n BASIC-TRANSLATIONAL REVIEW

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1 n BASIC-TRANSLATIONAL REVIEW At the Bedside: The emergence of group 2 innate lymphoid cells in human disease R. Stokes Peebles Jr. Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA RECEIVED AUGUST 7, 2014; REVISED NOVEMBER 12, 2014; ACCEPTED NOVEMBER 13, DOI: /jlb.3BT R < SEE CORRESPONDING ARTICLE ON PAGE 455 ABSTRACT ILC2s have been primarily identified at environmentalmucosal interfaces and can be activated quickly by environmental antigens and pathogens to produce large quantities of IL-5 and IL-13. As a result of the production of these cytokines, ILC2s have been implicated in the host response to allergens, viruses, and parasites. However, the exact role of ILC2s in any human disease state is presently unknown, as specifically eliminating these cells is not possible, given that potentially targetable cell-surface markers are shared with other immune cells. Likewise, selectively and completely inhibiting ILC2 activation is also not currently possible, as several activating cytokines, IL-25, IL-33, and TSLP, act in redundancy or are not specific for ILC2 stimulation. Therefore, at this point, we can only identify the relative abundance of ILC2s in organs and tissue identified as being involved in specific diseases, and the contribution of ILC2s in human disease can only be inferred from mouse studies. Given these limitations, in this article, we will review the studies that have examined the presence of ILC2s in human disease states and speculate on their possible role in disease pathogenesis. The intent of the review is to identify priority areas for basic research based on clinical research insights. J. Leukoc. Biol. 97: ;2015. Introduction The discovery of ILC2s is one of the most exciting developments in allergic disease pathogenesis in many years. The events leading to the discovery of ILC2s and the basic biology of these cells are outlined in greater detail in the accompanying review article by Taylor Doherty. In brief, ILC2s are resident primarily in the lung, skin, and gut mucosal surfaces [1 5]. ILC2s from these sites have been activated by the cytokines IL-25, IL-33, and TSLP, produced Abbreviations: BAL = bronchoalveolar lavage, CLA = cutaneous lymphocyte antigen, CRS = chronic rhinosinusitis, cyslt1 = cysteinyl leukotriene receptor 1, DP2 = chemoattractant receptor-homologous molecule expressed on Th2 lymphocytes, GvHD = graft versus host disease, HSCT = hematopoietic stem cell transplantation, ILC2 = group 2 innate lymphoid cell, IPF = idiopathic pulmonary fibrosis, KLRG1 = killer cell lectin-like receptor G1, lin 2 = lineage negative, PSP = primary spontaneous pneumothorax, TSLP = thymic stromal lymphopoietin by airway epithelium and other immune cells, most likely in response to danger-associated molecular patterns, such as allergens and infections with viruses or helminths [2 5]. After activation, ILC2s produce large quantities of IL-5 and IL-13, cytokines also produced by CD4 + Th2 cells, hence, the name ILC2s [6]. In regard to allergic disease, IL-5 is the most important eosinophil chemotactic, activating, and survival factor, whereas IL-13 is a central mediator of airway epithelial cell mucus metaplasia and airway responsiveness [7]. As a result of their elaboration of these cytokines, ILC2s have been shown to make important contributions in mice to allergen-induced inflammatory responses and anti-helminth defense, for which the immune response is somewhat similar to allergic disease [8, 9]. However, the contribution of ILC2s to allergy and defense against parasites in humans is, at this point, unknown, as there is no way of specifically depleting ILC2s in people. Therefore, the exact role of ILC2s in human allergic diseases can only be inferred at this time. In this article, we will review data that suggest that ILC2s may have a role in human allergy and also examine reports of the presence of ILC2s in other diseases. The exact role of ILC2s in any human disease state is presently unknown, as eliminating these cells specifically is not possible, given that potentially targetable cell-surface markers are shared with other immune cells DEFINING HUMAN ILC2 ILC2s are lin 2, meaning that they do not express receptors that define T cells, B cells, NK cells, NK T cells, dendritic cells, macrophages, mast cells, and basophils. In humans, these cells are excluded with the use of flow cytometric or magnetic bead strategies by use of antibodies against CD3, CD4, CD8, CD14, CD19, CD56, CD11c, CD11b, Fc«RI, TCR-gd, TCR-ab, and CD123. There are differences between investigative groups in regard to cellsurface markers that define human ILCs. Many groups define human ILC2 as expressing CD25 (IL-2Ra), CD127 (IL-7Ra), ST2 (IL-33R), the PGD 2 R DP2, or a combination of these markers. 1. Correspondence: T-1218 Medical Center North, Vanderbilt University Medical Center, st Ave., South, Nashville, TN , USA. stokes.peebles@vanderbilt.edu /15/ Society for Leukocyte Biology Volume 97, March 2015 Journal of Leukocyte Biology 469

