Clinical and immunological evaluation of cat‐allergic asthmatics living with or without a cat

Abstract Background Characterising the clinical and immunological impact of daily cat exposure in cat‐allergic subjects with asthma who live with cats (WC) and those who do not (WoC) may provide understanding of the drivers of the allergic response. Methods Clinical and immunological characteristics (skin prick test, spirometry, symptom assessments, immunological markers) were compared between asthmatic subjects WC (n = 10) and WoC (n = 9). Results WC subjects had greater use of long‐acting beta agonists (p < .05) and high‐potency corticosteroids. No differences were observed in lung function, nasal and ocular symptoms, or asthma control between the groups. Cat dander‐ and Fel d 1‐specific IgG4 concentrations were higher in WC than WoC subjects (both p < .05). Total IgE and cat dander‐, Fel d 1‐ and Fel d 7‐specific IgE concentrations were similar, but Fel d 4‐sIgE was higher in WC subjects (p < .05) versus WoC. Basophil sensitivity to cat dander extract and Fel d 1 was lower in WC versus WoC subjects (p < .05) and correlated with higher IgG4 concentrations (r = 0.63; p = .009). Fel d 1‐specific CD4+ T‐cell responses polarised toward Th2A responses in WC versus WoC subjects; Fel d 1‐specific IgE correlated with surface expression of CRTH2 and CD200R (both p ≤ .05). Conclusion Immunological differences observed in WC versus WoC did not reflect clinical tolerance with natural cat exposure. The ability to live with a cat despite allergy could be driven by higher preventative medication use. This study may support design of novel therapeutics for allergy management.

to be effective with some allergens, it can require treatment for up to 5 years, may be associated with adverse events, and has equivocal clinical efficacy for cat allergy. 6 Natural cat exposure has been implicated in conveying partial clinical and immunological tolerance, potentially mediated by IgG. 7 However, the role of IgG in cat allergy is complex; although some studies showed that accumulation of IgG 4 by chronic activation of the Th2 response may contribute to low level tolerance, 8 other AIT studies suggested that the blocking potency of native IgG may be heterogeneous and not efficient. 9 An explanation could be that both quantity and specificity of IgG may be important factors in attenuating the allergic response, as high affinity allergen-blocking monoclonal IgG antibodies reduced the allergic response in animal models and cat-allergic subjects. 9 Consequently, evaluation of management strategies for cat allergy relies on understanding baseline clinical and immunological drivers of the allergic response. Since such an understanding is critical in the design of studies for evaluating new therapeutic approaches, the objective of this study was to characterise the impact of daily cat exposure by exploring relationships between clinical symptoms and immune mechanisms in cat allergic individuals with asthma who live with cats and those who do not live with cats.

| Study design and subjects
This exploratory, observational study compared clinical and immunological characteristics between asthmatic subjects diagnosed with cat allergy who live with cats (WC) and those who live without cats (WoC) (1:1 ratio). For this study, WC was defined as living with close, regular contact with a cat for at least 1 year, and WoC were subjects who generally avoid cats and have little or no cat exposure. The study consisted of three in-clinic visits (screening, Day 1, and Day 28) and at-home completion of diaries and assessments as described below for the specific outcomes. The study received approval from the Benaroya Research Institute (BRI) Institutional Review Board (IRB07109-519), and all subjects provided written informed consent and Health Insurance Portability and Accountability Act authorisation prior to participation.
Subjects were identified from the BRI General Allergy Registry Biorepository and Virginia Mason allergy clinic, and were recruited by IRB approved in-clinic flyers, phone and email requests. For inclusion, subjects were required to be adults 18-65 years old, inclusive, with a history of cat-triggered allergic rhinitis and asthma for

G R A P H I C A L A B S T R A C T
Exploratory study characterizing the impact of daily cat exposure in cat-allergic subjects living with/without cats (WC/WoC). Cat danderand Fel d 1-specific IgG4 concentrations were higher in WC versus WoC subjects (p < .05); lower basophil sensitivity to cat dander extract/ Fel d 1 in WC versus WoC subjects (p < .05) correlated with higher IgG4 concentrations (r = 0.63; p = .009). Immunological differences not reflected in clinical tolerance with cat exposure. Fel d 1, major cat allergen; Ig, immunoglobulin; WC, with cat; WoC, without cat.

