- Email: [email protected]
Immunological Memory of Group 2 Innate Lymphoid Cells
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Review
Immunological Memory of Group 2 Innate Lymphoid Cells Itziar Martinez-Gonzalez,1,2 Laura Mathä,2,3 Catherine A. Steer,2,3 and Fumio Takei1,2,* Immunological memory has long been described as a property of the adaptive immune system that results in potent responses on exposure to an antigen encountered previously. While this definition appears to exclude cells that do not express antigen receptors, recent studies have shown that innate immune cells, including natural killer (NK) cells, macrophages, and, more recently, group 2 innate lymphoid cells (ILC2s) can record previous activations and respond more vigorously on reactivation. Here we review the similarities and differences between these forms of memory and the underlying mechanisms. Based on these insights, we propose to revise the definition of immunological memory, as the capacity to remember being previously activated and respond more efficiently on reactivation regardless of antigen specificity.
Trends Allergen-experienced group 2 innate lymphoid cells remember previous activation and respond strongly to secondary challenge in an antigen-nonspecific manner. Natural killer cells have both antigenspecific and antigen-nonspecific memory. Macrophages can be trained to become highly responsive to secondary infection. Innate memory and adaptive T cell memory have common key features.
Immunological Memory In immunological memory (see Glossary), information (antigen encounter) is encoded in antigen-stimulated naïve cells and stored by long-lived memory cells. On re-encounter with the same antigen, memory cells recall the information and respond more rapidly and effectively than naïve cells. Therefore, antigen specificity has long been considered an essential feature of immunological memory [1–3]. Although immunological memory is thought to be a hallmark of adaptive immunity, discoveries with memory NK cells have shown that innate lymphocytes also have antigen-specific memory [4]. Moreover, recent studies have suggested that macrophages have some features of memory, termed ‘trained immunity’, that have antigen specificity. More recently, we have shown that ILC2s can remember being previously activated and respond more efficiently during secondary encounters with allergens. Unlike T cells, ILC2s do not recognize antigens but rather are activated by cytokines, and therefore their memory is antigen nonspecific. Nevertheless, ILC2s show an enhanced recall response similar to T cells. Here, by focusing on recent evidence for ILC2 memory and comparing the underlying mechanisms for the different forms of memory outlined above, we propose to expand the concept of immunological memory to include any cell that can remember a previous state of activation and mount a more efficient response during secondary challenges.
ILC2 Memory ILC2s belong to the family of ILCs (Box 1), which are thought to be tissue-resident lymphocytes. ILCs develop early in life from common progenitors and seed various tissues [5]. In naïve adult mice, ILC2s are found in the lung, skin, small intestine, and adipose tissue, while they are very rare in the thymus, spleen, lymph nodes (LNs), and peripheral blood. Mouse ILC2s lack lineage markers (Gr-1, Ter119, CD19, CD3e, CD11b, CD11c, NK1.1) but express Thy1 (CD90), CD127 (IL-7Ra), and CD25 (IL-2Ra). Naïve mouse lung ILC2s also express ST2 (IL-33R [6]) and ICOS
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Similarities between innate and adaptive memory highlight how we can broaden the notion of immunological memory to reflect how exposure to antigens and cytokines shapes future immune responses.
1
Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC V6T 2B5, Canada 2 Terry Fox Laboratory, British Columbia Cancer Agency, 675 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada 3 Interdisciplinary Oncology Program, University of British Columbia, Vancouver, BC V5Z 1L3, Canada
*Correspondence: [email protected] (F. Takei).
http://dx.doi.org/10.1016/j.it.2017.03.005 © 2017 Elsevier Ltd. All rights reserved.
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Box 1. ILCs ILC2s belong to the family of ILCs, which includes NK cells, lymphoid tissue inducer (LTi) cells, and the cytokineproducing helper-like ILCs. Helper-like ILCs do not express antigen-specific receptors and derive from a common progenitor that expresses the transcription factor PLZF. Helper-like ILCs are divided into three groups – namely, ILC1, ILC2, and ILC3–based on their expression of transcription factors and production of cytokines. ILC1s express T-bet and produce IFNg and TNFa. ILC2s express GATA3 and produce IL-5 and IL-13 and ILC3s express RORgt and produce IL22 and IL-17. ILCs play roles in resistance to pathogens and maintenance of tissue homeostasis: ILC1s respond to intracellular pathogens, ILC2s are involved in tissue repair mechanisms and helminth expulsion, and ILC3s protect against extracellular fungi and bacteria. For more extensive descriptions of ILCs, please refer to recent reviews [56,57].
[7] but only low amounts of the IL-25 receptor (IL-25R), whereas ILC2s in the small intestine are ST2 [304_TD$IF] and IL-25R+ [8,9]. ILC2s play a role in helminth expulsion, wound healing, tissue repair after virus infection [10], obesity [11] and thermogenesis [12,13]. ILC2s can also be pathological, as allergen-activated ILC2s induce type 2 allergic inflammation both in the lung and the skin. Several allergens, including papain [14], fungal protease allergen [15], chitin [16], house dust mite extract [17], and the fungus Alternia alternata [18], are known to activate lung ILC2s. However, ILC2s do not express antigen receptors and do not directly recognize helminths or allergens. Instead, ILC2s are activated by the alarmins IL-33, IL-25, and thymic stromal lymphopoietin (TSLP), which are released by allergen-stimulated or damaged epithelium [19]. Lipid mediators including prostaglandins such as PGD2 and leukotrienes such as LTD4 can attract ILC2s and induce cytokine production and synergize with IL-33 in ILC2 activation [20]. Activated ILC2s produce IL-5 and IL-13. IL-5 induces eosinophil differentiation and recruitment while IL-13 drives mucus hyperproduction and airway hyper-responsiveness. Thus, lung ILC2 activation by allergens results in T cell-independent type 2 lung inflammation [21]. Furthermore, IL-13 produced by ILC2s promotes allergen-specific Th2 cell differentiation in the lung-draining mediastinal LN (mLN), which leads to IgE production and allergic lung inflammation [8,21]. While ILC2s are early responders to epithelial damage, analysis of their phenotype [306_TD$IF]and function in the long term demonstrated that they can acquire immunological memory. Intranasal injections of allergens, including papain and fungal protease, or recombinant IL-33 into naïve mice induced a 10–100-fold increase in the numbers of lung ILC2s [14,15,17]. This was followed by a contraction phase and the resolution of inflammation; however, lung ILC2 numbers in allergen- or IL-33-injected mice remained higher than those in naïve mice for several months, suggesting that previously activated ILC2s persist. IL-33-stimulated lung ILC2s labeled with BrdU were detected for more than 2 months [15], indicating the persistence of the same ILC2s that were previously activated. [307_TD$IF]The lung-draining mLN contains few ILC2s in naïve mice, but this population underwent expansion, activation, and contraction phases much like lung ILC2s. Although it may be possible that activated lung ILC2s migrated into the mLN, the relationship between lung and mLN ILC2s remains unclear. When mice injected with allergens or IL-33 were challenged with an unrelated allergen several months later, lung ILC2s responded more intensely than those in naïve mice. These ‘allergenexperienced’ ILC2s proliferated more, produced greater amounts of IL-5 and IL-13, and induced more severe allergic inflammation than naïve cells [15]. In vivo transplantation and ex vivo experiments showed that the high responsiveness of previously activated ILC2s was due to cell-intrinsic changes, which was confirmed by increased IL-25R expression on memory ILC2s. Whether there is any contribution from the lung environment remains to be determined. These results indicated that lung ILC2s are capable of ‘remembering’ a previous activation and becoming ‘memory ILC2s’, which are long lived (6 months or longer in mice) and able to mount an enhanced response [308_TD$IF]upon a secondary stimulation.
