CD4⁺ T helper 2 cells – microbial triggers, differentiation requirements and effector functions

Abstract

Over the past 10 years we have made great strides in our understanding of T helper cell differentiation, expansion and effector functions. Within the context of T helper type 2 (Th2) cell development, novel innate-like cells with the capacity to secrete large amounts of interleukin-5 (IL-5), IL-13 and IL-9 as well as IL-4-producing and antigen-processing basophils have (re)-emerged onto the type 2 scene. To what extent these new players influence αβ⁺ CD4⁺ Th2 cell differentiation is discussed throughout this appraisal of the current literature. We highlight the unique features of Th2 cell development, highlighting the three necessary signals, T-cell receptor ligation, co-stimulation and cytokine receptor ligation. Finally, putting these into context, microbial and allergenic properties that trigger Th2 cell differentiation and how these influence Th2 effector function are discussed and questioned.

Introduction

Governed by unique T-cell receptors (TCR), αβ⁺ CD4⁺ T helper (Th) cells provide antigen-specific and location-specific responses following TCR engagement. Responsible for mobilizing innate cells; providing help to B cells for class switching and antigen-specific immunoglobulin production; providing cues to local tissue and promoting wound healing and repair, CD4⁺ Th cells are fully operational conductors of immune activation, resolution and tissue repair. With such influence, CD4⁺ Th cells are tightly regulated throughout their development from the bone marrow, liver and thymus, through to their peripheral differentiation, activation, effector function and long-term survival. Despite multiple checkpoints and layers of highly evolved immune regulation, CD4⁺ Th cell dysfunction can arise, leading to hyper-inflammatory conditions causing local tissue damage and culminating in autoimmune or allergic diseases. Conversely, if CD4⁺ Th cells fail to develop, mature or differentiate, individuals can be left with insufficient immunological protection with equally catastrophic outcomes, such as life-threatening severe immunodeficiency.

Relatively unchallenged for almost 20 years, it was widely accepted that CD4⁺ Th cells differentiate into two distinct effector populations, interferon-γ (IFN-γ)-producing Th1 cells and interleukin-4 (IL-4) -producing Th2 cells.¹ It is now customary to acknowledge at least five, if not six, CD4⁺ T-cell subsets including Th1, Th2, Th17, T follicular helper (T Fh) and regulatory T (Treg) cells plus the yet to be fully accepted at the time of print Th9 cells.^2,3 With the exception of T Fh and Treg cells, effector CD4⁺ Th subsets are characterized by their cytokine expression profile and up-stream transcription factor usage. Beyond the usefulness for communication among scientists, pigeonholing T cells into such categories may be over-simplifying Th cell biology. The initial description of Th1 and Th2 cells described the outgrowth of irreversibly committed IFN-γ-producing or IL-4-producing T-cell clones over several weeks, a bench mark yet to be met for Th17 or Th9 cells. Plasticity between the subsets is widely documented (reviewed by Murphy and Stockinger⁴) with studies identifying Th2 (GATA3⁺ IL-4⁺) cells that co-express Th1 (T-bet and IFN-γ) -defining,⁵ Th17 (RoRγt and IL-17A) -defining⁶ markers or IL-9-secretion³ (Fig. 1). Despite the potential shortcomings of these studies (using in vitro-polarized or transgenic T-cell systems) these observations throw into question the biological and physiological relevance of subsets – Th1, Th2, and ‘Th2⁺1’ or ‘IL-17–Th2’ as the authors justly deride. Nevertheless, for the benefit of communication and until a more useful system is established, throughout this review we will subscribe to the current nomenclature and tie together recent advances in our understanding of Th2 cells, highlighting where possible the unique features of Th2 cells.

Figure 1.

T helper (Th) cell distinction. T helper cell differentiation of naive cells develops along a reversible continuum. Initial polarizing cytokines induce phosphorylation of signal transducer and activator of transcription (STAT) molecules and activation of lineage promoting transcription factors.

