2.1 Study design and study population

This prospective, single center study took place at the Department of Dermatology and Allergy of the University Hospital Bonn from 2019 until 2021. Patients were included according to the following inclusion criteria: age ≥18 and active diagnosis of AD and comorbid ARC.

AD was diagnosed based on the criteria of Hanifin and Rajka⁴⁵ and on the UK working party diagnostic criteria.^{46, 47} ARC was diagnosed based on the patient symptom history, the Rhinitis Control Assessment Test (RCAT ≤ 17), and sIgE (≥ 5 kU/L). Exclusion criteria were comorbidities such as malignomas, immunodeficiency, severe chronic diseases and pregnancy. All donors provided their written informed consent before the beginning of the study. The study followed the ethical principles of the Declaration of Helsinki and was approved by the local Ethics Committee (approval number: 267/19).

Initially, 44 patients were enrolled. Five patients discontinued the study protocol for personal reasons. In seven patients, the clinical parameters of the ARC symptoms based on the RCAT did not match with the sIgE levels of their birch pollen and grass pollen senitization, so we were not able to determine with certainty if the ARC symptoms were related to the seasonal allergens of birch pollen (rBet v1; Betula verrucosa, allergen 1, recombinant) or timothy grass pollen (rPhl p1 or p5; Phleum pratense, allergen 1 or 5, recombinant).

Two observation groups were formed: the index group and the control group. The index group included 21 patients (13 females and 8 males; age range: 18–69 years; median age: 29 years) with AD and comorbid ARC that received an anti-IL-4Rα antibody therapy (demographic data presented in Table 1). All patients treated with the monoclonal anti-IL-4Rα antibody were grouped according to their exposure to rBet v1, rPhl p1/rPhl p5, as well as according to their ARC symptoms (based on the RCAT). The control group consisted of 11 patients (seven females and four males; age range: 24−59 years; median age: 31 years) with AD and comorbid ARC that were treated with daily sublingual AIT (demographic data presented in Table 1). Patients treated with AIT were grouped according to their sIgE levels, their ARC symptoms and their allergen-specific treatment for birch and grass pollen.

To classify the observable effects of the monoclonal anti-IL-4Rα antibody on allergen-specific immune responses, we selected AD patients with AIT-treated ARC as a control group, since the effects of AIT on the IgE levels and the effector cells have already been examined in previous studies.^40-44

The observational time was 16 weeks, based on data from a registration study of a monoclonal anti-IL-4Rα antibody,¹⁸ in which the clinical parameters associated with skin lesions and the quality of life were shown to improve significantly during this period. Moreover, significant changes could be indicated more easily at the beginning of the treatment without disruptive factors or external triggers.

The observational period was assessed at three timepoints: (i) before the start of any systemic treatment (w0), (ii) at 4 weeks (w4) after the start of a systemic treatment and (iii) at 16 weeks (w16) after the start of a systemic treatment. All patients filled in a questionnaire based on the Dermatology Life Quality Index (DLQI), the RCAT and the Eczema Area and Severity Index (EASI); the answers to this questionnaire were evaluated to assess the efficacy of the treatment, to ensure the correct handling and to detect nonresponders. The DLQI was used so as to estimate the effects of skin diseases on daily life routine and their impact on the quality of life.^{48, 49} Additionally, questions based on the RCAT were used to objectify the ARC symptoms, while the EASI was raised so as to classify the expression and severity of AD.⁴⁸

Blood samples were obtained (by using Serum Monovette; 7.5 mL; Sarstedt AG & Co KG) from patients to determine the total IgE serum levels through an electrochemiluminescence immunoassay (by using Cobas® e801; Roche Diagnostics)⁵⁰ and the sIgE serum levels for birch (rBet v1) and grass (rPhl p1, rPhl p5) pollen through a fluoroenzyme immunoassay (by Phadia 250; Thermo Fisher Scientific, Phadia AB),⁵¹ the activation level of basophils⁵² and the T lymphocyte proliferation. Carboxyfluorescein-diacetate-succinimidyl-ester (CFSE) T cell allergen proliferation assays were evaluated in 11 donors treated with a monoclonal anti-IL-4Rα antibody and in 10 patients treated with AIT. Proliferation assays were performed in individuals sensitized to rBet v1 or rPhl p1/rPhl p5.