2 PGD 2 is particularly important in allergic diseases, as it is the primary PG produced by mast cells, a cell that is critical in driving immediate-type hypersensitivity responses. Other cells that produce PGD 2 in the airway during allergic inflammation include macrophages and eosinophils. The role of PGD 2 in human ILC2 function will be discussed at length in the Asthma and Allergic rhinitis below. ILC2s are lin 2, meaning that they do not express receptors that define T cells, B cells, NK cells, NK T cells, dendritic cells, and macrophages. ASTHMA Asthma is a chronic inflammatory disorder of the airways [10]. The inflammation in asthma results from airway epithelial cell injury and mast cell activation, which produce an influx of lymphocytes, eosinophils, and neutrophils into the airway wall and lumen. This airway inflammation is conjectured to be critical in the cardinal features of the disease, which include airway responsiveness, airway mucus, and limitation of air flow, leading to symptoms of chest tightness and shortness of breath. Many persons with asthma have allergic disease, yet viral infections are a common trigger for exacerbations. A common finding in people who have asthma and in animal models of this disease is the presence of cytokines, such as IL-5 and IL-13, in the airways. The relationship of ILC2 with inflammatory cells that are involved in asthma pathogenesis is unknown. Do ILC2s have a role in maintenance of mast cells and basophils or in the differentiation of CD4 + cells toward the Th2 pathway? Alternatively, do the products of these inflammatory cells have an important function in the proliferation and function of ILC2? IL-5 and IL-13 are produced in large quantities by ILC2s and have been identified in fetal and adult lungs, as well as in the BAL from recipients of transplanted lungs, suggesting that these cells could possibly have a pathophysiologic role in lung diseases, such as asthma [4, 5]. Barnig and colleagues [11] examined the presence of ILC2s in the peripheral blood of subjects stratified based on asthma severity and were defined as lin 2 CD127 + DP2 +.Therewasno difference in the percentage of lin 2 CD127 + DP2 + cells between healthy subjects ( %), mild asthmatics ( %), and severe asthmatics ( %). As DP2 was expressed on the surface of ILCs, the ability of ILC2s to produce IL-13 in response to PGD 2 was tested. PGD 2 induced IL-13 production by ILC2s by 73%, which was comparable with stimulation with IL-25 and IL-33. When ILC2s were cultured with PGD 2 in combination of IL-25 and IL-33, there was a synergistic increase in ILC2 production of IL-13 by 500%. Further studies revealed that the ILC2 increase in IL-13 mediated by PGD 2 was specific fordp2andnotdp1. The mechanism by which there was a synergistic increase in ILC2 production of IL-5 and IL-13 when PGD 2, IL-33, and IL-25 were added to ILC2 culture is likely explained by the findings of Xue and colleagues [12], who reported that PGD 2 signaling through DP2 increased human ILC2 expression of the IL-33R subunit (ST2) and the IL-25R subunit (IL-17RA). Thus, the increased IL-33R and IL-25R expression driven by PGD 2 likely heightened sensitivities to those cytokines. In these experiments, ILC2s were obtained from human skin and peripheral blood and were defined as lin 2 CD45 high CD127 + DP2 +. These investigators also reported that PGD 2 and IL-33 promoted ILC2 migration in a dose-dependent manner, peaking at 100 nm for PGD 2 and 30 ng/ml for IL-33, with PGD 2 having an almost 5-fold greater effect on ILC2 migration compared with IL-33. The effect of PGD 2 was mediated through DP2 signaling, as it was blocked by the DP2 antagonist TM IL-25 had almost no effect on ILC2 migration. PGD 2 also induced human ILC2 production of IL-4, IL-5, and IL-13 to a greater degree than IL-33 or IL-25, and PGD 2, in combination with IL-25, synergistically