Widely cited as being required for anti-helminth immunity, Th2 cells have only clearly been demonstrated to expel intestinal helminth infections.^7,8 Th2 cells are certainly observed during blood (Schistosome), lymphatic (Filarial) and tissue (Trichinella) dwelling helminth infections, and correlate with reduced re-infection in treated individuals, but whether or how Th2 cells control, or simply conciliate, helminth pathogens are unclear. Similarly, Th2 cells fit the description of a prime suspect during the development of atopy and subsequent allergic reactions, but their sole involvement and subsequent targeting for allergy therapy (which has only achieved modest success⁹) is unlikely. Hence, neither the Th2 cell, at a particular snapshot in time of analysis, or its associated cytokine profile after unphysiological stimulation in vitro, should be thought of alone, but rather in the context in which it is acting. These rather obvious reminders are often not observable during in vitro Th2 experiments or are not reported during complex in vivo studies. Yet to accurately report a Th2-dependent gene, to hypothesize and test the function of Th2 features and to ascribe some relevant meaning requires an appropriate environment. Th2 cells and their responses are often vaguely described as type 2 microenvironments, expanding the single Th2 cell to a multi-cytokine and multi-cell mélange including alternatively activated macrophages, eosinophils, basophils, mast cells and recently described innate-like cells. We will attempt to strip down these broad interpretations and draw attention to what we know and do not know about the type-2 namesake, αβ⁺ CD4⁺ Th2 cells.

CD4⁺ Th2 differentiation –in vitro necessity and in vivo redundancy

The activation of the il4 gene in CD4⁺ Th cells is the conventional marker for Th2 differentiation similar to the activation of the ifng gene for Th1 differentiation (Fig. 1). These markers have been used to identify the specific requirements for Th2, or Th1, differentiation in vitro, in vivo, in situ and ex vivo. Most of our current understanding of Th2 differentiation is therefore based upon the activation of this single gene. What about cells that do not activate il4, either naturally or through genetic manipulation of the il4 gene or il4 receptor, but display other Th2 markers? Are they still Th2 cells? Indeed, IL-4-independent Th2 differentiation has been reported^10–12 and will be discussed in more detail below. Reductionist in vitro experiments have been invaluable, forging ahead and undressing Th2 (and other CD4⁺ Th) cell differentiation down to three essential signals, (i) TCR engagement, (ii) appropriate co-stimulation, and (iii) cytokine receptor ligation (Fig. 2). Needless to say, discrepancies exist between in vitro and in vivo requirements for each Th subset.

Figure 2.

αβ⁺ CD4⁺ T helper type 2 (Th2) cell differentiation. A series of signals via the T-cell receptor, co-stimulatory receptors and cytokine receptors activate the appropriate transcription factors for il4 transcription and Th2 cell differentiation.

Signal 1 TCR engagement

T-cell receptor engagement, activating nuclear factor of activated T cell (NFAT) and GATA-binding protein-3 (GATA 3)¹³ may be the first signal to nudge CD4⁺ Th cells down a Th2 path. In seminal studies by Constant et al.¹² and Hosken et al.¹⁴ a low-strength TCR signal (0·5 pg/ml of peptide) in naive (16A^hi, now known as CD45RB^hi), but not total, transgenic CD4 T cells induced IL-4 secretion. In contrast, higher doses (≥ 0·5 μg/ml) promoted IFN-γ production. Mechanistically, low-strength TCR activation led to weak and transient extracellular signal-regulated kinase (ERK) activation and GATA-3 stabilization, triggering activation of il4. Interleukin-2 was also induced,¹⁵ which fed back in an autocrine manner, activating signal transducer and activator of transcription 5 (STAT-5) and providing a necessary survival and enhancing factor bypassing the requirement for exogenous IL-4.

The first signal, via the TCR, during Th2 cell polarization (TCR > GATA-3 > IL-4) highlights the central role for GATA-3 in Th2 cell differentiation in vitro. Beyond Th1 and Th2 cells, it would be interesting to know where Th17, T Fh and Treg cells fit on the signal strength continuum. However, greater questions remain, including which antigen-presenting cell would/could provide a low TCR signal and which cell provides co-stimulation and local cytokines required for Th2 cell differentiation.

