Initiation of type 2 immunity at barrier surfaces

Abstract

Although seemingly unrelated, parasitic worms, venoms, and allergens all induce a type 2 immune response. The effector functions and clinical features of type 2 immunity are well-defined, but fundamental questions about the initiation of type 2 immunity remain unresolved. How are these enormously diverse type 2 stimuli first detected? How are type 2 helper T cells primed and regulated? And how do mechanisms of type 2 initiation vary across tissues? Here, we review the common themes governing type 2 immune sensing and explore aspects of T cell priming and effector reactivation that make type 2 helper T cells a unique T helper lineage. Throughout the review, we emphasize the importance of non-hematopoietic cells and highlight how the unique anatomy and physiology of each barrier tissue shape mechanisms of type 2 immune initiation.

INTRODUCTION

Type 2 immune responses mediate protective immunity against parasitic worms (helminths) in mammals and are largely focused on tissue remodeling. For example, increased mucus production, smooth muscle hypersensitivity, skin thickening, and fibrosis are all designed to impede helminth migration and expel them when they take up residence. Type 2 immunity also encompasses immunoglobulin (Ig)E-mediated mast cell degranulation and the resulting vascular fluid leak, smooth muscle contraction, and tissue swelling that occurs within minutes and can lead to anaphylaxis. Why such rapid and potentially harmful responses are elicited by non-replicating helminths remains unclear. It has been proposed that acute type 2 immune responses instead evolved to protect against toxins, venoms, and biting insects^1,2. Indeed, some of these widespread immunologic features of type 2 immunity, such as mast cell degranulation and IgE production, have been associated with increased resistance to subsequent toxin exposure^3,4. Unfortunately, type 2 immunity is also often directed at harmless environmental stimuli and is the cause of the growing prevalence of asthma and allergy. The complete list of type 2 stimuli is, therefore, astonishingly diverse, incorporating everything from macroscopic helminths to soluble enzymes to inert particles. Thus, from the perspective of immune sensing and initiation, we are left with the question of how such diverse stimuli are sensed by the host and how these signals are interpreted to achieve a similar downstream immune response.

Following initiation of type 2 immune responses, both humoral and cell-mediated responses are activated. The details of antibody-mediated immune responses to allergens and helminths have been reviewed elsewhere^5,6 and are discussed only briefly here. Similar to other immune responses, follicular helper CD4 T cells (Tfh) in the lymph node (LN) are needed for germinal center reactions and subsequent B-cell antibody isotype switching. Although IgE-mediated activation of mast cells is central to allergic disease, the role of antibodies in anti-helminth immunity is likely parasite and tissue specific. Early studies found that IgG antibodies against Trichinella spiralis could provide protection against subsequent oral infection⁷, but not intestinal stage parasite⁸. B-cell deficient mice have no problem clearing Nippostrongylus brasiliensis, which infects multiple tissue sites; however, antibodies are critical for expulsion of intestinal-dwelling Heligmosomodies polygyrus⁹. Furthermore, IgM is necessary for both primary and secondary clearance of Brugia pahangi¹⁰. Further studies have proposed that antibodies produced in response to helminth infection may not be functionally beneficial for the host^11,12, as helminth molting can result in loss of exposed epitopes in a matter of days.

On the cellular side, group 2 innate lymphoid cells (ILC2s) and type 2 CD4 T helper cells (Th2s) act as a central hub for type 2 immunity. Their protective function in helminth infection, and detrimental role in allergy, have been repeatedly demonstrated^13–15. ILC2s are among the first immune cells activated in a primary type 2 immune response and are an important early source of interleukin (IL)-5, IL-9, and IL-13 in the tissue. In some contexts, tissue-resident natural killer T cells or γδ T cells also act as type 2 sentinels and are important sources of IL-4 and IL-13^16–18. Once activated, Th2s become the dominant source of these cytokines as well as IL-4, which collectively coordinate hallmarks of type 2 immunity in the tissue, as well as recruitment of cells like eosinophils and basophils that can further amplify the response. In this review, we will focus on mechanisms of immune sensing that lead to activation of ILC2s and Th2s.

Throughout the review, we will also highlight two important conceptual themes. First, certain aspects of type 2 immunity are highly tissue specific. Airway cells predominately contact inhaled particles. Skin epithelial cells are subject to venoms and toxins, and many potential allergens pass through damaged skin. The intestinal tract is constantly exposed to food allergens and is the primary niche of helminths (although helminth lifecycles can affect other barrier tissues, too). These different environmental exposures, combined with the unique anatomy and physiology of each barrier, drive the emergence of tissue-specific mechanisms governing type 2 immunity. Second, coincident detection of multiple signals is critical for regulatory control of type 2 immunity. Given the diversity of type 2 stimuli, evolving and maintaining germline-encoded receptors for each insult is unrealistic. In contrast, sensing relatively common structures or processes (e.g. cell death) risks non-specific activation. Coincident detection of non-redundant signals can provide regulatory control, and there are numerous instances where this occurs during initiation of type 2 immunity.

We will start this review by considering immune sentinels and the mechanisms by which they detect type 2 stimuli. Next, we will describe how ILC2s integrate signals from these sentinels for early type 2 responses. We will end by discussing how Th2s are primed in LNs and licensed for effector function in the tissue.