2.2 BAT

EDTA-blood (collected in a 9-mL tube using S-Monovette; Sarstedt AG & Co KG) was used to verify the activation of basophil cells via the detection of surface markers (CD203c and CD63) through the undertaking of BAT according to the standardized instruction manual (Bühlmann Laboratories AG).⁵² Data were acquired by using a flow cytometer emitting at 488 nm (FACSCanto; Becton Dickinson GmbH). An example of the gating strategy and the allergen-specific basophil activation is visualized in Figure 1A-C, as generated by FlowJo version 10.8.0.⁵³

In an attempt to ensure the high quality of the undertaken BAT, the three typical leukocyte populations (i.e., lymphocytes, monocytes and granulocytes) had to be detected in the blood samples before gating as a criterion of blood samples' quality. The absolute number of basophils ranged between 200 and 600 and the percentage of basophils in the negative control was determined as <5%.⁵² BAT assays were performed within the first 24 h after the blood collection, according to the standardized instruction manual (Bühlmann Laboratories AG).⁵² If the blood samples had to be stored, they were kept protected from light and refrigerated at 2°C–8°C.

2.3 Isolation of peripheral blood mononuclear cells (PBMCs)

Peripheral blood was collected into sodium heparin tubes (9-mL Heparin S-Monovette; Starstedt AG & Co KG) and PBMCs were isolated with the use of a Lymphoprep™ (Sigma-Aldrich) density gradient after centrifugation at 2100 rpm, for 30 min, at room temperature (25°C). After centrifugation, the mononuclear cell layer was collected, washed twice and counted by using an automated cell counter (NucleoCounter NC-200; ChemoMetec).

2.4 Allergens

The undertaken BAT and antigen-specific T cell proliferation assay were induced by freeze-dried allergens obtained by Bühlmann Laboratories AG (Schönenbuch, Switzerland). Each allergen was prepared as an aqueous extract in 250 μL of stimulation buffer, until the extract was dissolved. The allergens for BAT were used at different dilutions to rule out any dose-dependent effects (1 = 100 ng/mL; 1:1 = 50 ng/mL, 1:10 = 10 ng/mL, and 1:100 = 1 ng/mL).

2.5 CFSE labeling

CFSE mixed isomers (Vybrant CFDA-SE Cell Tracer Kit; Invitrogen) were used for the CFSE labeling. In brief, CFSE was stored frozen as a 10-mM stock solution until used. A pellet of 1 × 10⁶ cells was resuspended in 1 mL with 1% fetal calf serum (FCS)–phosphate buffered saline (PBS) solution and a CFSE labeling solution of a concentration ranging from 0.5 to 10 μM to 37 nM and were incubated for 8 min, at room temperature, in the dark. After incubation, 1 mL of FCS per 2 × 10⁶ cells was added to each sample, followed by a 10min incubation. Cells were then washed twice with a 2% FCS-PBS solution and were centrifuged at 1350 rpm for 10 min, before resuspension in RPMI.⁵⁴ PBMCs stained with CFSE were analysed before and after 7 days of cell culturing, through flow cytometry (FACSCanto; Becton Dickinson GmbH). Analysis was performed by using the BD FACSDiva software version 5.0.3 and the FlowJo version 10.8.0 for Mac OS X software.^{53, 55}

2.6 Cell viability determination

After the CFSE labeling, cells were stained with 7-amino actinomycin D (7-AAD; 0.2 μg/10⁶ cells) (Sigma-Aldrich) and were incubated for 5 min before their analysis through flow cytometry.⁵⁴ 7-AAD-positive cells were considered as dead and discarded for T cell proliferation analysis.

2.7 Cell culture

PBMCs were cultured in 96-well flat-bottomed plates (Corning Inc.) at a density of 0.2 × 10⁶ cells/well, in RPMI-1640 medium supplemented with 1 mM sodium pyruvate, 2 mM l-glutamine, 50 μg/mL gentamicin and 10% heat-inactivated FCS. Cells were incubated at 37°C, in a 5% CO₂ humidified chamber. Antigen-specific stimulation was performed by adding the corresponding allergen in diluted concentrations (1 = 100 ng/mL, 1:1 = 50 ng/mL, 1:10 = 10 ng/mL, and 1:100 = 1 ng/mL). Likewise, the cells were co-stimulated at day 0 in the presence or absence of MACSiBead™ (4 × 10⁸ particles) being conjugated to the monoclonal anti-biotin human antibodies against CD2 (CD2-biotin; 100 μg/mL), CD3 (CD3-biotin; 100 μg/mL) and CD28 (CD28-biotin; 100 μg/mL) (T Cell Activation/Expansion Kit, human; Miltenyi Biotec). Five experimental conditions were analysed to determine the T cell proliferation index after the in vitro allergen stimulation in both groups of donors. CFSE-labeled cells were cultured for 7 days with the corresponding allergen and were co-stimulated with MACSiBeads™ (at a ratio of 1:10 per 1 × 10⁶ cells) and with of recombinant human IL-2 (PeproTech; at a ratio of 50−100 UI/mL per 1 × 10⁶ cells, every 2 days). Moreover, CFSE-labeled PBMCs were cultured for 10 days with human recombinant IL-2 (50−100 UI/mL, every 48 h) and the related allergen dilutions. Additionally, cells were cultured only with monoclonal anti-biotin human antibodies against CD2, CD3, and CD28 (100 μg/mL) and with IL-2 (50−100 UI/mL per 1 × 10⁶ cells, every 2 days) and they were harvested after 7 and 10 days of culturing to serve as cell stimulation positive controls. Furthermore, cells were cultured only with IL-2 (50–100) and without any costimulation (RPMI). The fluorescence of the stimulated CD4+ CFSE-diluted cells was measured after 7 and 10 days of cell culturing, through flow cytometry.

