AllergoOncology: Opposite outcomes of immune tolerance in allergy and cancer

2.1 Tolerance induction with allergen immunotherapy

As part of tolerance induction, AIT has been correlated with enhanced regulatory T- and B-cell responses,3, 8 and regulatory DCs,9 all potential sources of the immunomodulatory cytokine IL-10. A classical hallmark of AIT is the induction of allergen-specific IgG1 and anti-inflammatory IgG4. Allergen-specific IgGs may (i) act as blocking antibodies, trapping allergens before they can cross-link allergen-specific IgE; (ii) interact with inhibitory IgG receptor FcγRIIb and downregulate IgE-mediated signalling10; or (iii) promote differentiation of tolerogenic M2b macrophages.11 While investigation into immune tolerance in allergy provided some insights, it is unclear which of these mechanisms may drive tolerance or which specific biomarkers could be used to predict clinical response to AIT.12

2.2 The opposite side of the coin: clues into breaking tolerance in cancer

Tumour cells and tumour-infiltrating immune cells can help to shape a strong tolerogenic microenvironment that promotes tumour growth and allows metastatic spread. The question of how to break tumour tolerance has recently become more pertinent, catalysed by the clinical efficacy of checkpoint inhibitors in some tumour types, including melanoma.

Two checkpoint molecules, the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and the programmed cell death protein 1 (PD-1), play key roles in restricting T-cell activation and inducing immunological tolerance that contribute to immunosuppressive environments in cancer by multiple mechanisms. They restrict recruitment, activation and proliferation of cytotoxic CD8+ T cells,13 and their effects are amplified by tumour cells expressing CTLA-414 or PD-1 ligand (PD-1L).15 These findings opened new avenues for intervention by so-called checkpoint inhibitors.

The anti-CTLA-4 monoclonal ipilimumab was the first clinically applied antibody that conferred tangible improvements in the treatment of metastatic melanoma,16 with long-term survivors after 3 and 5 years.17 The anti-PD-1 inhibitor antibodies, nivolumab and pembrolizumab, also approved for the treatment of malignant melanoma, also demonstrated significant progression-free and overall survival benefits for patients with advanced melanoma. Toxic side-effects of checkpoint inhibitors in human patients include severe autoimmunity,18 underlining the dangers when interfering in immune regulation. Clinical trials in other malignant diseases are performed with anti-PD-1 antibodies nivolumab and pembrolizumab based on better efficacy and less toxicities compared with ipilimumab. Nivolumab is approved for metastatic non-small-cell lung cancer (NSCLC), renal cell carcinoma (RCC) and classical Hodgkin lymphoma (cHL)19; pembrolizumab for NSCLC, head and neck squamous cell carcinoma (HNSCC) and classical Hodgkin lymphoma (cHL).20 Furthermore, anti-PD-L1 (PD-ligand1) antibody avelumab is applied for the treatment of NSCLC,21 and others such as atezolizumab and durvalumab22 are under clinical investigation.

OncoImmunology (alias ImmunoOncology) explores host immune responses to tumours for uncovering insights that can lead to novel treatments.23 Similarly, the study of Th2-type immune responses in cancer in the emerging field of AllergoOncology24-26 may also uncover new approaches for future treatment interventions.

3.1 Dendritic cells

DCs are professional antigen-presenting cells with the unique ability to induce differentiation of naïve T cells into tumour antigen–specific or allergen-specific T helper cells or CTLs. Maturation, upregulation of costimulatory molecules (CD40, CD80, CD86) and secretion of pro-inflammatory cytokines (IL-1β, IL-12, IL-6, TNFα) are required for successful proliferative T-cell responses following DC-mediated antigen presentation.

However, DCs also exhibit regulatory functions. IL-10-treated DCs derived from atopic donors suppress the allergen-driven Th2-specific response or convert T cells into Tregs.27 In particular in the tumour microenvironment, myeloid DCs are converted into tolerogenic phenotypes promoting Tregs rather than T-effector cells as an escape mechanism from immune clearance,28 supported by TGFβ and IL-10.29 Tolerogenic DCs show low expression of costimulatory molecules, secrete low levels of IL-12 and produce IL-10. Therefore, while useful tolerance in allergen immunotherapy, antigen presentation by tolerizing DCs is harmful in cancer as it can prevent antitumour T-cell responses.

