New insights into ILC2 memory

Group 2 Innate Lymphoid Cells (ILC2s) are innate lymphocytes involved in type 2 immunity. ILC2s are abundant at the barrier tissues and upon allergen exposure, respond to epithelial‐derived alarmins by producing type 2 cytokines (e.g., IL‐5 and IL‐13). Upon activation, some of these activated ILC2s acquire immunological memory and can mount enhanced responses upon further allergen encounters. Here, we review recent findings of the cellular and molecular mechanisms underlying immune memory in ILC2s both in mice and humans and discuss the implications of memory ILC2s in the context of allergic diseases.


| G ROUP 2 INNATE LYMPHOID CELL (ILC 2) DEFINITI ON AND FUN C TI ON
ILC2s are abundant at the barrier tissues and are the first sensors of changes in the microenvironment of the tissue they reside in.
In contrast to their T cell counterparts, ILC2s do not express rearranged receptors and therefore lack antigen recognition.Instead, ILC2s express the receptors for alarmins IL-33, thymic stromal lymphopoietin (TSLP) and IL-25, the receptors for the neuropeptides Neuromedin U (NMU) and vasoactive intestinal polypeptide (VIP), the receptors for the lipid mediators leukotriene D 4 (LTD4) and prostaglandin D 2 (PGD2) and the common gamma chain (γc) family of cytokine receptors including IL-2R and IL-7R.In response to all these factors, ILC2s proliferate and produce type 2 cytokines, including IL-5 and IL-13 and growth factors such as granulocyte macrophage colony stimulating factor (GM-CSF) and amphiregulin (AREG). 1,2ILC2s regulate tissue homeostasis.For instance, they mediate adipose tissue browning which may prevent the development of metabolic abnormalities. 3 Moreover, they are key players during parasite infections as ILC2-derived IL-13 is necessary for helminth expulsion and subsequent tissue repair. 4[7] On the contrary, ILC2s can be pathogenic and induce an allergic immune response upon allergen encounter in several tissues including the skin, lung, and intestine.][10][11][12][13][14] ILC2s have been considered the initiators of these allergic responses as they are the first sensors of the tissue-derived alarmins.In a second step, the adaptive immune responses, namely T helper type 2 cells (Th2) cells and IgE, take over the later phases of the allergic responses. 1DOI: 10.1111/imr.13323