Innate sensing of type 2 stimuli: common themes

Innate sensing is the first obligate step of any immune response. In the context of viral, bacterial, and fungal infections that trigger type 1 and type 3 immunity, two innate sensing strategies predominate. First is pattern recognition, where germline-encoded receptors have evolved to bind microbe-encoded ligands that are common to many microbes and essential for their survival or pathogenesis (e.g. bacterial and fungal cell walls or viral nucleic acid)¹⁹. The second is detection of pathogenic activities, or so-called “patterns of pathogenesis” (e.g. invasion of the cytosol or inhibition of translation)²⁰. As discussed below, there is evidence that both strategies also apply to the detection of type 2 stimuli. For example, proteolysis and mechanical tissue damage are activities associated with many type 2 stimuli and detected by type 2 sentinels. At the same time, pattern recognition receptors and non-classical chemosensory pathways also contribute to innate type 2 responses (Fig. 1).

Fig. 1.

Themes of type 2 stimuli sensing across barrier tissues. Allergens, venoms, and helminths penetrate the epithelium, resulting in direct and indirect cellular damage. Direct cellular damage is mediated by allergic proteases and helminths, but proteases themselves can also be detected by protease-activated receptors. These signals broadly result in alarmin production by the epithelium. Proteases further act on neurons in the skin, leading to the release of neuropeptides. In the airways, allergens are inhaled, where they collect around airway branch points. Helminths in circulation can also burst through lung capillaries to invade and damage alveoli. Intestinal helminths cause overt physical damage to the epithelium, but they can also activate classical pattern recognition receptors and chemosensing pathways. Despite differences in anatomical niches, alarmins, neuropeptides, and direct receptor binding activate dendritic cells and ILC2s to trigger downstream type 2 immune responses. DC = dendritic cell; ILC2 = group 2 innate lymphoid cells.

Sensing of cell death and tissue damage

Detection of cell death caused by helminths and allergens is a critical component of type 2 immunity, although the mechanisms leading to cell death can vary greatly. Helminths are large, multi-cellular parasites that crawl through tissues and can either directly lyse cells or secrete enzymes that cause tissue damage. In the case of allergens, cellular damage is often due to the activity of allergic proteases. Derp1, found in house dust mite (HDM) allergen, is one example of a protease that disrupts intracellular tight junctions²¹. Protease activity in Aspergillus cultures has also been linked to nasal epithelial damage in asthma models²². In the absence of serine proteases, Aspergillus stimulation does not disrupt the actin cytoskeleton of epithelial cells. Other inert allergens cause tissue damage in the absence of any protease activity. Diesel exhaust particles (DEPs), for example, consist of hydrophobic molecules that diffuse through cell membranes and trigger the generation of free radicals that can lead to DNA damage and inflammatory cytokine production²³. Silica crystals, aluminum salts, and microparticles are also associated with direct damage to epithelial cells²⁴ or indirect tissue damage through macrophage activation^25–28.

Sensing of cell death in the context of type 2 immunity hinges predominantly on the release of bioactive IL-33, which is normally sequestered in the nucleus of viable cells²⁹. Following its release from dying cells, IL-33 can be cleaved extracellularly to its more active mature form by neutrophil proteases and calpains^30,31. IL-33 signals via its receptor, ST2, expressed by numerous cell types, including ILC2s and Th2s and amplifies type 2 immune responses. In the case of allergy, epithelial damage leading to IL-33 release generally exacerbates allergic disease. Indeed, IL-33 release due to epithelial necroptosis was found to worsen Aspergillus extract-induced asthma³². Mouse models with exaggerated use of the necroptosis pathway also have exacerbated HDM-induced allergy³³, although the role of necroptosis-induced IL-33 was not examined in this study. Another model suggests that allergens acting on the airway epithelium can induce caspase-8 activity leading to the intracellular production of mature IL-33³⁴. In this model, mature IL-33 release preceded cell death suggesting a novel form of IL-33 secretion exists. Indeed, several studies have suggested that IL-33 may be released from either gasdermin C³⁵ or gasdermin D pores³⁶. Inert allergens, including DEPs, uric acid, chitin, and microparticles, induce release of IL-33, thereby exacerbating allergic models as well^27,37–40. Independently of IL-33, death of CX3CR1+ myeloid cells resulting from DOCK8 deficiency can also skew Th2 responses⁴¹. This was attributed to release of IL-1β, which amplified T cell production of granulocyte-macrophage colony-stimulating factor (GM-CSF). Adenosine triphosphate (ATP) released from dying cells can also promote type 2 responses. Airway tuft cells (a.k.a. brush cells) can sense ATP through P2Y2 receptor signaling⁴², while A2BAR expressed on the intestinal epithelium senses adenosine metabolized from extracellular ATP during helminth infection^43,44.

Cell damage independent of cell death has also been implicated in skewing type 2 immune responses. Phospholipase A₂ (PLA₂) is a major allergen in bee venom⁴⁵ and acts by cleaving membrane phospholipids. PLA₂ activity led to production of IgE as well as IL-33, which activated Th2s and ILC2s⁴⁶. Interestingly, immunization with bee venom PLA₂ protects against a subsequent near-lethal dose of PLA₂, suggesting that type 2 immunity to venoms is a protective immune response.