increased ILC2 cytokine production. Whereas ILC2s are defined by their production of Th2-like cytokines, PGD 2 also induced ILC2 production of IL-3, IL-8, IL-9, IL-21, GM-CSF, and CSF-1 in a dose-dependent manner. The concept that ILC2s could produce IL-8, the most important cytokine in neutrophil activation; chemotaxis; and survival is important, as this would be an important distinction between ILC2s and Th2 cells, which do not produce this cytokine. IL-17A, IL-17F, and IFN-g were not produced by ILC2s in these experiments. The effects of PGD 2 on ILC2 migration and cytokine production could be recapitulated when supernatants from mast cells that had been activated through Fc«RI were added to the culture, and this was blocked by a DP2 antagonist, confirming that human mast cell-derived PGD 2 triggers ILC2s through DP2. Interestingly, montelukast, a cyslt1 antagonist, also inhibited ILC2 cytokine production from mast cell supernatants, suggesting that human ILC2s express cyslt1 and might be activated by a cyslt, similar to mouse ILC2s [13]. What is the role of other PGs in ILC2 function? Whereas PGD 2 augments ILC2 cytokine secretion, do PGs that increase camp, such as PGI 2, inhibit ILC2 function? The understanding of the effects of other prostanoids, such as PGE 2,PGF 2a, and thromboxane A 2,in regulating ILC2 activity will be important in developing therapeutics targeting this cell type. Barnig and colleagues [11] also reported that ILC2s also expressed the lipoxin A4R, termed ALX/formyl peptide receptor 2, and the resolvin E1R, chemokine-like receptor 1. Lipoxin A4 and resolvin E1 have been implicated in the resolution of inflammation after allergen challenge in mouse models [14, 15]. Lipoxin decreased IL-13 release from human ILC2s, stimulated with PMA plus a divalent cation ionophone (A23187). Lastly, this group reported that ILC2s were present in human lungs. By immunofluorescence microscopy, they defined ILC2s as CD117 + CD161 + tryptase cells to distinguish ILC2s from mast cells that do produce tryptase. CD161 is a NK cell marker. There was no description as to whether the lungs examined were from healthy controls or asthma, so there is no information as to whether there is an increase in ILC2s in asthmatics compared with normal subjects. PGD 2 is an important stimulus for ILC2 migration and cytokine production. The fact that ILC2s produce IL-5 and IL-13 suggests that these cells could be important in asthma pathophysiology, but the exact role of ILC2 in asthma pathogenesis is because ILC2 cannot be specifically depleted. 470 Journal of Leukocyte Biology Volume 97, March

3 BASIC-TRANSLATIONAL REVIEW Peebles At the Bedside: ILC2 in human disease ALLERGIC RHINITIS Patients with rhinitis have at least 1 of the following symptoms: nasal congestion, rhinorrhea, sneezing, and itching [16]. Rhinitis is most commonly allergic in nature, and the inflammation resulting from an environmental exposure that causes symptoms predominantly consists of mast cells, eosinophils, and lymphocytes, although neutrophils may also be present. Doherty and colleagues [17] examined whether the number of peripheral blood ILC2s changed with nasal cat-allergen challenge in subjects who had rhinitis symptoms on exposure to cats and who were skin prick-test positive to this allergen. In this study, subjects were first challenged to diluent intranasally and then returned 2 12 weeks later for intranasal cat-allergen challenge. Peripheral blood was obtained to assess the number of ILC2s present before and 4 h after the intranasal diluent and cat-allergen challenges. ILC2s were defined as lin 2 DP2 +. As part of this study, the investigators also sought to determine whether novel phenotypic markers of ILC2s could be identified. They found that ILC2s also expressed CD84 at a significantly higher per-cell level that did Th2 cells isolated from allergic subjects. CD84 had been defined previously as a CD2 signaling lymphocyte activation molecule that was involved in T lymphocyte B lymphocyte cognate interactions [18]. Following diluent challenge, there was no change in the percentage of ILC2s in peripheral blood from baseline ( vs ); however, 4 h after cat-allergen challenge, there was a significant increase in the percentage of ILC2s ( vs ) [17]. Therefore, cat-allergen challenge promoted an increased number of peripheral blood ILC2s. The reason for this increase was not defined in this study; however, possible mechanisms include ILC2 recruitment