Allergen- specific IgG show distinct patterns in persistent and transient food allergy

Background: Immediate food- allergic reactions are IgE- mediated, but many individuals with detectable allergen- specific IgE do not react to the food. Allergen- specific IgG may interfere with allergen- IgE interaction and/or through intracellular inhibitory signalling to suppress mast cell and basophil response to food allergens. We aimed to understand the role of allergen- specific IgG in food allergy and natural tolerance. Methods: IgG and IgG isotypes specific to peanut, cow's milk and egg were measured using ImmunoCAP and ELISA respectively in samples of children with suspected food allergies. Expression of IgE and IgG and their receptors and expression of activation markers following allergen stimulation were measured on basophils and mast cells by flow cytometry, with and without blockade of Fc γ RII α or Fc γ RII β receptors. Results: The levels of peanut- specific IgG, IgG1, IgG2, IgG3 and IgG4 in ELISA were higher in peanut- allergic than in non– peanut- allergic children. No difference in allergen- specific IgG isotypes was observed between allergic and non- allergic children to milk or egg, except for milk- specific IgG4 that was higher in non– cow's milk-allergic than in cow's milk- allergic children. Basophils and LAD2 cells expressed IgG receptors, but IgG and IgA were not detected on the surface of either cell type and blocking Fc γ RII α or Fc γ RII β did not modify basophil or


| INTRODUC TI ON
Immediate allergic reactions to foods are mediated by allergenspecific IgE (sIgE) bound to FcεRI on the surface of basophils and mast cells. sIgE concentrations in serum can be used with the clinical history to confirm food allergy, but there are many individuals with detectable sIgE who do not react on oral food challenge (OFC). 1 In IgE-sensitized individuals who are tolerant to the food, allergenspecific antibodies of isotypes other than IgE may be involved in the absence of mast cell and basophil reactivity, despite the presence of IgE. One candidate of interest has been IgG4, which is typically produced together with IgE in Th2-type immune responses. Levels of allergen-specific IgG4 have been shown to increase in patients who outgrow food allergies and in patients who undergo allergenspecific immunotherapy (AIT); at the same time, these patients become less sensitive to the allergen during treatment. 2 Previously, we showed that peanut-sIgG4 in the plasma of patients treated with peanut AIT was able to suppress the activation of mast cells and basophils sensitized with plasma from peanut-allergic patients and stimulated with peanut allergens. 3 We confirmed the involvement of IgG4 through the depletion of IgG4 from the plasma of sensitized but tolerant subjects and the consequent reduction in this inhibitory effect. However, this reduction was only partial, despite the complete depletion of IgG4 from the plasma samples, suggesting that other factors may contribute to the suppressed mast cell response to food allergens. We hypothesized that such factors could be allergenspecific IgG antibodies of different subclasses other than IgG4 and that such inhibitory allergen-specific antibodies could suppress allergen-induced mast cell and basophil activation by either competing with IgE for binding to the allergen or by co-cross-linking 4 of receptor-bound IgE and IgG inducing inhibitory signalling inside the effector cells.
In other studies, allergen-specific IgG antibodies have shown to be able to modulate basophil and mast cell activation by acting as blocking antibodies, such as those induced during AIT to respiratory, venom and food allergens and more recently during a trial of monoclonal cat-specific IgGs. 3,[5][6][7] Monoclonal antibodies of different isotypes but the same specificity could equally suppress allergen-induced basophil activation in an in vitro model of grass pollen allergy; 8 thus, allergen-specific antibodies of different isotypes can share blocking activity. In in vitro studies and in mouse models, co-cross-linking of receptor-bound IgE and IgG led to phosphorylation of ITIM motifs and resulting inhibitory intracellular signalling dampened the stimulatory signalling downstream of the IgE receptor. [10][11][12]97 Using samples from a cohort of cat-allergic individuals treated with AIT, antibodies blocking FcγRIIα and FcγRIIβ increased basophil sensitivity to the allergen. 13 FcγRIIβ blocking antibodies also increased effector cell activation upon food challenge in a murine model of food allergy and in human mast cells derived from humanized mice and sensitized with plasma from peanut-allergic patients treated with AIT with and without blocking of FcγRII receptor. 14 To our knowledge, the inhibitory mechanisms of IgG have not been explored in the context of natural tolerance to food. We aimed to study the mechanisms by which IgG antibodies modulate mast cell and basophil responses to food allergens in IgE-sensitized children.