Sensing of protease activity

As described above, protease activity can be sensed indirectly when it leads to cell damage/death and release of IL-33, but there are also examples where proteases are detected independently of cell death. Many allergens are themselves proteases or enter the body together with proteases, although protease sensing is neither necessary nor always sufficient to induce type 2 immunity. Host cells can directly sense protease activity through protease-activated receptors (PARs), whose activity generally leads to exacerbation of allergic disease. Derp1 found in HDM is a well-studied example of this, but cockroach and mold extracts contain proteases as well. Derp1 cleaves and activates PAR-2 on respiratory epithelial cells, leading to secretion of IL-6 from in vitro cultures⁴⁷. The role of PAR-2 activation in mouse models of HDM allergy is unclear, as PAR-2 deficient mice still develop allergic responses and IgE⁴⁸. This may be partially due to the ability of Derp1 to cleave other surface molecules on epithelial cells, such as CX3CL1, leading to PAR-2-independent immune regulation⁴⁹. PAR-2 deficiency does protect against allergic responses to other proteases, including subtilisin and German cockroach frass^50,51.

For other allergic proteases, the sensing mechanism remains to be elucidated. Airway club cells can sense protease activity of Aspergillus through an unknown receptor, leading to TRPV4 mediated inflammation⁵². As many allergic extracts contain multiple immunostimulatory factors, the cysteine protease papain from papayas is often used to selectively study protease sensing. In this case, basophils and macrophages both detect papain dependent on its enzymatic activity⁵³. Papain, as well as allergic proteases in HDM, can also directly act on TRPV1+ sensory neurons, leading to their release of the neuropeptide Substance P^54,55. Substance P, in turn, stimulates both innate and adaptive type 2 immune responses by signaling mast cells and dendritic cells⁵⁵.

Perhaps the ‘guard theory’ of immune activation can provide a lens through which to consider sensing of protease activity. This theory suggests that insults leading to disruption of normal cellular functions can be sensed by ‘guard proteins’ that initiate immune defenses. Such mechanisms have been described for protease sensing in plant immunity⁵⁶ and type 1 immunity in mammals⁵⁷. Gasdermin D, which was recently described to form pores mediating IL-33 secretion in response to protease activity, might therefore serve as a guard protein for type 2 immunity³⁶. Ultimately, both direct and indirect sensing of proteases likely contributes to initiation of type 2 immunity against some allergens, underscoring the complexity of responses by type 2 sentinels and the potential to integrate multiple signals.

Pattern recognition

A final theme in sensing type 2 stimuli by the host immune system is direct sensing of allergen- or helminth-encoded ligands. Classical pattern recognition receptors (PRRs) are widely appreciated for their roles in sensing bacteria, viruses, and fungi⁵⁸. Some evidence exists to suggest that PRRs also play a role in allergen and helminth sensing. HDM extract contains numerous proteins and glycans apart from the Derp1 protease, and these glycans have been found to activate the C-type lectin receptor (CLR) Dectin-2 on CD11c+ alveolar macrophages in the lung, leading to production of leukotrienes^59–61. HDM-derived glycans can also stimulate airway epithelial cell production of CCL20, which is important for dendritic cell (DC) recruitment⁶². HDM also contains proteins that resemble myeloid differentiation protein 2, which is known to bind toll-like receptor 4 (TLR4) during lipopolysaccharide sensing. Indeed, HDM can activate TLR4 on stromal cells leading to production of various alarmins and GM-CSF⁶³. Type 2 inflammation following Cryptococcus neoformans infection was recently attributed to a secreted effector targeting non-canonical TLR4 signaling⁶⁴. Uncovering why TLR4 is a common target of type 2 pattern recognition remains to be elucidated. In particular, it is unclear how a receptor with well-defined roles in initiation of type 1 immunity can be repurposed for type 2 immunity, but this is likely an example of signal integration (e.g. TLR and cell death) determining context-specific outcomes. Indeed, macrophages, in particular, seem to require multiple signals for activation during type 2 inflammation. Surfactant in the lung, C1q in the peritoneal cavity, and apoptotic cells all provide critical signals that pair with IL-4 for alternative activation of macrophages^65,66.

Recognition of helminths by classical PRRs has largely been studied in the context of Schistosome mansoni sensing by CLRs. Adult S. mansoni lay eggs in their human hosts, which migrate to the intestine and directly skew T cell responses away from Th1, and toward Th2⁶⁷. To study how this occurs, homogenate of these eggs, called soluble egg antigen (SEA), has been used. SEA is comprised of numerous proteins and glycans that can interact with CLRs, including mannose receptor and DC-SIGN⁶⁸. Omega-1, an RNase secreted by S. mansoni and found in SEA, is internalized by host cells via the mannose receptor⁶⁹. This leads to disruption of protein synthesis by DCs, which is linked to their capacity to prime Th2s. Fucose expressed by S. mansoni has also been found to activate DC-SIGN, leading to suppression of proinflammatory responses through activation of BCL3⁷⁰. One exception to this use of CLRs to sense helminths, comes from the filarial nematode Acanthocheilonema viteae. This nematode produces a secreted product, ES-62, that binds to TLR4⁷¹. Interestingly, binding to TLR4 in this context negatively regulates inflammatory cytokine production and is lipopolysaccharide independent. More recently, serum amyloid A1 (SAA1) was also implicated in receptor-mediated initiation of type 2 immunity. Fatty acid binding proteins (FABP) derived from allergenic mites were found to bind SAA1, which is normally bound to highdensity lipoprotein⁷². FABP binding to SAA1 activated formyl peptide receptor 2, inducing the release of IL-33 from nasal epithelial cells.