from the bone marrow or that ILC2s migrate from the nasal mucosa into the blood. Another possibility is that PGD 2, which is newly formed by mast cells following allergen challenge, might be acting through DP2 to increase the migration of ILC2s into the blood and eventually to the nasal tissue, where the PGD 2 was released by mast cell degranulation. Chang and colleagues [19] reported that ILC2s from allergic subjects had increased chemotaxis and chemokinesis to PGD 2 compared with PGD 2 -mediated migration, exhibited by ILC2s from healthy subjects. Interestingly, ILC2 migration to PGD 2 exhibited a dose-responsive relationship within a 5 to 25 nm range for cells from allergic donors and healthy controls; however, the magnitude of migration was greater from ILC2s from allergic donors compared with healthy controls. CRS Cat-allergen challenge increased the number of peripheral blood ILC2s in subjects who had rhinitis symptoms on exposure to cats and who were skin prick-test positive to cat. The effect of current rhinitis medications on the number and function of ILC2 in this disease state is unknown. CRS occurs when the epithelial lining of the nose and sinuses is inflamed for.12 weeks [16]. Often, but not universally, patients with CRS will have nasal polyps. The mechanisms behind the development of nasal polyps are unknown. Mjösberg and colleagues [4] examined the presence of ILC2s in the nasal polyps of subjects with CRS. Investigation of the presence of ILC2s in polyps was spurred by the high levels of IL-5 and IL-13 and the large number of eosinophils in these tissues. Nasal polyps from CRS subjects contained larger proportions of ILC2s, defined as lin 2 CD127 + CD161 + DP2 +, compared with nasal tissue from healthy nonallergic donors, suggesting that ILC2s may contribute to the pathophysiology of CRS with nasal polyps. ILC2s, isolated from nasal mucosa, responded to IL-25 and IL-33 stimulation with heightened production of IL-13 [20]. Nasal tissue from healthy subjects and from those with CRS with nasal polyps produced comparable amounts of IL-33 and TSLP mrna. In vitro, TSLP induced ILC2 production of IL-13, and this was increased synergistically when TSLP and IL-33 were added in combination, with the finding that TSLP induced the expression of IL-33R subunit ST2. IL-25, particularly in concert with IL-33, induced significant ILC2 production of IL-13. The combination of IL-25 and TSLP also synergistically increased ILC2 production of IL-13 but not to the same level that was seen when TSLP was combined with IL-33. Shaw and colleagues confirmed that the number of ILC2s, defined as lin 2 CD117 + CD127 +, was increased in the nasal mucosal tissue from subjects with CRS with nasal polyps compared with CRS without nasal polyps [21]. They found significant induction of IL-33 when epithelial cells derived from subjects with CRS with nasal polyps were stimulated with Aspergillus fumigatus extract compared with IL-33 produced by epithelial cells from subjects that had CRS without nasal polyps. These results suggested that the ability of epithelial cells to generate IL-33 might control the number of ILC2s in the nasal tissue, which in turn, regulated the amount of tissue IL-5 and IL-13. Miljkovic and colleagues [22] extended these findings by characterizing the relationship between ILC2s and both CD4 + Th2 cells and the cytokines produced by these cells in subjects with CRS. In this study, they defined ILC2s as lin 2 CD127 + CD161 + DP2 + CD They created single-cell suspensions from sinonasal tissues and performed flow cytometry to assess the ILC2 and CD4 + Th2 cell populations. They found that sinonasal tissue from CRS with nasal polyps had a significantly greater percentage of ILC2s (0.0569% of CD45 + cells) compared with CRS without nasal polyps (0.0260% of CD45 + cells) or control subjects who had nasal mucosal tissue biopsied during pituitary surgery (0.0256% of CD45 + cells). The subjects were also stratified for their allergic status based on serology or skin testing. Although the difference did not reach statistical significance (P =0.07),therewas a numerical increase in the percentage of ILC2s in allergic subjects (0.051% of CD45 + cells) compared with nonallergic subjects (0.028% of CD45 + cells). Interestingly, there was no correlation between ILC2s and CD4 + Th2 cells in the nasal tissue biopsies. The number of ILC2s is increased significantly in the nasal tissue of persons with CRS and nasal polyps compared with healthy controls; however, it is not known how ILC2s modulate CRS pathogenesis. ATOPIC DERMATITIS Atopic dermatitis is characterized by intensely pruritic, erythematous papulovesicular lesions of the skin that usually present Volume 97, March 2015 Journal of Leukocyte Biology 471