| Study population and procedures
Children with suspected allergy to either peanut, egg, or CM were recruited into one of two diagnostic studies (REC 10/H0802/044 and 17/LO/029). Some patients were assessed for more than one food allergy ( Figure E1). Suspected food allergy was defined as: 1. reported history of an immediate allergic reaction upon exposure to the food; 2. absence of regular consumption of the food or active food avoidance; and/or 3. evidence of IgE sensitization to the food, as measured by skin prick test greater than or equal to 1 mm and/or sIgE greater than or equal to 0.10 KU/L. Individuals stopped anti-histamine treatment prior to evaluation, and treatment with immunomodulators, such as AIT or biologics, was an exclusion criteria. Study participants underwent clinical assessment, SPT, blood collection and OFC, as clinically indicated. For instance, allergen-specific IgE was measured to egg white, cow's milk and peanut extracts for children assessed for egg, cow's milk and peanut allergies respectively: 39 egg allergic, 21 egg non-allergic, 24 cow's milk allergic, 8 non-cow's milk allergic, 44 peanut allergic and 41 non-peanut allergic. Food allergy was defined as a positive OFC in the majority of participants. Patients who did not undergo OFC to foods tested in this study were diagnosed as allergic if they had an unequivocal history of an immediate-type allergic reaction to the food in the past year and showed evidence of sIgE on skin prick test and/or serum sIgE; and as tolerant if they had been consuming an age-appropriate amount of the food regularly in the diet without developing any allergic symptoms. Whole blood was used for basophil activation test (BAT) within 4 h of blood being taken. Plasma was separated from whole blood and stored at −80℃ until use in ELISA or mast cell activation tests (MATs). All participants' parents or carers with parental responsibility provided written consent prior to any study procedures.

Key Messages
Allergen-specific IgG shows distinct patterns in persistent (peanut) versus transient (milk and egg) food allergies, which could be a reflection of the different mechanisms of IgE production and dysregulation. Although allergenspecific IgG can compete with IgE for allergen binding and reduce allergen-induced basophil and mast cell activation and degranulation, we have found no evidence that additional suppression could be mediated by inhibitory IgG receptors on the surface of basophils and mast cells in natural food tolerance.

| B-cell isolation from tonsils and staining for flow cytometry
One adult patient undergoing tonsillectomy was recruited and consented at Guy's hospital for use of the discarded tonsil tissue (REC 08/H0804/94). The tonsil tissue was minced mechanically in a Petri dish and washed in RPMI. Supernatant was filtered to yield a singlecell suspension, and a Ficoll (Gibco) density centrifugation separation was performed to isolate peripheral blood mononuclear cells, which were then incubated with an equal volume of sheep red blood cells (Thermo Fisher) at 4℃ for 30 min to aggregate T cells and NK cells. The supernatant was centrifuged, the interface was removed, and a second Ficoll density centrifugation separation was performed to isolate tonsil B cells. B cells were washed in PBS containing 0.5% BSA and 2 mM EDTA and resuspended to a concentration of 1 × 10 6 cells/ml. B cells were stained at 4ᵒC for 30 min with αCD19-PECy7 (Biolegend), αIgG-APC and/or αIgA-FITC. Flow cytometry data collection and analyses were performed as above.

| Statistical analyses
Statistical analyses were performed using the GraphPad Prism v7.0 (GraphPad Software). Normally distributed data were analysed by the two-tailed t tests or paired t tests. Non-normally distributed data were analysed using the two-tailed Mann-Whitney test, while nonparametric data were analysed using the Wilcoxon signed ranked tests.

| Study population
Seventy-one children were assessed for egg allergy (EA), 72 for cow's milk allergy (CMA) and 79 for peanut allergy (PA). Table 1 represents their clinical and serological features. Overall, their ages ranged between 9 months and 9.75 years (median = 2.58 years) and 68.49% of children were male. Allergen-specific IgE was detectable in all allergic patients and in a subset of non-allergic children (n = 18/21 for egg, n = 8/8 for cow's milk and 30/41 for peanut). Allergen-sIgE concentrations were higher in allergic than in non-allergic patients, but there was an overlap between the groups ( Figure 1A-C).

| Allergen-specific IgG isotypes were elevated
in peanut-allergic patients, and IgG4 was elevated in children with resolved cow's milk allergy No statistically significant difference was seen in the concentration of peanut-specific IgG between peanut-allergic and non-peanutallergic individuals as measured by ImmunoCAP, but there was a trend towards higher levels in PA (p = .056; Figure 1D). Higher levels of peanut-sIgG1 (p < .001), sIgG2 (p < .001), sIgG3 (p = .003) and sIgG4 (p < .001) as measured by ELISA were observed in peanutallergic compared with non-peanut-allergic subjects ( Figure 1E-H).