The long-standing notion that dendritic cells (DCs) are the primary antigen-processing and antigen-presenting cells and that IL-4 came from a separate innate cell recently merged, with basophils reported to be necessary and sufficient to single-handedly induce Th2 cell differentiation and effector function. A trio of back-to-back papers supported previous observations that basophils could provide an early IL-4 signal,^16–18 but also that basophils were essential for antigen presentation and Th2 cell priming,^19–21 hence acting as both antigen-presenter and cytokine-provider. Following helminth infection of DC-restricted MHC-II-expressing mice¹⁹ or papain injection of basophil-depleted mice¹⁷ impaired Th2 differentiation was reported. Restricting MHC II sufficiency to basophils, or DC depletion, had no impact on Th2 priming, suggesting that basophils played a non-redundant role in Th2 priming in vivo. However, the use of depleting antibodies that target CD200R3, a proposed basophil-specific marker, may have also removed an inflammatory DC population, demanding re-interpretation of some of these experiments. Refuting the basophil claims, DC depletion significantly impaired Th2 responses following papain injection or helminth infection,^22–25 reclaiming the role of antigen presentation to DCs. Whether basophils or DCs are the definitive antigen-presenting cell for Th2 differentiation is still debated; however, the above-mentioned studies did not dissect spatial separation of these cells, mucosal delivered antigens compared with tissue delivered antigens or the absolute number of each particular cell type in these locations. A recent paper indicated that basophils interact with antigen-experienced T cells in the periphery and not within lymphoid tissue.²⁶ It is therefore conceivable that a collaboration between DCs and basophils may develop, as previously suggested,²⁷ or that each cell provides optimal signals for Th2 cell differentiation, expansion or effector function. Answers to some of these questions may clarify the important role played by each of these cells in Th2 cell development and re-activation.

Signal 2 co-stimulation

Th2 induction via low strength TCR stimulation can by-pass the requirement for exogenous IL-4 but requires a second signal, via CD28 co-stimulation.²⁸ This simple observation highlights the importance of additional TCR-independent cell–cell interactions. Cognate antigen presented on MHC II molecules alone is usually insufficient to fully stimulate αβ⁺ CD4⁺ T cells. For optimal activation, a TCR–MHC II synapse forms, re-arranging the local extracellular and intracellular landscape on both antigen-presenting cell and the responding T cell to allow additional cell-to-cell interactions. Of particular importance, B7 molecules (B7-1, CD80 and B7-2, CD86) on the antigen-presenting cell associate with CD28, and other members of the CD28 superfamily including inducible co-stimulator protein (ICOS), cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed death-1 on the responding T cell. As mentioned above, along with TCR stimulation, CD28 ligation is necessary, but also sufficient to stimulate il4 transcription.

Inducible co-stimulator protein, another member of the CD28 superfamily, is expressed on naive αβ⁺ CD4⁺ T cells and is up-regulated on activated cells. In the absence of ICOS, Th2 differentiation is also abrogated.²⁹ In the absence of CD28, ICOS can provide co-stimulation for Th2 cells, albeit at a much lower efficiency than CD28, and rescue Th2 cell development. These studies suggest a hierarchy of co-stimulation, with a critical requirement for CD28 and a less important role for ICOS.

CTLA-4 also interacts with B7 molecules on the antigen-presenting cell, but unlike CD28, which provides a stimulatory signal, CTLA-4 provides an inhibitory signal. CTLA-4-deficient mice die of Th2-associated lymphoproliferative disorders,³⁰ suggesting that CTLA-4 provides a critical inhibitory signal to Th2 cell development. In loss-of-function studies using CTLA-4-deficient TCR transgenic mice, Th2 differentiation in vitro was significantly enhanced following TCR and CD28 ligation.³¹ This was supported by in vitro gain-of-function experiments where Th2 polarization was inhibited following stimulation of T cells with anti-CTLA-4 agonist antibodies during Th2 polarization.³²

In vivo ligation of CD28 on T cells provides a lethal stimulation in CTLA-4-deficient mice driving IL-4 production³³ and Th2-mediated lymphoproliferation. These data further support the notion that CTLA-4 is a potent inhibitor of Th2 cell development. However, using anti-CTLA-4 blocking antibodies in vivo, Th2 cell responses appeared to develop normally following infection with the filarial nematode, Litomosoides sigmodontis.³⁴ The apparent conflict in results may be because of the TCR transgenic system used, reductionist in vitro systems not translating to in vivo scenarios where additional co-stimulation may compensate for the lack of CTLA-4. Alternatively, insufficient CTLA-4 blockade, or the indirect impact of anti-CTLA-4 treatment on CD8 T cells³⁵ may render in vivo CTLA-4 blocking antibody treatment difficult to interpret.

