Group 2 ILCs: A way of enhancing immune protection against human helminths?

Summary Group 2 innate lymphoid cells (ILC2s) play crucial roles in type 2 immune responses associated with allergic and autoimmune diseases, viral and helminth infections and tissue homoeostasis. Experimental models show that in helminth infections ILC2s provide an early source of type 2 cytokines and therefore are essential for the induction of potentially protective type 2 responses. Much of our knowledge of ILC2s in helminth infections has come from experimental mouse models with very few studies analysing ILC2s in natural human infections. In attempts to harness knowledge from paradigms of the development of protective immunity in human helminth infections for vaccine development, the role of ILC2 cells could be pivotal. So far, potential vaccines against human helminth infections have failed to provide effective protection when evaluated in human studies. In addition to appropriate antigen selection, it is apparent that more detailed knowledge on mechanisms of induction and maintenance of protective immune responses is required. Therefore, there is need to understand how ILC2 cells induce type 2 responses and subsequently support the development of a protective immune response in the context of immunizations. Within this review, we summarize the current knowledge of the biology of ILC2s, discuss the importance of ILC2s in human helminth infections and explore how ILC2 responses could be boosted to efficiently induce protective immunity.

schistosome-infected children and are restored to levels observed in children who are exposed to infection but remain uninfected following curative antihelminthic treatment. 11 Within this review, we will discuss the current knowledge of the biology, function and regulation of ILC2s, their "potential" importance in human helminth infections and possibilities of utilizing ILC2 to boost protective immune response induced following treatment and vaccination. This knowledge could inform helminth control efforts as calls for helminth vaccine development escalate in the light of global mandates such as "Sustainable Development Goal 3" advocating for eradication or elimination of helminth infection.