2.8 T cell proliferation assay

T lymphocyte proliferation was monitored by establishing a dose-response curve with the culprit allergen. Therefore, a dose-response curve using four allergen concentrations (1 = 100 ng/mL; 1:1 = 50 ng/mL, 1:10 = 10 ng/mL, and 1:100 = 1 ng/mL) was established after 7 days of cell culturing. T cell proliferation was evaluated through the undertaking of flow cytometry and the obtained data were analysed with the use of FlowJo version 10.8.0.⁵³ Representative histograms of the allergen-specific T cell proliferation are presented in Figure 2A−D and Figure S1a−d.

T cell proliferation assays were analysed through the calculation of the average number of divisions that all responding cells have undergone since the initiation of the culturing process (proliferation index). Similarly, the cell growth was evaluated by calculating the number of divisions for all cells in the original culture (division index) versus the number of undivided cells after 7 days of allergen stimulation.⁵⁶

2.9 Statistics

All collected data were analysed with one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test by using GraphPad Prism version 9.1.2 for MacOS. Differences were considered as statistically significant when the test yielded p values that were <.05.

For the analysis of the index group (treated with the anti-IL-4Rα antibody; n = 21), patients with ARC symptoms exhibiting ≤17 score points (based on the RCAT) and/or sIgE serum levels ≥5 kU/L were included. For the analysis of the control group (receiving an AIT; n = 11), seven patients receiving a birch pollen-specific AIT and four patients receiving a grass pollen-specific AIT were analysed. For patients that dropped out or for those whose diagnosis of seasonal-related ARC was not ensured, their data were excluded. Finally, 32 patients were included into the statistical analysis.

For the BAT analysis the calculation of the CD-sens was based on a dose-response curve adapted from Hoffmann et al.³⁹ and Santos et al.⁵⁷ The data were analysed with one-way ANOVA followed by a Friedman test.

The results of the proliferative capacity of responding T cells are shown in units deriving from the division index parameter as the total number of divisions per number of cells at the beginning of the culturing process.

The figures were generated through GraphPad Prism version 9.1.2. and FlowJo version 10.8.0.⁵³

3.1 Decrease in the total IgE and sIgE serum levels was observed in patients treated with an anti-IL-4Rα antibody, but not in patients receiving an AIT

The levels of the total IgE decreased significantly after 4 and 16 weeks in patients treated with an anti-IL-4Rα antibody (w0–w4: p < .0001; w0–w16: p < .0001; w4–w16: p < .01; Table 2A and Figure 3A). Additionally, the serum levels of sIgE decreased significantly after 4 and 16 weeks under an anti-IL-4Rα antibody treatment (rBet v1: w0–w4, p < .05; w0–w16, p < .001; w4–w16, p < .001; rPhl p1: w0–w4, p < .05; w0–w16, p < .0001; w4–w16, p < .0001; rPhl p5: w4–w16, p < .01; Table 2A and Figure 3B−D). In contrast, the levels of total IgE and sIgE did not change significantly until week 16 in patients receiving an AIT (Table 2H and Figure 3E−G).

3.2 Allergen-specific basophil activation and sensitivity increased in patients treated with a monoclonal anti-IL-4Rα antibody, but decreased in patients under birch pollen-specific AIT

To investigate the effects of a monoclonal anti-IL-4Rα antibody and of an AIT on early effector cells, we analysed the in vitro activation response on CD203c + /CD63 + basophils at four different allergen concentrations (1 = 100 ng/mL, 1:1 = 50 ng/mL, 1:10 = 10 ng/mL, and 1:100 = 1 ng/mL). In an attempt to detect dose-dependent effects, four different allergen dilutions were used for the induction of allergen stimulation. We calculated the basophil sensitivity (CD-sens = 1/EC50 × 100) based on a dose-response curve adapted from Hoffmann et al.³⁹ and Santos et al.⁵⁷

In patients treated with the monoclonal anti-IL-4Rα antibody and sensitized toward rBet v1, the in vitro basophil activation in response to the birch pollen allergen increased significantly depending on the allergen concentration.