4 during early infancy but can persist or start during adulthood [23]. Because of the intense, pruritic nature of the condition, the patient scratches his or her skin, often leading to excoriation, and with a resulting serous exudate. Atopic dermatitis may often be the first manifestation of allergic disease in patients who eventually have another allergic disorder, such as asthma, rhinitis, or food allergy. IL-5- and IL-13-producing cells are often found in the lesional skin of patients with atopic dermatitis, implicating cells that produce these cytokines as being pathogenic in the disease. Kim and colleagues [24] identified ILC2s, defined as lin 2 CD25 + ST2 +, in skin tissue from both healthy subjects and in lesions from subjects with atopic dermatitis. They found that there were significantly more ILC2s in lesional atopic dermatitis skin than in skin from healthy controls. The skin from the lesions from atopic dermatitis subjects expressed DP2 and CD161, whereas these markers were not expressed by the lin 2 CD25 + ST2 + ILC2s from healthy subjects. These results suggest that the ILC2s in the lesional skin from atopic dermatitis subjects are a distinct population of ILC2s or are in a different activation state from healthy skin. Salimi and colleagues [25] investigated ILC2 biology in skin biopsies and peripheral blood of humans, with and without atopic dermatitis. In this study, they defined ILC2s as lin 2 CD45 high CD127 + DP2 +. First, these investigators sought to determine whether there were differences in the blood and skin compartments in ILC2 phenotype or function. ILC2s were enriched in normal human skin compared with the blood ( % vs %,respectively).Inaddition,the transcript levels of the amphiregulin gene were greater in skin ILC2s compared with blood ILC2s, indicating that there is differential regulation in the activation state of ILC2s depending on the anatomic site. They next sought to determine if there were differences in ILC2 number between subjects with atopic dermatitis compared with healthy controls.thereweresignificantly more ILC2s detected in the lesional skin of subjects with atopic dermatitis compared with the skin of healthy individuals, yet there was a similar frequency of circulating ILC2s in the peripheral blood of both groups. In the skin of atopic dermatitis subjects, there was an increase in the ILC2 mrna expression of subunits of the receptors for IL-33 (ST2), IL-25 (IL-17BR), and TSLP (TSLPR) compared with healthy individuals. The ILC2 mrna expression of these cytokine receptors was also increased in the lesional skin from atopic dermatitis subjects compared with nonlesional skin. IL-33 and IL-25 gene expression was increased in the lesional skin of atopic dermatitis subjects, suggesting that these cytokines might have a role in ILC2 activation at this anatomic site. Indeed, IL-33 stimulated ILC2 production of IL-5, IL-6, and IL-13 protein but little IL-4 and no IL-17A or IL-22. In contrast, IL- 25 failed to induce IL-13 production by skin-derived ILC2s. In addition, IL-33, but not IL-25, induced ILC2 migration in a transmigration assay. Only very high concentrations of TSLP induced ILC2 migration in vitro. ILC2s from freshly isolated atopic dermatitis lesional skin also expressed the KLRG1 to a greater level than skin from healthy controls. KLRG1 binds to the cell-adhesion molecule E-cadherin, whichisexpressedathighlevelsonkeratinocytesand Langerhans cells, and consequently inhibits cell proliferation. Interestingly, E-cadherin expression is decreased in the skin of patients with atopic dermatitis; therefore, loss of E-cadherin expression might reduce the expression of a restraint on ILC2 cytokine production. To test this possibility, this group activated ILC2s in the presence or absence of plate-bound E-cadherin, which inhibited IL-5 and IL-13 protein expression by ILC2s activated by IL-33 and IL-25. Thus, when E-cadherin is decreased in atopic dermatitis, an inhibitory signal on ILC2 function is reduced or lost, thus representing a mechanism for increased allergic inflammation as a result of reduced skin-barrier sensing. To determine the in vivo relevance for these in vitro findings, the authors used a