Functional Recognition Theory and Type 2 Immunity: Insights and Uncertainties

The immune system evolved to protect hosts from pathogens and toxins, as well as promote tissue repair (1, 2). To combat the extensive diversity of pathogens and toxins, the adaptive immune system developed the capacity to generate lymphocytes with an enormous number of clonally diverse Ag receptors (1). Consequently, the adaptive immune system possesses the ability to respond to virtually any threat, regardless of the antigenic composition. Beyond Ag recognition, adaptive immunity provides host protection only if it couples Ag recognition with an effector program that successfully targets the specific pathogen or toxin. To combat classes of pathogens and toxins, distinct immune modules have evolved that generate unique effector mechanisms (3). Each immune module possesses lymphocyte effector cells from both the innate and adaptive immune systems, which contribute to the response through production of specialized cytokines. Type 1 immunity is induced by intracellular pathogens and orchestrated by type 1 innate lymphoid cells (ILC1s) and CD4⁺ Th1 cells, as well as CD8⁺ cytotoxic T cells, which all produce the cytokine IFN-γ. Type 2 immunity is induced by helminths, toxins, and allergens, being orchestrated by ILC2s and CD4⁺ Th2 cells, which produce the cytokines IL-4, IL-5, IL-9, and IL-13. Type 3 immunity is induced by extracellular pathogens and orchestrated by ILC3s and CD4⁺ Th17 cells, which produce the cytokines IL-17 and IL-22. The cytokines from each immune module provide help to innate immune cells, as well as structural cells and neurons, to promote a targeted host defense response. In addition, the differentiation of specialized CD4⁺ T follicular helper (Tfh) cells provides B cell help and promotes class-switch recombination to specific Ab isotypes, thereby dictating the Ab-mediated effector response (4). Notably, although one module is generally dominant during an immune response, most immune responses exhibit a spectrum of activated modules in vivo.

The innate immune system controls the induction of all adaptive immune modules with type 1 and type 3 immune modules using distinct receptors, but a shared structural-based approach of sensing (3). Pathogens inducing type 1 and type 3 immunity are recognized via pattern recognition receptors (PRRs), which bind evolutionarily conserved pathogen-associated molecular patterns (PAMPs) (3). PRR sensing of PAMPs by innate immune cells leads to an initial innate effector response. In addition, PAMP detection by dendritic cells (DCs) at barrier surfaces leads to DC maturation and trafficking to the draining lymph nodes with Ag presentation, costimulation, and skewing cytokines instructing a specific CD4⁺ T cell differentiation program (5). For example, the intracellular bacterium Listeria monocytogenes possesses multiple PAMPs, including lipoproteins, flagellin, and unmethylated CpG sequences, among others, which promote CD4⁺ Th1 cell differentiation (6). Candida albicans possesses distinct PAMPs, such as α-mannan molecules, which are structurally recognized by Dectin-2 on DCs, leading to CD4⁺ Th17 cell differentiation (7, 8). Consequently, structural recognition theory serves as the conceptional foundation for the induction of type 1 and type 3 immunity. Type 1 and type 3 immunogens can be organized into classes based on their respective PRR, which provides a clear framework of immune sensing.