The immunosuppressive features of DCs may be overwritten by inflammatory signals. IgE-mediated antigen sampling is a particularly potent mechanism to activate primary T-cell responses,30 not inducing IL-10, and dampening LPS-induced IL-12 production31; irrespectively, it activates CTLs via cross-presentation and improves antitumour responses.30 Therefore, the current literature implies that IgE-mediated antigen presentation supports DC-based immunity rather than leading to DC-mediated tolerance.

3.2 Macrophages

The different functional phenotypes of macrophages depend on signals from the surrounding environment. A general classification mirroring the Th1/Th2 paradigm of classically pro-inflammatory (M1) and alternatively anti-inflammatory (M2) macrophages is broadly accepted.

Pro-inflammatory M1 cells are activated by mainly IFNγ, LPS and other TLR ligands. M1 were observed in exacerbation of lung injury and airway remodelling in allergic asthma via nitric oxide production.32 The presence of M1 macrophages in the tumour microenvironment has been associated with extended survival of cancer patients.

M2 macrophages comprise different activation phenotypes: M2a, M2b and M2c. M2a are triggered by IL-4 and IL-13 and positively correlate with the severity of airway inflammation in allergic asthma.33 In cancer, a low M1/M2a ratio was associated with poor prognosis in a variety of murine and human malignancies.

M2b and M2c are involved in immune regulation, tissue remodelling, angiogenesis and tumour progression. M2b are induced by IgG immunoglobulin complexes and LPS and are reported in the context of allergy as well as cancer.34 The inhibitory IgG receptor FcγRIIb (CD32) is a critical player for signal transduction and upregulation of IL-10 and CCL1 expression, while reducing IL-12. CCL1 secretion is critical to maintain the M2b phenotype in mice and humans.35 M2c are induced by glucocorticoids, TGFβ and IL-10, and support induction of Tregs,36 which in tumours correlates with disease progression and poor prognosis.

Immune complexes with an antitumour IgE antibody or cross-linking of surface-bound IgE can polarize monocytes and macrophages to upregulate CD80 and the pro-inflammatory mediator TNFα. TNFα can then stimulate production of the macrophage chemoattractant protein 1 (MCP-1) by both monocytes and tumour cells, and trigger recruitment of macrophages into tumour lesions and restriction of tumour growth.37 This macrophage TNFα/MCP-1 signalling has been reported in IgE-mediated clearance of parasites by macrophages and brings an additional dimension to the functions of IgE antibodies.38

Based on these multifaceted phenotypes of macrophages, blocking M1 pro-inflammatory or M2 anti-inflammatory macrophages may lead to opposite effects in cancer and allergy. Targeting macrophage functions with tumour antigen-specific IgE antibodies or vaccines that trigger IgE responses against cancer would require careful examination.

3.3 Tregs

The main mechanisms underpinning Treg immune effects include production of inhibitory cytokines (IL-10, TGFβ and IL-35), effector cell cytolysis (via secretion of granzymes A and B), direct targeting of DCs via the inhibitory PD-1 and CTLA-4 cell surface checkpoint molecules and metabolic disruption of effector cells (CD25, cAMP, adenosine, CD39 and CD73).39 FoxP3+-induced Tregs (iTregs) are mandatory, while thymic-derived natural (FoxP3+) nTregs are dispensable for oral tolerance. In addition, iTreg cells rather than nTreg cells are involved in the control of mucosal Th2 responses.40

In contrast in cancer patients, Tregs contribute to an immunosuppressive tumour microenvironment. Accumulation of Tregs in tumours can occur through their recruitment via chemokines (eg, CCL-17, CCL-22, CCL-5, CCL-28), while their expansion in situ or conversion of conventional T cells can be promoted by high local levels of cytokines, such as TGFβ.41 Specifically, the smoke compound acrolein promotes regulatory cells via the arylhydrocarbon receptor (AhR), thereby increasing malignant growth, while decreasing the risk for allergic sensitization.42 High numbers of infiltrating Tregs are associated with poor prognosis in many cancers, including ovarian, pancreatic, lung cancer, glioblastoma or melanoma.41 Therefore, cancer immunotherapy targeting Tregs while breaking tumour tolerance can also break tolerance to self,43 highlighting the fine balance between antitumour immune activation and the risk of autoimmunity.