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Glossary Alarmins: constitutively expressed endogenous molecules that are rapidly released on non-programmed cell death. Immune cells can also be induced to produce and release alarmins without dying. They play a role in the orchestration of tissue homeostasis by recruiting and activating innate immune cells that express the receptors for the alarmins. They include cytokines such as IL-33, IL-25, TSLP, and IL1a and other molecules such as uric acid and HMGB1. Immunological memory: the ability of immune cells to recall previous exposure to an antigen and respond more rapidly and vigorously to a subsequent encounter with the same antigen. This definition can be widened to include the capacity of an immune cell to recall a previous activation and display enhanced effector functions on reactivation. Trained immunity: the ability of innate immune cells such as macrophages to mount resistance to re-infection in a non-antigen-specific manner. It is orchestrated by epigenetic reprogramming and does not involve permanent changes. Trained cells have undergone global gene expression modifications on primary infection that enable them to strongly react during secondary infections. Type 2 cytokines: cytokines released by Th2 cells; include IL-4, IL-5, IL-9, and IL-13. Other immune cells, such as ILC2s, mast cells, and basophils, also secrete type 2 cytokines. Type 2 immunity: immunity mediated by type 2 cytokines, often involving class switching to IgE and high-titer antibody production; provides protection against helminths and toxins, mediates resolution of type 1 inflammation, and is involved in wound healing and tissue repair. In addition to Th2 cells, alternatively activated M2 macrophages and ILC2 s are known to mediate type 2 immunity. Type 2 inflammation: caused by excessive type 2 immune responses and characterized by eosinophil, rather than neutrophil, recruitment, IgE, and mucus production; may result in allergy.
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Lung effector ILC2s purified 4 days after IL-33 treatment showed changes in their expression of a wide range of genes. Most genes were associated with cell activation and proliferation or [309_TD$IF]encoded cytokines, chemokines, and their respective receptors. The expression of the large majority of those genes reverted back to the level of those in naïve ILC2s as the ILC2 population contracted. Lung memory ILC2s differed from naïve lung ILC2s in their expression of a
Table 1. List of Genes Differentially Expressed in Naïve and Memory Lung ILC2sa Genes that are more highly expressed in memory ILC2s than in naïve ILC2s Gene
Protein
Function
[29_TD$IF]Refs
Bcl2a1b
Bcl2a1b
[42]
Bcl2a1d
Bcl2a1d
Members of the Bcl2 family of [23_TD$IF]antiapoptotic genes
Ier3
IEX1
Inhibits apoptosis induced by Fas or TNFa
[24_TD$IF][43]
Il1r2
IL1R2
A decoy receptor that negatively regulates IL-1 family signaling
[25_TD$IF][44]
Il2
IL-2
Involved in CD4+[298_TD$IF] T cell expansion and survival, memory CD8+ T cell generation, and Treg cell development
[27_TD$IF][45]
Il17rb
IL-17Rb (IL-25R)
Receptor for IL-17[28_TD$IF]B and IL-17E (IL-25)
[46]
Il6
IL-6
A [29_TD$IF]proinflammatory cytokine involved in neutrophil trafficking, T cell activation and differentiation, [30_TD$IF]and B cell maturation; also promotes macrophage and megakaryocyte differentiation
[31_TD$IF][47]
Tnfsf18
TNFSF18
A cytokine expressed in endothelial cells[32_TD$IF]; provides signals for T cell survival by regulating [3_TD$IF]the NFkB signaling pathway
[34_TD$IF][48]
Syne1
Nesprin-1
[35_TD$IF]Establishes nuclear–cytoskeletal interactions by connecting the nuclear envelope to F-actin
[36_TD$IF][49]
Genes that are more highly expressed in naïve ILC2s than [30_TD$IF]in memory ILC2s
a
Cd2
CD2
Cell adhesion molecule expressed on T cells and [38_TD$IF]NK cells; thought to be important in T cell activation and cell[39_TD$IF]-lytic activity of NK cells
[40_TD$IF][50,51]
Cd7
CD7
Expressed on thymocytes, T cells[41_TD$IF], and NK cells; involved in T cell co-stimulation and integrin-mediated adhesion
[42_TD$IF][52]
Il6st
IL-6 signal transducer
[43_TD$IF]Interacts with IL-6R, formation of highaffinity IL-6-binding site, and transduces IL-6 signals
[4_TD$IF][53]
S1pr1
S1PR1
Receptor for S1P[45_TD$IF]; critical for lymphocyte egress from secondary lymphoid organs and thymus
[46_TD$IF][54]
Sell
L-selectin
Involved in the [47_TD$IF]rolling of lymphocytes on high endothelial venules[48_TD$IF]; highly expressed by central memory T cells and thus [49_TD$IF]used as a marker for central memory T cells
[50_TD$IF][55]
Normal B6 mice received three intranasal injections of recombinant IL-33 and lung ILC2s (Lin-Thy1+[30_TD$IF]CD127+ST2+) were purified by cell sorting 4 months later as memory ILC2s. Naïve ILC2s were purified from B6 mice that received PBS injections in place of IL-33. Gene expression was analyzed by Affymetrix microarray (Mouse Gene 2.0ST).