At allergen dilution 1 (100 ng/mL) no significant changes were observed after 16 weeks of an anti-IL-4Rα antibody treatment (Table 2B and Figure 4A). In contrast, at the 1:1 (50 ng/mL; w0−w16, p < .05) and the 1:10 (10 ng/mL; w0−w16, p < .001) allergen dilutions, a significant increase of the basophil reactivity was found (Table 2B and Figure 4B,C). At the 1:100 (1 ng/mL) allergen dilution no statistical differences were detected (Table 2B and Figure 4D).

In patients with grass senitization (rPhl p1 and rPhl p5) that received an anti-IL-4Rα treatment, an increase of the basophil activation to the grass pollen allergen was observable within 16 weeks depending on the allergen concentration. At the 1 (100 ng/mL) and the 1:1 (50 ng/mL) allergen dilutions, no significant changes were observed (Table 2C and Figure 4E,F). In contrast, at the 1:10 (10 ng/mL; w0−w16, p < .01) and the 1:100 (1 ng/mL; w0−w16, p < .01) allergen dilutions, a significant increase of the basophil activation was detected (Table 2C and Figure 4G,H).

On the other hand, the basophil activation decreased in patients treated with an anti-IL-4Rα antibody after a basophil stimulation with fMLP (positive control; w0−w16: p < .05; Table 2D and Figure 4I), but increased after the allergen stimulation. Additionally, basophils displayed an increasing tendency of activation after a stimulation by FcεRI (positive control), but the observed effect was not determined as statistically significant (Table 2D and Figure 4J).

Since a significant increase of the basophil activation after the allergen stimulation could be observed, we hypothesized that through an IL-4Rα blockade induced by a monoclonal anti-IL-4Rα antibody, the activation of basophils were modified depending of the activating stimulus.

Additionally, we calculated the CD-sens based on a dose-response curve. In patients receiving the monoclonal anti-IL-4Rα antibody and being sensitized toward birch pollen, the EC50 value decreased (w0−w16, p < .05; Table 2B) and, correspondingly, the CD-sens increased significantly after 16 weeks of treatment (w0−w16, p < .05; Table 2B and Figure 4K). A significant decrease of the EC50 from week 4 onward (w0−w16, p < .001; w4−w16, p < .01; Table 2C) and, correspondingly, a significant increase of the CD-sens was identified in patients with grass pollen senitization that were treated with a monoclonal anti-IL-4Rα antibody (w0−w16, p < .001; w4−w16, p < .01; Table 2C and Figure 4L).

In patients receiving an AIT, a significant decrease of the basophil activation after stimulation with the birch pollen allergen was observed at week 16 at the allergen dilution 1 (100 ng/mL; w0−w16, p < .05; Table 2I and Figure 5A).

At the 1:1 (50 ng/mL), 1:10 (10 ng/mL) and 1:100 (1 ng/mL) allergen dilutions, no significant effects on the basophil activation were identified (Table 2I and Figure 5B−D).

In patients receiving an AIT and being sensitized to the grass pollen allergen, no significant differences in basophil activation were observed within 16 weeks (Table 2J and Figure 5E−H).

On the other hand, the basophil activation did not change significantly in patients receiving an AIT after a basophil stimulation with fMLP and FcεRI (Table 2K and Figure 5I,J). Due to the fact that the basophil activation did not change significantly in response to the fMLP- and the IgE-mediated stimulation, but decreased after the birch pollen allergen stimulation, the suppressive effect of AIT was considered to be allergen-specific.

In AIT-patients with birch and grass pollen senitization, a decreasing tendency of the CD-sens was observable, but due to the small number of patients recruited, the effect was not statistically significant (Table 2I,J and Figure 5K,L).

Overall, an IL-4Rα blockade by a monoclonal anti-IL-4Rα antibody significantly increased the basophil reactivity and the basophil sensitivity depending on the activating stimulus, whereas the AIT decreased the basophil responsiveness.

3.3 T cell proliferation in response to seasonal allergens was found decreased in patients treated with an anti-IL-4Rα antibody or with an AIT

To investigate the effect of the IL-4Rα blockade through the administration of a monoclonal anti-IL-4Rα antibody versus that of an AIT on specific T cells deriving from AD patients with comorbid ARC, we analysed the proliferative response in parallel to the basophil activation and the sIgE levels.