| D ISCOVERY OF MEMORY ILC 2s
Lung ILC2s proliferate upon exposure to allergens.The number of ILC2s remain elevated in the lung long after allergen inhalation.This finding led us to the hypothesis that perhaps ILC2s could have a role in subsequent allergen encounters.A single intranasal administration of the alarmin IL-33 a month after the initial exposure to the protease allergen papain, induced a strong expansion and activation of lung ILC2s and eosinophilia in the lung, while little response was observed in naïve mice exposed to a single IL-33 challenge, suggesting that allergen experienced ILC2s are more sensitive to stimuli than naive ILC2s.This acquired ability to enhance their response to stimuli suggested immunological memory in ILC2s. 15We demonstrated that the acquisition of immunological memory was a cell intrinsic feature of ILC2s, as transfer of memory ILC2s into naive mice induced an enhanced response to allergen challenge compared to naive ILC2 transfer.
Moreover, purified memory ILC2s exposed to suboptimal amounts of IL-33 + TSLP in vitro resulted in more production of IL-5 and IL-13 than naive ILC2s.However, we could not exclude a contribution from the environment.One possibility could be that the first exposure to an allergen induces changes in the lung epithelium to constantly release IL-33 and that during the secondary challenge, ILC2s had higher accessibility to IL-33, explaining their enhanced response.However, IL-33 KO mice treated with IL-33 and challenged 1 month later with IL-33 displayed an enhanced ILC2 activation and lung inflammation.Therefore, the maintenance of memory ILC2s could be independent of epithelial-derived IL-33.Whether other changes in the lung environment happening after the first exposure to allergens have a role in the activation of memory ILC2s during the secondary challenge needs to be further explored.One approach may be to administer an allergen into an ILC2 KO mice, which will induce changes in the lung and then challenging 1 month later after transferring memory or naive ILC2s.
An enhanced response in the mice transferred with memory ILC2s would suggest that the lung environment is not as important in this process.
It has been long thought that only adaptive lymphocytes can acquire immunological memory as antigen specificity is thought to be a key characteristic of immunological memory.However, the definition of immunological memory can be revised as the capacity of a cell to remember a previous activation state regardless of the way of activation.[17] Currently, the field has generally accepted that immune memory can also be a characteristic of an innate immune cell.9][20][21] Indeed, NK cell memory has been extensively investigated and the mechanisms behind it have been elucidated. 22,23[26]
Three consecutive intranasal administration of IL-33, but not IL-25, into naive mice strongly activates lung ILC2s.The majority of lung ILC2s stimulated by intranasal IL-33 or allergens express the proliferation marker Ki67, proliferate and become cytokine-producing effector ILC2s.Effector ILC2s quickly stop proliferating and stop producing cytokine within 2 weeks after the activation, and ILC2 numbers continuously decline over the following 4 weeks while memory ILC2s persist for more than 6 months. 15Our microarray transcriptome analyses of naive, effector (4 days after IL-33 treatment) and memory ILC2s (4 months after IL-33 treatment) showed upregulation of a large number of transcripts, mostly those associated with cell activation and proliferation, in effector ILC2s.Most of those activation-associated genes were downregulated to the level of naive ILC2s in memory ILC2s.However, a small number of those transcripts remained higher in memory ILC2s than naive ILC2s.Among them is Il17rb.Memory, but not naive, ILC2s in the lung express IL17RB (IL-25R) and strongly react to a single intranasal challenge with IL-25. 15Preliminary unpublished results from our group show that memory responses of ILC2s are diminished in the IL-25R KO suggesting that this alarmin is important for memory ILC2 responses.In vitro stimulation of purified memory ILC2s with IL-25 does not result in IL-5 and IL-13 production, indicating that activation of memory ILC2s require other co-stimulatory signals.Our unpublished results demonstrate that memory ILC2s need cell contact with dendritic cells and T cell-derived IL-2 in addition to IL-25 to mount full memory responses.These data suggest that memory ILC2s may relocate to specific tissue niches that contribute to their enhanced responsiveness.
Naive ILC2s express IL-5 and IL-13 mRNA but secrete very little cytokines.Upon activation, ILC2s upregulate those mRNAs and produce high levels of these cytokines.Memory ILC2s, which are in "rest", maintain elevated IL-5 and IL-13 mRNA, but only secrete cytokines when they are activated.This suggests translational regulation of cytokine production in memory ILC2s.

Further analysis of our microarray data of naïve and memory lung
ILC2s, revealed that transcriptional regulation mediated by RUNX family of transcription factors may be involved in memory generation (Figure 1A).It has been shown that Runx1 influences effector and memory T CD8+ cell formation. 27Miyamoto et al. showed that during allergic inflammation, the absence of Runx1 impairs the ability of ILC2s to proliferate and produce effector Th2 cytokines and chemokines.Instead, functional deletion of Runx1 induces the expression of exhaustion markers, such as IL-10 and TIGIT, on ILC2s.Thus, suggesting that Runx1 protects ILC2s from exhaustion during allergic inflammation. 28Other pathways that seem important for memory ILC2 formation is the TCF/LEF/β-catenin pathway and adherent junction (Figure 1B,C).These pathways regulate cell-cell contacts.As mentioned before, memory ILC2s interact with DCs to fully respond to IL-25.
Verma et al. 29 have recently reported ILC2 memory in RAG-KO mice.In a chronic asthma model of Alternaria, memory ILC2s were responsible for asthma-like symptoms including airway hyperresponsiveness.They further characterized the epigenome landscape and found that Activator Protein 1 (AP-1), a major transcriptional activator, was one of the top DNA-binding motifs of memory ILC2s.
They suggest that memory ILC2s express both suppressors' genes such as the transcription factor JunD and "preparedness" genes such as Four-and-a-half LIM domain protein 2 (Flh2) and that after challenge become active and downregulate the repressor genes, allowing ILC2s to strongly respond.Transcriptomic and epigenomic analyses of memory ILC2s have been done with bulk ILC2s and future studies should analyze memory ILC2s at the single cell level.
In a follow-up paper, the same authors showed that NFkb1 inhibits memory formation but supports the effector function of ILC2s in memory-driven asthma. 30