In addition to the CD28 superfamily, the tumour necrosis factor receptor family consists of an increasing number of receptor–ligand pairs.³⁶ With regard to Th2 cell differentiation and polarization two members have received attention, OX40 and glucocorticoid-induced tumour necrosis factor receptor-related protein (GITR). OX40 is up-regulated on recently activated T cells following CD40 ligand stimulation. OX40 ligand (OX40L) -expressing DCs, but not other cells, provides a critical return signal to the Th2 survival or expansion.³⁷ For initial priming of T cells OX40 does not appear to be required, indicated by experiments using OX40L-deficient DCs. However, for proliferation, re-activation of effector function and cytokine secretion, OX40 ligation was required. GITR is also up-regulated on activated αβ⁺ CD4⁺ Th cells and regulatory T cells. Super-physiological stimulation through GITR can enhance Th2 cell frequency,³⁸ exacerbate Th2-associated airway inflammation^39,40 and also potentiate Th1 cell responses.⁴⁰ However, in the absence of GITR ligation Th2 cells still develop following helminth infection.³⁸ GITR may therefore be a redundant co-stimulatory molecule for Th2 development in vivo, and may act to fine-tune Th2 cell differentiation and expansion, along with other co-stimulatory/inhibitory signals.

Finally, the third families of co-stimulatory molecules involved in T-cell activation are the Notch-Jagged/Delta interactions. Of the four Notch receptors (Notch 1–4) and five ligands (Jagged 1, 2 and Delta 1, 3 and 4) several interactions have been studied in the context of Th2 differentiation. In two independent studies using genetic manipulation, Notch-signalling in the T cell was found to target GATA-3, independent of exogenous IL-4.^41,42 Whereas Jagged two does not appear to be the necessary ligand for Notch,⁴³ Jagged-1⁴⁴ and Delta-4^45,46 both appear to enhance Th2 responses. However, Delta-1-expressing and Delta-4-expressing DCs can also inhibit Th2 differentiation.⁴⁷ The precise pairing of ligands and receptors is still not clear and may involve a combination of several ligands and receptors playing an appropriate Th2 ‘chord’. In summary, the complete narrative regarding co-stimulation, beyond the above-mentioned interactions, for Th2 cell differentiation may never be fully realized, but so far we can certainly enhance and inhibit Th2 cell differentiation, and differentiate or disarm Th2 effector functions when necessary. As more advanced imaging and genetic tools become commonplace, our understanding of Th2 cells and the co-stimulatory requirements will become more refined and in course more able to be manipulated.

Signal 3 cytokine micro-environment

The third signal received, not in this particular order, is provided by soluble cytokines. As mentioned above, Th2 differentiation has traditionally been induced in vitro by exogenous IL-4. Although this observation is still solid, IL-4-independent pathways have recently been identified, with cytokines such as IL-25, IL-33 and thymic stromal lymphopoietin (TSLP) added to the list of Th2-promoting factors. Nevertheless, IL-4 remains on top of the pile as a dominant Th2-promoting molecule. Interleukin-4 receptor α and the common gamma chain provide the necessary type I IL-4 receptor for IL-4 binding. Signalling is transduced by the transcription factor STAT-6, which in combination with TCR-derived signals and other transcription factors, activates GATA3. These signals trigger transcription of il4 and other Th2-associated cytokines within the il4 locus, including il5 and il13. In addition, IL-2 produced as a result of TCR triggering leads to STAT-5-induced IL-4 production.⁴⁸ GATA3 is both necessary and sufficient for Th2 development and lies at the heart of Th2 cell differentiation and proliferation. Transgenic over-expression of gata3 induces il4⁴⁹ whereas its absence abolishes il4 transcription.^7,50 GATA3 also serves to stabilize chromatin rearrangement within the il4 locus during Th2 differentiation and represses IL-12-mediated STAT4 expression and Th1 development (A more detailed review of the il4 locus and GATA3 can be found in refs ^51,52). Based upon in vitro observations with IL-4, it stood to reason that in vivo IL-4 would be the signal necessary for Th2 differentiation. The precise source of early IL-4 in vivo eluded immunologists for a long time with innate cells such as natural killer T cells initially proposed.⁵³ Despite observations made approximately 20 years ago that basophils can produce IL-4 and are present in lymphoid organs⁵⁴ a flurry of recent papers re-invigorated the basophil and identified the influx of IL-4^gfp+ basophils into lymph nodes following infection with Nippostrongylus brasiliensis^23,55 or Schistosoma mansoni¹⁹ or immunization with papain.¹⁷ However, reiterated throughout this review is the idea that in vitro observations provide likely, but not guaranteed, hypotheses for in vivo scenarios. Interleukin-4 is the perfect example of in vitro dependence and in vivo redundancy. Th2 cells can differentiate in vivo in the complete absence of IL-4. Normal in vivo Th2 responses were observed in IL-4- and STAT6-deficient mice infected with N. brasiliensis suggesting that Th2 cells can differentiate independent of IL-4–STAT-6 signalling.^10,56 Of note, GATA3 appears to be essential for in vitro and in vivo generation of Th2 cells.⁵⁰