KEYMESSAGES
• Cat-allergic asthmatics who live with cats have significantly higher medication requirements to control symptoms compared with cat-allergic asthmatics who do not live with cats.
• Basophils are less sensitive to Fel d 1 stimulation in those who live with cat, which may be due to higher serum concentrations of allergen-specific IgG4.
• Fel d 1-specific Th2A responses are higher in those who live with cats relative to those who do not, and correlate with Fel d 1 sIgE levels.
at least 2 years that had been diagnosed by an allergist and subsequently confirmed by the principal investigator. Subjects were also required to demonstrate immunological responses that included a skin prick test (SPT) mean wheal diameter ≥5 mm than negative control to standardised cat hair extract, and IgE ≥0.35 kU/L to cat dander. Additional inclusion criteria were the ability to stop antihistamine use for 5 days prior to each visit for SPT or basophil activation test (BAT), and possession of a vacuum that could accommodate an allergen collection adapter. Exclusion criteria were a history of cat AIT or any AIT within 6 months; concomitant dog allergy among subjects currently living with dog(s); significant mechanical nasal obstruction, or history of nasal or sinus surgery; a history of smoking within 6 months prior to screening; and use of systemic corticosteroids within 4 weeks prior to visit 2 (Day 1). While use of controller medications (leukotriene modifiers, long-acting bronchodilators, and daily inhaled corticosteroids up to 1000 mcg Fluticasone equivalent) was allowed, doses >1000 mcg Fluticasone equivalent of inhaled corticosteroids or regular oral corticosteroid was cause for exclusion, as was use of biological agents (e.g., omalizumab, dupilumab, mepolizumab) within 6 months prior to screening.

| Clinical measures
To determine allergen exposure, samples from each subject's mattress/bedroom were collected on Days 1 and 28 using the Indoor  the CSMS had a maximum score of 24 with higher scores indicative of a greater symptom burden.

| Immunological and biomarker assessments
Imunological and biomarker assessments were conducted during the study visits on Days 1 and 28, and included SPT; serum sIgE, sIgG 4 , and total IgE assessments; blood eosinophil count, BAT; and cat allergen-specific T-cell analysis.
The SPT (Jubilant HollisterStier Allergy) was conducted via a standard protocol 15 using allergen extracts and controls reported in the supplementary methods of the Online Respository.

| Statistical analysis
No power calculations were considered to determine sample size.
Furthermore, because of the variety of assessments, the enrolment goal was 10 WC subjects and 10 WoC subjects.
Between-group comparisons were conducted using t-tests or

| Population and allergen exposure
The analysis included 10 asthmatic WC individuals and 9 WoC subjects; 1 WoC subject was excluded because of a prior history of cat AIT discovered after study completion. All subjects had a history of cat allergy-induced allergic rhinitis and asthma symptoms of at least 5 years. Demographics were similar between the two groups except for dog ownership, which was reported by 4 WoC subjects and one WC subject (see Table S1). Of the WC subjects, 5 had 1 cat, 4 had 2 cats, and 1 had 10 cats, and while one subject had been living with a cat for 1.5 years, all other WC subjects had been living with cat(s) for ≥3 years (Table S2). All WC subjects reported that the cats were indoors >20 h per day.
Home cat allergen exposure, expressed as µg/g of dust collected, was significantly higher among WC subjects relative to WoC, as indi-  (Table S3), likely reflecting the dog ownership reported by 4 of these subjects. The value of indoor allergen assessment was limited, however, by high variability between samples from the two time points.

| Medication burden
The WC subjects had greater preventative medication requirements, including significantly higher long acting beta agonist use (p < .05) and generally used more potent corticosteroids at higher doses. The higher medication burden was further manifested by significantly higher mean scores on the DMS (4.0 ± 0.7 vs. 2.5 ± 1.8) and CSMS (7.8 ± 2.2 vs. 5.0) ± 2.3 (both p < .05). Asthma severity classification was higher among WC due to the increased requirement for preventative medications (Table S4). No significant differences were observed between the two groups in lung function including FEV1, FEV1% predicted, and in-clinic PNIF ( Figure 1A and Table S4). Similarly, there were no differences for nasal and ocular symptoms scores (TNSS, TOSS), or ACQ between the two groups.
These similarities between the two groups, especially lung function, may be partly accounted for by the higher medication burden among WC subjects, since the medications likely ameliorated clinical symptoms. However, WC subjects reported higher scores on the RQLQ (mean 2.1 ± 0.9 vs 1.0 ± 0.8; p < .05) with a difference in score that exceeded the minimal clinically important difference indicating that this difference is clinically meaningful.