| AC TIVATI ON -INDUCED MI G R ATI ON , MEMORY, AND E XHAUS TI ON OF LUNG ILC 2
2][33] The ILC2 turnover rates in adult mice vary between tissues, lung ILC2s having the lowest (30%) and the skin ILC2s the highest rate (80%). 34o weeks after birth, mouse lung ILC2 numbers increase to a level higher than those in naive adult mice.It is due to an intrinsic wave of IL-33 production in the lung, which seems to drive functional maturation of lung ILC2s. 35In the skin, ILC2s seem to be less responsive than in other tissues.Topical application of calcipotriol induces atopic dermatitis-like symptoms in the mouse skin, and TSLP is an important mediator in this process. 368][39] ILC2 repopulation of empty niches after irradiation was also shown by parabiosis mouse analysis. 40We have previously shown that intranasal administration of IL-33 or papain into mice increases ILC2 numbers in blood at day 7 with a subsequent accumulation in the liver. 41rabiosis mouse analyses showed that the accumulation of ILC2s in the liver was due to migration of activated ILC2s from the treated lung.The liver is also a reservoir for memory NK cells. 42However, we have recently found that these ILC2s accumulated in the liver upon allergen inhalation do not live long and do not respond to i.v.injection of IL-33 more than naive liver ILC2s, indicating that they are not memory ILC2s (unpublished).Memory generation and migration are not the only fates of activated ILC2s.Based on the rapid expansion and rapid contraction of ILC2 numbers upon allergen inhalation, it is likely that most of the expanded ILC2s undergo cell death, although it has not been well studied.It also seems that some of these activated ILC2s become exhausted. 28hausted ILC2s express killer cell lectin-like receptor G1 (KLRG1),

| MEMORY ILC 2s IN THE LUNG -DR AINING LYMPH NODE
ILC2s reside also in lymphoid organs in very low numbers.Upon intranasal administration of IL-33, ILC2s in the lung-draining mediastinal lymph node (mLN) greatly increase in numbers, and mLN ILC2s incorporate more BrdU than lung ILC2s.Purified ILC2s from the mLN of IL-33-treated mice show an enhanced proliferation rate in vitro compared to lung derived ILC2s (unpublished data).It seems likely that some activated lung ILC2s migrate to the draining mLNs through the lymphatics and proliferate within the mLN.Increased numbers of ILC2s in other tissues draining LNs such as the skin and the gut upon inflammation have also been reported. 43,44Role of ILC2s in the LNs is still unknown.ILC2s in mLN can also become memory cells and they differ from lung memory ILC2s both at the transcriptomic and protein levels. 15In contrast to memory ILC2s in the lung, mLN memory ILC2s maintain high levels of intracellular staining of IL-5 and IL-13 for a long time (more than 6 months).However, it is unlikely that those ILC2s in the mLN secrete IL-5 and IL-13 at this stage as eosinophils are no longer present in the mLN.Purified memory ILC2s from the mLN stimulated with IL-33 plus TSLP produced more IL-5 and IL-13 than memory ILC2s from the lung (unpublished).These findings suggest that memory ILC2s in the lung and mLN are different, reminiscent of central and tissue resident effector memory T cells. 45[48]