Chemosensing, another form of pattern recognition, can also trigger type 2 immune responses. In the late 90s, an α-gustducinexpressing cell in the rodent intestine was found to have similar structural features as taste cells⁷³. Indeed, these cells were later found to broadly express taste-signaling genes, including the ion channel TRPM5⁷⁴. More recently, these solitary chemosensory cells, now commonly known as tuft cells, were found to play a major role in the sensing and clearance of helminths. Following infection, tuft cells sense helminths in a TRPM5-dependent manner to produce IL-25 and cysteinyl leukotrienes^75–78. These tuft cell products act on the broader type 2 immune system, namely ILC2s, to drive the production of IL-13, which acts back on intestinal stem cells to bias their differentiation toward the tuft cell lineage^75–77. This feed-forward loop, called the tuft-ILC2 circuit, is required for clearance of helminth infections. The tuft-ILC2 circuit is also induced in response to protist colonization⁷⁶, whereby tuft cells directly sense protist-derived succinate^79,80. Although some helminths also produce succinate, succinate receptor signaling is dispensable for tuft cell activation in this context^79–81. The proximal signal leading to tuft cell detection of helminths remains to be discovered, but clues to its identity may be found in the receptor repertoire of these cells.

Tuft cells present at other mucosal surfaces, such as the airways and trachea, use canonical taste receptors to detect microbial ligands^82–84, but it is less clear whether and how these tuft cells interface with type 2 immunity. Tuft cells in all tissues express IL-25 and are capable of leukotriene synthesis, but a feed-forward tuft-ILC2 circuit and the associated tuft cell hyperplasia have not been clearly demonstrated outside the small intestine. As mentioned, tuft cells in the upper airways can sense ATP during aeroallergen challenge and have been linked to type 2 immune responses in the distal airways^42,85, but type 2 responses to the lung stage of hookworm infection proceed normally in the absence of tuft cells⁷⁸. At the same time, acetylcholine released from airway tuft cells leads to mast cell degranulation via induction of neuronal Substance P release⁸⁶. Gallbladder tuft cells respond to bile acids and propionate to regulate neutrophilia, gallbladder emptying, and mucus secretion but have not been linked to type 2 immunity^87,88. Given the importance of coincident detection of multiple type 2 signals, it appears likely that tuft cells in these organs could contribute to type 2 immune responses when paired with other damage signals.

Expanding role of neurons in type 2 initiation?

Given that Substance P activates both mast cells and DCs, it is plausible that other sensing mechanisms leading to neuronal Substance P release also contribute to type 2 immunity. For example, TRPV1 has been reported to act as a sensor of noxious stimuli in neurons. In the cornea, TRPV1 expression by TRPV8+ cold-sensing neurons is responsible for cold-induced nociception via release of Substance P⁸⁹. Ethanol can also induce the activation of TRPV1+ neurons, leading to bronchoconstriction and gastric injury dependent on Substance P release^90,91. Whether nociception plays a role in allergy or asthma has not been directly examined, but a correlative study suggests that asthmatics are predisposed to cold-induced respiratory symptoms⁹². It should be noted, however, that this is another instance where integration of signals is critical to ensure that not every neuronal stimulus triggers a type 2 response. Indeed, a new paradigm of type 2 immunity has been proposed, in which damage-associated molecular patterns such as IL-33 paired with release of neuropeptides are needed to elicit type 2 immune responses⁹³.

Furthermore, while neurons are type 2 sentinels, they can also act as type 2 effectors through a number of mechanisms. For example, IL-31 produced by Th2s and DCs has previously been found to act on TRPV1+ TRPA1+ neurons to induce transient itch response⁹⁴. The alarmin thymic stromal lymphopoietin (TSLP) produced by keratinocytes can also act on TRPA1+ neurons to promote an acute itch response⁹⁵. Again, this production of TSLP was dependent on protease activation of PAR-2 on keratinocytes. Chronic itch, meaning itch lasting days or weeks, has also been associated with type 2 cytokine signaling. In this case, IL4Rα signaling on TRPV1+ TRPA1+ neurons leading to JAK1 activation was responsible for chronic itch during atopic dermatitis⁹⁶. Interestingly, IL-4 and IL-13 were insufficient to induce itch alone; rather, IL4Rα signaling is thought to sensitize neurons to other pruritogens present during atopic dermatitis⁹⁶. IL-5 signaling on sensory neurons has also been found to induce release of vasoactive intestinal peptide (VIP)⁹⁷, which can amplify allergic responses. In sum, it appears the crosstalk between neurons and type 2 immune cells is a critical component of both the sensing and effector stages of type 2 immunity, as is discussed further in the context of ILC2s below.

