Numbers Of Innate Lymphoid Cells Research Articles

Role of the Innate Immune System in the Development of Hypersensitivity Pneumonitis

Hypersensitivity pneumonitis (HP) is an extrinsic allergic alveolitis disease that is constantly affecting the farmers. It is triggered by the continuous exposure of environmental antigens, specifically antigens that grow in hot humid conditions in hay like Saccharopolyspora rectivirgula (SR). While a role of T cells has been established in the development of this diseases, the role of the innate immune system is still largely unknown in the development of lung inflammation leading to HP. To study this disease, we used a mouse model of HP, induced by SR, along with Rag−/− mice which only have an innate immune system and lack an adaptive immune system (and thus have no T cells). We hypothesized that the development of Hypersensitivity pneumonitis requires the presence of T (and B) cells, at the same time, there will be a lower percentage and number of inflammatory cells in the lungs of Rag−/− mice upon exposure to SR. WT and Rag−/− mice were exposed to SR. Lungs, spleen and lymph nodes where collected to analyze their response. We examined the percentage and number of myeloid and Innate Lymphoid Cells (ILC), as well as their ability to produce cytokines. We found that there was no significant difference in the number of Innate Lymphoid cells and myeloid cells between SR‐exposed WT and Rag−/− mice. These results may be due to the low concentration of bacteria used for these experiments. In future experiments, we propose to use a higher concentration of SR to induce inflammation.Support or Funding InformationThis work was supported in part by grants from the NIH‐AI38570, NIH‐AI20701, and NIH‐AI129422. NIH‐NIGMS #2R25GM096955 (RISE). VIP‐Veterinary Investigation Program.

Recovery of Innate Lymphoid Cells after Allogeneic Stem Cell Transplant in a Pediatric Population: A Single Institution Pilot Study.

<h3>Background</h3> Innate lymphoid cells (ILCs) are a subset of innate immune cells that mimic the functions of T lymphocytes, but lack rearranged antigen receptors or clonal expansion in response to antigens. They are characterized in human peripheral blood as Lin⁻ (CD3, CD1a, CD4, CD14, CD16, CD19, CD34, CD94, CD303, FceRI) CD127⁺ cells. ILCs are divided into three sub-groups: ILC type 1, 2 and 3. The recovery of these cells after allogeneic hematopoietic stem cell transplant (aHSCT) in the pediatric population has not been described. <h3>Objectives</h3> The primary objective is to study the recovery of ILCs after aHSCT in pediatric patients. The secondary objective was to determine if recovery of ILCs correlated with the incidence or severity of graft-versus-host disease (GVHD). <h3>Methods</h3> We are conducting a prospective study, enrolling consecutive patients undergoing aHSCT at our institution. After approval from local IRB, the study opened in May 2019. Blood samples are drawn from patients prior to the conditioning regimen, and then weekly starting on day 0 until day +30 (+/- 3 days except day 0), then on day +60, day +90 and day +180. All blood samples are analyzed on the same day by flow cytometry along with a healthy control (thawed frozen peripheral blood mononuclear cells [PBMCs]), serving as the positive control. <h3>Results</h3> All eligible patients have consented to the study. Six have been enrolled through September 2019; with the following demographics: median age is 9 years (range 21 months-18 years); 2 malignant, 4 non-malignant; matched sibling (n = 1), unrelated (n = 4 complete match, n=1 mismatched 9/10). All patients received donor bone marrow. Conditioning regimens were variable depending on the indication for transplant, with fludarabine, busulfan and individualized dose rATG being the most commonly utilized regimen (67%). GVHD prophylaxis for all patients was tacrolimus with mini-methotrexate. We observed substantial drop in number of ILCs in response to conditioning regimen in all patients, with nadir on day +14 that plateaued through day +30, followed by recovery by day +60 (figure 1). The one patient who has day +90 sample had a complete recovery of total number of ILCs. Similar trend was seen in type 2 ILCs (Lin⁻ CD127⁺ CRTH2⁺) and Type 3 ILCs (Lin⁻ CD127⁺ CRTH2-ve CD117⁺) while type 1 ILCs (Lin⁻ CD127⁺ CRTH2⁻ CD117⁻) showed a slower drop, reaching a nadir on day +21 with slow recovery by day +60. Interestingly, patients who had GVHD (n = 3), had a higher number of ILCs compared to patients who did not develop GVHD (n = 3) on day 0 (p = 0.0781, 2way ANOVA) (Figure 2). This pattern was statistically significant in type 1 ILCs between the two groups of patients on day 0 (p = 0.0062, 2way ANOVA) (Figure 3). <h3>Conclusion</h3> It is feasible to prospectively study the recovery of ILCs in the post aHSCT pediatric population. We plan to enroll additional patients allowing us to better evaluate a possible correlation between ILCs and GVHD.

