For respiratory allergies, allergen immunotherapy (AIT) is the only etiological treatment option which can provide long-term relief of symptoms even after treatment cessation (1). The production of allergen-specific IgG4 response is regarded as a ‘protective’ factor of AIT (2, 3). During AIT, B-cells show a shift from producing the allergenic IgE to IgG4. In terms of allergen binding to effector cells (including mast cells and basophils), IgG4 competes with IgE to prevent these cells from activation and subsequent degranulation (4). Despite the potentially protective effect of IgG4, many studies failed to demonstrate the correlation of IgG4 and clinical response to AIT (5, 6).

Component-resolved diagnosis (CRD)is a method recently developed to detect IgE antibodies with the allergen components. CRD makes it possible to obtain the individual sensitization profile to a certain allergen (7, 8). It also enables us to observe the longitudinal evolution of complex sIgE and sIgG4 components’ profiles. A growing spectrum of molecules, representing single allergens of clinical relevance, have been identified and produced for commercial in vitro assays (9). Take house dust mite (HDM) a perennial allergen for example, it is known as a major risk factor for allergic diseases, including asthma, allergic rhinitis (AR), and atopic dermatitis (10). So far, 82 HDM allergen components have been identified (11). The sensitization profile of HDM components may correlate with disease severity and outcome (10). For example, in our previous study, we found that sensitization to both Derp1 and Derp2 may be a risk factor for developing asthma in HDM-AR patients (12).

The allergen sensitization profile may also have an impact on AIT. The quality of HDM allergen extracts used for AIT is usually standardized by the major components such as Der p 1 and Der p 2 but not all components (13). It is possible that the allergen components in HDM extracts are not well-matched with the HDM sensitization profile of an individual, which may lead to attenuated response to AIT. It has been estimated that non-responder may comprise up to 30% of patients treated with AIT (14). We hypothesize the HDM component sIgE and sIgG4 profile plays an important role in AIT. However, the data of longitudinal evolution of sIgE and sIgG4 against HDM components during AIT is limited. Some studies provided cross-sectional IgG4 data rather than longitudinal profiles. Therefore, the aim of this study was to investigate the profiles of sIgEs and sIgG4s generated against HDM components over 18 months of AIT and to assess if they could be potential biomarkers to evaluate the AIT responses.

In this study, we investigated the profile of sIgE and sIgG4 against HDM components at different time-points of AIT under real-life conditions and their correlation with AIT responses. Here we presented the data of AIT with two commercially standardized HDM extracts. We found that the levels of sIgE saw an early increase at up-dosing period and a late decrease at maintenance period while the levels of sIgG4 showed a continuous increase during 18 months of AIT. We also found the numbers of positive sIgE and sIgG4 to HDM components increased after AIT. However, the levels of sIgE and sIgG4 against HDM component didn’t show correlations with AIT responses, which implies the IgG4 against allergen components may not qualify as a biomarker to evaluate the response of AIT.

In line with other studies, we found the majority of the HDM-induced AR patients were sensitized to the HDM major components Der f 1, Der p 1, Der p 2 and Der f2. Meanwhile, less than 33.3% of these patients were sensitized to Der p 7, Der p 10, Der p 21, which was in accord with our expectation as these 3 components had been reported as minor HDM allergens in many studies. Der p 23 is a recently identified HDM major allergen which was supposed to cause asthmatic disease with relatively high sensitization rates (ranging from 70% to 87%) in European countries (15). However, we found only 40% of the patients were sensitized to Der p 23 in our study. Interestingly, the sensitization rate of Derp23 was also relatively low in another small-size Chinese population (16).The difference of Der p 23 sensitization profile may be attributed to the HDM species in different countries, but it also reflected the different immune responses among an array of ethnic groups.