3.4 B cells and Bregs

B cells play key roles in inflammation, antigen presentation and antibody production in the context of allergy. Their contributions in cancer are less well delineated.

Tumour-infiltrating B cells (TiBCs) have been detected in human solid malignancies, including melanoma,44, 45 breast, ovarian, cervical, colorectal and non-small-cell lung cancer. As in allergy,46 TiBCs show evidence of antigen-driven expansion and somatic hypermutation, implying active in situ antibody affinity maturation in the cancer tissue.45 The presence of TiBCs, and B cells in tertiary lymphoid structures (TLSs), is associated with improved prognosis in different cancer types. Increased survival has been observed when CD8+ cells are also detected in the same tumours, suggesting synergies between T and B cells and induction of adaptive immune response. TiBCs may mediate immune responses against tumours by several mechanisms: (i) TiBC-derived antibody activities, (ii) direct cytotoxicity by B-cell-secreted mediators, (iii) immunomodulation of other TILs and promotion of TLSs, or antigen presentation.47

B regulatory cells (Bregs) can mediate allergen tolerance by IL-10-dependent and IL-10-independent mechanisms.48 In cancer, Bregs may function in a similar manner to promote immune tolerance or potentiate Treg responses, leading to tumour progression.47, 49, 50 The latter is consistent with TiBCs found in close proximity to FoxP3+ T cells in melanoma lesions and in other tumour types.44 Specific compartments and actions of B cells may thus be targeted to improve treatment of allergic or malignant diseases.

3.5 Innate lymphoid cells (ILCs)

Innate lymphoid cells (ILCs) broadly mirror helper T-cell subsets, but they do not express specific antigen receptors. Based on their lineage-specific transcription factor and cytokine production, they are classified in 3 groups.51 ILC1s phenotypically like Th1, respond to IL-12, IL-15 and IL-18, and are defined by the production of IFNγ and expression of transcription factor T-bet. NK cells expressing eomesodermin and producing cytotoxic granzymes and perforin also belong to that group. ILC2s, which resemble Th2 cells, respond to epithelium-derived cytokines, such as IL-33, IL-25, TSLP, eicosanoids and IL-1β. The cells are defined by production of type 2 cytokines IL-4, IL-5, IL-9 and IL-13 and by the expression of the transcription factor GATA-3.

ILC2s are involved in the initiation of innate allergic inflammation and in its enhancement by interacting with other immune cells. They are stimulated by epithelial cells (through IL-33, IL-25, TSLP) or by proximal mast cells (via IgE-mediated eicosanoid release) that induce type 2 cytokine production from human ILC2s.51 On the other hand, ILC2s are negatively regulated by IL-33-activated mast cells that suppress them via Treg cell expansion or by KLRG1 (produced by ILC2 after stimulation with IL-33 or TSLP)/E-cadherin (expressed by keratinocytes) axes.

In cancer, IL-33 secreted by macrophages stimulates ILC2s and, in turn, the secretion of IL-13 and IL-5, which have pro-tumoral effects. ILC2s can also establish an immunosuppressive tumour microenvironment by amphiregulin secretion.52

ILC3s resemble Th17 and Th22 cells. They respond to IL-1β and IL-23 and are defined by the production of IL-17A and IL-22 and by the expression of RORγt.53 Furthermore, cells of the ILC3 subtype secrete IL-22 upon IL-23 stimulation by macrophages and have tumorigenic effects. On the other hand, ILC3s could induce tolerance by increasing IL-10 and retinoic acid secretion by DCs upon stimulation by microbiota and macrophages,54 or by enabling T-cell tolerance through the expression of MHC Class II in the absence of costimulatory molecules.55 Thus, among the ILC type, especially the ILC3s could favour tumour growth and tolerance.