Yeats4 drives ILC lineage commitment via activation of Lmo4 transcription.

Innate lymphoid cells (ILCs) play critical roles in defending infections and maintaining mucosal homeostasis. All ILCs arise from common lymphoid progenitors (CLPs) in bone marrow. However, how CLPs stratify and differentiate into ILC lineages remains elusive. Here, we showed that Yeats4 is highly expressed in ILCs and their progenitors. Yeats4 conditional KO in the hematopoietic system causes decreased numbers of ILCs and impairs their effector functions. Moreover, Yeats4 regulates α4β7 + CLP differentiation toward common helper ILC progenitors (CHILPs). Mechanistically, Yeats4 recruits the Dot1l-RNA Pol II complex onto Lmo4 promoter through recognizing H3K27ac modification to initiate Lmo4 transcription in α4β7 + CLPs. Additionally, Lmo4 deficiency also impairs ILC lineage differentiation and their effector functions. Collectively, the Yeats4-Lmo4 axis is required for ILC lineage commitment.

Fingolimod Alters Tissue Distribution and Cytokine Production of Human and Murine Innate Lymphoid Cells.

Sphingosine-1 phosphate receptor 1 (S1PR1) is expressed by lymphocytes and regulates their egress from secondary lymphoid organs. Innate lymphoid cell (ILC) family has been expanded with the discovery of group 1, 2 and 3 ILCs, namely ILC1, ILC2 and ILC3. ILC3 and ILC1 have remarkable similarity to CD4+ helper T cell lineage members Th17 and Th1, respectively, which are important in the pathology of multiple sclerosis (MS). Whether human ILC subsets express S1PR1 or respond to its ligands have not been studied. In this study, we used peripheral blood/cord blood and tonsil lymphocytes as a source of human ILCs. We show that human ILCs express S1PR1 mRNA and protein and migrate toward S1P receptor ligands. Comparison of peripheral blood ILC numbers between fingolimod-receiving and treatment-free MS patients revealed that, in vivo, ILCs respond to fingolimod, an S1PR1 agonist, resulting in ILC-penia in circulation. Similarly, murine ILCs responded to fingolimod by exiting blood and accumulating in the secondary lymph nodes. Importantly, ex vivo exposure of ILC3 and ILC1 to fingolimod or SEW2871, another S1PR1 antagonist, reduced production of ILC3- and ILC1- associated cytokines GM-CSF, IL-22, IL-17, and IFN-γ, respectively. Surprisingly, despite reduced number of lamina propria-resident ILC3s in the long-term fingolimod-treated mice, ILC3-associated IL-22, IL-17A, GM-CSF and antimicrobial peptides were high in the gut compared to controls, suggesting that its long term use may not compromise mucosal barrier function. To our knowledge, this is the first study to investigate the impact of fingolimod on human ILC subsets in vivo and ex vivo, and provides insight into the impact of long term fingolimod use on ILC populations.

CARMA3 Mediates Allergic Lung Inflammation in Response to Alternaria alternata.

The airway epithelial cell (AEC) response to allergens helps initiate and propagate allergic inflammation in asthma. CARMA3 is a scaffold protein that mediates G protein-coupled receptor-induced NF-κB activation in airway epithelium. In this study, we demonstrate that mice with CARMA3-deficient AECs have reduced airway inflammation, as well as reduced type 2 cytokine levels in response to Alternaria alternata. These mice also have reduced production of IL-33 and IL-25, and reduced numbers of innate lymphoid cells in the lung. We also show that CARMA3-deficient human AECs have decreased production of proasthmatic mediators in response to A. alternata. Finally, we show that CARMA3 interacts with inositol 1,4,5-trisphosphate receptors in AECs, and that inhibition of CARMA3 signaling reduces A. alternata-induced intracellular calcium release. In conclusion, we show that CARMA3 signaling in AECs helps mediate A. alternata-induced allergic airway inflammation, and that CARMA3 is an important signaling molecule for type 2 immune responses in the lung.