| HUMAN MEMORY ILC 2s
To study memory in human ILC2s is more complicated as humans are exposed to environmental triggers since birth and therefore it seems unlikely that all ILC2s in healthy individuals are naïve.During inflammation, ILC2 numbers increase in both circulation and tissues from patients with type 2 inflammatory conditions such as asthma, chronic rhinosinusitis with nasal polyps (CRSwNP), [49][50][51] and atopic dermatitis. 12,52,53These ILC2s have changed their transcriptome and epigenome and have a cell activation signature.In vitro activation of human ILC2s isolated from peripheral blood (PB) evoked substantial H3K4Me2+ epigenome expansion around genes involved in lipid metabolism, cytokine/chemokine signaling, and asthma. 54Recently, the human counterparts for the mouse inflammatory ILC2s 55 have been defined as Lin-CD127+ CD45RO+ ILC2s in both PB and nasal polyps from CRSwNP patients. 56 transcription factor (BATF) transcription factors.Moreover, inflammatory CD45RO+ ILC2s are steroid resistant compared to naive CD45RA+ ILC2s.In a follow-up study, Golebski et al. 57 showed that the abundance of CD45RO+ ILC2s in PB of patients with CRSwNP is associated with a rapid response to dupilumab, which inhibits ILC2 activation by blocking IL-4/IL-13 alpha receptor.Dupilumab has been shown to be a safe and effective treatment in severe and uncontrolled CRSwNP patients. 58C2s from PB from healthy individuals are mostly CD45RA+ and considered to be naive.We have now described CD127-CD45RO+ memory ILC2s. 59We purified CD45RA+ ILC2s from healthy tissues (PB, skin) and cultured them in two ways, (1) in the presence of IL-2 + IL-7 or (2) IL-2 + IL-7 + IL-33 + TSLP for 5 days.The former maintained CD45RA+ ILC2s without activation whereas in the latter, CD45RA+ ILC2s were activated and became CD45RA-CD45RO+ and maintained CD45RO expression long after they became resting.We then added IL-33 for 24 h into both cultures.The CD45RO+ ILC2s produced more IL-5 and IL-13 than the CD45RA+ ILC2s.CD127 (IL7R) is the canonical marker for human ILCs used for identification and isolation of ILC2s from several human tissues.
We have observed that activated ILC2s reduced the expression of CD127.We found CD127-CD45RO+ ILC2s in healthy tissues including the skin and upon ex vivo stimulation they showed a quicker and more robust response than CD127+ CD45RA+ ILC2s, characteristics of immunological memory.Memory ILC2s are likely present in healthy skin as is a tissue constantly exposed to the environment.Memory ILC2s expressed many activation-associated genes although they are in a resting state.They were marked by the expression of Nuclear Receptor subfamily 4 group A member 1 (Nr4a1) and genes from the AP-1 pathway such as FosB and JunD.A very recent publication identifies a distinct activation subset of ILC2s in the mouse inflammatory lung that expressed Nr4a1 and shared a gene signature with memory T cells. 60Verma et al. transferred FLH2+ ILC2s from PB of asthma patients into immunodeficient mice and found that they induced airway hyperreactivity upon alternaria administration, in contrast, FLH2-ILC2s had no effect.Whether memory ILC2s in humans can be identified by the expression of FLH2 needs to be further validated.ILC2s from adult PB express cMaf whereas cord blood ILC2s are cMaf negative. 61,62In mice, naive ILC2s are cMaf-and it is induced upon activation and remains highly expressed in memory ILC2s.The deletion of cMaf in memory ILC2s inhibits their enhanced response, suggesting that this transcription factor may be important in memory ILC2s.
These findings indicate dynamic changes in CD127 expression that may reflect a continuum of activation and memory states of ILC2s.This phenomenon has been recently shown for memory T cells. 63C chemokine receptor 3 (CXCR3) expression in CD8+ T cells correlates with the differentiation degree after encountering an antigen.The higher the expression CXCR3 is, the more differentiated and less responsive T cells are.CXCR3 low T cells show enhanced capacity to respond to stimuli, suggesting that they are memory cells.
It has been suggested that upon allergen challenge in humans, ILC2s from the circulation are recruited into the airways and fill the alveolar space. 64These ILC2s in the bronchoalveolar lavage (BAL) had a gene activation profile linked to lower expression of IL7R, suggesting they could be memory ILC2s, which were found elevated in the BAL after the allergen challenge.