| Lung function
Assessment of lung function showed high intra-subject consistency between the at-home and in-clinic assessments, regardless of whether the subject was living with a cat, although the at-home mean values were slightly lower, and WC subjects had higher variability ( Figure 1A). At-home spirometry resulted in generally similar values at the morning and evening assessments ( Figure 1B) and minimal daily variability over the 28 days (data not shown). However, WC subjects manifested greater variability in both morning and evening assessments and mean values that were slightly lower than WoC ( Figure 1B). The trend to lower overall average FEV1% predicted measured over the 28 days study period compared to in-clinic measurements reflects the generally lower FEV1 morning values.

| IgG 4 levels
In WC subjects, serum IgG 4 concentrations toward cat dander and Fel d 1 were ~3-and 4.5-fold higher, respectively, than concentrations in WoC subjects (both p < .05; Figure 2 and Table S5); measurements were consistent at the two evaluated time points. Fel d 4-and Fel d 7-sIgG 4 levels were not significantly different between the two groups, likely due to smaller sample size and larger variability of the results. Neither were there significant differences between the two groups for total serum IgE levels, or cat dander-, Fel d 1-or Fel d 7-sIgE. However, Fel d 4-sIgE was significantly higher in WC subjects (p < .05). In addition, WC subjects had significantly higher Fel d 1 sIgG 4 /sIgE ratio (ng/ml) compared with WoC (p < .001), although there was no significant difference between the 2 groups for cat dander sIgG 4 /sIgE ratio ( Figure 2 and Table S5). Individual subject data for serum immunoglobulin concentrations are presented in Tables S6 and S7. It is worth noting that allergen-specific IgE and IgG 4 measurements on Days 1 and 28 were highly reproducible ( Figure S1A). While SPT results were comparable at the two time points (Figure S1B), they were not as reproducible as the serum sIgE measurements; there was no significant difference between the two groups in SPT for standardised cat hair extract (data not shown). Subjects had variable sensitivities to other allergens, but the two groups were not statistically different except for dog allergen (p = .02), since there were more dog-allergic WC than WoC subjects (data not shown).

| Basophil sensitivity to Fel d 1
To further evaluate the impact of daily cat exposure, basophil activation was evaluated ex vivo in the presence of cat major allergens (Tables S6 and S7 Interestingly, CD63 and CD203c expression correlated, suggesting that a certain level of activation (i.e., CD203c) is needed before degranulation (i.e., CD63; Figure 3B). As shown in Figure 3C, WC subjects had significantly decreased basophil sensitivity (higher EC 50 ) to cat dander extract and to Fel d 1 compared to WoC subjects,   Time  Figure S2). Reduced sensitivity in subjects who lived with cats correlated with accumulation of specific IgG 4 , which has the capacity to attenuate IgE mediated allergic symptoms ( Figure 3D).
Importantly, longitudinal consistency was observed for CD63 and EC 50 values ( Figure S3), indicating robustness of the findings.

| T-cell reactivity to Fel d 1 and Fel d 4
CD154/CD137 up-regulation assay was used to assess Fel d 1 and Fel d 4-reactive CD4+ T effector (cTeff) and T-regulatory (cTreg) cell responses. WoC subjects tended to have higher frequencies of cTeff and cTreg responses to Fel d 1 and Fel d 4, but the differences between groups were not statistically significant ( Figure 4A and Figure S4).  Figure 4D).

No correlation was observed between basophil reactivity and T-cell
responses against individual cat allergens (data not shown).

| Cytokine profiles
To  Figure S5). Interestingly, co-staining for cytokines in Fel d 4 tetramerpositive cells showed that IL-4-producing cells were mutually exclusive from IFNγ-producing CD4 + T cells ( Figure S5).   with AIT or during passive administration of monoclonal antibodies also increase the IgG/IgE ratio, which has been reported to correlate with clinical symptom improvement. 9,25 Consistent with these studies, we observed that living with a cat was associated with increased

| CON CLUS IONS
Cat allergen exposure modulates the relationship between primary effector basophils and allergen-specific blocking IgG 4 , with Fel d 1 appearing to be the primary allergen. Even though we observed immunological changes in WC subjects relative to WoC subjects, the clinical results do not support tolerance with natural cat exposure.
Despite the small sample size, which is the primary limitation of this study, the results showed low intra-subject variability and remarkable robustness in longitudinal measurement of clinical assessments and immunological assays. We also observed immunological differences that could distinguish between the two groups with statistical significance. These immunological differences suggest the importance of designing studies for evaluation of allergy management strategies that specifically reflect the population of interest and their baseline clinical/immunological characteristics. The data also imply a need to stratify the cat allergic population based on living with a cat. Finally, the clinical and immunological markers that we evaluated in this study should be further explored as surrogate measure of therapeutic effects in future clinical trials, and also as biomarkers for mechanistic studies of the allergic response.

ACK N OWLED G EM ENTS
The

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.