The JAK inhibitor ruxolitinib reduces inflammation in an ILC3-independent model of innate immune colitis.

Innate immunity contributes to the pathogenesis of inflammatory bowel disease (IBD). However, the mechanisms of IBD mediated by innate immunity are incompletely understood and there are limited models of spontaneous innate immune colitis to address this question. Here we describe a new robust model of colitis occurring in the absence of adaptive immunity. RAG1-deficient mice expressing TNFAIP3 in intestinal epithelial cells (TRAG mice) spontaneously developed 100% penetrant, early-onset colitis that was limited to the colon and dependent on intestinal microbes but was not transmissible to co-housed littermates. TRAG colitis was associated with increased mucosal numbers of innate lymphoid cells (ILC) and depletion of ILC prevented colitis in TRAG mice. ILC depletion also therapeutically reversed established colitis in TRAG mice. The colitis in TRAG mice was not prevented by interbreeding to mice lacking group 3 ILC, nor by depletion of TNF. Treatment with the JAK inhibitor ruxolitinib ameliorated colitis in TRAG mice. This new model of colitis, with its predictable onset and colon-specific inflammation will have direct utility in developing a more complete understanding of innate immune mechanisms that can contribute to colitis and in pre-clinical studies for effects of therapeutic agents on innate immune mediated IBD.

CHARACTERIZATION OF INTERLEUKIN-17 PRODUCING CELLS IN THE PERIPHERAL BLOOD OF PATIENTS WITH TUBERCULOSIS

Abstract Tuberculosis (TB), an infection caused by Mycobacterium tuberculosis, is one of the ten main causes of death by infectious disease. When the mycobacteria are inhaled, they interact with immune cells in the respiratory tract in a specialized structure known as granuloma. Interleukin 17 (IL-17) contributes to formation and size of the granuloma, and it correlates with the recruitment of neutrophils. It has been demonstrated that other innate lymphocytes, such as Tγδ cells, contribute to the production of IL-17 during TB. Innate lymphoid cells (ILCs) are other innate lymphocytes that are classified into groups (ILC1, ILC2 and ILC3) according to their cytokine patterns. ILC3 produce IL-17, but it is not known if these cells participate in the response to TB infection. We evaluated, by flow cytometry, the frequency and IL-17 production of Th, Tgd, NK and ILCs (ILC1, ILC2 and ILC3) in the peripheral blood of healthy individuals (negative and positive for the Quantiferon [QTF] test, which detects latent TB), and of patients with active pulmonary TB. The numbers of Th cells, NK cells and ILCs (ILC1, ILC2 and ILC3) were decreased in TB patients, compared to healthy individuals. Peripheral blood was stimulated with M. tuberculosis soluble extract (MTSE) for 5 h in the presence of brefeldin A. After stimulation, Th cells from QTF-positive healthy individuals and TB patients produced IL-17. Tγδ cells also produced IL-17, although the percentage of these cells did not change after activation with MTSE. In some cases, NK, ILC2 and ILC3 from TB patients produced IL-17, but this production did not change after MTSE activation. These results indicate that Th and Tγδ cells contribute to IL-17 production during TB. This project was funded by SIP-IPN-México (20180254).

Lack of Type 2 Innate Lymphoid Cells Promotes a Type I-Driven Enhanced Immune Response in Contact Hypersensitivity