suction blister model to deliver house dust mite to the subepidermis as a model of allergenprovoked skin. Twenty-six hours after house dust mite or vehicle administration, there was a significant increase in ILC2s in the fluid from the blister site that had been challenged with house dust mite. This increase in ILC2s after house dust mite challenge was associated with an increase in IL-4, IL-5, and IL-13 protein levels in the blister fluid of the atopic individuals compared with the healthy controls. Allergen challenge in the skin to an allergen to which a person has hypersensitivity increases the number of ILC2s at the site of challenge. However, the extent to which ILC2s play a role in allergic skin diseases is undefined. OTHER DISEASES PSP is an abnormal accumulation of air in the pleural space, which is the area between the parietal pleura, which lines the inner surface of the chest wall, and the visceral pleura, the pleura that covers the lungs. One of the characteristic features of pleural fluid obtained from patients experiencing PSP is the presence of eosinophils. The mechanisms by which eosinophils appear in the pleural fluid as a result of PSP are incompletely understood. Whereas IL-5 was found to be increased in the pleural fluid of subjects with PSP, the source of IL-5 was not determined [26]. Kwon and colleagues [27] found that neither IL-5-producing CD4 + nor CD8 + T cells were increased in the pleural fluid from subjects with PSP compared with control subjects. However, there was a significant correlation between IL-33 levels in pleural fluid and the concentrations of both IL-5 and eotaxin, as well as with the number of eosinophils in the pleural fluid. Based on the fact that IL-33 activates ILC2s to secrete IL-5, the percentage of ILC2s in pleural fluid from both control subjects with those with PSP was determined. In subjects with PSP, there was also a significant increase in the percentage of ILC2s in pleural fluid (average ;20% with a range from undetectable to ;60%) compared with an almost completely undetectable percentage of ILC2s in the pleural fluid of control subjects. 472 Journal of Leukocyte Biology Volume 97, March

5 BASIC-TRANSLATIONAL REVIEW Peebles At the Bedside: ILC2 in human disease Furthermore, both IL-33 and pleural fluid of patients with PSP induced ILC2 production of IL-5. These results suggest that the increase in IL-33 that occurs with PSP may induce IL-5 production directly from ILC2s in the pleural cavity, leading to the migration of eosinophils and eosinophilic pleural effusion. There is an increase in IL-33 and ILC2 in the pleural fluid of subjects with PSP compared with controls. The relationship between ILC2 and the pleural fluid eosinophilia seen in this condition has yet to be defined. Pulmonary fibrosis Pulmonary fibrosis is a chronic, progressive disease in which there is fibrosis of the lungs [28]. This disease predominantly occurs in older adults and is limited to the lungs. The inflammation is usually mild in nature and most often consists of lymphocytes and plasma cells that occur in association with hyperplasia of type 2 pneumocytes and the bronchiolar epithelium. There is no clear, causative etiology for the development of this disease. Hams and colleagues [29] reported that they identified ILC2s in the BAL and lung tissue of subjects with IPF. They found that there was a significant increase in IL-25 in the BAL of subjects with IPF compared with control subjects who had early-stage erythema nodosum without fibrosis. In the subjects with IPF, the BAL concentration of IL-25 further increased, 1 year after the initial sampling. Th1, Th2, and Th17 cytokines were not different in the BAL from IPF subjects compared with controls at the initial measurement but were significantly increased 1 year later. There was a significant increase in ILC2s in BAL from IPF subjects compared with controls, and ILCs were defined as lin 2 DP2 + ST2 + CD45 + ICOS + IL- 7Ra + IL-17RB +. ILC2s secrete a protein known as amphiregulin that is involved in the normal wound-repair process. Perhaps dysregulation of ILC2 production of amphiregulin may be part of the pathophysiology of IPF, although this has not been studied in people. There was a significant increase in IL-25 and ILC2 in the BAL of subjects with IPF compared with control subjects who had early-stage erythema nodosum without fibrosis. The determination of whether ILCs have a role in IPF pathogenesis might lead to a treatment option