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surprisingly small number of genes (Table 1). Genes that were more highly expressed in memory ILC2s included antiapoptotic genes (Ier3, Bcl2a1b, Bcl2a1d), cytokine genes (Il2, Il6, Tnfsf18), and cytokine receptor genes (Il1r2, Il17rb). Naïve ILC2s, by contrast, showed greater expression of some genes that are involved in cell adhesion and migration (Cd2, Cd7, S1pr1, Sell). Il5 and Il13 were highly expressed in both naïve and memory ILC2s and only slightly increased in effector ILC2s. By contrast, the IL-5 or IL-13 protein was detected only in effector and not in naïve or memory ILC2s, suggesting that cytokine expression is regulated at the translational level. Among the genes upregulated in memory ILC2s, Il17rb, which encodes the receptor for IL-25, was of particular interest. Il17rb mRNA expression and, accordingly, IL-25R protein levels were low in naïve ILC2s, increased on activation, and remained high in memory ILC2s. A single intranasal injection of recombinant IL-25 stimulated memory ILC2s in the lung and in the mLN but did not activate naïve ILC2s. As IL-25 is known to be secreted by allergen-stimulated epithelial cells [22], it is likely to play a critical role in the enhanced response by memory ILC2s. Thus, activation of lung ILC2s induces upregulation of IL-25R, which is maintained in ‘memory’ ILC2s as a record of a previous activation, allowing them to be more prone to activation than naïve ILC2s on challenge.
ILC2 Memory and T Cell Memory ILC2s lack antigen-specific receptors and therefore memory ILC2s do not have antigen specificity, which is thought to be an essential feature of immunological memory. Nevertheless, memory ILC2s and memory T cells share many characteristics. On activation, naïve ILC2s and T cells expand and become effector cells, followed by a contraction phase in which most of the effector cells die leaving a few memory cells that survive for a long time. Both memory T cells and memory ILC2s are generally quiescent and do not display effector functions until they are reactivated. On reactivation, they both rapidly become effector cells with enhanced functions, although the nature of the effector molecules produced is the same in primary and memory responses. The main difference is the method of activation. T cells are activated by the ligation of antigen-specific receptors resulting in clonal expansion and the generation of antigenspecific memory T cells. By contrast, ILC2s are activated by cytokines, resulting in antigennonspecific memory ILC2s. However, it has also been shown that T cells can mount antigennonspecific (also called bystander) memory responses [23]. Guo et al. showed that antigenspecific Th2 cells could be activated by IL-33, and in vivo-generated ovalbumin-specific Th2 cells responded to papain in an MHC-independent manner [24]. Thus, both memory T cells and memory ILC2s remember being previously activated regardless of whether the activation is via antigen-specific receptors or cytokine receptors. As discussed by Bedoui et al. [25], ILC2s and T cells in the naïve state differ from each other. Naïve ILC2s are already committed and poised to become type 2 cytokine-producing effectors on activation. Memory ILC2s are similarly committed for the same effector functions. By contrast, naïve T cells are uncommitted for specific effector lineages (e.g., Th1, Th2) and have to undergo differentiation processes involving changes in the expression of a broad range of genes to become effector and memory T cells. These differences are likely to explain why cell-surface markers including CD44 that differentiate memory T cells from naïve and effector T cells are not differentially expressed in memory ILC2s. Nevertheless, Gene Set Enrichment Analysis (GSEA) suggested that the gene expression profile of memory ILC2s is similar to that of memory T cells. Mainly, cell cycle-related genes including Top2a, Mki67, Cdk1, Ccna2, and Cdkn3 were found to be expressed at lower levels in memory than in naïve ILC2s and CD8+[305_TD$IF] T cells [15].
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ILC2s and T cells also differ in the sites of naïve cell activation and memory cell generation. Circulating naïve T cells are mostly activated in the LN. Memory T cells can be divided into different subsets based on their localization and functions. Central memory T cells reside in lymphoid tissues and effector memory T cells circulate between various non-lymphoid tissues, whereas tissue-resident memory T cells remain in the non-lymphoid tissues ready to respond to the next encounter with the antigen. By contrast, naïve ILC2s are tissue resident [26] and lung ILC2s are activated in the lung by epithelium-derived alarmins. Some activated lung ILC2s seem to migrate and persist as memory ILC2s in the draining mLN. Interestingly, unlike memory ILC2s in the lung, mLN ILC2s remain positive for intracellular cytokines for more than 5 months, suggesting that mLN memory ILC2s may become cytokine-secreting effectors more rapidly than those in the lung. These differences between lung and mLN ILC2s suggest that, similar to memory T cells, there might be different subsets of memory ILC2s. It remains to be determined whether memory ILC2s in the mLN migrate to the lung when mice receive secondary intranasal allergens/IL-33 challenges. It is also unknown whether activated ILC2s circulate and settle and persist in other tissues as memory ILC2s[310_TD$IF].
ILC2 Memory and NK Cell Memory ILC2 memory shares many features with NK cell memory. Both naïve ILC2s and naïve NK cells are primed for effector functions. Naïve NK cells are committed to cytotoxicity and IFNg production and highly express effector genes including Ifng, Gzma, and Gzmb but not their respective proteins [27], similar to naïve ILC2s. Naïve NK cells are activated by two distinct pathways; namely, recognition of ligands by receptors such as NKG2D or Ly49H and the binding of cytokines including IL-12,IL-15, and IL-18 to the receptors on naïve NK cells. On activation, NK cells undergo expansion and contraction and acquire immunological memory [28]. NK cells can mount specific memory responses to haptens, influenza virus, vaccinia virus,
(A)
Memory ILC2s
Cytokine producon
m m m m Enhanced immune response m
Challenge
Sensizaon Upregulaon of IL-25R e e e
n m
n
Time
Effector molecules
(B)
Trained immunity t
First infecon
Figure 1. [302_TD$IF]Memory Group 2 Innate Lymphoid Cells (ILC2s) versus Trained Immunity. (A) Allergen-activated ILC2s expand and produce type 2 cytokines. At the same time, activated ILC2s upregulate the receptor for IL-25 (IL-25R). Then, they undergo a contraction phase and ILC2s become resting memory cells. On a secondary challenge, memory ILC2s proliferate more and produce more type 2 cytokines than in the first allergen encounter. (B) Monocytes or macrophages undergo epigenetic changes and become trained cells after an infection. Trained cells respond strongly during secondary encounters with pathogens.