Allergic contact dermatitis and its animal model, contact hypersensitivity, are T-cell-mediated inflammatory skin diseases that require activation of the innate immune system. Here we investigate the role of innate lymphoid cells (ILCs) during the elicitation phase of 2,4,6-trinitrochlorobenzene-induced contact hypersensitivity using EomesGfp/+ x Rorc(γt)-CreTg x Rosa26RYfp/+ reporter mice. Ear swelling responses, cutaneous ILC numbers, and cytokine production were determined at different time points. Functional analyses were performed in a CD90.1/.2 congenic adoptive transfer model that allowed selective antibody-mediated depletion of ILCs before hapten challenge, and in Rorasg/floxIl7rCre/+ mice, which lack ILC2. Hapten challenge induced early increases of natural killer cells in skin and ear draining lymph nodes corresponding to the peak ear swelling response. In contrast, ILC1, 2, and 3 showed a delayed increase in numbers corresponding to the contact hypersensitivity resolution phase. Hapten challenge induced increased marker cytokines in all ILC subtypes and an activated phenotype in ILC2. Depletion of all ILC resulted in a significantly enhanced ear swelling response. Similarly, ILC2-deficient mice (Rorasg/floxIl7rCre/+) displayed increased ear swelling responses on hapten challenge, suggesting that ILC2 act as negative regulators in the type 1-dominated immune response of contact hypersensitivity.

BCG vaccination drives accumulation and effector function of innate lymphoid cells in murine lungs

The tuberculosis (TB) vaccine bacille Calmette-Guérin (BCG) prevents disseminated childhood TB; however, it fails to protect against the more prevalent pulmonary TB. Limited understanding of the immune response to Mycobacterium tuberculosis, the causative agent of TB, has hindered development of improved vaccines. Although memory CD4 T cells are considered the main mediators of protection against TB, recent studies suggest there are other key subsets that contribute to antimycobacterial immunity. To that end, innate cells may be involved in the protective response. In this study, we investigated the primary response of innate lymphoid cells (ILCs) to BCG exposure. Using a murine model, we showed that ILCs increased in number in the lungs and lymph nodes in response to BCG vaccination. Additionally, there was significant production of the antimycobacterial cytokine IFN-γ by ILCs. As ILCs are located at mucosal sites, it was investigated whether mucosal vaccination (intranasal) stimulated an enhanced response compared to the traditional vaccination approach (intradermal or subcutaneous). Indeed, in response to intranasal vaccination, the number of ILCs, and IFN-γ production in NK cells and ILC1s in the lungs and lymph nodes, were higher than that provoked through intradermal or subcutaneous vaccination. This work provides the first evidence that BCG vaccination activates ILCs, paving the way for future research to elucidate the protective potential of ILCs against mycobacterial infection. Additionally, the finding that lung ILCs respond rigorously to mucosal vaccination may have implications for the delivery of novel TB vaccines.

Innate Lymphoid Cells in HIV/SIV Infections.

Over the past several years, new populations of innate lymphocytes have been described in mice and primates that are critical for mucosal homeostasis, microbial regulation, and immune defense. Generally conserved from mice to humans, innate lymphoid cells (ILC) have been divided primarily into three subpopulations based on phenotypic and functional repertoires: ILC1 bear similarities to natural killer cells; ILC2 have overlapping functions with TH2 cells; and ILC3 that share many functions with TH17/TH22 cells. ILC are specifically enriched at mucosal surfaces and are possibly one of the earliest responders during viral infections besides being involved in the homeostasis of gut-associated lymphoid tissue and maintenance of gut epithelial barrier integrity. Burgeoning evidence also suggests that there is an early and sustained abrogation of ILC function and numbers during HIV and pathogenic SIV infections, most notably ILC3 in the gastrointestinal tract, which leads to disruption of the mucosal barrier and dysregulation of the local immune system. A better understanding of the direct or indirect mechanisms of loss and dysfunction will be critical to immunotherapeutics aimed at restoring these cells. Herein, we review the current literature on ILC with a particular emphasis on ILC3 and their role(s) in mucosal immunology and the significance of disrupting the ILC niche during HIV and SIV infections.

Brief Report: Inflammatory Colonic Innate Lymphoid Cells Are Increased During Untreated HIV-1 Infection and Associated With Markers of Gut Dysbiosis and Mucosal Immune Activation.