| IMPLI C ATI ON S OF MEMORY ILC 2s IN DISE A SE S
We have previously shown that priming mice airways with the protease allergen papain and challenging with the fungus Aspergillus induced a stronger memory ILC2 activation and allergic lung inflammation than in the mice receiving Aspergillus challenge without papain priming. 15Asthmatic patients usually react to many different allergens.Diagnosis is done using skin prick tests, where skin is exposed to suspected allergy-causing substances and is then observed for signs of an allergic reaction.There are many false negatives, which are explained by the test's lack of sensitivity.However, these tests are based on antigen-specific reactions as there will be allergic reactions only if the individual presents with the specific IgE.It may be possible that antigen nonspecific memory ILC2s are responsible for the IgE-independent allergy.ILC2s also play a role in allergic reactions in other tissues, such as the skin and the intestine.Skin biopsies from patients with atopic dermatitis are enriched with ILC2s and mouse models of atopic dermatitis demonstrate a role for ILC2s in the pathogenesis.In food allergy, the role of ILC2s is less clear but it has been suggested that ILC2-derived IL-4 suppresses regulatory T cells and activates mast cells in the gut facilitating allergic reactions in the intestine in a mouse model of food allergy. 65e allergy journey starts in childhood with the appearance of atopic dermatitis followed by food allergy and continues with respiratory allergies such as rhinitis and asthma.This clinical observation is referred to as "atopic march" and the immune mechanisms behind it are not known. 66As the allergens that cause the reactions at the different tissues are usually not the same, it is hard to place T cells as the main responsible factor.On the contrary, previously activated ILC2s which enter the circulation may seed other tissues or be recruited by them when they encounter an allergen.Elucidating the role for memory ILC2s in the atopic march process will unveil novel targets for an early intervention to stop more serious allergic diseases such as asthma from developing later in life.Emerging evidence shows that memory T cells can also mount antigen nonspecific responses. 67,68Upon differentiation, Th2 cells express ST2 (IL-33R) and therefore become responsive to this alarmin.However, in our mouse models of allergic lung inflammation involving unrelated allergens, the contribution of T cells to the overall IL-5 and IL-13 production in the lung was minimal and the majority of IL-5 and IL-13 positive cells were ILC2s.Memory ILC2s produced more cytokines per cell than Th2 cells.We have previously shown that memory ILC2s enhanced Th2 responses, 15 suggesting that memory ILC2s are not only important for the allergen nonspecific inflammation but they also potentiate Th2 cell responses.
Whether memory ILC2 numbers reflect disease conditions in allergic patients is not known.It is also unknown whether memory ILC2s are steroid resistant and sensitive to dupilumab like inflammatory ILC2s.Correlation between the abundance of memory ILC2s and response to treatment needs to be determined.It is possible that memory ILC2s could be used as a biomarker to guide a more personalized therapeutic approach.

| CON CLUDING REMARK S
Growing evidence indicates that the five classifical subtypes of ILCs (NK cells, ILC1s, ILC2s, ILC3s, and LTi) are not as well-defined as we originally thought.Plasticity is a common phenomenon in ILCs and almost every ILC can transition into other ILC subtype in the appropriate tissue microenvironmental conditions.Furthermore, upon activation, ILCs can acquire several cell states, that is, inflammatory, memory, regulatory etc.Indeed, in some contexts, single cell transcriptomic analysis cluster ILCs based on their cellular state rather than their lineage origin.Acknowledging these blurry boundaries in mature ILCs may be important when approaching future studies in the field.
The finding of immunological memory on ILCs has opened a new field of research with many questions.What are the implications of having memory ILCs in our organism?Would memory ILCs protect us better to unrelated infections?Would they become faster in repairing our tissues with accumulating episodes of tissue damage?
Given their antigen nonspecific nature and their enhanced sensitivity to stimuli, would memory ILCs connect apparently unrelated diseases?These compelling questions warrant future studies.