If Th2 responses can develop in the absence of IL-4, IL-4Rα and STAT-6, then which cytokine signals compensate in the absence of this pathway? Several candidates have been identified. Firstly, TSLP, a cytokine produced by a variety of cells including epithelial cells,⁵⁷ mast cells,⁵⁸ DCs⁵⁹ and basophils,^17,60 can be induced by Toll-like receptor 3 (TLR3), IL-4 or IL-13 signalling.⁶¹ TSLP can act on both DCs and T cells, up-regulating OX40L⁶² and inducing CCL17 and CCL22 secretion⁵⁷ from DCs and simultaneously suppressing IL-12 production.⁶² These effects on DCs confer the ideal Th2-inducing characteristics. Furthermore, TSLP can enhance IL-4⁺ basophil recruitment, facilitating Th2 priming or expansion¹⁹ and providing all the necessary components for Th2 differentiation. In addition to these indirect effects on Th2 cells, via DCs and basophils, TSLP can act directly on T cells, signalling via STAT-5 and enhancing T-cell survival.^63,64 Taken together, one would suspect that TSLP would be an integral part of Th2 differentiation, however, there is redundancy in TSLP in several systems. Following infection with several different helminths (Heligmosomoides polygyrus, N. brasiliensis and S. mansoni) Th2 responses developed normally with only modest changes in TSLP- or TSLPR-deficient mice.^65,66 So far TSLP appears to contribute to Th2 differentiation in several settings and is sufficient to drive Th2 differentiation, but Th2 differentiation may not be critically dependent upon the action of TSLP, or any single molecule, with multiple layers of redundancy.⁶⁷

Interleukin-25, produced by mast cells, eosinophils, basophils⁶⁸ or epithelial cells following allergen stimulation,^69,70 helminth infection,^71,72 or IL-4⁷³ can also contribute to Th2 development, possibly via TSLP and OX40L.⁷⁴ Interleukin-25 can also be produced by Th2 cells, re-enforcing the Th2 cell lineage by up-regulating GATA3.⁷⁴ The primary target of IL-25, however, appears to be novel innate-like cells (Nuocytes),⁷⁵ multi-potent progenitors,⁷⁶ fat-associated lymphoid cells⁷⁷ or non-B, non-T cells⁷³) with the capacity to release a storm of type 2 cytokines.^78,79 Whether these newly identified cells are the same cells, related to each other or stem from different origins has not been clarified.^80,81 The IL-25-mediated effects on Th2 cells in vivo may therefore involve IL-25-responsive innate-like cells; however, the relationship between innate-like cells and Th2 cell differentiation, effector function or memory stability is also unclear. Is IL-25 dispensable for Th2 cells and broader type-2 responses in vivo? Infection of il25-deficient mice with N. brasiliensis⁷¹ or Trichuris muris⁷² delays, or prevents, worm expulsion, respectively, suggesting a clear non-redundant role for IL-25 for Th2-dependent mucosal helminth immunity.