Apart from the different immune responses that may be ethnicity-related, our study of the longitudinal profiles of IgE against HDM allergens Der p 1, Der f 1, Der p 2, Der f 2, Der p 21, and Der p 23 also showed a similar trend of increase in the up-dosing period and decrease in the maintenance period of AIT, although only Der f 2 and Der p 21 have statistical difference in the NHD group. Just as several studies have demonstrated that AIT could induce a transient rise in sIgE levels before a decline and a continuing increase of sIgG4 during treatment,¹⁴ our findings were in line with two AIT studies in China in which the longitudinal profile of IgE against Der p 1 and Der p 2 was observed for 1 year (16, 17). Different from sIgG4 that remained stable in the control group, the levels of IgG4 against HDM components increased continuously during the 18 months of AIT, except Der p 7, Der p 10 and Der p 23. In short, the longitudinal evolution of sIgE and sIgG4 against HDM components were very similar to the Der p and Der f detected by ImmunoCAP.

To further understand the significance of this evolution, we also analyzed the pretreatment IgE sensitization pattern and the molecular profile of the IgG4 responses during AIT. It was reported that B-cells show a shift from producing the allergenic sIgE to the ‘protective’ sIgG4 during AIT (18). Proliferating B-cells rearrange the constant region genes in the immunoglobulin heavy chain locus to switch from expressing IgM to others (such as IgE, IgG et. al.). Class switch produces an antibody with different effector properties, without altering its antigen specificity. One study suggested the IgE sensitization profile at baseline determined the molecular profile of the IgG4 during timothy grass pollen AIT (19). However, we didn’t find a significant correlation between sIgE against a certain HDM component at baseline and sIgG4 against the same HDM component after AIT. Interestingly, we couldn’t establish the correlations of pre-treatment IgE profile with the subsequent IgG4 responses neither in the ALK nor in the NHD group. Our result implied that the induced sIgG4 against HDM components was not restricted to the pre-treatment HDM component sIgE after AIT. We hypothesize that the mechanisms of sIgG4 generation were different after high-dose exposure to HDM through AIT. The generation of sIgG4 against specific epitopes in AIT is not only due to the class-switching of already sensitized epitopes, but also due to the emergence of new epitopes. The new epitopes may explain why there were no significant correlation between pre-treatment sIgE and post-treatment sIgG4.

It was reported that the sIgE response might expand from anoligomolecular sensitization stage to apolymolecular sensitization stage in many allergic children. This phenomenon has been defined as ‘molecular spreading’ (20). Naturally, there is always a deep concern that AIT may induce or boost new sensitization to other components of a given allergen. In our study, we found that numbers of sensitization HDM components especially the major HDM components statistically increased after AIT, both in the ALK and the NHD group. Meanwhile, the HDM components sensitization didn’t correlate with the efficacy of AIT. Our findings implied the new sensitization to allergen components during AIT was of less clinical relevance. One explanation might be the competitive allergen-binding effects from sIgG4. When IgG4 was removed from the serum, we found that more sIgE could bind to the HDM components fixed on the chip.

The production of IgE and IgG4 were intricately regulated by CD4⁺ T cells, especially allergen specific CD4⁺T cells. In our study, PBMCs of the AIT patients were incubated with HDM allergen to stimulate the CD4⁺T cells. Although unrelated CD4⁺T cells might exist, the majority of HDM stimulated-CD4+T cells were antigen specific (21). Following in vitro stimulation with allergen, several surface markers are specifically expressed on activated CD4+ Th cells (22). We didn’t find statistical differences of CD4⁺ T cell subsets (Th1, Th2, Tfh and Treg) after AIT. However, the changes of Tfh were correlated with sIgE and Treg correlated with sIgG4. Our results suggested that the switch of IgE to IgG4 after AIT may be due to the conversion of the subsets of CD4⁺T cells. In the FLOWSOM analysis, the cluster cells (CD3⁺CD4⁺CXCR5⁺) decreased, which could induce B-cell activation and share functional properties with Tfh cells (23). The decline of peripheral CD4⁺CXCR5⁺ cells by AIT weakens the assistance to B cells. Our study also found a cluster cell (CD3⁺ CD4⁺CD25⁺) began to express PD-1 after AIT. The effect of this cluster cell (CD3⁺ CD4⁺ CD25⁺PD-1⁺) was not reported in AIT before. However, it was identified in tumor microenvironment as PD-L1^hi Tregs to predict the response to PD-1/PD-L1 blockade immunotherapy (24). The effect of PD-1^hi Tregs in AIT will be further studied in our following work. Our results indicated that Tfh plays an important role in sIgE production and Treg correlates with sIgG4 production.