for a disease that currently has no effective ones. Psoriasis Psoriasisisaninflammatory skin disease that often relapses and remits [30]. The main classifications of psoriasis include plaque psoriasis, guttate psoriasis, and pustular psoriasis. Plaque psoriasis is characterized by demarcated red, scaly plaques. Guttate psoriasis is an acute eruption of small papules that may result following a streptococcal infection. Pustular psoriasis consists of pustules that may be localized or generalized. Teunissen and colleagues [31] found ILC2s in skin from both healthy subjects and in skin from subjects with psoriasis. In this study, they defined ILC2s as lin 2 CD127 + DP2 + and found that the enzymes used to prepare dermal-free cell suspensions dispase II and collagenase did not affect the cell-surface expression of CD127 or DP2. Skin ILC2s highly expressed CD161 and had variable expression of CD117. ILC2s produced IL-13 in response to stimulation with IL-33 plus TSLP. These investigators were able to produce ILC2 cell lines, which maintained expression of DP2, CD127, and CD117, yet no markers for T lymphocytes or NK cells. Whereas Mjösberg and colleagues [20] reported that ILC2s isolated from polyps and peripheral blood had synergistically enhanced IL-13 production to IL-33 and TSLP, Teunissen and colleagues [31] found that ILC2s in the skin were regulated primarily by TSLP and IL-25, with IL-33 not as important. This is in contrast to IL-33 and IL-25 being the predominant ILC2-inducing cytokines when ILC2s are obtained from lesional skin of atopic dermatitis subjects [25]. Furthermore, in contrast to peripheral blood and polyp ILC2s, ILC2s from skin did not produce IL-4, IL-9, or GM-CSF. ILC2s isolated from the peripheral blood from subjects with psoriasis expressed the CLA, a surface molecule important in skin homing. There was no difference in the proportion of ILC2s in theskinofhealthysubjectsandinlesionalskinfromsubjects with psoriasis. ILC2s isolated from the peripheral blood from subjects with psoriasis expressed the CLA. The mechanism by which ILC2s migrate to the skin still needs to be defined. HSCT Munneke and colleagues [32] examined the longitudinal effects of induction chemotherapy, conditioning radiotherapy, and allogeneic HSCT on ILC2 number and function (Tables 1 and 2). They found that induction chemotherapy significantly reduced the number of ILC2s and that the recovery of ILC2s was slow compared with the recovery of other innate immune cells, such as neutrophils and monocytes. Patients who developed less GvHD after HSCT had higher proportions of circulating, activated ILC2s after induction chemotherapy and before HSCT, suggesting that these cells may have a protective effect against conditioning therapy-associated tissue damage, preventing the development of GvHD. One possible explanation for this finding is that ILC2s produce amphiregulin, a member of the epidermal growth factor family that aids tissue repair. However, the role of amphiregulin in the protection against GvHD was not examined in this study. The authors suggest that their results may warrant providing patients who undergo allogeneic HSCT with in vitro-expanded ILC2s to determine definitely if ILC2s protect against GvHD. ILC2s have been identified in the pleural fluid of persons with spontaneous pneumothorax, the lung tissue of patients with pulmonary fibrosis, the skin of patients with psoriasis, and the blood of patients who had undergone allogeneic HSCT. However, the contribution of ILC2s to any of these diseases states has not been proven. Volume 97, March 2015 Journal of Leukocyte Biology 473

6 TABLE 1. Identification of ILC2 in disease states Disease Tissue compartment Comments Phenotypic marker used Reference Asthma Blood ILC2 present in asthmatics lungs lin 2 CD117 + CD161 + tryptase 2 [11] Healthy Skin and blood PGD 2 promoted ILC2 migration lin 2 CD45 high CD127 + DP2 + [12] CRS Nasal polyps ILC2 in nasal polyps. normal tissue lin 2 CD127 + CD161 + DP2 + [4] CRS Nasal polyps ILC2 in nasal polyps. normal tissue lin 2 CD117 + CD127 + CRS Nasal polyps ILC2 in nasal polyps. normal tissue lin 2 CD127 + CD161 + DP2 + CD123 2 [21] Allergic rhinitis Blood Allergen challenge increased ILC2 lin 2 DP2 + [17] Allergic rhinitis Blood Increased DP2 chemotaxis in lin 2 CD127+DP2+ [19] allergics Atopic dermatitis Skin Increased in lesional skin lin 2 CD25 + ST2 + CD161 + DP2 + [24] Atopic dermatitis Skin Allergen increased ILC2 in skin lin 2 CD45 high