In contrast with the essential role of PRRs in the induction of type 1 and 3 immunity, the type 2 immune module can be regulated by PRRs without absolutely requiring structural recognition. Type 2 immunogens can contain PRR ligands that play an important regulatory role in the host response. For instance, house dust mites contain the TLR4 ligand LPS, and the house dust mite allergen Der p 2 exhibits structural homology with MD-2, the LPS-binding component of the TLR4 signaling complex, which can directly promote TLR4 complex signaling (9, 10). Furthermore, house dust mites contain glycans that can activate Dectin-1 and Dectin-2 signaling (11). However, these PRRs can exhibit remarkably variable influence over type 2 immunity depending on the context. TLR4 expression in structural cells has been reported to promote type 2 immunity to house dust mites, whereas others have found that TLR4 is not required (9, 12). Numerous reports have suggested that the level of TLR4 signaling dictates the variable responses in vivo, with low-dose LPS promoting type 2 immunity, whereas high-dose LPS inhibits type 2 immunity (13–16). LPS activity appears to switch from promoting to inhibiting type 2 immunity depending on the production of GM-CSF and TNF-α by monocyte-derived cells (12). Similarly, Dectin-1 can play distinct roles in type 2 immunity with engagement of Dectin-1 by house dust mites promoting DC migration to the draining lymph node and induction of CD4⁺ Th2 immunity, whereas Dectin-1 expression in lung epithelial cells inhibits CD4⁺ Th2 immunity (17, 18). Such findings indicate that PRRs can play a regulatory role but are not required for the induction of type 2 immunity. In support of such a model, type 2 immunogens can induce type 2 immunity in the absence of structural recognition. For instance, the papaya-derived allergen papain, which lacks Dectin-1 or Dectin-2 ligands, induces type 2 immunity in vivo in Tlr2/Tlr4 double-knockout mice and MyD88-deficient mice (19). Rather than requiring structural recognition, papain-induced type 2 immunity is completely dependent on its cysteine protease activity (19). Notably, there are data suggesting that proteases from Aspergillus orzyzae cleave fibrinogen, with the cleavage products activating TLR4 to promote allergic immunity (20). Nevertheless, even if PRRs are used, the essential mechanisms of sensing type 2 immunogens appear to be distinct from structural recognition used by the type 1 and 3 immune modules.

How does the immune system sense the enormous number of structurally distinct type 2 immunogens? In contrast with structural recognition, the immune system senses type 2 immunogens through their functional properties, which include the ability to promote the release of damage-associated molecular patterns and activate sensory neurons (3, 21, 22). Functional recognition theory has arisen as the paradigm for the initiation of type 2 immunity. However, the vast array of structurally unrelated type 2 immunogens and numerous experimental models make it challenging to advance our understanding of type 2 immunity in vivo. In this article, we review functional recognition theory and organize type 2 immunogens into distinct classes based on how each class fits into the foundational concepts of functional recognition. Lastly, we discuss areas of uncertainty in functional recognition theory, including how Ags lacking intrinsic adjuvanticity generate an adaptive type 2 immune response, how innate and adaptive arms of the type 2 immune module can be differentially activated in vivo, and how functional recognition at barrier sites changes after previous type 2 inflammation. Our goal is to provide a framework to help further define the logic of type 2 immunity in host protection and immunopathology.

Type 2 immunity evolved, in part, to provide host protection against parasitic helminths, including either mediating parasite expulsion or tolerance to the helminth in peripheral tissues (23). For instance, in mice, Nippostrongylus brasiliensis primary expulsion and Heligmosomoides polygyrus secondary expulsion require CD4⁺ Th2 cells (24). In contrast with the host protective functions of type 2 immunity in the context of helminth infection, inappropriate type 2 immunity to noninfectious Ags drives allergic diseases. However, macroscopic helminths and microscopic allergens are very distinct immunogens, raising the question as to the shared mechanism(s) inducing type 2 immunity. Although type 2 immunogens lack conserved structural features, many exhibit the shared property of being noxious to the host (22). Even seemingly harmless environmental allergens often exhibit enzymatic activity, which is required for their ability to induce type 2 immunity (19, 25). The toxin hypothesis of allergic disease posits that type 2 immunity evolved to combat harmful toxins with Ag-specific, adaptive immunity providing protection to future exposures (26). In support of the toxin hypothesis, mice treated with low doses of bee or snake venom are protected from the effects of secondary challenges with sublethal and lethal doses of venom in an IgE and mast cell–dependent manner (27–29). As a result, these findings suggest that molecules capable of causing a specific type of tissue “stress” activate the type 2 immune module. Furthermore, it is well established that the effector mechanisms employed by type 2 immunity promote tissue repair pathways, suggesting that this immune module evolved to couple recognition of tissue stress to immune effector mechanisms capable of expelling noxious Ags while maintaining tissue integrity and promoting repair (2, 30). Damage or stress to tissues promotes the release of a class of molecules termed damage-associated molecular patterns (DAMPs), which transmit the signal of tissue injury to the innate immune system (31). In murine models, DAMPs such as IL-33, IL-25, and thymic stromal lymphopoietin (TSLP) play an important role in the initiation of type 2 immunity (3, 32). In addition, genome-wide association studies in humans have found that polymorphisms in IL-33 and the IL-33 receptor are associated with the development of allergic asthma (33, 34). Although there are multiple cellular sources of DAMPs, epithelial cells and adventitial stromal cells represent major sites of DAMP production, indicating specialized roles of these structural cells in sensing noxious stimuli and tissue stress (35, 36). Notably, while the above DAMPs play an important role in type 2 immunity, genetic deletion of individual DAMPs causes a reduction, but not complete abrogation, of type 2 immunity (27, 37–41). Even triple-knockout mice lacking the IL-33 receptor, TSLP receptor, and the cytokine IL-25 exhibit no defect in initial CD4⁺ Th2 cell differentiation in the lymph node on infection with the helminth N. brasiliensis, although effector CD4⁺ Th2 cell function at barrier sites was significantly impaired (42). As a result, the three conventional type 2 DAMPs promote type 2 immunity, but there are additional sensing pathways to initiate the type 2 immune module.