CO N FLI C T O F I NTE R E S T S TATE M E NT
Authors declare no conflicts.
Skin ILC2s proliferate as they increase Ki67 expression (unpublished data), but they produce very little IL-5 and IL-13.Even purified skin ILC2s need longer time to respond to stimuli in vitro compared to lung ILC2s.This also happens for ILC2s isolated from the bone marrow.It is possible that skin ILC2s might be dysfunctional due to constant exposure to environmental factors.Alternatively, skin ILC2s might be similar to bone marrow ILC2s and functionally immature.Interestingly, a secondary calcipotriol treatment induces ILC2 activation both in vivo and in vitro, and these ILC2s produce IL-5 and IL-13 (not shown).This observation led us to hypothesize that the high turning over rate of skin ILC2s accounted for their immature state and explains why it takes more than one stimulus to induce functional maturation of ILC2s in the skin.Tissue ILC2s do not circulate or repopulate empty niches at steady state, however, upon activation, a minor subset of ILC2s

F I G U R E 1
Functional enrichment in memory ILC2s.Gene enrichment analysis on differentially expressed genes between memory and naïve ILC2s using Enrichr.(A) Enrichment of RUNX regulated genes in memory ILC2s.(B, C) Pathway enrichment analysis, quering KEGG (B) and Reactome (C) databases, show overrepresentation of cell-cell contact and B-catenin related genes in memory ILC2s compared to naïve ILC2s.Bar graphs represented the significance of the enrichment (p value).programmed cell death protein 1 (PD-1), and glucocorticoidinduced TNFR-related (GITR) and are unresponsive to subsequent stimuli.However, it should be noted that most effector ILC2s express KLRG1 and PD-1, therefore, they are not specific markers for exhausted ILC2s.In our recent studies, we gave multiple intranasal allergen administrations into mice over 5 weeks and found that lung ILC2s become less responsive to intranasal IL-33 than those stimulated only once, suggesting that ILC2s are exhausted by chronic allergen treatments.Flow cytometry analysis of those dysfunctional ILC2s showed that they are heterogeneous, mostly expressing PD-1, some expressing T Cell Immunoreceptor with Ig and ITIM Domains (TIGIT) and a minor double negative population.When purified and stimulated in vitro with IL-33 plus IL-7, both PD-1+ and PD-1 − ILC2s were vigorously activated and produced cytokines, whereas TIGIT+ ILC2s were not activated, suggesting that they are dysfunctional (unpublished).The relationship between effector, memory, migratory, and exhausted ILC2s remain to be elucidated.Whether memory ILC2s develop from effector cells, or they develop separately remains to be determined (Figure 2).Identifying the molecular mechanisms dictating the fate of an ILC2 after being activated will enable future interventions towards modifying the fate of ILC2s when necessary.
CD45RO+ ILC2s can be generated from naive CD45RA+ ILC2s in the presence of IL-33 and TSLP.The CD45RA-CD45RO conversion is tightly linked to Signal transducer and activator of transcription 5 (STAT5) activation and upregulation of the interferon regulatory factor 4 (IRF4)/basic leucine zipper F I G U R E 2 Hypothetical scheme of generation of memory ILC2s and relationship between various subsets of activated ILC2s.Lung ILC2s in naïve mice do not express PD-1 and IL-17RB (IL-25R) and remain in the lung as tissue resident lymphocytes.Intranasal administrations of allergens or the alarmin IL-33, but not IL-25, into naïve mice activate lung ILC2s.Most, but not all activated lung ILC2s express PD-1 and IL-17RB, proliferate and become cytokine producing effector ILC2s.A small subset of activated lung ILC2s becomes migratory and exits the lung, circulate and mostly settle in the liver, but they are short-lived in the liver.Effector ILC2s stop producing cytokines within days and slowly decline in numbers, presumably due to activation-induced cell death (AICD).Another small subset of activated PD-1 lo/− IL-17RB + ILC2s persist in the lung for months.Weeks after the initial allergen/IL-33 treatment, most lung ILC2s are PD-1 −/lo and some IL-17RB + memory ILC2s.Those memory ILC2s are vigorously activated by intranasal administration of allergen, a single dose of IL-33 or IL-25 and become effector and migratory ILC2s while some remain memory ILC2s.
The authors would like to thank Dr. Sergio Martinez-Høyer for data and figure support.IMG is financially supported by Wallenberg Academy Fellow (KAW 4-239/2020), Swedish Research Council and Karolinska Institute (2018-02377).FT is supported by grants from the Canadian Institute of Health Research (Grant numbers: 153304 and 173434).