CD127 + DP2 + [25] Pneumothorax Pleural fluid Increased ILC2 compared with lin 2 CD45 + CD117 + DP2 + ST2 + [27] controls Pulmonary fibrosis Lung and BAL ILC2 present lin 2 DP2 + ST2 + CD45 + ICOS + CD127 + [29] Psoriasis Skin and blood ILC2 in blood expressed CLA lin 2 CD127 + DP2 + [31] Stem cell transplant Blood ILC2 may protect against GvHD lin 2 CD127 + [32] SUMMARY Clinical review for basic researchers Whereas ILC2s are potent producers of cytokines that are critical in the pathophysiology of allergic disease, the importance of ILC2s to human allergic disease pathogenesis is unknown, as there is no method currently available to eliminate or inhibit specifically the function of these cells without also directly targeting other effector cells. Until such techniques are developed, the contribution of ILC2s to any disease state can only be inferred indirectly. There are currently clinical trials underway that block cytokines, such as TSLP, that are important in activating ILC2s; however, such cytokines also have important immunostimulatory effects on other cells types as well. As animal models have shown that ILC2s may amplify allergic responses through their interaction with CD4 + Th2 cells, the therapeutic potential of effective inhibition of ILC2s for allergen-induced diseases is great. Now, we just need the means to accomplish this task. TABLE 2. Research questions 1. The exact role of ILC2s in any human disease state is presently unknown, as eliminating these cells specifically is not possible, given that potentially targetable cell-surface markers are shared with other immune cells. 2. The relationship of ILC2 with inflammatory cells that are involved in asthma pathogenesis is unknown. Do ILC2s have a role in maintenance of mast cells and basophils or in the differentiation of CD4 + cells toward the Th2 pathway? Alternatively, do the products of these inflammatory cells have an important function in the proliferation and function of ILC2? 3. What is the role of other PGs in ILC2 function? Whereas PGD 2 augments ILC2 cytokine secretion, do PGs that increase camp, such as PGI 2, inhibit ILC2 function? The understanding of the effects of other prostanoids, such as PGE 2, PGF 2a, and thromboxane A 2,in regulating ILC2 activity will be important in developing therapeutics targeting this cell type. 4. PGD 2 is an important stimulus for ILC2 migration and cytokine production. The fact that ILC2s produce IL-5 and IL-13 suggests that these cells could be important in asthma pathophysiology, but the exact role of ILC2 in asthma pathogenesis is because ILC2 cannot be specifically depleted. 5. Cat-allergen challenge increased the number of peripheral blood ILC2s in subjects who had rhinitis symptoms on exposure to cats and who were skin prick-test positive to cat. The effect of current rhinitis medications on the number and function of ILC2 in this disease state is unknown. 6. The number of ILC2s is increased significantly in the nasal tissue of persons with CRS and nasal polyps compared with healthy controls; however, it is not known how ILC2s modulate CRS pathogenesis. 7. Allergen challenge in the skin to an allergen to which a person has hypersensitivity increases the number of ILC2s at the site of challenge. However, the extent to which ILC2s play a role in allergic skin diseases is undefined. 8. There is an increase in IL-33 and ILC2 in the pleural fluid of subjects with PSP compared with controls. The relationship between ILC2 and the pleural fluid eosinophilia seen in this condition has yet to be defined. 9. There was a significant increase in IL-25 and ILC2 in the BAL of subjects with IPF compared with control subjects who had early-stage erythema nodosum without fibrosis. The determination of whether ILCs have a role in IPF pathogenesis might lead to a treatment option for a disease that currently has no effective ones. 10. ILC2s isolated from the peripheral blood from subjects with psoriasis expressed the CLA. The mechanism by which ILC2s migrate to the skin still needs to be defined. 11. ILC2s have been identified in the pleural fluid of persons with spontaneous pneumothorax, the lung tissue of patients with pulmonary fibrosis, the skin of patients with psoriasis, and the blood of patients who had undergone allogeneic HSCT. However, the contribution of ILC2s to any of these diseases states has not been proven. 474 Journal of Leukocyte Biology Volume 97, March

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