Over the last several years, growing evidence supports a central role for sensory neuron-derived neuropeptides in the regulation of both innate and adaptive type 2 immunity (21, 22). First, allergens directly activate sensory neurons to induce itch and pain in mice (43). Sensory neuron activation promotes avoidance behaviors in the organism, as well as plays a central role in initiating type 2 immune responses. Activated sensory neurons release neuropeptides that can cross-talk with local immune cells, leading to innate effector responses. For example, sensory neurons intertwine with mast cells in peripheral tissues with these two cell types forming physical synapses poised for intercellular communication (21, 44–49). Release of substance P and vasoactive peptide (VIP) from activated sensory neurons directly promotes mast cell degranulation (50–53). This may be sufficient for the IgE-independent mast cell protection against primary venom exposure, which requires degranulation and release of carboxypeptidase A and other proteases that degrade venom components (54). In addition, neuropeptide-induced mast cell degranulation may also act to coordinate type 2 inflammatory responses. Primary exposure to house dust mite allergen directly activates sensory neurons to promote release of substance P, which promotes mast cell degranulation and local inflammatory responses (55). In contrast, models of contact hypersensitivity suggest that the neuropeptide calcitonin gene–related peptide inhibits mast cell function, indicating that sensory neurons can tune the responsiveness of mast cells (52, 56). Along with mast cells, neuropeptides regulate the function of other innate type 2 immune cells. ILC2s express the receptor for the neuropeptide neuromedin U, which promotes ILC2 production of IL-5 and IL-13 (57–59). Furthermore, VIP also directly promotes ILC2 production of IL-5, although the functional role of VIP in vivo remains less clear (60, 61). Similar to mast cells, calcitonin gene–related peptide plays an immunomodulatory role in ILC2 activation in response to DAMPs, suggesting that distinct neuropeptides can differentially regulate ILC2 effector function (62, 63).

In addition to the innate effector response, neuropeptides also play a role in initiating the adaptive immune response to type 2 immunogens through their effects on DCs. A specialized population of IRF4- and KLF4-dependent, type 2 conventional DCs (cDC2s) is required for CD4⁺ Th2 cell differentiation (64–66). Unlike CD4⁺ Th1- and CD4⁺ Th17-skewing DCs, CD4⁺ Th2-skewing cDC2s do not appear to respond directly to allergens. In the skin, sensory neuron-derived substance P stimulates CD4⁺ Th2-skewing cDC2s via the receptor MRGPRA1, promoting migration to the draining lymph node and the induction of CD4⁺ Th2 cell differentiation (43). Notably, substance P alone is insufficient for CD4⁺ Th2 cell differentiation, suggesting that additional signals, such as DAMPs, collaborate with substance P to promote CD4⁺ Th2 cell differentiation (43). In summary, although type 2 immunogens lack shared structural features, they are recognized functionally via release of DAMPs and the activation of sensory neurons (Fig. 1). However, it remains unclear how the immune system integrates signals from DAMPs and neuropeptides: do they simply act to amplify a generic activation signal, or do they activate and instruct specific type 2 immune outcomes? This second possibility is supported by observations from type 1 responses, where DAMP release leads to ILC2 activation to promote tissue repair without inducing an adaptive type 2 response (67–69). Such observations suggest the existence of a DAMP/neuropeptide code for the induction of specific type 2 immune responses.