| Basophils and LAD2 cells differentially express FcγRIIα and FcγRIIβ
Expression of IgE and IgG receptors was assessed by flow cytometry (Table 2). FcεRI was constitutively expressed on LAD2 cells and primary blood basophils (Figure 2A), as expected. FcγRI (CD64) was detected on LAD2 cells ( Figure 2B) but was virtually undetectable on basophils. FcγRII (CD32) was constitutively expressed on both LAD2 and primary basophils ( Figure 2A+B). FcγRIII (CD16) did not seem to be expressed by basophils or LAD2 cells ( Figure 2A+B).
FcγRII has two known subtypes, FcγRIIα and FcγRIIβ, with the latter being implicated in the inhibition of basophil and mast cell activation via ITIM motifs. 14 Efficient binding of FcγRIIα and FcγRIIβ blocking antibodies was confirmed by the loss of detection of pan-CD32 on LAD2 and basophil surfaces ( Figure E4). FcγRIIβ was preferentially expressed on basophils and at greater levels than LAD2 cells (p < .001 Figure 2C+D). Conversely, LAD2 cells expressed FcγRIIα at higher levels than that of FcγRIIβ (p < .001; Figure 2C+D and E5).

| Basophils and sensitized LAD2 cells exhibit IgE, but little IgG or IgA, on their surface
The expression of surface-bound IgE was observed in both basophils ( Figure 3A) and sensitized LAD2 cells ( Figure 3B). The ratio of fluorescence measured for IgE between sensitized and non-sensitized LAD2 cells was not significantly different between peanut-allergic TA B L E 1 Clinical and serological characteristics of the study population. Median and interquartile range shown for age and allergen-specific IgE  and non-peanut-allergic children ( Figure 3C), indicating similar levels of IgE bound to receptors on the surface of the effector cells.
Conversely, IgA and IgG were not detected on the surface of basophils ( Figure 3A) or LAD2 cells ( Figure 3B). IgG and IgA were, however, expressed on the surface of B cells isolated from human tonsils, which was tested in a parallel control experiment ( Figure 3D+E).

| Blocking FcγRIIα or FcγRIIβ does not change basophil or mast cell responses to allergen
To assess the role of FcγRIIα or FcγRIIβ during the basophil response to food allergy, primary blood basophils from patients were stimulated with CM, EW or peanut extract (PE) following the incubation  Figure 1N-P shows the results using the unpaired t tests, in which *p < .05. Allergen-specific IgE and peanut-specific IgG were measured by ImmunoCAP (Thermo FcγRIIβ receptors prior to sensitization did not attenuate LAD2 cell activation in response to peanut allergen neither in cells sensitized with plasma from peanut-allergic patient ( Figure 5A) nor in cells sensitized with plasma from non-peanut-allergic children ( Figure 5B).    IgG for binding to the allergen has previously been demonstrated using the inhibition of mast cell and basophil activation assays, in which effector cells sensitized with plasma from allergic subjects were stimulated with allergen that had previously been incubated with test plasma, and therefore allowing for antibodies present in human serum to compete with allergen prior to exposure to sensitized cells. 3,6 Furthermore, it is possible that the quality of both of the IgG and IgE antibodies themselves plays a role in the differences in effector cell response observed. 30,31 Although the number of patients tested on BAT to peanut was limited and one out of three peanut-allergic patients had basophils who did not respond to allergen, more patients were tested on the MAT to peanut and on the BAT to milk and egg, and overall, the findings were consistent across allergens and cell type.

| DISCUSS ION
The proposed mechanism for intracellular suppression of allergic responses via FcγRIIβ requires cross-linking of FcεRI and FcγRIIβ [32][33][34] and was demonstrated in mouse and in vitro studies with monoclonal antibodies and hybrid protein constructs combining the Fc portion of an IgG with allergen. 10 Investigations should now be directed towards further characterizing the interaction of IgG with allergen, including IgG specificity and avidity for allergen, together with potential IgG-allergen complex interactions with other cells of the immune system, such as IgE+B cells and dendritic cells, to better understand whether allergen-specific IgGs could be effective, not only in passive immunization, but also in actively inducing a tolerogenic response to food allergens.

ACK N OWLED G EM ENTS
The authors wish to thank Professors Gideon Lack and George Du Toit and the Paediatric Allergy team at the Evelina London Children's Hospital for their contribution and continued work to generate samples vital for this study. The authors also wish to thank Dr. David Fear and Thomas Moore for tonsil-derived B cells.

F I G U R E 5
Mast cell activation (measured as proportions of CD63+ LAD2 cells) to peanut allergen after anti-FcγRIIα or anti-FcγRIIβ treatment of LAD2 cells prior to sensitization with plasma from peanut-allergic (A, n = 10) or non-peanut-allergic (B, n = 10) children. Boxplots express range, median and IQR. Wilcoxon signed-rank tests were performed to compare no blocking, alpha blocking and beta blocking under all conditions, but no significant differences were observed

PEER R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1111/pai.13567.