 between WT and ICOSL -/- mice in any tissue, indicating that this pathway is not required for ILC homeostasis at these sites. These data were compared with CD80 -/-CD86 -/- mice given evidence of CD28 expression by some ILC and ILC crosstalk with activated T cells. Notably, the absence of CD28 ligands resulted in a significant increase in ILC2 and ILC3 numbers in the intestine. Conclusions: Together, these data provide new insight into ILC composition in different tissues in both WT and genetically modified mice where key costimulatory pathways are genetically deleted, providing a useful resource for further research into ILC biology.

Characterisation of innate lymphoid cell populations at different sites in mice with defective T cell immunity

Background: Innate lymphoid cells (ILCs) have now been identified within most tissues of the body and current evidence indicates that this family of cells play a fundamental role in maintaining tissue homeostasis. However, few studies have compared the ILC populations between several tissues. Methods: We sought to generate a comprehensive characterisation of the ILC populations in different tissues of C57BL/6 WT and genetically modified mice targeting costimulatory pathways, using transcription factor expression to define specific groups. Results: Consistent with studies individually describing the ILC composition in different tissues, our analysis revealed different ILC groups dominate the ILC population in different tissues. Additionally, we observed a population of IL-7Rα +Id2 + cells lacking expression of lineage markers but also lacking expression of GATA-3, RORgt or T-bet. This population was most evident in ear skin where it outnumbered the defined ILC groups, however, further experiments demonstrated that detection of these cells was influenced by how the tissue was digested, raising concerns as to its real nature. Since both ILC2 and ILC3 express ICOS, we then investigated the requirement for ICOS:ICOSL interactions in the homeostasis of ILC populations at these sites. Surprisingly, no significant differences were detected in the number of ILC1, ILC2 or ILC3 between WT and ICOSL -/- mice in any tissue, indicating that this pathway is not required for ILC homeostasis at these sites. These data were compared with CD80 -/-CD86 -/- mice given evidence of CD28 expression by some ILC and ILC crosstalk with activated T cells. Notably, the absence of CD28 ligands resulted in a significant increase in ILC2 and ILC3 numbers in the intestine. Conclusions: Together, these data provide new insight into ILC composition in different tissues in both WT and genetically modified mice where key costimulatory pathways are genetically deleted, providing a useful resource for further research into ILC biology.

Characterisation of innate lymphoid cell populations at different sites in mice with defective T cell immunity

Background: Innate lymphoid cells (ILCs) have now been identified within most tissues of the body and current evidence indicates that this family of cells play a fundamental role in maintaining tissue homeostasis. However, few studies have compared the ILC populations between several tissues. Methods: We sought to generate a comprehensive characterisation of the ILC populations in different tissues of C57BL/6 WT and genetically modified mice targeting costimulatory pathways, using transcription factor expression to define specific groups. Results: Consistent with studies individually describing the ILC composition in different tissues, our analysis revealed different ILC groups dominate the ILC population in different tissues. Additionally, we observed a population of IL-7Rα+Id2+ cells lacking expression of lineage markers but also lacking expression of GATA-3, RORgt or T-bet. This population was most evident in ear skin where it outnumbered the defined ILC groups, however, further experiments demonstrated that detection of these cells was influenced by how the tissue was digested, raising concerns as to its real nature. Since both ILC2 and ILC3 express ICOS, we then investigated the requirement for ICOS:ICOSL interactions in the homeostasis of ILC populations at these sites. Surprisingly, no significant differences were detected in the number of ILC1, ILC2 or ILC3 between WT and ICOSL-/- mice in any tissue, indicating that this pathway is not required for ILC homeostasis at these sites. These data were compared with CD80-/-CD86-/- mice given evidence of CD28 expression by some ILC and ILC crosstalk with activated T cells. Notably, the absence of CD28 ligands resulted in a significant increase in ILC2 and ILC3 numbers in the intestine. Conclusions: Together, these data provide new insight into ILC composition in different tissues in both WT and genetically modified mice where key costimulatory pathways are genetically deleted, providing a useful resource for further research into ILC biology.

Characterisation of innate lymphoid cell populations at different sites in mice with defective T cell immunity

Background: Innate lymphoid cells (ILCs) have now been identified within most tissues of the body and current evidence indicates that this family of cells play a fundamental role in maintaining tissue homeostasis. However, few studies have compared the ILC populations between several tissues. Methods: We sought to generate a comprehensive characterisation of the ILC populations in different tissues of C57BL/6 WT and genetically modified mice targeting costimulatory pathways, using transcription factor expression to define specific groups. Results: Consistent with studies individually describing the ILC composition in different tissues, our analysis revealed different ILC groups dominate the ILC population in different tissues. Additionally, we observed a population of IL-7Rα+Id2+ cells lacking expression of lineage markers but also lacking expression of GATA-3, RORgt or T-bet. This population was most evident in ear skin where it outnumbered the defined ILC groups, however, further experiments demonstrated that detection of these cells was influenced by how the tissue was digested, raising concerns as to its real nature. Since both ILC2 and ILC3 express ICOS, we then investigated the requirement for ICOS:ICOSL interactions in the homeostasis of ILC populations at these sites. Surprisingly, no significant differences were detected in the number of ILC1, ILC2 or ILC3 between WT and ICOSL-/- mice in any tissue, indicating that this pathway is not required for ILC homeostasis at these sites. These data were compared with CD80-/-CD86-/- mice given evidence of CD28 expression by some ILC and ILC crosstalk with activated T cells. Notably, the absence of CD28 ligands resulted in a significant increase in ILC2 and ILC3 numbers in the intestine. Conclusions: Together, these data provide new insight into ILC composition in different tissues in both WT and genetically modified mice where key costimulatory pathways are genetically deleted, providing a useful resource for further research into ILC biology.