Although the cellular sensors of type 2 immunogens are becoming clearer, whether these cellular pathways apply to all type 2 immunogens in vivo remains a mystery. For instance, while the enzymatic activity of protease allergens may imbue them with inherent adjuvanticity, many food allergens are seemingly inert and characterized mainly by their stability to heat and acid degradation. Modeling food allergy in vivo often relies on the use of exogenous adjuvants like cholera toxin (CT), but is this relevant to food allergy in humans? In addition, during type 1 or type 3 immune responses to pathogens, type 2 immunity can be activated to promote tissue repair or host defense. How are type 2 immunogens sensed and regulated during a dominant type 1 or 3 immune response? Lastly, although functional recognition is generally studied in the naive state in mice, there is growing evidence that induction of type 2 immunity at a barrier site induces tissue-resident memory that durably changes local functional recognition. How does functional recognition at a barrier site change after previous type 2 inflammation? One challenge to addressing these areas of uncertainty is the significant breadth of type 2 immunogens and murine models available for mechanistic studies. To further define such biology, we will review the unique features of different classes of type 2 immunogens and place them in the conceptual framework of functional recognition.

Allergenic proteins from plants, animals, and fungi are marked by significant structural heterogeneity that makes any classification scheme imprecise. However, by classifying allergens by function, which provides enzymatically active allergens with their inherent adjuvanticity, instead of structural or phylogenetic relatedness, certain major themes become apparent (Table I) (70, 71). The first theme is the enrichment of enzymatically active proteins as allergens, specifically those that are active against proteins (proteases) and carbohydrates (carbohydrate-active enzymes [CAZymes]). Protease allergens include cysteine, serine, and aspartic proteases, as well as metalloproteases. These allergenic proteases display a variety of targeted peptide sequences and are enriched in dust mite, cockroach, and fungi, although they can also be detected in some food plants (kiwi, papaya, pineapple, and muskmelon) and environmental pollens (ragweed, timothy grass, Bermuda grass). The enzymatic activity of cysteine and serine protease allergens has been clearly shown to act as an allergic adjuvant (19, 72, 73). How proteases exert this adjuvant activity is not entirely clear, but allergic proteases have been shown to degrade tight junction proteins and activate protease-activated receptors, indicating possible routes for indirect and direct adjuvant effects (74, 75). Finally, cysteine protease allergens have been shown to cleave CD25, leading to the hypothesis that it could target CD25^hi regulatory T cells (76). However, the relevance of this in vitro finding remains unclear in the setting of in vivo data supporting a role for cysteine protease allergens in the indirect activation of CD25^hi ILC2s and mast cell production of IL-2, which promotes regulatory T cell function in vivo (77, 78). Clearly, there remains much to be determined about how protease allergens drive allergic inflammation.

CAZymes include the well-described chitinase family (found in dust mite, cockroach, and food plants), as well as lysozymes from chicken egg and milks, and the pectin lyases found mainly in pollens (ragweed, mugwort, juniper) (79). CAZymes can be exogenous, as in the form of allergens, or endogenous. It is well described that worm or fungal chitins can induce the expression of mammalian chitinase enzymes leading to type 2 inflammation (80). Finally, CAZymes are also found in venom allergens, which are enriched in hyaluronidases that can act as type 2 adjuvants (81). In addition to these major classes are those enzymes involved in metabolic pathways, specifically the enolase and arginine kinase allergens, as well as those with detoxification roles (82). Although little is known about how these allergens might be recognized, the enrichment of enzymes in allergens across plant, animal, and fungal kingdoms underscores the potential role for their functional activity in driving their allergenicity.

Enzymes easily lend themselves to functional grouping, but many other immunodominant allergens, even if lacking in enzymatic activity, have functional activities that may be related to their allergenicity. Many food allergens are characterized by their stability to heat (cooking) and acid (digestion), but these same allergenic proteins also share in common the ability to bind bioactive lipids and small molecule ligands (83). The ubiquitous ligand binding proteins such as albumins and globulins, found in both plant food and environmental animal allergens (e.g., cat and dog), do not have enzymatic activity but can exert major physiological effects through the binding and release of bioactive small molecules and xenobiotics. Lipid transfer proteins from plants and lipocalins from animals get their name from their ability to bind bioactive lipid mediators and hormones (84). Furthermore, there is evidence that the ubiquitous PR-10 family of allergenic proteins in both pollens and plant foods bind phytohormones and flavonoids in a shared hydrophobic cavity (85). These findings raise the possibility that otherwise “inert” proteins may exert a functional effect on the host by either binding host-derived bioactive small molecules or, alternatively, that they may carry small molecular adjuvants that exert functional effects on the host.