HIV-1 infection is associated with intestinal inflammation, changes in the enteric microbiota (dysbiosis), and intestinal epithelial cell damage. NKp44 innate lymphoid cells (ILCs) play an important role in epithelial barrier maintenance through the production of interleukin (IL)-22 but also display functional plasticity and can produce inflammatory cytokines [eg, interferon gamma (IFNγ)] in response to cytokine milieu and stimulatory signals. The objective of this pilot study was to enumerate frequencies of IL-22 and IFNγ-expressing colonic NKp44 ILCs during untreated, chronic HIV-1 infection. A cross-sectional study was performed to compare numbers of cytokine-expressing ILCs in colonic biopsies of untreated, chronic HIV-1 infected (n = 22), and uninfected (n = 10) study participants. Associations between cytokine ILC and previously established measures of virological, immunological, and microbiome indices were analyzed. Multicolor flow cytometry was used to measure the absolute number of colonic CD3NKp44CD56 ILCs expressing IL-22 or IFNγ after in vitro mitogenic stimulation. Numbers of colonic NKp44 ILCs that expressed IFNγ were significantly higher in HIV-1 infected versus uninfected persons and positively correlated with relative abundances of dysbiotic bacterial species in the Xanthomonadaceae and Prevotellaceae bacterial families and with colonic myeloid dendritic cell and T-cell activation. Higher numbers of inflammatory colonic ILCs during untreated chronic HIV-1 infection that associated with dysbiosis and colonic myeloid dendritic cell and T-cell activation suggest that inflammatory ILCs may contribute to gut mucosal inflammation and epithelial barrier breakdown, important features of HIV-1 mucosal pathogenesis.

Reduced numbers of circulating group 2 innate lymphoid cells in patients with common variable immunodeficiency.

Recent studies identified an emerging role of group 2 and 3 innate lymphoid cells (ILCs) as key players in the generation of T-dependent and T-independent antibody production. In this retrospective case-control study, CD117+ ILCs (including the majority of ILC2 and ILC3) were reduced in patients with common variable immunodeficiency (CVID). The reduction in CD117+ ILCs was distinctive to CVID and could not be observed in patients with X-linked agammaglobulinemia. Patients with a more pronounced reduction in CD117+ ILC numbers showed significantly lower numbers of peripheral MZ-like Bcells and an increased prevalence of chronic, non-infectious enteropathy. Subsequent phenotyping of ILC subsets in CVID revealed that the reduction in CD117+ ILC numbers is due to a reduction in ILC2 numbers. In vitro expansion of CVID ILC2 in response to IL-2, IL-7, IL-25 and IL-33 was impaired. Furthermore, upregulation of MHCII and IL-2RA in response to IL-2, IL-7, IL-25 and IL-33 was impaired in CVID ILC2. Thus, our results indicate a dysregulation of ILC subsets with a reduction in ILC2 numbers in CVID, however, further studies are needed to explore whether ILC abnormalities are a primary finding or secondary to disease complications encountered in CVID.

Role of group 2 innate lymphoid cells (ILC2) in SHARPIN-deficient mice

Abstract SHARPIN is a component of the linear ubiquitination assembly complex and a key regulator of NFkB and integrin signaling. SHARPIN-deficient mice develop a phenotype known as chronic proliferative dermatitis (cpdm), characterized by progressive epidermal hyperplasia, apoptosis of keratinocytes, cutaneous and systemic eosinophilic inflammation, and hypoplasia of secondary lymphoid organs. We recently reported that the cutaneous inflammation in SHARPIN-deficient mice (Sharpincpdm) develops independently of B and T lymphocytes. We therefore sought to determine the role of innate lymphoid cells (ILCs) in the dermatitis of Sharpincpdm mice. ILCs were identified as a discrete population of CD45+ Lin−CD90.2hi cells in the skin and draining lymph nodes (LN) of wild type (WT) and Sharpincpdm mice. The number of ILCs was markedly increased in Sharpincpdm mice. The majority of cells were group 2 ILC (ILC2) as indicated by labeling for GATA3, IL5, and IL13. The skin of Sharpincpdm mice had increased expression of Il33 and Tslp mRNA. To determine the role of IL33, double mutant mice were generated in which the receptor for IL33 (Il1lr1 also known as ST2) was deleted. Loss of IL33-signaling greatly reduced the number of ILCs and reduced the severity of the dermatitis. These experiments suggest that the dermatitis in Sharpincpdm mice is driven by IL33-dependent ILC2. Supported by the NIH (AR049288).

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).