t Strong immune response t t
Challenge
Epigenec changes e n
e n t
Time
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vesicular stomatitis virus (VSV), HIV-1, and murine cytomegalovirus (MCMV) [4,29]. Lanier’s group showed that naïve NK cells expressing the stimulatory receptor Ly49H were activated by its ligand, the MCMV protein m157, and became memory NK cells that provided protection against subsequent MCMV re-infection but not against infection with an unrelated virus. When Ly49H+ NK cells were adoptively transferred into mice lacking this receptor, these Ly49H+ NK cells were readily detected and underwent robust antigen-driven expansion after MCMV infection. Thus, similar to memory T cells, the antigen specificity of MCMV-specific memory NK cells is mediated by the activation and proliferation of naïve NK cells expressing m157 receptors. Several genes and cell-surface proteins including Ly6C (Ly6c1) and CD49a (Itga1) are commonly expressed by memory NK cells and memory T cells [27]. Epigenetic modifications are also thought to play an important role in T cell and NK cell memory [30–32]. Memory NK cells have displayed epigenetic ‘imprinting’ at the IFNG locus, which resembles the same locus in CD8+ memory T cells and Th1 cells [31]. Cytokine stimulation of naïve NK cells has been shown to generate antigen-nonspecific memory NK cells. Naïve NK cells stimulated in vitro with a combination of IL-12, IL-15, and IL-18 and adoptively transferred into RAG-deficient mice persisted for more than 12 weeks and produced greater amounts of IFNg on re-stimulation with IL-12 and IL-15 than did naïve NK cells [33,34]. Thus, cytokine-induced memory NK cells are similar to memory ILC2s as both are activated in an antigen-nonspecific manner and respond more vigorously when reactivated by cytokine stimulation compared with the primary response. Currently, the molecular basis of antigen-nonspecific NK cell memory is unclear.
ILC2 Memory and Trained Immunity Activation of macrophages by pathogen-derived molecules including b-glucan results in their differentiation into highly functional macrophages that live up to 4 weeks and mediate antigennonspecific innate protection against subsequent infections [35]. Naïve macrophages are also trained by helminth infection and acquire the ability to kill larvae in a later infection [36]. The term trained immunity has been proposed for this form of immune memory. At present, research on trained immunity and ILC2 memory is limited and direct comparison is difficult. However, there are some similarities between these two types of memory (Figure 1). Both ILC2s and macrophages respond to a primary stimulus and increase in number either by proliferation or by recruitment. They are able to remember the state of activation, persist in increased numbers for a long period of time, and then respond more intensely to a secondary stimulus. However, naïve ILC2s are already committed to specific functions in the naïve state [15] whereas naïve macrophages acquire new functions to become trained [37]. However, while memory ILC2s have been shown to respond in vivo for at least 6 months after their generation, it is unknown whether trained macrophages can respond for a period longer than 45 days. For example, Chen et al. demonstrated that, on primary infection with the helminth Nippostrongylus brasiliensis, macrophages clear larvae poorly in the lungs, allowing them to transit to the gut. However, exposure to the larvae enables them to become ‘trained’. On a secondary exposure to the larvae, the trained macrophages upregulate genes encoding effector molecules that are not expressed in untrained macrophages, including Arg1, to induce a more appropriate and intense response to the larvae, effectively removing them from the lungs independently from the adaptive immune system [36]. Mechanistic studies have demonstrated that training is mediated by epigenetic modifications rather than genetic changes [37,38]. We have shown that naïve and memory ILC2s differ in their expression of only a small set of genes and proteins. However, it remains to be studied whether ILC2 memory also involves epigenetic changes.
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Key Figure
Outstanding Questions
Immunological Memory
Is IL-33 sufficient for the generation of memory ILC2s or does it require costimulatory signals?
Naive
Effector
Is the upregulation of IL-25R solely responsible for the high responsiveness of memory ILC2s? Do ILC2s in IL-25- or IL-25R-deficient mice still become memory ILC2s?
Memory
Acvaon
ILC2s
(Alarmin) What is the role of memory ILC2s in the mLN in allergic lung inflammation? How do the functions of tissue versus LN memory ILC2s parallel those of central and resident memory T cells?
Acvaon/ differenaon
T cells
(Angen) Are memory ILC2s important during helminth infection or only in allergic diseases?
Macrophages
Acvaon
Are ILC1s and ILC3s also capable of acquiring memory features?
(β-glucan/infecon)
Figure 2. Group 2 innate lymphoid cells (ILC2s) and macrophages have now been shown to have memory similar to adaptive T cells. Naïve ILC2s expand and become effector cells on encounter with alarmins and some remain as memory ILC2s, which differ from naïve cells (top row). Antigen-specific naïve T cells differentiate to become effector cells on antigen encounter and some remain as antigen-specific memory T cells (middle row). Macrophages undergo epigenetic modifications on first infection and persist as trained cells (lower row). Immunological memory can now be defined as the ability to remember a previous activation state and is not exclusive to adaptive antigen-specific cells.
Memory ILC2s in Allergic Lung Diseases ILC2 memory has important implications for allergic lung diseases including asthma. As ILC2s are antigen nonspecific, the generation of memory ILC2s can sensitize individuals to a broad range of allergens. Since ILC2s do not produce much IL-4, ILC2-mediated T cell-independent type 2 inflammation does not involve production of IgE, a hallmark of allergy. Thus, memory ILC2s may drive non-allergic asthma in patients who show negative skin tests with allergens and do not have elevated IgE [39]. Moreover, memory ILC2s may also play a role in allergic asthma. It was previously shown that IL-13 derived from activated ILC2s promoted the differentiation of naïve CD4+[31_TD$IF] T cells into Th2 cells [21]. More recently, we have shown that memory ILC2s promote Th2 cell responses more efficiently than naïve ILC2s [15], possibly due to greater amounts of IL-13 production. Therefore, memory ILC2s may also enhance the development of Th2 cell-dependent allergic asthma. Patients with asthma have been found to have more ILC2s in their blood than non-asthmatic individuals [40,41]. As very few naïve ILC2s circulate, ILC2s in the peripheral blood of asthma patients may be previously activated memory ILC2s. It remains to be investigated whether other ILCs also have the capacity to acquire memory functions.
Concluding Remarks Whereas antigen specificity has been thought to be a hallmark of immunological memory, which only T and B cells have, the discovery of hapten- and virus-specific NK cell memory has
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shown that innate lymphocytes also have immunological memory. Recent findings of memory in innate cells have further widened our understanding of immunological memory to include antigen-nonspecific memory (Figure 2, Key Figure). In general, memory is defined by the capacity to encode, store, and retrieve information [35]. In accordance, immunological memory can now be defined by the ability of immune cells to remember information recorded from a previous activation state and to have stronger effector functions on challenge than during the primary activation. Whether it occurs through the ligation of antigen-specific receptors, cytokine receptors, or Toll-like receptors, activation of naïve cells induces changes in gene expression, some of which are maintained for a long time as record of previous activation states in memory cells. Future research in the field focused on the factors that give rise to memory in ILC2s, as well as potentially in other ILC subsets (see Outstanding Questions), should allow us to further define the expanding concept of immune memory and the sweeping effects of experience in immune responses. Acknowledgments The authors thank Reyes Gonzalez-Aller for illustrating Figure 2. They are supported by CIHR, MSFHR, UBC, and Vanier CGS.