The last major class of functional allergens includes those that may impact the structural or membrane integrity of eukaryotic cells. This group includes the actin-binding profilins, which integrate membrane signaling with cytoskeletal dynamics and can even activate PI3K/Akt signaling pathways when encountered extracellularly (86). It also includes the plasma membrane toxins in allergenic venoms such as bee venom phospholipase A₂ (bvPLA₂) (Api m 1) and the pore-forming toxin melittin (Api m 4). In addition, given their described role in membrane targeting and disruption, we consider the defensin subset of allergens seen in both food plants and pollens to be another member of this group (87). Each of these groups has the ability to directly cause toxicity to membrane integrity, signaling pathways, and cytoskeletal structure, but some members may act to amplify another group’s functions. For example, the pore-forming toxin melittin has been shown to increase the toxicity of bvPLA₂ by facilitating its entry into membranes (88).

If allergens can be classified based on their functional principles, then certain predictions can be made. First, allergens that share functional activity, and not structural features or phylogenetic origin, will lead to similar DAMP release, neuropeptide release, and immune outcomes. There are data supporting this between the cysteine proteases (e.g., Der p 1 and papain), but data are mixed concerning families within the same functional class (e.g., cysteine versus serine proteases) (43, 55). The second prediction is that secreted products, such as bacterial toxins, that come from these functional classes may promote a type 2 immune pathway. This has recently been illustrated for the LasB metalloprotease toxin from Pseudomonas aeruginosa (89). The third prediction is that these broad classes of allergens would be sensed by shared pathways that detect alterations in host homeostasis or damage. However, these fundamental pathways may be different between the protease allergens and other classes. DAMPs consist of one such shared pathway for the functional detection of protease allergens and the membrane toxin bvPLA₂ (27, 77). In this case, the allergen’s activity, and therefore adjuvanticity, is relayed through a host-derived molecule in response to cellular damage. But how does the DAMP system detect “inert” proteins or their small molecule ligands? Could mammals have a conserved pathway to detect altered self through sacrificial decoy proteins? If such a pathway existed in mammals, it could provide the detection mechanism driving allergic responses to naturally occurring small molecules, as well as xenobiotics like penicillin.

As outlined earlier, many type 2 immunogens exhibit intrinsic adjuvanticity, offering a mechanism that couples induction of adaptive immunity with antigenic recognition. However, it remains less clear how Ags without intrinsic adjuvanticity, such as many food allergens, induce type 2 immunity (22). In such circumstances, the Ag is sensed in association with some noxious stimulus that may not be defined. The latter substance may not be a protein and consequently serves as an adjuvant without being an Ag. Given that food Ags promote oral tolerance, murine models of food allergy aiming to induce sensitization via the oral route require addition of an adjuvant such as CT or staphylococcal enterotoxin B (90). Although CT has been shown to induce IL-33 release in the gut, it remains unclear whether oral adjuvants used in murine models are truly recapitulating the biology of type 2 immunity to food Ags that occurs in humans (91). Specifically, growing evidence suggests that early-life allergen exposure through the skin induces Ag-specific type 2 immunity, whereas early oral ingestion promotes immune tolerance (92). For example, a number of genetic mutations that disrupt skin barrier function are associated with atopic dermatitis, which is associated with an increased risk for allergic sensitization to foods (93–95). In addition, among children with high-risk atopic disease, early oral introduction of peanut significantly decreases the frequency of the development of peanut allergy (96). Even in the absence of skin barrier dysfunction, the skin has distinct features of type 2 immunity from other barrier sites. In the murine skin, basal IL-13 from dermal ILC2 promotes the differentiation of CD4⁺ Th2-skewing cDC2s in a STAT6- and KLF4-dependent manner (97). In the absence of IL-13 signaling, dermal cDC2s were unchanged in number but exhibited a reduced CD4⁺ Th2-skewing phenotype, as well as reduced ability to promote CD4⁺ Th2 cell differentiation, whereas CD4⁺ Th17 cell differentiation was increased (97). Such an IL-13–dependent mechanism for promoting CD4⁺ Th2-skewing cDC2s did not exist in the lungs or small intestine (97). In support of these findings, human cDC2s from the skin also exhibit an IL-4 and IL-13 signaling gene signature, which was absent in cDC2s from the blood, spleen, or lungs (97). As a result, the threshold of functional recognition and initiation of adaptive type 2 immunity may be distinct in the skin compared with other barrier tissues. Ags without intrinsic adjuvanticity may be more likely to be sensed in association with another noxious stimulus and trigger adaptive type 2 immunity in the skin, particularly in the context of barrier disruption, rather than other barrier sites such as the gut. Consequently, defining the unique features of functional recognition in the skin may yield new insight into the mechanisms whereby Ags without intrinsic adjuvanticity induce adaptive type 2 immunity.