Interleukin-33, similar to IL-25, can also promote TSLP expression and activate innate-like cells.^77,82 However, IL-33 has long been associated with Th2,^83,84 mast cell and basophil^85–87 activation. Interleukin-33, signalling through its receptor ST2, is a chemoattractant⁸⁸ for Th2 cells, and, in collaboration with TSLP or other STAT5 activators, can induce Th2 cell activation independent of TCR ligation.^83,89 Despite these all-round Th2 activating properties, there is controversy regarding the requirement for IL-33-ST2 for Th2 cell development. Using T1/ST2 neutralizing antibodies or ST2-deficient mice, Th2 cell development in vivo was curtailed following pulmonary antigen challenge.^90–92 However, similar experiments, but using a different ST2-deficient mouse, indicated that Th2 cells developed normally in vitro and in vivo.⁹³ These studies are open to broader interpretation if ST2 is shared by other ligands. One study has reported il33-deficient mice that develop milder airway inflammation following allergen challenge;⁹⁴ however, a detailed analysis of Th2 cell development in vitro or in vivo was not reported.

In addition to other cytokines, which most likely contribute to Th2 cell differentiation, so far IL-4, TSLP, IL-25 and IL-33 have all been associated with differentiation, activation and/or recruitment of Th2 cells. Whether a context-specific hierarchy of importance for these molecules can be drawn up or not is unclear. There appears to be significant overlap and redundancy, from the current literature. Whether this is true redundancy, or a failure on our part to dissect Th2 cells at sufficient resolution is not clear. For example, are naive or differentiated Th2 cells that are exposed to IL-4, TSLP, IL-33 and or IL-25 similar? Adding one more dimension, such as variable TCR signal strength, are these cells still similar? Further still, adding a third dimension of co-stimulation, do these polarizing cytokines still act in similar ways? And so on. We hypothesize that there is significant heterogeneity within the Th2 spectrum, so much so that there is overlap into what may appear to be Treg, Th9, Th17 or Th1 cells, depending on the signals received and lineage-defining markers used. As briefly mentioned above, T helper cell plasticity is slowly being unravelled and is smudging the lines between the current subsets. Current Th cell nomenclature, such as Th1 and Th2 will make a half-century but as we delve deeper into the molecular machinery of Th cell biology unique properties of Th cells in the context of disease are appearing. This has led to two schools of thought (i) fractionating the Th subsets further still into unique subsets, or (ii) grouping the Th cells together with an appreciation of plasticity depending upon the environment. As more data are reported, support for a plasticity model is gaining weight, but presumably this too has a limit. Can a fully polarized IFN-γ-producing cell with TCR re-arrangement, chromatin remodelling of the ifng gene and tissue-specific homing markers ever turn on IL-4, IL-5 and IL-13? Would it ever need to in vivo?

Microbial stimuli inducing Th2 cell differentiation

The interactions between microorganisms and antigen-presenting cells, via pathogen-associated molecular patterns and pathogen recognition receptors leading to induction of Th1 responses are well documented.^95,96 Progress is being made to elucidate helminth products, allergens and their cognate receptors expressed by DCs that lead to the induction of Th2 responses.^97,98 Excretory–secretory products from parasitic helminths consist mainly of proteins and glycans that are sensed by TLRs, C-type lectins and scavenger receptors leading to modulation of DC structure and function (reviewed in ref. 99). For instance, the glycoprotein omega-1 has been identified as the major Th2-inducing component of soluble egg antigen of S. mansoni (SEA) in vitro.¹⁰⁰ Other components of SEA such as the glycoprotein IL-4-inducing principle of S. mansoni eggs (IPSE or alpha-1) and the glycoconjugate, lacto-N-fucopentose III, play a contributory role in inducing Th2 responses in vivo.^101–103 The C-type lectins DC-SIGN, mannose receptor and macrophage galactose-type lectin have also been implicated in the uptake of SEA and its components by rapid internalization and targeting to MHC II lysosomal compartments.¹⁰⁴ Rzepecka et al.¹⁰⁵identified a low-density lipoprotein, calreticulin, secreted by tissue-phase intestinal H. polygyrus larvae that functions as a pathogen-associated molecular pattern. A Class A scavenger receptor expressed by DCs can bind calreticulin and mediate adjuvant-independent induction of IL-4 in vivo. Uptake of excretory–secretory products from other helminths such as N. brasiliensis and T. muris can influence DC function in vivo⁹⁹ and polarize Th2 cells, independent of Th2 polarizing mediators^65,106,107 or directly induce Foxp3⁺ Treg cell development.¹⁰⁸ However, the composition of these products and uptake mechanism is yet to be identified. In T. muris, ES-mediated DC modulation was found to be dependent on TSLP–TSLP-R interaction,⁶⁵ suggesting that ES composition may directly influence the nature of T helper cell differentiation.