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Review
Immunological Memory of Group 2 Innate Lymphoid Cells Itziar Martinez-Gonzalez,1,2 Laura Mathä,2,3 Catherine A. Steer,2,3 and Fumio Takei1,2,* Immunological memory has long been described as a property of the adaptive immune system that results in potent responses on exposure to an antigen encountered previously. While this definition appears to exclude cells that do not express antigen receptors, recent studies have shown that innate immune cells, including natural killer (NK) cells, macrophages, and, more recently, group 2 innate lymphoid cells (ILC2s) can record previous activations and respond more vigorously on reactivation. Here we review the similarities and differences between these forms of memory and the underlying mechanisms. Based on these insights, we propose to revise the definition of immunological memory, as the capacity to remember being previously activated and respond more efficiently on reactivation regardless of antigen specificity.
Trends Allergen-experienced group 2 innate lymphoid cells remember previous activation and respond strongly to secondary challenge in an antigen-nonspecific manner. Natural killer cells have both antigenspecific and antigen-nonspecific memory. Macrophages can be trained to become highly responsive to secondary infection. Innate memory and adaptive T cell memory have common key features.
Immunological Memory In immunological memory (see Glossary), information (antigen encounter) is encoded in antigen-stimulated naïve cells and stored by long-lived memory cells. On re-encounter with the same antigen, memory cells recall the information and respond more rapidly and effectively than naïve cells. Therefore, antigen specificity has long been considered an essential feature of immunological memory [1–3]. Although immunological memory is thought to be a hallmark of adaptive immunity, discoveries with memory NK cells have shown that innate lymphocytes also have antigen-specific memory [4]. Moreover, recent studies have suggested that macrophages have some features of memory, termed ‘trained immunity’, that have antigen specificity. More recently, we have shown that ILC2s can remember being previously activated and respond more efficiently during secondary encounters with allergens. Unlike T cells, ILC2s do not recognize antigens but rather are activated by cytokines, and therefore their memory is antigen nonspecific. Nevertheless, ILC2s show an enhanced recall response similar to T cells. Here, by focusing on recent evidence for ILC2 memory and comparing the underlying mechanisms for the different forms of memory outlined above, we propose to expand the concept of immunological memory to include any cell that can remember a previous state of activation and mount a more efficient response during secondary challenges.
ILC2 Memory ILC2s belong to the family of ILCs (Box 1), which are thought to be tissue-resident lymphocytes. ILCs develop early in life from common progenitors and seed various tissues [5]. In naïve adult mice, ILC2s are found in the lung, skin, small intestine, and adipose tissue, while they are very rare in the thymus, spleen, lymph nodes (LNs), and peripheral blood. Mouse ILC2s lack lineage markers (Gr-1, Ter119, CD19, CD3e, CD11b, CD11c, NK1.1) but express Thy1 (CD90), CD127 (IL-7Ra), and CD25 (IL-2Ra). Naïve mouse lung ILC2s also express ST2 (IL-33R [6]) and ICOS
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Similarities between innate and adaptive memory highlight how we can broaden the notion of immunological memory to reflect how exposure to antigens and cytokines shapes future immune responses.
1
Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC V6T 2B5, Canada 2 Terry Fox Laboratory, British Columbia Cancer Agency, 675 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada 3 Interdisciplinary Oncology Program, University of British Columbia, Vancouver, BC V5Z 1L3, Canada
*Correspondence: [email protected] (F. Takei).
http://dx.doi.org/10.1016/j.it.2017.03.005 © 2017 Elsevier Ltd. All rights reserved.
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Box 1. ILCs ILC2s belong to the family of ILCs, which includes NK cells, lymphoid tissue inducer (LTi) cells, and the cytokineproducing helper-like ILCs. Helper-like ILCs do not express antigen-specific receptors and derive from a common progenitor that expresses the transcription factor PLZF. Helper-like ILCs are divided into three groups – namely, ILC1, ILC2, and ILC3–based on their expression of transcription factors and production of cytokines. ILC1s express T-bet and produce IFNg and TNFa. ILC2s express GATA3 and produce IL-5 and IL-13 and ILC3s express RORgt and produce IL22 and IL-17. ILCs play roles in resistance to pathogens and maintenance of tissue homeostasis: ILC1s respond to intracellular pathogens, ILC2s are involved in tissue repair mechanisms and helminth expulsion, and ILC3s protect against extracellular fungi and bacteria. For more extensive descriptions of ILCs, please refer to recent reviews [56,57].
[7] but only low amounts of the IL-25 receptor (IL-25R), whereas ILC2s in the small intestine are ST2 [304_TD$IF] and IL-25R+ [8,9]. ILC2s play a role in helminth expulsion, wound healing, tissue repair after virus infection [10], obesity [11] and thermogenesis [12,13]. ILC2s can also be pathological, as allergen-activated ILC2s induce type 2 allergic inflammation both in the lung and the skin. Several allergens, including papain [14], fungal protease allergen [15], chitin [16], house dust mite extract [17], and the fungus Alternia alternata [18], are known to activate lung ILC2s. However, ILC2s do not express antigen receptors and do not directly recognize helminths or allergens. Instead, ILC2s are activated by the alarmins IL-33, IL-25, and thymic stromal lymphopoietin (TSLP), which are released by allergen-stimulated or damaged epithelium [19]. Lipid mediators including prostaglandins such as PGD2 and leukotrienes such as LTD4 can attract ILC2s and induce cytokine production and synergize with IL-33 in ILC2 activation [20]. Activated ILC2s produce IL-5 and IL-13. IL-5 induces eosinophil differentiation and recruitment while IL-13 drives mucus hyperproduction and airway hyper-responsiveness. Thus, lung ILC2 activation by allergens results in T cell-independent type 2 lung inflammation [21]. Furthermore, IL-13 produced by ILC2s promotes allergen-specific Th2 cell differentiation in the lung-draining mediastinal LN (mLN), which leads to IgE production and allergic lung inflammation [8,21]. While ILC2s are early responders to epithelial damage, analysis of their phenotype [306_TD$IF]and function in the long term demonstrated that they can acquire immunological memory. Intranasal injections of allergens, including papain and fungal protease, or recombinant IL-33 into naïve mice induced a 10–100-fold increase in the numbers of lung ILC2s [14,15,17]. This was followed by a contraction phase and the resolution of inflammation; however, lung ILC2 numbers in allergen- or IL-33-injected mice remained higher than those in naïve mice for several months, suggesting that previously activated ILC2s persist. IL-33-stimulated lung ILC2s labeled with BrdU were detected for more than 2 months [15], indicating the persistence of the same ILC2s that were previously activated. [307_TD$IF]The lung-draining mLN contains few ILC2s in naïve mice, but this population underwent expansion, activation, and contraction phases much like lung ILC2s. Although it may be possible that activated lung ILC2s migrated into the mLN, the relationship between lung and mLN ILC2s remains unclear. When mice injected with allergens or IL-33 were challenged with an unrelated allergen several months later, lung ILC2s responded more intensely than those in naïve mice. These ‘allergenexperienced’ ILC2s proliferated more, produced greater amounts of IL-5 and IL-13, and induced more severe allergic inflammation than naïve cells [15]. In vivo transplantation and ex vivo experiments showed that the high responsiveness of previously activated ILC2s was due to cell-intrinsic changes, which was confirmed by increased IL-25R expression on memory ILC2s. Whether there is any contribution from the lung environment remains to be determined. These results indicated that lung ILC2s are capable of ‘remembering’ a previous activation and becoming ‘memory ILC2s’, which are long lived (6 months or longer in mice) and able to mount an enhanced response [308_TD$IF]upon a secondary stimulation.