Group 2 innate lymphoid cells integrate multiple tissue cues

ILC2s are among the first immune cells activated when a host encounters type 2 stimuli, but unlike the immune sentinels discussed above (e.g. epithelium, neurons), ILC2s do not detect type 2 stimuli directly. Instead, ILC2s are a prime example of signal integration in type 2 immunity. As tissue-resident cells at all barrier surfaces, ILC2s receive signals from immune sentinels and coordinate downstream type 2 immune responses.

ILCs share many phenotypic and functional properties with CD4+ Th cells of similar lineage (e.g. ILC1 and Th1, ILC2 and Th2, ILC3 and Th17). In the case of ILC2s and Th2s, these similarities have been traced to the level of the transcriptome and epigenome⁹⁸, highlighting how Th2s converge with ILC2s as they acquire effector function in the tissue. The key distinction between these cells, however, is that ILCs do not express a T cell receptor (TCR) and therefore do not respond to stimuli in an antigen-specific manner. Despite lacking a TCR, ILC2s similarly rely on NFkB, AP-1, and NFAT, which they mobilize by integrating multiple signals derived from cytokine, lipid, and neuropeptide receptors. Epithelial- and stromal-derived alarmins (e.g. IL-33, IL-25) are largely responsible for activation of NFkB and AP-1 in ILC2s⁹⁹. More recently, leukotriene signaling leading to NFAT activation has also been appreciated to play a critical role in both lung and intestinal ILC2 function^78,85,100. Synergy of alarmin and leukotriene signaling is critical for optimal cell activation, leading to proliferation and production of IL-5 and IL-13. In the context of helminth infection, both alarmin and leukotriene signaling on ILC2s is important for worm clearance^75,77,78. Although the importance of ILC2s in early and T cell-independent type 2 responses is well established in mice, their importance in later immune responses (when adaptive immunity has been engaged) and in humans is less clear^101,102. One study suggested that the lack of major complications in patients lacking both innate and adaptive lymphocytes but receiving only adaptive lymphocytes from a bone marrow transplant demonstrates that ILCs are dispensable. It must be noted, however, that a lack of allergic disease in the absence of ILC2s would not be noted clinically, and it is unlikely that this patient cohort was exposed to helminth infection. Recent development of ILC2-deficient mice¹⁰³ will help shed light on potential redundancies arising with age and history of allergic exposure.

ILC2s are also regulated by neuronal-derived signals in multiple tissues. The neuropeptide neuromedin U (NMU) signals through NMUR1 to promote ILC2 activation in response to helminths and allergens^104,105. Mechanistically, NMUR1 signaling activates ERK and NFAT, as well as upregulates ILC2 expression of alarmin receptors¹⁰⁵, rendering them more sensitive to epithelial signals. Neuronal-derived VIP also potentiates intestinal ILC2 effector function by activating the cAMP pathway^106,107. VIP was further found to induce expression of glycolysis-associated genes¹⁰⁶, supporting the idea that metabolic tuning of ILC2s is an important consequence of signal integration. Neuronal support of skin ILC2s has also recently been described, whereby neuronal production of IL-18 mediates ILC2 activation¹⁰⁸. Other neurotransmitters can negatively regulate ILC2s. Signaling through beta-2 adrenergic receptors broadly suppresses proliferation and cytokine production of ILC2s¹⁰⁹. Neuromedin B (NMB) also suppressed ILC2 function following helminth infection¹¹⁰. Calcitonin gene-related peptide (CGRP) activates cAMP and specifically suppresses intestinal ILC2 production of IL-13 while enhancing IL-5 production¹¹¹. It is unclear whether additional pathways are uniquely activated by VIP and CGRP to mediate their selective effects on ILC2 effector function. Together, this suggests neuronal signals fine-tune ILC2 activation by altering their transcriptional and metabolic landscape.

Despite a defined set of transcriptional rules governing ILC2 activation in all tissues and a shared reliance on tissue-derived signals, the precise mechanisms of ILC2 activation vary greatly across tissues. To begin, ILC2 expression of certain receptors is tissue specific. Although airway and adipose-associated ILC2s express high levels of ST2 (IL-33 receptor) and low levels of IL17RB (IL-25 receptor)^112,113, intestinal ILC2s express more IL17RB than ST2¹¹⁴. Following helminth infection, ILC2s from the lung and intestine can be found in circulation, perhaps because their expansion has exceeded the niche capacity¹¹⁵. While in circulation, ILC2s retain their original tissue-specific alarmin receptor expression but adopt a new tissue phenotype when they enter a different organ^115,116. This suggests that tissue-specific features of ILC2s (and likely also Th2s) are the result of available signals within different tissues rather than being developmentally imprinted. Skin ILC2s express both ST2 and IL17RB; however, their steady-state production of IL-5 is also dependent on IL-18¹¹⁷. During atopic dermatitis, skin ILC2 activation is also partially dependent on TSLP signaling¹¹⁸, highlighting the plasticity of ILC2 responses to different alarmins in multiple contexts. Importantly, human ILC2s show similar tissue-specific imprinting of alarmin receptors as has been described in mice¹¹⁹.