| THE BIOLOGY OF GROUP INNATE LYMPHOID CELLS
In mice, ILC2s were originally identified as a type 2 cytokine expressing cell subset, which could not be classified by conventional lineage markers for T cells, B cells, NK cells, macrophages, dendritic cells, neutrophils, eosinophils, basophils or mast cells, but expressed the common leucocyte antigen (LCA) CD45 and their morphology resembled those of typical lymphocytes. 1,2,12 Early studies identified the markers IL-17 receptor B, in combination with IL-17RA forming the IL-25 receptor, the IL-33 receptor (T1/ST2) with varying expression of the stem cell factor c-kit (CD117). 1,2 These innate lymphoid-like cells were given various names including nuocytes, 1 innate helper type 2 cells (Ih2) 2 or natural helper cells. 13 They were enriched in mesentery 13 and have been shown to express the common gamma chain (γ c , CD132)associated receptors CD25 (IL-2Rα) and CD127 (IL-7Rα). IL-7 be has been shown to play an essential role in the development and survival of ILC2s and ILC3s. [14][15][16] Human ILC2s were initially described by Mjosberg et al. 17 as being similar to murine ILC2s in lacking the expression of classical lineage defining markers, but being positive for the leucocyte marker CD45 and the IL-7Rα (CD127). In addition, human ILC2s express the "chemokine receptor homologous molecule expressed on TH2 cells" (CRTH2=CD294), 17 a marker well characterized for its expression on human CD4+ T H 2 cells, 18 the NK cell receptor NKR-P1A (CD161) 17 and ST2 19 (a member of the IL-1 family receptors), which is part of the IL-33 receptor complex. 20 A combination of these markers is frequently used for identifying human ILC2s as Lin-CD45+CD127+CRTH 2+CD161+(ST2+) 11,17,21-23 as we depict in the flow chart for analysing human ILC2 by flow cytometry (Figure 1).
Apart from the IL-7Rα, ILC2s express the IL-2Rα (CD25) 17 and both IL-2 and IL-7 are indispensable for the development, homoeostasis and activation of ILC2s. 13,24,25 The IL-7Rα chain forms a heterodimer with the "thymic stromal lymphopoietin" (TSLP) receptor 26 a further characteristic marker of human ILC2s. 25 TSLP is able to activate cytokine production by ILC2s, but works more efficiently in combination with IL-2 and has synergistic effects with IL-33. 25 IL-33 (or IL-1F11) is a IL-1 family member and acts via the IL-33 receptor. 20 Furthermore, IL-25 activates cytokines production by ILC2s signalling via the IL-25 receptor, a heterodimer of IL-17RB and IL-17RA. IL-25, IL-33 and TSLP can be seen as the classical ILC2 activating cytokines and often referred to as alarmins (alarm signals). Hematopoietic cells can produce alarmins, but the primary sources are nonhematopoietic cells. IL-33 is primary produced by endothelial and epithelial cells, [27][28][29] but can be released by macrophages 30 or dendritic cells. 31 In contrast, tuft cells, a subset of epithelial cells of the small intestine with previously more or less unknown function, were identified as a major source of IL-25, 32-34 which is required for ILC2 homoeostasis. The numbers of tuft cells increase significantly when exposed to intestinal parasites.
ILC2s express a variety of additional receptors involved in the activation and homoeostasis. Expression of the IL-4Rα (CD124) was shown in mice, and basophil-derived IL-4 can positively control ILC2s. 35 As IL-4 is secreted by ILC2s, IL-4 could potentially act as an F I G U R E 1 Identification of human ILC2s by flow cytometry as conducted in our studies. PBMCs were isolated from human peripheral blood and analysed by multifluorochrome-based flow cytometry. PBMCs were gated on leucocytes (A), single cells (B) and live cells using a viability dye (C). Live single cells were gated on lineage negative (CD3, CD14, CD16, CD19, CD20, CD56, CD123, CD11c, αβTCR γδTCR), CD45+ (D), CD127+ (E) and CD161+CRTH2+ cells (F), which finally leads to the identification of lin-CD45+CD127+CRTH2+CD161+ ILC2s autocrine feedback mechanism for activation of ILC2s. However, the exact role of IL-4 in controlling activation of human ILC2s is currently unknown. ILC2s are also the main source of IL-9, another common γ chain (γ c ) cytokine, 36,37 with expression of IL-9 receptor being essential for ILC2 activation, survival of activated ILC2s and finally for efficient helminth worm expulsion in mouse experimental models. 36 IL-9 released by lung resident ILC2s plays a central role in the epithelial response to murine N. brasiliensis infection by inducing IL-5 and IL-13 production. 38 Gene expression analyses indicated that the IL-9 receptor is expressed on murine ILC2s, and in humans, expression of this receptor has been shown on blood and lung ILC2s. 21 The CRTH2 is a crucial marker for the identification of human ILC2s 17 and for classical T H 2 cells. 18,39 The agonist for CRTH2 is prostaglandin (PG)D2, a well-characterized mediator of allergic asthma 40 released by activated mast cells. PGD2 is crucial for chemotaxis of T H 2 cells 41 and drives accumulation of ILC2s in inflamed tissues. 42 Murine ILC2s isolated from lymph nodes and the spleen, and to a less extent, ILC2s from the peritoneal or broncho-alveolar lavage, express major histocompatibility complex class-II (MHC-II) molecules.
They also express the co-stimulatory molecules CD80 and CD86. 43 Expression of MHC-II in combination with co-stimulatory molecules allows a direct interaction with CD4+ T cells and can drive CD4+ Tcell expansion and activation and T H 2 polarization and is important for efficient worm expulsion in murine infections of N. brasiliensis.
Accordingly, it had been demonstrated that human ILC2s isolated from peripheral blood express high levels of HLR-DR, CD80 and CD86. 43 Similar to all other immune responses, the function of ILC2s needs counter-regulation allowing control of their function. Type 1 and type 2 interferons can negatively regulate ILC2s, 23,44 and both types of interferons are long known to inhibit helminth-driven T H 2 responses. Additionally, ILC2s can be suppressed by IL-27 45 [32][33][34] Myeloid cells (dendritic cells (DC) or macrophages) can also release IL-33 and thereby activate ILC2s. 30,31 ILC2 activation is maintained and multiplied by IL-4 and IL-9 (acting in an autocrine manner) 36 and require IL-2 and IL-7 for homoeostasis and activation. ILC2s secrets type 2 cytokines upon activation. IL-5 induces eosinophilia, 139,151 and IL-4 triggers B cells and induces isotype switching to IgE. Furthermore, IL-13 can activate mucus secretion by goblet cells, 1,16,152 act on mast cells (potentially in conjunction with IL-9 152 ) and regulate DC migration. 153 IL-4 and IL-13 can also induce alternative activated macrophages (AAM). 154 ILC2s also secrete amphiregulin (Areg) important for tissue repair. 9 Furthermore, ILC2s interact with T H 2 CD4+ T cells (T H 2), which induces T H 2 immune response 43,155 and IL-2 secreted by T cells could further sustain ILC2 responses and further affect generation of T-cell memory, 156 which is altered in chronic helminth infections. 84 Helminth can induce regulatory T cells (Treg), which potentially can dampen the development of full protective immune response 118