Another area of uncertainty involves defining how innate type 2 immunity can be activated during type 1 or type 3 immunity to promote tissue repair without inducing an Ag-specific type 2 response. One hypothesis is that DAMP production promotes ILC2 activation, but the concomitant presence of PAMPs inhibits the induction of an adaptive type 2 response (2). Such a mechanism would allow the host to use the tissue reparative functions of innate type 2 immunity without engaging an adaptive type 2 response that may be inappropriate to combat a pathogen that is eliminated by type 1 or type 3 immunity. In support of such a model, high-dose LPS exposure, as would occur during an infection with an endotoxin-producing bacteria, is capable of inhibiting an adaptive type 2 immune response (12–16, 98). However, one challenge to the high-dose PAMP-inhibition model in adaptive type 2 immunity is that toxin-producing bacteria such as Staphylococcus aureus induce Ag-specific IgE, demonstrating that adaptive type 2 immunity can be activated even in the presence of high levels of PAMPs (99). Notably, Staphylococcus aureus induces a specialized subset of CD4⁺ Tfh cells that produce IL-13 (99). Tfh13 cells represent a unique population of CD4⁺ Tfh cells that are induced by allergens, but not helminths (100, 101). Tfh13 cells coexpress the canonical CD4⁺ Tfh and CD4⁺ Th2 cell transcription factors Bcl6 and Gata3, respectively, and are required for the instruction of high-affinity IgE, which causes anaphylaxis in the context of allergic disease (100, 101). The observation that both allergens and toxins, but not helminths, induce Tfh13 cell differentiation is intriguing. The fact that all of these type 2 immunogens induce DAMP release suggests that some other sensing mechanism dictates the differentiation of Tfh13 cells. Given that allergens directly activate sensory neurons and sensory neuron–derived neuropeptides play an important role in inducing adaptive type 2 immunity, it is possible that neuropeptides play an important role in engaging an Ag-specific type 2 immune response even in the presence of high levels of PAMPs. Could the presence of DAMPs and either activating or regulatory neuropeptides dictate engagement of adaptive type 2 immunity (21)? Defining how the immune system integrates signals from PAMPs, DAMPs, and neuropeptides to regulate type 2 immunity represents an area of central importance to the field.