Tissue-specific receptor expression by ILC2s is likely determined by anatomical differences in barrier tissues that establish unique microenvironments for ILC2 residence and activation. The existence of such microenvironments is supported by the localization of resting ILC2s. For example, in the intestine, ILC2s are widely distributed throughout the lamina propria, while lung ILC2s cluster at airway branch points. Neuronal contacts with ILC2s in the densely innervated lamina propria are thought to support this spatial organization in the intestines¹²⁰. In the lung, ILC2s reside in close association with adventitial stromal cells (ASCs)¹²¹ and pulmonary neuroendocrine cells, as well as neurons^105,122. ASCs provide IL-33 and TSLP to airway ILC2s, which in turn secrete IL-13 to drive reciprocal ASC expansion¹²¹. Pulmonary neuroendocrine cells produce CGRP, which plays an important role in ILC2 activation during allergic asthma¹²². In adipose tissue, white adipose tissue-resident multipotent stromal cells play a similar role in maintaining an ILC2 niche by providing IL-33 and ICAM-1, and receiving ILC2-derived IL-4 and IL-13^123,124. Interestingly, many of these tissue-specific signals are available constitutively, and indeed many ILC2s constitutively secrete IL-5. How then is further activation of ILC2s achieved during type 2 inflammation? Increased levels of constitutive signals (e.g. IL-33) certainly contribute, but the addition of rapidly synthesized signals that are absent at steady-state is also critical. Leukotrienes, for example, do not regulate ILC2 homeostasis but can be synthesized in minutes and synergize with cytokines like IL-25 and IL-33 during helminth infection¹⁰⁷.

Although intestinal, airway, and skin ILC2s can all be activated by IL-25, the source of IL-25 appears to be driven by anatomical differences in these organs as well. Tuft cells are the source of IL-25 in the intestines^75–77 and upper airways¹²⁵, while keratinocytes provide this signal in the skin¹²⁶. There are, however, no IL-25-expressing cells in the lower regions of the murine lung where most airway ILC2s reside. This suggests that different signaling networks exist in the lung to facilitate airway ILC2 activation. Recently, tracheal tuft cells have been suggested as a source of IL-25 and leukotrienes for lung ILC2s⁸⁵. The very short half-life of leukotrienes¹²⁷ suggests that additional sources in closer proximity to ILC2s may also exist. Following lung damage, such as during influenza infection or bleomycin treatment, airway tuft cells have been reported to emerge from KRT5+ scars¹²⁸. Whether these cells play a role in ILC2 maintenance or activation during subsequent type 2 challenges will require future studies, but it suggests the interesting possibility that different tissue networks may exist following perturbations in homeostasis. Indeed, a recent study highlighted the spatial redistribution of ILC2s that occurs following activation¹²⁹.

Th2 cell priming

Our understanding of ILC2 regulation has advanced significantly since their discovery just 10 years ago. In contrast, there is much we still do not understand about the pathways that lead to Th2 priming. In general, antigen-presenting cells such as DCs are activated by innate immune sensing, take up foreign material in the periphery, and migrate to the draining LN to present foreign peptides to naïve T cells. Antigen-specific T cells then differentiate into effector Th subsets based on various microenvironmental cues¹⁹. In the case of type 2 immunity, Th2s are the key T-cell subset involved in effector responses. How Th2s are primed is still relatively unclear, but new paradigms are beginning to emerge.

As with all T cell priming, DCs play a critical role in Th2 differentiation (Fig. 2). Specifically, in the absence of IRF4+ conventional DCs, Th2s do not develop in response to either helminth or allergen exposure¹³⁰. Dermal immunization models have been particularly informative when studying Th2 priming, as the process of antigen administration, as well as subsequent antigen draining to LNs, is well-defined and easy to track and manipulate. These models have identified the CD301b+ DC subset to be critical in Th2 priming¹³¹, as well as shed light on the signals that regulate DC activation and migration to the LN. Following allergen immunization, alarmins produced by epithelial cells, as well as neuropeptides like Substance P, act on dermal DCs to induce their activation, migration, and upregulation of the costimulatory molecule OX40 ligand (OX40L)^54,132,133. In the airway, a similar pathway has been defined, where epithelial release of CSF1 drives conventional DC2 migration to the LN¹³⁴. These data suggest that epithelial cells and neurons must both sense type 2 stimuli and produce signals that together drive optimal DC activation and subsequent Th2 priming. Here then, is another example where type 2 immunity gains specificity by integrating at least two coincident signals.

Fig. 2.

DCs integrate hematopoietic and non-hematopoietic cues to prime Th2s. Neuronal, ILC2, and epithelial signals (alarmins) are integrated by DCs to trigger their migration to draining LNs. In the skin, an IRF4+ CD301b+ DC subset is the critical focal point of these signals. Activation by these signals following allergy exposure induces the upregulation of CCR8 on DCs, which, together with CCR7, allows them to enter the afferent lymphatics and subcapsular sinus of the LN. CXCR5-dependent migration of DCs further allows their entrance into the LN parenchyma. B cell-derived LT acts on stromal cells in the parenchyma to produce CXCL13, which positions CXCR5+ DCs outside of the T cell zone. These DCs engage naïve T cells via classical MHCII-T cell receptor interactions, as well as OX40L. IL-4 derived from T cells themselves, or an undefined cellular source plays a critical role in overall type 2 helper T cell differentiation. CCR = C-C chemokine receptor; CXCL = chemokine ligand; CXCR = chemokine receptor; DC = dendritic cell; IFNAR1 = interferon-α/β receptor 1; IL = interleukin; ILC2 = group 2 innate lymphoid cells; IRF4 = interferon regulatory factor 4; LN = lymph node; LT = lymphotoxin; OX40L = OX40 ligand.