The value of sIgE and sIgG4 antibodies’ profiles in predicting the efficacy of AIT has been debated for many years. The sIgE level, sIgG4 or IgE/IgG4 ratio as biomarker of successful AIT had been proposed in many studies but always failed to be validated in other studies (25). It is believed that stratifying HDM allergic patients according to molecular sensitization profiles and molecular monitoring of AIT-induced IgG responses may enhance success of AIT (26). AIT performed with HDM extracts inducing IgG antibodies only to Der p 1 and Der p 2 was reported to benefit patients sensitized exclusively to these allergens (27).In our study, the CSMS were significantly improved both in the NHD and the ALK group. However, the improvement of CSMS had no correlations with HDM component sIgE, sIgG4 and sIgE/sIgG4 ratio of the eight HDM components. Meanwhile, although the profiles of IgE and IgG4 responses to AIT had some differences, the clinical efficacy in the two groups were the same. Our findings suggested the changes in sIgE and sIgG4 HDM components levels couldn’t efficiently work as biomarkers for AIT.

There were some limitations in our study. Firstly, the study population in each group was relatively small. Secondly, the observational period was relatively short (18 months) whereas AIT was suggested to last for 3 to 5 years according to the current guidelines. In addition, our study was based on the patients who finished 18-month AIT. Some patients dropped out due to less efficacy and their data were not included in the analysis, which possible slightly biased interpretation of data. Thirdly, the pattern of IgE reactivity at baseline of the control group and the two AIT groups were different. However, it may not be really important to our study, as the role of control group was not to compare with the AIT groups directly, but to investigate the changes of IgE and IgG4 patterns over a relatively long term (18 months) of natural HDM exposure. With the control group, we could ensure the data observed in the AIT groups were attributed to the treatment but not to HDM exposure. Finally, the aim of our study was to investigate the changes of IgE and IgG4 profiles during AIT, although the direct comparison between the two AIT groups may not be really important in this study, it would be interesting to know if different allergen extracts could induce similar or different antibody responses. However, the sIgE against Der f 2 was lower, but sIgE against Der p 21 was higher at baseline in the NHD group compared to the ALK group (data not shown here). We found the post-treatment sIgE and sIgG4 patterns were slightly different in the two AIT groups, but we couldn’t figure out if it was caused by the different allergen components of the two HDM extracts or by the imbalance of IgE pattern at baseline.

In summary, our study revealed longitudinal evolution of sIgE and sIgG4 against HDM components during AIT shown in an early increase and a late decrease of IgE level and a continuous increase of sIgG4 level. However, the pretreated IgE sensitization patterns couldn’t determine the molecular profile of the IgG4 responses during AIT. The post-treated sIgE and sIgG4 profiles had no correlations with AIT efficacy. Future studies combining antibodies functional binding, allergen epitope sensitization and clinical manifestations may help to elucidate the mechanism of AIT.

TJ-IRB20210761

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Medical ethics committee of Tongji Hospital, Tongji Medical College of Huazhong University of science and technology. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

RZ and BS were responsible for the work as a whole. LY analyzed the HDM components data in the study and wrote the first draft of the manuscript. YY, WL, YW, and DM were responsible for CSMS data. QX and XL were responsible for data analysis. WZ and QJ contributed to IgE test by ImmunoCAP. All co-authors have contributed to the interpretation of the data and provided advice and approval of the final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.