It is now evident that the uptake of a majority of helminth products by DCls does not induce classical maturation but instead limits their activation, promoting conditions that lead to Th2 differentiation. This may favour parasite longevity in the host as well as limiting the induction of inflammatory Th1 and Th17 responses. It has been demonstrated that potent IL-4R-independent Th2 polarization mediated by omega-1 corresponds with its ability to inhibit IL-12 release by DCs. Using a CD40L-expressing cell line to mimic T-cell interaction, omega-1 was found to reduce dendritic cell production of IL-12p70 at a concentration 50-fold less than total SEA. This effect was also observed when recombinant omega-1 was used, albeit with reduced potency when compared with natural omega-1.¹⁰⁰ Furthermore, studies have demonstrated that recruitment of natural and inducible regulatory CD4⁺ T cells provide global regulatory responses, which control tissue immunopathology in vivo (reviewed in ref. 109).

Most allergens induce DC maturation, either indirectly by contaminating bacterial products such as lipopolysaccharide (reviewed in ref. 110) or, as recently described for the mite allergens Der p 2 and Der f 2 which bear a similar structure to MD-2, via the LPS-binding site of TLR-4.¹¹¹ Such allergens trigger TLR-4-dependent Th2 priming by the concerted activity of lung epithelial cells and DCs.¹¹² Other allergens, such as papain, are comprised of cysteine proteases that can promote Th2 differentiation and proliferation via DCs and basophils.^23,111 Danger and stress signals following allergen encounter or parasite invasion can invoke danger-associated molecular patterns (DAMPs) such as ATP.^113–115 ATP, in addition to TLR signalling, can potently activate the inflammasome leading to IL-1β processing, which has been shown by several groups to enhance Th2 effector responses.^89,116–118 Interestingly, blood dwelling schistosomes posses ATP-catabolizing enzymes on their tegument surfaces that breakdown ATP to adenosine, potentially interfering with this pathway.¹¹⁹

Th2 effector function

Following differentiation, Th2 cells are distinguishable from Th1 cells by more than just cytokine gene activation. For example, Th2 cells lose the ability to sustain calcium flux ¹²⁰ resulting in reduced tyrosine phosphorylation.¹²¹ Th2 cells also have an unconventional synapse, relative to Th1 and naive T cells, and fail to form a ‘bulls-eye’ structure.¹²² These apparent differences may be because of reduced CD4 and increased CTLA-4 expression, as suggested by others.¹²³ The consequences of these structural differences between Th1 and Th2 cells are unclear. Unlike IFN-γ, which is secreted directionally in the immunological synapse, IL-4 can be secreted multi-directionally influencing many surrounding cells.^124,125 Whether this is a result of altered synapse formation or not has not been reported. Also, whether IL-5 and IL-13 are indiscriminately secreted multi-directionally within the reactive lymph node has not been reported. The precise activation signals received by differentiated Th cells, stimulating their effector function are rather vague. For example it may not be desirable for a Th2 cell, or Th1, Th17 or Th9 cell, to release their payload of potent cytokines, beyond polarizing IL-4, in the case of Th2, within the T-cell zones of lymphoid tissue. Therefore, restricted re-activation via peptide-loaded MHC-II-expressing cells or other activating signals at the site of infection, allergy or action must take place. What these additional signals are is surprisingly unclear. Following Th2 differentiation, chromatin remodelling at conserved non-coding sequence (CNS)-1, DNase I hypersensitivity (DHS) site, CNS-2 and the conserved intron 1 sequence of IL-4 (CIRE) in the il4 locus facilitates rapid cytokine transcription.^126–128 Poised in such a state, it may only require a ‘tickle’ to induce translation and secretion of these cytokines. An elegant study by Mohrs et al.,¹²⁹ using a dual reporter system to identify transcription and secretion of IL-4, discovered that although IL-4 was transcribed in lymphoid and non-lymphoid tissue, secretion was only observed in non-lymphoid tissue upon antigen encounter. This study is in slight contradiction to a recent paper from the same group identifying the widespread influence of IL-4 in the reactive lymph node.¹²⁵ This study suggested that T cells secrete IL-4 throughout the reactive lymph node leading to STAT-6 activation, although the different IL-4-producing cells were not the main focus of this paper. What is not clear is the influence of the different IL-4-producing cells within the lymph node. Do basophils secrete IL-4 multi-directionally and T cells secrete focused IL-4? If IL-4 secretion is representative of other effector cytokine secretions, the former study¹²⁹ supports the notion that cytokines are only secreted at the site of antigen re-encounter, spatially separating differentiation from effector function. Whether peptide–MHC complexes are the final or only trigger activating effector Th2 cells in non-lymphoid tissue or if signals with or without TCR engagement can trigger effector function is not clear. The local cytokine environment, including IL-33⁸⁴ and TSLP,¹³⁰ can enhance Th2 cytokine secretion, but whether ST2 and TSLP-R ligation also requires TCR engagement is not clear. Furthermore, the cross-talk between damaged stroma following invasion, tissue damage, or danger signals and their direct impact on Th2 cells has not been reported.