Long-term Persistence of Innate Lymphoid Cells in the Gut After Intestinal Transplantation.

Little is known about innate lymphoid cell (ILC) populations in the human gut, and the turnover of these cells and their subsets after transplantation has not been described. Intestinal samples were taken from 4 isolated intestine and 3 multivisceral transplant recipients at the time of any operative resection, such as stoma closure or revision. ILCs were isolated and analyzed by flow cytometry. The target population was defined as being negative for lineage markers and double-positive for CD45/CD127. Cells were further stained to define ILC subsets and a donor-specific or recipient-specific HLA marker to analyze chimerism. Donor-derived ILCs were found to persist greater than 8 years after transplantation. Additionally, the percentage of cells thought to be lymphoid tissue inducer cells among donor ILCs was far higher than that among recipient ILCs. Our findings demonstrate that donor-derived ILCs persist long-term after transplantation and support the notion that human lymphoid tissue inducer cells may form in the fetus and persist throughout life, as hypothesized in rodents. Correlation between chimerism and rejection, graft failure, and patient survival requires further study.

ILC regs : The new kid in class

ILC regs are a population of innate lymphoid cells that control innate intestinal inflammation through IL-10.

Basic Science Liver

Basic Science Liver

Innate lymphoid cells (ILCs) from septic patients produce higher levels of cytokines than ILCs from healthy controls

Abstract Sepsis is a systemic inflammatory response to infection, which leads to life-threatening organ dysfunction. During sepsis, many cells of the innate and adaptive immune systems have functional alterations; in particular, monocytes produce reduced levels of pro-inflammatory cytokines in response to microbial products (MAMPs). Innate lymphoid cells (ILCs) are cells with lymphocyte morphology that lack antigen-specific receptors and respond directly to cytokines and MAMPs; it is not known if ILCs are affected during sepsis. In this study, we used flow cytometry to evaluate the frequency of ILCs in the peripheral blood of septic patients and healthy controls, as well as the expression of CD56, CCR6 and NKp44 and the production of IL-17 and IFN-γ by ILCs. ILCs were defined as CD45+ lineage- CD127+ cells. The number of total lymphocytes and the number of ILCs were significantly lower in septic patients, compared to healthy controls, with ILCs representing around 0.5% of peripheral blood lymphocytes. In both healthy controls and septic patients, CD56 defines two populations of ILCs: a higher percentage of the CD56− cells express CCR6, compared to the CD56+ cells, while a higher percentage of the CD56+ cells express NKp44, compared to the CD56− cells. CD56− ILCs are the main producers of IL-17, while CD56+ cells are the main producers of IFN-γ, in both healthy controls and septic patients. The production of these two cytokines is increased in the ILCs from septic patients, compared to the ILCs from healthy controls. This is in contrast with the decreased cytokine production that is observed in monocytes and T lymphocytes from septic patients. This project was funded by CONACyT-México (219661).

Topical exposure to the quaternary ammonium compound didecyldimethylammonium chloride augments dermal type 2 innate lymphoid cell populations

Abstract Didecyldimethylammonium chloride (DDAC) is a dialkyl-quaternary ammonium compound used in commercial and industrial products for its antimicrobial properties. Clinical reports suggest that topical exposure to DDAC can resultin immediate and delayed type hypersensitivity, and recently our laboratory has confirmed that DDAC is a strong non-IgE mediated sensitizer in mice at relevant human exposure concentrations. Here, we further investigate the mechanism of DDAC induced hypersensitivity in mice, focusing on dermal immune responses. Gene expression analysis shows significant reduction in epithelial Cdh1, and increases in Tslp and Ccl17 following DDAC application to the skin, indicating that dermal type 2 innate lymphoid cells (ILC2s) may be involved in DDAC-induced hypersensitivity. Phenotypic analysis of dermal ILC2s shows that these cells are rapidly activated with increased expression of CD25, ICOS, and KLRG1 occurring by 24 hours post DDAC exposure. Increases in ILC2 number were observed following 2 weeks of DDAC exposure and found to persist for up to 2 weeks in the absence of DDAC and Tslp. Additionally, the phenotype of these cells suggests a potential for longevity (reduced KLRG1 and increased CD127) and enhanced activity (increased ICOS), which may influence future sensitization events. ILC2s have been previously implicated to play a role in IgE-independent allergic responses, but to our knowledge have not been investigated in chemical allergy. These results indicate that skin resident ILC2s may play a role in the development of the hypersensitivity response to DDAC and raise concerns about the use of this chemical and other quaternary ammonium compounds that may elicit similar effects.

Characterization of an alternatively activated IL-10 producing innate lymphoid type-2 effector cell population

Abstract Type-2 innate lymphoid (ILC2) cells share cytokine and transcription factor expression with CD4+ Th2 cells, but it is unknown if this ILC subtype undergoes functional diversification. Here, we report in vivo induction of a molecularly distinct subset of activated lung ILC2 cells, termed ILC210 cells, that produce IL-10 and downregulate genes associated with inflammation. Signals that generate ILC210 cells were distinct from those that induced IL-13 production and, along with transcriptome data, suggested an alternative pathway of activation. In vivo, IL-2 greatly enhanced ILC210 cell generation by IL-33 and was associated with decreased eosinophil recruitment to the lung. Unlike the majority of activated ILC2 cells, the ILC210 cell population underwent contraction following cessation of IL-33 stimulation in vivo, but with maintenance of a subpopulation of cells that can be recalled by restimulation, analogous to T cell effector cell and memory cell generation. Together, these data demonstrate previously unappreciated heterogeneity in the ILC2 cell response.