CHARACTERIZATION OF INTERFERON-γ PRODUCING CELLS IN THE PERIPHERAL BLOOD OF PATIENTS WITH TUBERCULOSIS

Abstract Tuberculosis (TB) caused 1.4 million deaths in 2015. The infection starts when Mycobacterium tuberculosis is inhaled and interacts with immune cells in the respiratory tract. Alveolar macrophages, neutrophils and dendritic cells (DCs) engulf the mycobacteria; these DCs migrate to the draining lymph node and induce the activation of mycobacteria-specific Th cells. These Th cells migrate back to the lung and produce IFNγ and TNFα, two cytokines essential for mycobacterial control. In addition to Th cells, other cells, such as Tγδ and NK cells, produce IFNγ in response to mycobacteria; innate lymphoid cells (ILCs) are also able to produce IFNγ, but it still unknown if ILCs, which have been detected in human lungs during cancer, produce this cytokine in response to mycobacteria. We evaluated, by flow cytometry, the frequency and IFNγ production of Th, Tγδ, NK and ILCs (ILC1, ILC2 and ILC3) in the peripheral blood of healthy individuals (negative and positive for the Quantiferon [QTF] test, which detects latent TB), and patients with active pulmonary TB. The numbers of Th, Tγδ, NK cells and ILCs (ILC1, ILC2 and ILC3) were decreased in TB patients. Peripheral blood was stimulated with M. tuberculosis soluble extract for 5 h in the presence of brefeldin A. In healthy, QTF-negative individuals, IFNγ production was increased in Tγδ and ILC2 after stimulation; in healthy, QTF-positive individuals, IFNγ production was increased in Th, Tγδ and ILCs, and in TB patients, IFNγ production was increased in NK cells and ILC2. These results suggest ILCs produce IFNγ during TB infection. This project was funded by SIP-IPN-México (20170616).

395 Lack of type 2 innate lymphoid cells promotes a type I driven enhanced immune response in TNCB contact hypersensitivity

The role of innate lymphoid cells (ILC) in allergic contact dermatitis has not adequately been addressed. We investigated quantitative changes and functional relevance of ILCs during the elicitation phase of hapten-induced contact hypersensitivity (CHS). EomesGfp/+ x Rorc(γt)-CreTg x Rosa26RYfp/+ reporter mice were sensitized and challenged with the hapten TNCB. Ear swelling, ILC numbers and cytokine production were measured at different time points. For functional analysis we depleted ILCs in Rag1-/- mice before recieving sorted T-cells from sensitized donor mice (CD90.1) using either a CD90.2-specific or isotype mAb. In addition CHS was performed in Rorα+/floxIl7rCre mice, which selectively lack ILC2. The quantitative analysis of total cell numbers revealed early increases of natural killer (NK) cells in skin and skin draining lymph nodes 24h after allergen challenge, corresponding to the highest ear swelling response and leukocyte infiltrate in the skin. These cells produced high amounts of the type 1 cytokines IFNγ and TNFα. ILC1, 2 and 3 showed a delayed increase in total numbers starting from 48h after allergen challenge. ILC2 cells displayed an activated phenotype reflected by increased ICOS expression. Total ILC depletion through CD90.2 mAb administration resulted in a significantly enhanced ear swelling response. Finally, Rorα+/floxIl7rCre mice also displayed increased ear swelling responses after allergen challenge. In conclusion, our data support the concept of NK cells as main innate proinflammatory player and suggests that simultaneously activated ILC2 act as counter regulators in the type 1 dominated immune response of CHS.

Flt3 Ligand Regulates the Development of Innate Lymphoid Cells in Fetal and Adult Mice.

Flt3 ligand (Flt3L) promotes survival of lymphoid progenitors in the bone marrow and differentiation of dendritic cells (DCs), but its role in regulating innate lymphoid cells (ILCs) during fetal and adult life is not understood. By using Flt3L knockout and transgenic mice, we demonstrate that Flt3L controls ILC numbers by regulating the pool of α4β7(-) and α4β7(+) lymphoid tissue inducer cell progenitors in the fetal liver and common lymphoid progenitors in the bone marrow. Deletion of flt3l severely reduced the number of fetal liver progenitors and lymphoid tissue inducer cells in the neonatal intestine, resulting in impaired development of Peyer's patches. In the adult intestine, NK cells and group 2 and 3 ILCs were severely reduced. This effect occurred independently of DCs as ILC numbers were normal in mice in which DCs were constitutively deleted. Finally, we could show that administration of Flt3L increased the number of NKp46(-) group 3 ILCs in wild-type and even in Il7(-/-) mice, which generally have reduced numbers of ILCs. Taken together, Flt3L significantly contributes to ILC and Peyer's patches development by targeting lymphoid progenitor cells during fetal and adult life.