| Common γ c cytokine receptors
Common gamma chain (γ c ) (CD132) cytokine receptors play a central role in the development, homoeostasis and function of several immune cell lineages and are indispensable for the immune system itself.
Therefore, it is not surprising that common γ c cytokines and the corresponding receptors are also essential for the development of ILC2s.
The IL-7Rα chain, forming a heterodimer with the common γ c (also known as common IL-2 receptor gamma chain), was one of the first surface receptors identified as marker for ILCs, and the development of ILC2s was depending on the common γ c and IL-7. 13 The IL-2Rα is also a marker human ILC2s and provides an important co-stimulatory signal for the activation of ILC2s. 25 Using a reporter mouse strain, ILC2s, rather than CD4+ T cells, were also identified as main source of IL-9 in a model of airway inflammation. 37 More importantly, IL-9 acts as feedback signal enhancing the cytokine production by ILC2s. The importance of IL-9 as a feedback signal was subsequently confirmed in experimental infection with N. brasiliensis, in which IL-9 receptor expressing ILC2 is important for restoring tissue damage caused by the lung stage of N. brasiliensis. 36,38 Hence, common γ c receptors play a pivotal in the development, maintenance and activation of ILC2s.
In T cells, common γ c signalling is mainly mediated by three While the importance of common γ c cytokine receptors for ILC2s is well described for mice, the precise signalling pathways controlling the development and function of human ILC2s remain to be investigated.

| LOCATION OF ILC2S AND THE IMPLICATION FOR HUMAN HELMINTH INFECTIONS
ILC2s have been identified in various tissues. Using reporter mice in experimental models of N. brasiliensis infection, ILC2s were identified in the spleen, liver, mesenteric lymph nodes, the intestine, fatassociated lymphoid clusters 1,2,13 and skin. 62 In humans, ILC2s have been described in nasal polyps, tonsils, gastrointestinal tract, peripheral blood 17,25 and the lung. 9,17 ILC2 are also described in human skin 46,63 with their migration to the skin being associated with PGD2, the ligand for CRTH2, 63 and the skin-homing marker cutaneous lymphocyte antigen. 64 Overall, mucosa-associated tissues of the lung, ILCs derive from a common lymphoid precursor in bone marrow 74 expressing the integrin α 4 β 7 , mediating migration to endothelial venues and mucosal tissues, and chemokine receptor CXCR6 mediating migration to the intestine. 75 Additionally, a lineage-specific precursor has been identified for ILC2s. 24,47 However, it has been suggested that ILC2s proliferate within tissues and are rarely replenished from the bone marrow. 45 More committed progenitors were also identified in secondary lymphoid organs. 76 Therefore, the contribution of circulating ILC2s from peripheral blood to tissue-resident ILC2 pool needs to be studied in more detail to allow interpretation of immuno-epidemiological data based on human blood, as theoretically, blood ILC2s may be important in blood residing pathogens including schistosomes.

| ILC2S IN HUMAN HELMINTH INFECTION-WHAT WE DO "NOT" KNOW
So far there are very few studies that have analysed ILC2s in natural human helminth infection, a fact, which is not surprising considering the history and biology of ILC2s outlined above. ILC2s constitute a small fraction of human blood leucocytes. In our studies in a Zimbabwean population, a mean of 0.031% (median 0.023%, range 0.003-0.133, N=72) of live-gated leucocytes was denoted as ILC2s, a proportion which is comparable to data published elsewhere 17 and data in Caucasians (unpublished data). Of note, ILC2s are hardly detectable in peripheral blood of naïve mice. 1,2 In humans, proportions are slightly higher in the skin, ileum, lung and tonsils compared to peripheral blood and are increased in inflamed tissues such as inflamed nasal polyps and skin lesions of patients with atopic dermatitis. 17 In a different study, we evaluated ILC2s in context of natural infection with S. haematobium in Zimbabwean children. 11 Schistosomeinfected children aged 6-13 years (as diagnosed by parasite egg excretion) had a significantly lower frequency of ILC2s in the peripheral blood compared to same-age schistosome-uninfected children ( Figure 3A). In contrast, older infected children (aged 14-18 years) had comparable levels of ILC2s to uninfected children. 11 Proportions of ILC2s recovered following curative antihelminthic treatment ( Figure 3B). Of note is the difference in these age groups; children are exposed to schistosome infection very young and therefore acquire infection at a young age. 82 By the time they reach adolescence, they