Similar to viral and bacterial infection, DCs activated by type 2 agonists utilize CCR7 for migration to LNs, but they additionally upregulate CCR8¹³⁵. CCL8 secreted by allergen-activated LN resident macrophages provides a critical cue to pull DCs out of the subcapsular LN sinus and into the LN parenchyma. Once in the LN, activated DCs interact with naïve T cells. During type 2 priming, this appears to occur in a unique microenvironment near the B-cell follicle rather than deep in the T-cell zone. DC expression of CXCR5, which positions them in these B-cell zones, has therefore been described as necessary for Th2 priming following allergen immunization¹³⁶. Based on their positioning in the B-cell zone, B-cell derived factors have also been implicated in Th2 priming. B-cell derived lymphotoxin upregulates expression of CXCL13 by stromal cells, resulting in proper colocalization of DCs and T cells¹³⁶. B cells expressing B7 costimulatory molecules and secreting IL-2 are also necessary for Th2 differentiation^137,138. DC-derived cytokines are thought to skew Th cell polarization, but confoundingly, DCs have not been shown to secrete the hallmark Th2 polarization cytokine IL-4. Some studies have proposed that accessory cells (e.g. basophils or eosinophils) provide the necessary IL-4, but this has generally not been validated¹³⁹. NKT and γδ T cells are also not required¹⁴⁰. Instead, multiple studies have found that autocrine IL-4 signaling is sufficient for Th2 development^140,141. Notably, expression of OX40L has been shown to drive early IL-4 production by T cells and is important for Th2 differentiation¹⁴⁰. In some contexts, it seems IL-4 is even dispensable for Th2 differentiation¹⁴. In all, these studies suggest additional surface and/or secreted factors that DCs use to selectively prime Th2s. Although these factors remain to be clearly defined, antigen dose and affinity have both been implicated in tuning Th2 differentiation¹⁴².

ILC2s have also been suggested to support Th2 differentiation in several contexts, although challenges in specifically targeting ILC2s lead to caveats in the interpretation of these results. In one study, allergen-induced ILC2 activation led to enhanced Th2 priming dependent on IL-13 production¹⁴. IL-13 acting on CD40+ DCs promoted their migration to the draining LN, where they could prime Th2s. Cell-contact-dependent support of Th2s by ILC2s has also been suggested, dependent on ILC2 expression of MHCII^143,144. Yet subsequent studies have found no role for ILC2 expression of MHCII or ILC2s more generally in Th2 responses¹⁴⁵, so the exact interplay between these two cells remains to be elucidated.

Interferons (IFN) and IL-17 are classically involved in anti-bacterial responses, yet proper balance of these cytokines also plays an important role in Th2 priming. DCs activated following HDM or helminth exposure were found to express a unique IFN-I signature that was critical for their ability to prime a Th2 response^146,147. Although type-2 cytokines can actively suppress IL-17 production^148,149, enhanced pathology in response to helminths and allergens has been reported when both type-2 and IL-17 responses are present^150,151. Mechanistically, optimal Th2 priming requires suppression of IFNg production by IL-17A immediately following infection¹⁵². IL-17A has additionally been shown to enhance IL-13 mediated STAT6 activation¹⁵³, again increasing type 2 immune responses. These mechanisms perhaps underly the finding that DEP exacerbation of HDM was IL-17A dependent¹⁵⁴.

Tissue licensing of Th2 effector function

Following priming in the LN, Th2s return to peripheral inflamed tissues, where they mediate protective immune responses (Fig. 3). Canonically, primed T cells need only encounter their antigen to induce effector functions, but in the case of Th2s, additional signals are required to license their full effector function in the tissue. Specifically, effector Th2s require many of the same tissue-derived signals that activate ILC2s. In fact, alarmins produced following papain stimulation are sufficient to activate tissue-resident Th2s generated by a heterologous challenge¹⁵⁵. This antigen-independent activation of Th2s was instead dependent on IL-33 signaling. Effector Th2 function following HDM exposure was similarly dependent on the alarmin IL-25^156,157. Cytokine-dependent Th2 effector function has also been shown for primary helminth infection. Importantly, these tissue-derived cytokines (TSLP, IL-33, and IL-25) were not important for Th2 priming in the LN; rather, they specifically control the ability of Th2s to produce effector cytokines in the tissue¹⁴⁵. These data suggest that Th2s adopt similar cytokine-dependent features of ILC2s when in the tissue.

Fig. 3.