Most models of functional recognition focus on the naive state. However, prior type 2 immunity leads to long-term changes in functional recognition and the capacity to induce type 2 inflammation. For instance, ILC2s can be “trained” by previous inflammation to acquire memory-like properties (102, 103). On activation, ILC2s proliferate in situ and produce type 2 cytokines to induce inflammation (104). While the ILC2 population subsequently contracts, the number of memory-like ILC2s remains greater than the naive state (102, 105). In addition, ILC2 can acquire cell-intrinsic, memory-like properties, including an enhanced responsiveness to DAMPs via epigenetic changes that are responsible for a poised effector program (102, 105). ATAC-seq analysis has shown that trained ILC2s possess alterations in accessibility of Bach2 and AP1 motifs, which play an important role in the memory-like program that allows activation to previous subthreshold stimulation (105). Furthermore, induction of adaptive type 2 immunity in a particular barrier tissue produces long-term, tissue-resident memory. After allergic inflammation within the lungs, memory CD4⁺ Th2 cells establish residency and promote recurrent allergic inflammation in an allergen-specific manner (106–109). Tissue-resident memory CD4⁺ Th2 cells are transcriptionally distinct from their circulating counterparts and durably persist in the lung parenchyma without the need for replenishment from circulating memory CD4⁺ Th2 cells (108). Beyond canonical CD4⁺ Th2 cells, a distinct subset of CD4⁺ Th2 cells that acquire the capacity to express IL-9 (Th9 cells) are well described in human allergic diseases (110–112). IL-9 promotes type 2 immunity via activation of mast cells, enhancing eosinophil recruitment, activating macrophages, and promoting mucous metaplasia (113–115). Notably, IL-9 is predominantly expressed in highly differentiated CD4⁺ Th2 cells, indicating that chronic stimulation is necessary for induction of the Th9 program (110). In murine models of allergic lung inflammation, short-term allergen exposure protocols do not seem to induce a significant Th9 cell subset, whereas chronic allergen treatments yield a distinct subset of Th9 cells (109, 116). After chronic allergen exposure, IL-9 is predominantly derived from a population of tissue-resident memory CD4⁺ T cells that exhibit a distinct chromatin and transcriptional signature from CD4⁺ Th2 cells (109). Tissue-resident memory Th9 cells produced IL-9 in an allergen-specific manner, which is enhanced by IL-33 (109). Beyond CD4⁺ Th2 and Th9 cells, the induction of IgE significantly enhances the ability of mast cells to respond in an Ag-specific manner (117). Furthermore, both type 2 cytokines and IgE can regulate the function of nociceptive neurons that participate in type 2 immunity. For example, nociceptive neurons express IL-4Rα and the high-affinity IgE receptor FcεR1 and can directly respond to IL-4 and IgE (118, 119). Specifically, TRPV1⁺ sensory neurons respond to IgE-allergen immune complexes by releasing substance P, which, in turn, amplifies CD4⁺ Th2 cell production of IL-5 and IL-13 (118, 120–122). Consequently, the induction of Ag-specific type 2 immunity allows sensory neurons to broaden their sensing function to include Ag-specific recognition to regulate type 2 immunity. Along with changes in the responsiveness of cells involved in type 2 immunity, there are clearly changes in the niches supporting these cellular networks. For example, ILC2s and CD4⁺ Th2 cells persist in adventitial stromal niches that line the outermost regions of large vessels, ducts, and airways, which include specialized stromal cells that produce IL-33 and TSLP (123–126). In models of early-life skin inflammation, a population of CD4⁺ Th2 cells persists into adulthood via interactions with expanded fascial fibroblasts (127). Establishment of the microanatomical niche of CD4⁺ Th2 cells and CD4⁺ Th2-interacting fascial fibroblasts in the skin results in altered reparative responses to tissue injury (127). In sum, after type 2 immunity, ILC2s can acquire enhanced sensitivity to DAMPs; tissue-resident memory CD4⁺ Th2 and Th9 cells, as well as IgE, provide the barrier tissue with the capacity to broaden the mode of immunosurveillance from functional recognition to include Ag-specific recognition; and there are changes to the supporting microanatomical niches within tissues. Defining the cellular networks and niches that support type 2 immune cells in barrier tissues and how these niches durably change in response to various type 2 immunogens represents a critically important area in type 2 immunity.

Although the topics outlined earlier cannot be exhaustive, we believe such areas represent important gaps in our working model of functional recognition and type 2 immunity. Notably, it is clear that the rules of functional recognition exhibit shared and immunogen-, tissue-, and context-specific features. To help advance our understanding of type 2 immunity in vivo, we need clearer delineation as to whether particular pathways or mechanisms are specific to a model/context or represent a fundamental feature of functional recognition. Delineating both shared and context-specific mechanisms regulating type 2 immunity will be critical to advancing our insight of type 2 immunity in vivo.

Functional recognition theory serves as the conceptual foundation for understanding type 2 immune recognition. Although there have been many advancements in delineating the biology of type 2 immunity, several important areas of uncertainty remain. The dizzying array of stimuli and models remain a challenge for the field, impairing our ability to define the logic of type 2 immunity in vivo. Further defining the rules regulating shared and context-specific type 2 immunity will be critical to develop novel approaches to target the type 2 immune module to promote host protection and prevent or limit immune-mediated disease states.

C.L.S. is a paid consultant (expert witness) for Bayer and Merck and receives sponsored research support from GlaxoSmithKline.