The proliferation in response to the birch-induced stimulation decreased significantly in CFSE-treated CD4+ cells deriving from PBMCs of patients treated with a monoclonal anti-IL-4Rα antibody or with AIT, depending on the allergen concentration.

In patients treated with a monoclonal anti-IL-4Rα antibody, no significant differences in T cell proliferation were found after a birch-induced stimulation at the 1 (100 ng/mL) and the 1:1 (50 ng/mL) allergen dilutions (Table 2E and Figure 6A,B), but this was not the case for the 1:10 (10 ng/mL; w0−w16, p < .05) and the 1:100 (1 ng/mL; w0−w16, p < .01) allergen dilutions after 16 weeks (Table 2E and Figure 6C,D).

In contrast, the T cells of AIT-treated patients did not exhibit any differences in terms of their proliferation after a birch-induced stimulation from week 4 onward (Table 2K and Figure 6E−H).

As far as the grass pollen allergen is concerned, T cells did not show any significant diminished division at allergen dilution 1 (100 ng/mL) within 16 weeks after an anti-IL-4Rα antibody treatment (Table 2F and Figure 6I), but this was not the case at the 1:1 (50 ng/mL; w0−w16, p < .05), 1:10 (10 ng/mL; w0−w16, p < .01) and 1:100 (1 ng/mL; w0−w16, p < .05) allergen dilutions (Table 2F and Figure 6J−L).

In AIT-treated patients the CFSE-treated CD4+ cells decreased significantly from week 4 onward after a grass pollen allergen stimulation. A significant decrease in T cell proliferation was observed at the 1:1 (50 ng/mL) dilution (w4−w16, p < .01; Table 2M and Figure 6M,N). No significant changes in T cell proliferation were found at the 1 (100 ng/mL), the 1:10 (10 ng/mL) and the 1:100 (1 ng/mL) allergen dilutions tested (Table 2M and Figure 6O,P).

No statistical differences were observed with regard to the T cell proliferative indexes response after a birch- and a grass-induced stimulation with human recombinant IL-2 (Figure S2) and in cell stimulation positive controls with monoclonal anti-biotin human antibodies against CD2, CD3 and CD28, recombinant human IL-2 or RPMI. In summary, the anti-IL-4Rα antibody treatment, as well as the AIT decreased the allergen-specific T cell proliferation.

3.4 ARC symptoms

In patients treated with an anti-IL-4Rα antibody, the severity of ARC symptoms decreased significantly within 4 weeks, whereas no significant changes were observed in patients under an AIT.

In addition to the measurement of total IgE- and sIgE levels, we examined the severity of ARC symptoms based on the RCAT. In patients receiving an anti-IL-4Rα antibody, the ARC symptoms improved significantly (w4) within 4 weeks (Table 2G). In contrast, in patients receiving an AIT, no significant changes of the allergic symptoms were observed within the observational time of 16 weeks (Table 2N).

4.1 Strengths and limitations

Our study is the first attempting to analyse the impact of a monoclonal anti-IL-4Rα antibody on basophil activation and allergen-specific T cell proliferation in vitro, in patients with AD and comorbid ARC. We have, herein, selected the patients according to their sIgE levels (being ≥5 kU/L) and/or their ARC symptoms (based on the RCAT being ≤17) to assure a symptomatic comorbid ARC. We have also monitored the development of the parameters for 16 weeks, based on data from the registration studies of the monoclonal anti-IL-4Rα antibody.¹⁸ Moreover, significant changes could be indicated more easily at the beginning of the treatment, without the interference of disruptive factors or external triggers. We have chosen AD patients with AIT-treated ARC as a control group, because the effects of AIT on IgE levels and the basophil activation have been described in several studies and are, therefore, well known.^{40, 43-45} Furthermore, our patients filled in a questionnaire based on the DLQI as well as the RCAT and the EASI; this questionnaire enabled us to evaluate the efficacy of the monoclonal anti-IL-4Rα antibody and to ensure the correct handling, the detection of nonresponders and the minimizing of bias.

A limitational factor in our study was the fact that most of the patients were polysensitised and, therefore, the effects of external allergen triggers could not be excluded. Additionally, the observational time was only 16 weeks, but the treatment with a monoclonal anti-IL-4Rα antibody lasts longer in patients with AD, while the administration of an AIT should be conducted for a total of 3 years. Furthermore, the study took place during the COVID-19 pandemic and the recruitment of the patients had to be halted; as a result, only a small number of patients receiving an AIT were finally included in our study.