3.6 Mast cells

Mast cells are major players in allergy, but also accumulate in the stromal and intratumoral tissues of a diverse array of cancers. Mast cells are chemoattracted by different factors such as stem cell factor (SCF), vascular endothelial growth factor (VEGF), chemokines, prostaglandins, leukotrienes, histamine and osteopontin56 in the tumour microenvironment. The controversial role of mast cells in tumorigenesis and tumour progression could possibly depend on their micro-localization and the type of tumour. Their contribution to human tumour growth has mostly been assessed by relating the number of mast cells in cancer tissue to the stage and/or prognosis of the disease, while generally no markers for functional activity were taken into account.56, 57 The net outcome of mast cell accumulation for the tumour cells may be determined by the condition of the tumour micromilieu shaping local pH and oxygen tension.56, 58, 59 Upon activation, mast cells can release a great number of prestored and newly synthesized mast cell mediators, which can stimulate tumour growth (see below).

Furthermore, mast cells express cell surface molecules, which upon contact with target cells inhibit cell activation or may polarize mast cells towards tolerance induction. Expression of PD-L1 or PD-L2 on mast cells may contribute to inhibition of T-cell activation in the tumour microenvironment.60 Furthermore, co-culture of human mast cells with immature DCs stimulates their expression of PD-1 and programs DCs towards tolerogenicity supporting induction of regulatory T cells.61 The impact of the mast cells checkpoint inhibitors for tumour development requires further research.

3.7 Eosinophils

Eosinophils were found mainly antitumorigenic in solid tumours such as bladder, gastrointestinal, melanoma, prostate, with only some studies showing a detrimental effect in favour of tumour growth such as in the case of Hodgkin lymphoma.62 Mechanistically, CCL1 produced by tumour cells can attract eosinophils (via CCR3) that may interfere with tumour growth.63 Tumour cells can stimulate eosinophils to produce IL-18, which in turn facilitates eosinophil-cancer cell interactions leading to tumour cell death.64 Production and secretion of ROS and cytotoxic mediators, typical eosinophil mediators, were found antitumorigenic in many cancer studies.65 Nevertheless, the growth factor APRIL could support growth of malignant plasma cells in bone marrow in a multiple myeloma mouse model.66 More recent studies place eosinophils as first line “sensors” for the general antitumour immune response, showing for example, that intratumoral eosinophils are crucial for the recruitment of antitumour CTLs in melanoma.67 On the other hand, lung eosinophils were shown to induce Treg infiltration and consequently support lung metastasis.68 Lastly, eosinophils are involved in curative processes induced by checkpoint inhibitor immunotherapies (eg, ipilimumab or pembrolizumab69).

Hence, numerous findings suggest that eosinophils can be an immunotherapeutic effector cell either being activated by immunotherapies such as checkpoint inhibitors, or granulocyte-macrophage-colony-stimulating factor (GM-CSF)-based vaccines,70 or perhaps by adoptive transfer of these cells in an appropriate setting.71

3.8 Epithelial cells

The essential contribution of the epithelial barrier to a balanced immune response is well established. For oral antigens, the site of antigen uptake and its particulate nature is decisive for the subsequent immune response.72 Antigen passage through mucus-producing goblet cells has been specifically associated with tolerance induction.73 In allergy as well as cancer, the cell surface receptor repertoire of epithelial cells includes the high-74 and the low-affinity IgE Fc receptors facilitating antigen passage and directing antigen presentation.75 The epithelium is additionally an important cytokine source, profoundly modulating the immune response (Table S1). In the intestinal tract, extracellular vesicles physiologically produced from intestinal epithelial cells contribute to innate immunosuppression,76 which may contribute to oral tolerance—or cancer progression.