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Glossary Alarmins: constitutively expressed endogenous molecules that are rapidly released on non-programmed cell death. Immune cells can also be induced to produce and release alarmins without dying. They play a role in the orchestration of tissue homeostasis by recruiting and activating innate immune cells that express the receptors for the alarmins. They include cytokines such as IL-33, IL-25, TSLP, and IL1a and other molecules such as uric acid and HMGB1. Immunological memory: the ability of immune cells to recall previous exposure to an antigen and respond more rapidly and vigorously to a subsequent encounter with the same antigen. This definition can be widened to include the capacity of an immune cell to recall a previous activation and display enhanced effector functions on reactivation. Trained immunity: the ability of innate immune cells such as macrophages to mount resistance to re-infection in a non-antigen-specific manner. It is orchestrated by epigenetic reprogramming and does not involve permanent changes. Trained cells have undergone global gene expression modifications on primary infection that enable them to strongly react during secondary infections. Type 2 cytokines: cytokines released by Th2 cells; include IL-4, IL-5, IL-9, and IL-13. Other immune cells, such as ILC2s, mast cells, and basophils, also secrete type 2 cytokines. Type 2 immunity: immunity mediated by type 2 cytokines, often involving class switching to IgE and high-titer antibody production; provides protection against helminths and toxins, mediates resolution of type 1 inflammation, and is involved in wound healing and tissue repair. In addition to Th2 cells, alternatively activated M2 macrophages and ILC2 s are known to mediate type 2 immunity. Type 2 inflammation: caused by excessive type 2 immune responses and characterized by eosinophil, rather than neutrophil, recruitment, IgE, and mucus production; may result in allergy.
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Lung effector ILC2s purified 4 days after IL-33 treatment showed changes in their expression of a wide range of genes. Most genes were associated with cell activation and proliferation or [309_TD$IF]encoded cytokines, chemokines, and their respective receptors. The expression of the large majority of those genes reverted back to the level of those in naïve ILC2s as the ILC2 population contracted. Lung memory ILC2s differed from naïve lung ILC2s in their expression of a
Table 1. List of Genes Differentially Expressed in Naïve and Memory Lung ILC2sa Genes that are more highly expressed in memory ILC2s than in naïve ILC2s Gene
Protein
Function
[29_TD$IF]Refs
Bcl2a1b
Bcl2a1b
[42]
Bcl2a1d
Bcl2a1d
Members of the Bcl2 family of [23_TD$IF]antiapoptotic genes
Ier3
IEX1
Inhibits apoptosis induced by Fas or TNFa
[24_TD$IF][43]
Il1r2
IL1R2
A decoy receptor that negatively regulates IL-1 family signaling
[25_TD$IF][44]
Il2
IL-2
Involved in CD4+[298_TD$IF] T cell expansion and survival, memory CD8+ T cell generation, and Treg cell development
[27_TD$IF][45]
Il17rb
IL-17Rb (IL-25R)
Receptor for IL-17[28_TD$IF]B and IL-17E (IL-25)
[46]
Il6
IL-6
A [29_TD$IF]proinflammatory cytokine involved in neutrophil trafficking, T cell activation and differentiation, [30_TD$IF]and B cell maturation; also promotes macrophage and megakaryocyte differentiation
[31_TD$IF][47]
Tnfsf18
TNFSF18
A cytokine expressed in endothelial cells[32_TD$IF]; provides signals for T cell survival by regulating [3_TD$IF]the NFkB signaling pathway
[34_TD$IF][48]
Syne1
Nesprin-1
[35_TD$IF]Establishes nuclear–cytoskeletal interactions by connecting the nuclear envelope to F-actin
[36_TD$IF][49]
Genes that are more highly expressed in naïve ILC2s than [30_TD$IF]in memory ILC2s
a
Cd2
CD2
Cell adhesion molecule expressed on T cells and [38_TD$IF]NK cells; thought to be important in T cell activation and cell[39_TD$IF]-lytic activity of NK cells
[40_TD$IF][50,51]
Cd7
CD7
Expressed on thymocytes, T cells[41_TD$IF], and NK cells; involved in T cell co-stimulation and integrin-mediated adhesion
[42_TD$IF][52]
Il6st
IL-6 signal transducer
[43_TD$IF]Interacts with IL-6R, formation of highaffinity IL-6-binding site, and transduces IL-6 signals
[4_TD$IF][53]
S1pr1
S1PR1
Receptor for S1P[45_TD$IF]; critical for lymphocyte egress from secondary lymphoid organs and thymus
[46_TD$IF][54]
Sell
L-selectin
Involved in the [47_TD$IF]rolling of lymphocytes on high endothelial venules[48_TD$IF]; highly expressed by central memory T cells and thus [49_TD$IF]used as a marker for central memory T cells
[50_TD$IF][55]
Normal B6 mice received three intranasal injections of recombinant IL-33 and lung ILC2s (Lin-Thy1+[30_TD$IF]CD127+ST2+) were purified by cell sorting 4 months later as memory ILC2s. Naïve ILC2s were purified from B6 mice that received PBS injections in place of IL-33. Gene expression was analyzed by Affymetrix microarray (Mouse Gene 2.0ST).