Innate Lymphoid Cells Are Depleted Irreversibly during Acute HIV-1 Infection in the Absence of Viral Suppression

Innate lymphoid cells (ILCs) play a central role in the response to infection by secreting cytokines crucial for immune regulation, tissue homeostasis, and repair. Although dysregulation of these systems is central to pathology, the impact of HIV-1 on ILCs remains unknown. We found that human blood ILCs were severely depleted during acute viremic HIV-1 infection and that ILC numbers did not recover after resolution of peak viremia. ILC numbers were preserved by antiretroviral therapy (ART), but only if initiated during acute infection. Transcriptional profiling during the acute phase revealed upregulation of genes associated with cell death, temporally linked with a strong IFN acute-phase response and evidence of gut barrier breakdown. We found no evidence of tissue redistribution in chronic disease and remaining circulating ILCs were activated but not apoptotic. These data provide a potential mechanistic link between acute HIV-1 infection, lymphoid tissue breakdown, and persistent immune dysfunction.

Abstract A01: Innate lymphoid cells (ILCs) in murine melanoma: A new paradigm in cancer immunity

Abstract Introduction: Innate lymphoid cells (ILCs), have emerged as novel important immune effector cells, playing a critical role also in tumor immune-surveillance. Recently, it was shown that the number of ILCs in patients with different types of cancer is significantly reduced after chemotherapy/radiotherapy as compared to healthy donors. However, so far, no data on ILC frequency, subset distribution and function in patients with melanoma are available. Therefore, we analyzed the frequency, subset distribution and function of ILCs in a murine model of melanoma with/without treatment, a parameter that is of paramount importance to understand the role of ILCs during melanoma and to assess the impact of current treatments on this type of cancer. Methods: C57/BL6 mice were injected with B16F10 murine melanoma cell line (1 x 106 cells/mouse, subcutaneous). Tumor growth monitored. Once tumors reached 12mm in size, the mice were sacrificed and tumor cells subjected to flow-cytometry and cell culture to identify the ILC subclasses in the tumor. Results: Our findings establish, for the first time in melanoma at diagnosis, base-line reference values for ILC frequency and function, as well as the ILC subtype distribution. Conclusion: Our studies extend previously published work on changes in innate lymphoid cells. Our findings will help better understanding of tumor microenvironment and its interaction with immune system. These values indicate the potential of ILCs as crucial players in the treatment of cancer, specifically melanoma. Citation Format: Blake Christianson, James Walker, Marsha Kocherla, Xu Qin, Jack C. Yu, Babak Baban. Innate lymphoid cells (ILCs) in murine melanoma: A new paradigm in cancer immunity. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A01.

Increased prevalence of lymphoid tissue inducer cells in the cerebrospinal fluid of patients with early multiple sclerosis

Background: Inflammatory cytokines produced by cells of the immune system are believed to play a central role in the pathogenesis of multiple sclerosis (MS). Innate lymphoid cells (ILCs) have been shown to produce and secrete a wide range of the cytokines involved in MS pathogenesis; however, a possible implication of ILCs in MS development and disease progression has not been investigated. Objective: With this study, we aimed to clarify a potential role of ILCs in the early stages of MS. Methods and Results: Using flow cytometry, we analysed the prevalence and phenotype of ILCs in the cerebrospinal fluid (CSF) of patients experiencing their first or second demyelinating event. We found a substantial increase in both frequency and number of ILCs, in particular the LTi subset, as compared to healthy controls. We also found an association between CSF pleocytosis and an increased frequency of LTi cells in the CSF, suggesting a favoured recruitment of blood derived LTi cells. Conclusion: Our data suggests a role for ILCs, and in particular the LTi subset, in the early stages of MS. This finding represents an important contribution to the understanding of early inflammation in MS, and adds new knowledge beneficial for future MS therapies.