4.1 IgG4 antibodies

IgG4 is the least abundant subclass of IgG in normal human serum, but it may reach elevated levels following chronic exposure to antigen, or during inflammation. IgG4 has unique structural characteristics that are responsible for its inability to fix complement, low affinity for activating FcγRs, and relatively high affinity for the inhibitory Fc receptor FcγRIIb.26 IgG4 has a unique ability to undergo Fab-arm exchange, resulting in the formation of bispecific antibodies which are functionally monovalent, and are therefore not able to cross-link antigens to form large immune complexes.44 For these reasons, IgG4 are widely considered to be anti-inflammatory antibodies, playing a positive and protective role in allergy and helminth infections. On the other hand, in IgG4-related diseases, such as sclerosing pancreatitis and autoimmune pancreatitis (AIP), pathogenic roles for IgG4 cannot be fully precluded yet. In contrast, the anti-inflammatory properties of IgG4 may have a negative impact in the context of cancer. As tumours are characterized by chronic inflammation with prolonged exposure to tumour-associated antigens, elevated IgG4 has been detected in extrahepatic cholangiocarcinoma, pancreatic cancer, melanoma45 and glioblastoma.77 Indeed, these tumour types are characterized by Th2-biased expression of mediators known to trigger B cells to produce IgG4. Tumour-specific IgG4 may act as a blocking antibody competing with inflammatory antibodies such as IgE or IgG1 for binding to antigen, or in form of immune complexes engage the inhibitory receptor FcγRIIb and thus dampen the inflammation. IgG4 antibodies in cancer may thus have a darker side, as compared to their association with tolerance induction to allergens.

4.2 IgE and IgG repertoires in allergy and cancer

New-generation sequencing for immunoglobulin repertoire analysis78 was applied in subcutaneous AIT79 and longitudinal studies of cancer progression.80 Also the IgG repertoire of healthy individuals contained antibodies to tumour-associated carbohydrate antigens, with a possible function immunosurveillance.81

A study of IgE sequences in the blood and nasal biopsies of eight allergic rhinitis patients undergoing SIT79 compared the repertoires at baseline and after 2 months and 1 year of SIT with the library of paired allergen-specific IgE heavy- and light-chain variable region fragment (scFv) sequences selected at baseline by phage display. The allergen-specific IgE clones showed increased persistence, higher likelihood of clones related to other switched isotypes, and larger clonal families. Related IgG clones may represent the putative competitors for allergen responsible for the acquired allergen tolerance.82, 83

Studies of the immunoglobulin repertoire in cancer are rare. The study of a multiple myeloma patient not only confirmed the monoclonality of the myeloma cells, previously observed in conventional low-throughput sequencing, but also demonstrated certain pitfalls in the massively parallel sequencing technology.80 Another study of 15 B-cell acute lymphoblastic leukaemia (B-ALL) patients tracked the clonal diversification of the malignant cells and found that large numbers of small subclones, present at diagnosis, re-emerged at relapse alongside a dominant clone.84 The technology detected minimal residual disease with unprecedented sensitivity and framed early cyto-reduction, such as surgery and radiation to reduce the tumour bulk, as an important determinant of long-term survival.

Immunoglobulin repertoire analysis by new-generation sequencing can in future provide a rich source of information relating to immuno-biology and immuno-therapeutics in allergy as well as cancer.

4.3 Free light chains (FLC)

Free light chains (FLC) are produced and secreted in excess to immunoglobulin heavy chains during synthesis of tetrameric immunoglobulins by plasma cells and can be detected in all body fluids. Significant increases in systemic and local polyclonal FLC levels have been measured in many chronic inflammatory diseases, including allergic diseases (such as asthma, rhinitis), chronic obstructive pulmonary disease (COPD), multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, diabetes and cancer.57, 85 FLC can induce inflammatory responses by mediating antigen-specific mast cell activation86 and also reduce neutrophil apoptosis and stimulate their release of pro-tumorigenic IL-8. The expression of FLC was found to be a biomarker for poor prognosis in basal-like breast cancer.87 In a preclinical melanoma model, it was further demonstrated that FLC-induced mast cell activation-stimulated tumorigenesis.87 As FLC were found in the stromal tissues of a great number of human tumours, one could speculate that overall FLC may contribute to cancer growth and progression.