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surprisingly small number of genes (Table 1). Genes that were more highly expressed in memory ILC2s included antiapoptotic genes (Ier3, Bcl2a1b, Bcl2a1d), cytokine genes (Il2, Il6, Tnfsf18), and cytokine receptor genes (Il1r2, Il17rb). Naïve ILC2s, by contrast, showed greater expression of some genes that are involved in cell adhesion and migration (Cd2, Cd7, S1pr1, Sell). Il5 and Il13 were highly expressed in both naïve and memory ILC2s and only slightly increased in effector ILC2s. By contrast, the IL-5 or IL-13 protein was detected only in effector and not in naïve or memory ILC2s, suggesting that cytokine expression is regulated at the translational level. Among the genes upregulated in memory ILC2s, Il17rb, which encodes the receptor for IL-25, was of particular interest. Il17rb mRNA expression and, accordingly, IL-25R protein levels were low in naïve ILC2s, increased on activation, and remained high in memory ILC2s. A single intranasal injection of recombinant IL-25 stimulated memory ILC2s in the lung and in the mLN but did not activate naïve ILC2s. As IL-25 is known to be secreted by allergen-stimulated epithelial cells [22], it is likely to play a critical role in the enhanced response by memory ILC2s. Thus, activation of lung ILC2s induces upregulation of IL-25R, which is maintained in ‘memory’ ILC2s as a record of a previous activation, allowing them to be more prone to activation than naïve ILC2s on challenge.
ILC2 Memory and T Cell Memory ILC2s lack antigen-specific receptors and therefore memory ILC2s do not have antigen specificity, which is thought to be an essential feature of immunological memory. Nevertheless, memory ILC2s and memory T cells share many characteristics. On activation, naïve ILC2s and T cells expand and become effector cells, followed by a contraction phase in which most of the effector cells die leaving a few memory cells that survive for a long time. Both memory T cells and memory ILC2s are generally quiescent and do not display effector functions until they are reactivated. On reactivation, they both rapidly become effector cells with enhanced functions, although the nature of the effector molecules produced is the same in primary and memory responses. The main difference is the method of activation. T cells are activated by the ligation of antigen-specific receptors resulting in clonal expansion and the generation of antigenspecific memory T cells. By contrast, ILC2s are activated by cytokines, resulting in antigennonspecific memory ILC2s. However, it has also been shown that T cells can mount antigennonspecific (also called bystander) memory responses [23]. Guo et al. showed that antigenspecific Th2 cells could be activated by IL-33, and in vivo-generated ovalbumin-specific Th2 cells responded to papain in an MHC-independent manner [24]. Thus, both memory T cells and memory ILC2s remember being previously activated regardless of whether the activation is via antigen-specific receptors or cytokine receptors. As discussed by Bedoui et al. [25], ILC2s and T cells in the naïve state differ from each other. Naïve ILC2s are already committed and poised to become type 2 cytokine-producing effectors on activation. Memory ILC2s are similarly committed for the same effector functions. By contrast, naïve T cells are uncommitted for specific effector lineages (e.g., Th1, Th2) and have to undergo differentiation processes involving changes in the expression of a broad range of genes to become effector and memory T cells. These differences are likely to explain why cell-surface markers including CD44 that differentiate memory T cells from naïve and effector T cells are not differentially expressed in memory ILC2s. Nevertheless, Gene Set Enrichment Analysis (GSEA) suggested that the gene expression profile of memory ILC2s is similar to that of memory T cells. Mainly, cell cycle-related genes including Top2a, Mki67, Cdk1, Ccna2, and Cdkn3 were found to be expressed at lower levels in memory than in naïve ILC2s and CD8+[305_TD$IF] T cells [15].
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ILC2s and T cells also differ in the sites of naïve cell activation and memory cell generation. Circulating naïve T cells are mostly activated in the LN. Memory T cells can be divided into different subsets based on their localization and functions. Central memory T cells reside in lymphoid tissues and effector memory T cells circulate between various non-lymphoid tissues, whereas tissue-resident memory T cells remain in the non-lymphoid tissues ready to respond to the next encounter with the antigen. By contrast, naïve ILC2s are tissue resident [26] and lung ILC2s are activated in the lung by epithelium-derived alarmins. Some activated lung ILC2s seem to migrate and persist as memory ILC2s in the draining mLN. Interestingly, unlike memory ILC2s in the lung, mLN ILC2s remain positive for intracellular cytokines for more than 5 months, suggesting that mLN memory ILC2s may become cytokine-secreting effectors more rapidly than those in the lung. These differences between lung and mLN ILC2s suggest that, similar to memory T cells, there might be different subsets of memory ILC2s. It remains to be determined whether memory ILC2s in the mLN migrate to the lung when mice receive secondary intranasal allergens/IL-33 challenges. It is also unknown whether activated ILC2s circulate and settle and persist in other tissues as memory ILC2s[310_TD$IF].
ILC2 Memory and NK Cell Memory ILC2 memory shares many features with NK cell memory. Both naïve ILC2s and naïve NK cells are primed for effector functions. Naïve NK cells are committed to cytotoxicity and IFNg production and highly express effector genes including Ifng, Gzma, and Gzmb but not their respective proteins [27], similar to naïve ILC2s. Naïve NK cells are activated by two distinct pathways; namely, recognition of ligands by receptors such as NKG2D or Ly49H and the binding of cytokines including IL-12,IL-15, and IL-18 to the receptors on naïve NK cells. On activation, NK cells undergo expansion and contraction and acquire immunological memory [28]. NK cells can mount specific memory responses to haptens, influenza virus, vaccinia virus,
(A)
Memory ILC2s
Cytokine producon
m m m m Enhanced immune response m
Challenge
Sensizaon Upregulaon of IL-25R e e e
n m
n
Time
Effector molecules
(B)
Trained immunity t
First infecon
Figure 1. [302_TD$IF]Memory Group 2 Innate Lymphoid Cells (ILC2s) versus Trained Immunity. (A) Allergen-activated ILC2s expand and produce type 2 cytokines. At the same time, activated ILC2s upregulate the receptor for IL-25 (IL-25R). Then, they undergo a contraction phase and ILC2s become resting memory cells. On a secondary challenge, memory ILC2s proliferate more and produce more type 2 cytokines than in the first allergen encounter. (B) Monocytes or macrophages undergo epigenetic changes and become trained cells after an infection. Trained cells respond strongly during secondary encounters with pathogens.