Modulation of this signalling pathway in T cells has been shown for
various infectious diseases including tuberculosis. 97 Whether and to which degree common γ c signalling is modulated in ILC2s in particular during helminth infection remains elusive. Furthermore, modulation of signalling via the IL-9R and TSLPR could be regulated and may provide molecular targets for chemoprophylaxis or therapy.
The impact of nutrition, particularly micronutrients on the immune system, is well established in experimental models. Micronutrient deficiency is widespread in helminth-endemic areas with vitamin A deficiency being one of the most common. Interestingly, work in experimental studies indicates that vitamin A deficiency is characterized by an increase in ILC2 cells and increased production of IL-13 by these cells to maintain mucosal barrier immunity to helminth infection under malnutrition. 98 In addition, recent work has also highlighted that ILC2 cells predominantly depend on fatty acid (FA) metabolism during helminth infection. 99 The vast majority of the world's malnour- with a combination of regulatory and T H 2 response. However, the type 2 cytokine IL-13 also contributes to hepatic fibrosis. 106 In mouse models, it has been shown that ILC2s are a likely source for IL-13 in hepatic fibrosis. 107 Hepatic IL-33 triggered the expansion and activation of liver-resident ILC2s, which produced IL-13 and mediated fibrosis. 107,108 In human intestinal schistosomiasis, the majority of patients develop a less severe form of the disease, but about 5%-10% suffer from hepatosplenic schistosomiasis with progressive fibrosis. 109 To what extent, hepatic ILC2s contribute to the development of severe forms of schistosomiasis remains to be investigated. Furthermore, the impact of environmental enteropathy, which affects gut permeability, exacerbated by helminth infections has yet to be investigated. 110

| THE POTENTIAL IMPACT FOR TREATMENT STRATEGIES AND SUCCESSFUL VACCINATION
The  [112][113][114][115] ). To do so, helminths utilize immunosuppressive and immunoevasive mechanisms, mediated through various mechanisms. For instance, the importance of regulatory T cells has been shown for filarial 116,117 and schistosome infections 118,119 and excretory-secretory products released by helminth parasites can directly induce regulatory T cells. 120 In the cases of schistosomiasis suppression of immune responses induced by worms can delay the development of protective immunity. 121 Mechanisms of how the host eventually manages to express a resistance phenotype have been a subject of our research, leading to the description of the threshold hypothesis 122 ; that is, the host needs to experience a threshold of antigens to mount an effective immune response and that these antigens become available following worm death. We and others have also demonstrated the requirement of the ratio of regulatory vs effector cellular immune to favour effector responses for expression of resistance. 118 However, the precise mechanism of the induction of a

| MODULATING ILC2 RESPONSES
Common gamma γ c cytokines and their receptors are crucial for homoeostasis and activation of ILC2s and therefore are potential targets to boost ILC2 responses thereby potentially increase the effectiveness of vaccinations or I&T approaches. IL-2 therapy has a long history in antitumour therapy, 133 and therapies with low-dose IL-2 are currently tested in autoimmune disease such as hepatitis C virus-related vasculitis 134 and type 1 diabetes. 135,136 Early on, it has been recognized that IL-2 therapy can lead to increased plasma levels of IL-5 and eosinophila 137,138 an effect that, at least in mouse models, is caused by an activation of ILC2s. 139 Side effects of low doses of IL-2 are considered to be relatively safe, but in the context of autoimmune diseases are used to expand regulatory T cells (reviewed in 140 ), which may contradict attempts to trigger a protective response in helminth infections.
However, with detailed investigations of treatment regimes regarding the dose and duration of the IL-2 therapy might help to tackle this problem. For instance, regulatory T cells may expand only after a few weeks of IL-2 therapy, whereas ILC2 activation may occur quicker in particular if incorporated in I&T approaches or if applied with vaccinations. IL-7, another common gamma γ c cytokine, is also considered for use in cancer [141][142][143] and chronic viral infections, 144 highlighting the potential in immunotherapies. This is particularly important in the carinogenic trematodes, S. haematobium, Opisthorchis viverrini and Clonorchis sinensis where one of the pathological manifestations of these infections is cancer in different organs (bladder, bile duct and liver) 145 for which we currently do not have any therapeutic interventions beyond surgery. As IL-7 is crucial for the development and homoeostasis of human ILC2s, its potential to increase responses mediated by ILC2s in vaccination and/or I&T protocols should be investigated.
Apart from the direct use of cytokines in immunotherapies, molecules crucial for the downstream signalling induced by these cytokine could be targeted. Interestingly, the effects by IL-7 in the study on chronic viral infections were partially mediated by repression of SOCS3. 144 Hence, targeting the JAK/STAT or the MAPK/Erk pathway including SOCS inhibitors may have the potential to increase ILC2 activation, but also T H 2 responses in general. 53,146 The main trigger of the ILC2 activity are the alarmins IL-25, IL-33 and TSLP, but their potential as activators in immunotherapy has not been investigated in detail. However, blocking alarmins has been considered for treating allergic diseases, 147-149 but has not really gone beyond experimental testing with only initial studies in human. 150 ILC2 targeting alarmins could be also used in combination with common γ c cytokines. Overall, specific modulation of ILC2 activity to improve vaccine or I&T-induced protective immune responses is an exciting idea.
Precise treatment strategies need to be carefully approved to avoid induction of regulatory T cells or to avoid the induction of allergic immune responses. Before attempting ILC2 targeting strategies to build up protective immune responses, much more work needs to be done on dissecting mechanisms and signalling pathways in ILC2s.