4.4 Regulatory cytokines and chemokines

Without doubt among cytokines, IL-10 and TGFβ are pivotal in the establishment of immune tolerance, and their production by many different cell types is desirable in allergy3 but unwanted in cancer, as discussed above. For instance, CCL1 is a chemokine expressed by monocytes and especially by the tolerogenic M2b macrophage subpopulation, after engagement of FcyRIIb and LPS or by IL-1β stimulation.35, 88 CCL1 may be potentiated by the expression of its cognate receptor CCR8 on CCR8+ FoxP3+ Treg cells, and by the CCL1:CCR8 axis in immune regulation.89

Growing interest has recently emerged on the role of the CCL5:CCR5 axis (and other CCL5 receptors, CCR1 and CCR3; and CCR5 ligands, CCL3 and CCL4) in cancer progression, but also in the possibility to exploit this chemokine-receptor interaction to combat cancer. Depending on the tumour environment, CCL5 has been implicated in antitumour adjuvanticity but also in carcinogenesis. Intratumoral myeloid-derived suppressor cells (MDSCs) can mediate recruitment of Tregs through the secretion of CCL3, CCL4 and CCL5 acting on the CCR5.90 Specifically tailored interventions on CCR5 expressed by tumour cells or by tumour stromal cells resulted in antitumour effects.91, 92 Interestingly, CCL5 and CCR5 are involved in immune tolerance and in the recruitment and activation of MDSCs.93, 94 This CCL5:CCR5/MDSC axis may provide yet another important link between allergy and cancer. An example may be the association of chronic food allergy and the development of colorectal cancer via mast cell-mediated recruitment of MDSCs.95 Therefore, while the acute allergic reactions per se via the cytokine and chemokine systems should stimulate the immune system and elevate tumour surveillance, any prolonged chronic inflammation supports tumour development.

4.5 Mast cell mediators and receptors

As discussed above, despite their pro-inflammatory functions, mast cells can have a prominent role in the induction of immune tolerance. Among the prestored and newly synthesized factors, some stimulate tumour growth (eg, histamine, nerve growth factor [NGF], IL-8), neovascularization (eg, VEGF, heparin, TGFβ), and can restrict T-cell responses.96 On the other hand, mast cells can also secrete mediators which are detrimental to the tumour by inducing growth inhibition, cellular disruption and apoptosis (eg, TNFα, IFNγ, activation of PAR-1/2), and by attracting inflammatory leucocytes (eg, IL-8, TNFα), and inhibiting metastasis (eg, chondroitin sulphate). Of note, the mast cell-derived enzymes chymase and tryptase can be both beneficial and detrimental.

Histamine can modulate different immune cells via the histamine 2 (H2) receptor. This could have juxtaposed functions, such as enhanced mobilization of DCs, reduction in cytolytic activity of NK cells and modification of MDSCs.97 Interestingly, ranitidine, an H2 receptor antagonist, was inhibited lung metastasis and breast cancer development in mouse models.98 Mast cells can also release immunosuppressive IL-10 and TGFβ,97, 99 tryptophan hydroxylase-1 (thp-1) and amphiregulin.100, 101 thp-1 deficiency in mast cells results in slower tumour growth kinetics.100 The exact mechanism by which thp-1 regulates immune responses is still unclear. Amphiregulin stimulates the epidermal growth factor receptor (EGFR), enhances Treg function and contributes to the local immunosuppression in the tumour micromilieu.101 All these suggest an important role of mast cell mediators in immunoregulation.

4.6 Lipocalins

Many innate defence proteins are differentially regulated in allergy and cancer, for instance lipocalin-2 (LCN2; NGAL). LCN2 is highly expressed in the liver, bone marrow, spleen, salivary gland, the colon, the lung and the kidney. LCN2 is upregulated in various cancer types and has been proposed as a cancer biomarker.102-104 LCN2 binds iron through iron-chelating compounds.105 As such, it is involved in numerous processes including innate immunity, apoptosis and renal development. It can also form complexes with the matrix-metallopeptidase 9 (MMP9, a 92-kDa gelatinase), itself being a poor prognostic factor in several cancer types.106 Increased iron raises the cancer risk and as a participant in iron homoeostasis also LCN2 is upregulated in various cancers.107 Contrastingly, decreased LCN2 and serum iron are associated with allergy.108 Some exogenous antigens have a striking structural resemblance with human LCN2,109, 110 suggesting potential interference with the iron loading of endogenous LCN2 and affecting its immunomodulatory potency. Taken together, LCN2 could assume divergent immune roles in allergy and cancer.