Tissue reactivation of effector Th2s. Effector Th2s respond to cognate antigens presented by DCs, as well as alarmins produced by epithelial cells in an antigen-independent manner. Mechanistically, IL-33 signaling via ST2 mediates the dissociation of GATA3 from BCL6, allowing for GATA3-driven production of Th2 effector cytokines IL-4, IL-5, and IL-13. IL-10 signaling is also able to relieve BCL6-mediated suppression of GATA3 by activating BLIMP-1, which itself is a repressor of BCL6. IL-2 signaling can directly repress BCL6. IL-33 can also act on tissue-resident ILC2s to induce the expression of OX40L and PDL1. OX40L and PDL1 then act on Th2s to further enhance their effector cytokine production, as well as proliferation. Basophils also support type 2 helper T cell function in the tissues by a Notch-dependent mechanism that is incompletely understood. BCL6 = B-cell lymphoma 6; BLIMP-1 = B-lymphocyte-induced maturation protein-1; DC = dendritic cell; IL = interleukin; ILC2 = group 2 innate lymphoid cells; OX40L = OX40 ligand; PDL1 = programmed death ligand-1; Th2s = type 2 CD4 T helper cells; TSLP = thymic stromal lymphopoietin.

Mechanistically, IL-33 signaling diminishes the association between the repressor BCL6 and GATA3 loci, resulting in enhanced Th2 effector cytokine production¹⁵⁸. IL-10 produced during allergic disease has additionally been found to modulate BCL6 expression¹⁵⁹. In this case, IL-10 acting on T cells induces the expression of Blimp-1, which represses BCL6, allowing for increased GATA3 expression. In the absence of Blimp-1, Th2 expansion is abrogated, and subsequent type 2 immune responses are dampened. This mechanism can also explain the observation that IL-10 production during intestinal helminth infection promotes Th2 responses¹⁶⁰. IL-2 signaling is also a critical factor in development of tissue-resident Th2s following allergic sensitization¹⁶¹. This is likely due to IL-2-mediated suppression of the Tfh lineage and BCL6^161,162.

Physical interactions between Th2 and other cells within the tissues are also required for optimal Th2 effector function. ILC2s in the skin, lung, adipose tissue, and intestine have all been shown to play critical roles in eliciting Th2 function. Following IL-33 signaling, ILC2s upregulate OX40L¹⁶³ and PDL1¹⁶⁴, which are needed for tissue-specific Th2 proliferation and polarization, respectively. Th2s provide concomitant support for ILC2s in the form of IL-2¹⁴⁴, solidifying the mutualistic relationship between these cell types within the tissues. An interesting preprint recently found bronchus-associated macrophages interact with effector Th2s in the lung and enhance effector cytokine production from T cells in vitro¹⁶⁵. Basophils have also been implicated in supporting Th2 responses in the tissues. Stable interactions have been observed between Th2s and basophils in the lung following helminth infection¹³⁹, although the functional implications of these connections are unclear. In the intestine, basophil intrinsic Notch signaling is needed for Th2 expansion in the tissue¹⁶⁶, so it is possible that a similar mechanism plays a role in the lung.

An important caveat to the discussion of tissue-specific ILC2 and Th2 function is that neither cell is restricted to the tissue they were originally activated in or primed to defend. Migratory ILC2s that can seed multiple tissues via the blood have been defined following helminth infection¹¹⁶. Similarly, Th2 cells are found in nearly all tissues following an intestine-restricted helminth infection^167,168, and a central tenet of the atopic march is that Th2s primed in the skin mediate subsequent allergic disease in the lung and gut¹⁶⁹. How these cells traffic to distal tissues and contribute to immune responses in these tissues will be important questions for future studies.

CONCLUSION

The numerous and diverse stimuli leading to similar type 2 immune responses have made it challenging to define simple paradigms of type 2 immunity. However, by viewing type 2 stimuli in terms of the ways in which they impact the tissue rather than their diverse properties, we hope to simplify some of these threads. Cell death, protease sensing, and pattern recognition are common features resulting from exposure or infection with various type 2 stimuli. These features are largely sensed by epithelial and neuronal sentinels present at each barrier tissue, although the relevant contributions of these sentinels and how their signals are integrated remain to be fully defined. The next years will likely expand our mechanistic understanding of the ‘sensing’ itself. Much more work is also needed to understand Th2 priming and the signals that license and enhance their effector function back in the tissue. Despite some shared themes, each barrier tissue appears to have evolved a different cellular and chemical network to facilitate type 2 responses. Although significant work has been done to understand this network and the resulting pathway leading to initiation of type 2 immunity in the skin following allergen immunization, other barrier tissues are less well-defined. In particular, much remains unknown about the intestine and understudied sites of allergic inflammation, such as the female reproductive tract and the eye. Therapeutically, monoclonal antibodies and oral immunotherapies have been hugely successful, but the underlying causes driving increases in allergic disease remain poorly understood. Further study of type 2 immune sensing and priming promises to identify new strategies for disease prevention.

Abbreviations:

AP-1

activator protein 1

BCL

B cell lymphoma

cAMP

cyclic adenosine 3’−5’-monophosphate

DC-SIGN

dendritic cell-specific ICAM3 grabbing non-integrin

DOCK8

dedicator of cytokinesis

ERK

extracellular signal-regulated kinase

ICAM

intercellular adhesion molecule

IL4Ra

IL-4 receptor alpha

IRF4

interferon regulatory factor 4

JAK1

Janus activated kinase 1

MHC

major histocompatibility complex

NFAT

nuclear factor of activated T cells

NFkB

nuclear factor kappa B

TRPA1

transient receptor potential ankyrin 1

TRPM5

transient receptor potential melastatin 5

TRPV1

transient receptor potential vanilloid 1.