The impact and role of Th2-derived cytokines has been widely reported. It is undisputable that IL-4 is required for optimal IgG1 and IgE class switching in B cells,¹³¹ alternative activation of macrophages¹³² and Th2 stability; IL-5 mobilizes, matures¹³³ and recruits eosinophils¹³⁴ and IL-13 induces goblet cell differentiation, mucus secretion and tissue repair.¹³⁵ Th2 cells can certainly provide this trio of potent cytokines, but they are not the only ones. The recently reported type-2 innate-like cells seem more than capable of fulfilling this role as cytokine providers but they do not appear to be controlled by antigen specificity.

In addition to overlapping cues for the development of Th2 cells, their functional properties may also have overlap and redundancy. For example, infection of IL-5, IL-9 and IL-13 compound cytokine-deficient mice with N. brasiliensis demonstrated the ability of IL-4 to mediate worm expulsion,¹³⁶ although these mice have not been extensively studied. Nevertheless, intestinal helminth infection models have unanimously identified mechanisms of protection optimally mediated by αβ⁺ CD4⁺ Th2 cells activating a suite of innate cells. The inflammatory phenotype seen in Th2-driven asthma is also characterized by the release of IL-4, IL-5, IL-13 and IL-9.¹³⁷ These features of disease have focused researchers for many years on developing strategies to perturb Th2 development and effector function to benefit allergies and to identify ways of enhancing Th2 functions to protect against helminths, or at least, the intestinal-dwelling helminths. Therapeutic approaches that involve the use of biological modifiers such as monoclonal antibodies that target Th2-associated cytokines are being tested (reviewed in ref. 138). Interestingly, such intervention studies have shown that selective inhibition of IL-4 is not effective for the treatment of asthma. This is not surprising as IL-4 has been shown to be important mainly for allergen sensitization (atopy) and IgE production while IL-13 plays a more central role in development of airway hyper-responsiveness and tissue-remodelling.⁹ It has been suggested that targeting IL-13 alone or in combination with IL-4 may be more useful in combating asthma.¹³⁹ Also, a mutated IL-4 that targets IL-4Rα, thereby blocking the effects of IL-4 and IL-13, is also being developed.¹⁴⁰ Other strategies that target IL-5 and tumour necrosis factor-α have been proposed, but the benefits of using biological modifiers need to be weighed against the risks of unwanted effects before they can be put into clinical use.

Conclusion and forward thinking

The type-2 microenvironment has been re-structured over the past 5 years with the born-again basophil providing early IL-4 and with the capacity to process and present antigen to Th cells. At 90 degrees to this interaction is the discovery of innate-like cells with the capacity to secrete large amounts of IL-5, IL-13 and IL-9, triggering type-2 responses, presumably before the clonal expansion of antigen-restricted Th2 cells. Finally, the observation that Th2 cells can develop into Th1,⁵ Th17⁶ or ‘Th9’³ cells with the appropriate environmental cues suggest a great degree of plasticity within the Th cell populations. However, while these newer discoveries fill in the gaps of the type 2 environment and have tended to down-grade the Th2 cell into a co-star role, there is still a great deal we do not know about Th2 cells. If antigen specificity and memory Th responses are required for improved vaccine efficacy, either directly or via antibody production, and if allergen-reactive T cells are responsible for atopic disorders, then investigating how these newer discoveries impact Th2 cell development and their effector function in this context remains an important area of research.