t Strong immune response t t
Challenge
Epigenec changes e n
e n t
Time
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vesicular stomatitis virus (VSV), HIV-1, and murine cytomegalovirus (MCMV) [4,29]. Lanier’s group showed that naïve NK cells expressing the stimulatory receptor Ly49H were activated by its ligand, the MCMV protein m157, and became memory NK cells that provided protection against subsequent MCMV re-infection but not against infection with an unrelated virus. When Ly49H+ NK cells were adoptively transferred into mice lacking this receptor, these Ly49H+ NK cells were readily detected and underwent robust antigen-driven expansion after MCMV infection. Thus, similar to memory T cells, the antigen specificity of MCMV-specific memory NK cells is mediated by the activation and proliferation of naïve NK cells expressing m157 receptors. Several genes and cell-surface proteins including Ly6C (Ly6c1) and CD49a (Itga1) are commonly expressed by memory NK cells and memory T cells [27]. Epigenetic modifications are also thought to play an important role in T cell and NK cell memory [30–32]. Memory NK cells have displayed epigenetic ‘imprinting’ at the IFNG locus, which resembles the same locus in CD8+ memory T cells and Th1 cells [31]. Cytokine stimulation of naïve NK cells has been shown to generate antigen-nonspecific memory NK cells. Naïve NK cells stimulated in vitro with a combination of IL-12, IL-15, and IL-18 and adoptively transferred into RAG-deficient mice persisted for more than 12 weeks and produced greater amounts of IFNg on re-stimulation with IL-12 and IL-15 than did naïve NK cells [33,34]. Thus, cytokine-induced memory NK cells are similar to memory ILC2s as both are activated in an antigen-nonspecific manner and respond more vigorously when reactivated by cytokine stimulation compared with the primary response. Currently, the molecular basis of antigen-nonspecific NK cell memory is unclear.
ILC2 Memory and Trained Immunity Activation of macrophages by pathogen-derived molecules including b-glucan results in their differentiation into highly functional macrophages that live up to 4 weeks and mediate antigennonspecific innate protection against subsequent infections [35]. Naïve macrophages are also trained by helminth infection and acquire the ability to kill larvae in a later infection [36]. The term trained immunity has been proposed for this form of immune memory. At present, research on trained immunity and ILC2 memory is limited and direct comparison is difficult. However, there are some similarities between these two types of memory (Figure 1). Both ILC2s and macrophages respond to a primary stimulus and increase in number either by proliferation or by recruitment. They are able to remember the state of activation, persist in increased numbers for a long period of time, and then respond more intensely to a secondary stimulus. However, naïve ILC2s are already committed to specific functions in the naïve state [15] whereas naïve macrophages acquire new functions to become trained [37]. However, while memory ILC2s have been shown to respond in vivo for at least 6 months after their generation, it is unknown whether trained macrophages can respond for a period longer than 45 days. For example, Chen et al. demonstrated that, on primary infection with the helminth Nippostrongylus brasiliensis, macrophages clear larvae poorly in the lungs, allowing them to transit to the gut. However, exposure to the larvae enables them to become ‘trained’. On a secondary exposure to the larvae, the trained macrophages upregulate genes encoding effector molecules that are not expressed in untrained macrophages, including Arg1, to induce a more appropriate and intense response to the larvae, effectively removing them from the lungs independently from the adaptive immune system [36]. Mechanistic studies have demonstrated that training is mediated by epigenetic modifications rather than genetic changes [37,38]. We have shown that naïve and memory ILC2s differ in their expression of only a small set of genes and proteins. However, it remains to be studied whether ILC2 memory also involves epigenetic changes.
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Key Figure
Outstanding Questions
Immunological Memory
Is IL-33 sufficient for the generation of memory ILC2s or does it require costimulatory signals?
Naive
Effector
Is the upregulation of IL-25R solely responsible for the high responsiveness of memory ILC2s? Do ILC2s in IL-25- or IL-25R-deficient mice still become memory ILC2s?
Memory
Acvaon
ILC2s
(Alarmin) What is the role of memory ILC2s in the mLN in allergic lung inflammation? How do the functions of tissue versus LN memory ILC2s parallel those of central and resident memory T cells?
Acvaon/ differenaon
T cells
(Angen) Are memory ILC2s important during helminth infection or only in allergic diseases?
Macrophages
Acvaon
Are ILC1s and ILC3s also capable of acquiring memory features?
(β-glucan/infecon)
Figure 2. Group 2 innate lymphoid cells (ILC2s) and macrophages have now been shown to have memory similar to adaptive T cells. Naïve ILC2s expand and become effector cells on encounter with alarmins and some remain as memory ILC2s, which differ from naïve cells (top row). Antigen-specific naïve T cells differentiate to become effector cells on antigen encounter and some remain as antigen-specific memory T cells (middle row). Macrophages undergo epigenetic modifications on first infection and persist as trained cells (lower row). Immunological memory can now be defined as the ability to remember a previous activation state and is not exclusive to adaptive antigen-specific cells.
Memory ILC2s in Allergic Lung Diseases ILC2 memory has important implications for allergic lung diseases including asthma. As ILC2s are antigen nonspecific, the generation of memory ILC2s can sensitize individuals to a broad range of allergens. Since ILC2s do not produce much IL-4, ILC2-mediated T cell-independent type 2 inflammation does not involve production of IgE, a hallmark of allergy. Thus, memory ILC2s may drive non-allergic asthma in patients who show negative skin tests with allergens and do not have elevated IgE [39]. Moreover, memory ILC2s may also play a role in allergic asthma. It was previously shown that IL-13 derived from activated ILC2s promoted the differentiation of naïve CD4+[31_TD$IF] T cells into Th2 cells [21]. More recently, we have shown that memory ILC2s promote Th2 cell responses more efficiently than naïve ILC2s [15], possibly due to greater amounts of IL-13 production. Therefore, memory ILC2s may also enhance the development of Th2 cell-dependent allergic asthma. Patients with asthma have been found to have more ILC2s in their blood than non-asthmatic individuals [40,41]. As very few naïve ILC2s circulate, ILC2s in the peripheral blood of asthma patients may be previously activated memory ILC2s. It remains to be investigated whether other ILCs also have the capacity to acquire memory functions.
Concluding Remarks Whereas antigen specificity has been thought to be a hallmark of immunological memory, which only T and B cells have, the discovery of hapten- and virus-specific NK cell memory has
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shown that innate lymphocytes also have immunological memory. Recent findings of memory in innate cells have further widened our understanding of immunological memory to include antigen-nonspecific memory (Figure 2, Key Figure). In general, memory is defined by the capacity to encode, store, and retrieve information [35]. In accordance, immunological memory can now be defined by the ability of immune cells to remember information recorded from a previous activation state and to have stronger effector functions on challenge than during the primary activation. Whether it occurs through the ligation of antigen-specific receptors, cytokine receptors, or Toll-like receptors, activation of naïve cells induces changes in gene expression, some of which are maintained for a long time as record of previous activation states in memory cells. Future research in the field focused on the factors that give rise to memory in ILC2s, as well as potentially in other ILC subsets (see Outstanding Questions), should allow us to further define the expanding concept of immune memory and the sweeping effects of experience in immune responses. Acknowledgments The authors thank Reyes Gonzalez-Aller for illustrating Figure 2. They are supported by CIHR, MSFHR, UBC, and Vanier CGS.
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