Allergen-specific immunotherapy (AIT) is an effective treatment for many allergens including house dust mite allergens (Wang et al., 2006; Durham et al., 2012; Nelson, 2014), which are a major cause of allergic rhinitis and allergic asthma in China (Li et al., 2009). The therapeutic mechanisms of AIT involve modulation of cellular reactions and related antibody responses as well as inhibition of anaphylactic cell release of their mediators (Till et al., 2004).

Cellular changes include generation of allergen-specific regulatory subsets of T and B cells and inhibition of allergen-specific T helper type 2 (Th2) cells (Wachholz et al., 2002). Antibody responses are involved in the induction of allergen-specific immunoglobulin G (IgG) antibodies, in particular, IgG4 antibodies. IgG4 antibodies may have blocking activities, as they compete with specific immunoglobulin E (sIgE) for binding to the allergen. This inhibits allergen–immunoglobulin E (IgE) complex formation on sIgE receptor–expressing cells such as mast cells and basophils. The responses of basophils to allergen stimulation are dose dependent. Reactivity is defined as the maximal response plateau to allergen stimulation located on the dose-response curve. Cellular sensitivity represents the threshold of allergen sensitivity under submaximal allergen concentration stimulation (MacGlashan, 1993). The basophil activation test (BAT) is a useful tool for allergy diagnosis (Gonzalez-Munoz et al., 2008; Santos et al., 2014; Imoto et al., 2015); studies have provided evidence for its usefulness in monitoring the induction of immune tolerance by immunotherapy (Bidad et al., 2014; Kepil Ozdemir et al., 2014). Potapinska et al. demonstrated that BAT has high sensitivity and specificity values in the diagnosis of atopic diseases (Potapinska et al., 2009; Ozdemir et al., 2011). In a peanut immunotherapy study, Santos et al. found that depletion of IgG4 reduced the inhibitory effect of peanut-induced basophil activation (Santos et al., 2015). Our previous study demonstrated that IgG4 responses are prominent during Dermatophagoides pteronyssinus (Der p) subcutaneous immunotherapy (SCIT) (Lai et al., 2013). In this study, we investigated the relationship between basophil response and IgG4 antibodies to demonstrate that Der p SCIT might reduce basophil reactivity and/or sensitivity through induction of IgG4 in dust mite–sensitive subjects.

In this study of SCIT with Der p extract, we found that SCIT could significantly improve asthma symptoms and reduce medication requirements starting after 12 weeks of treatment. We demonstrated that allergen-induced basophil activation decreased after 16 weeks of AIT at submaximal allergen concentrations compared to week 0. SCIT-induced sIgG4 antibodies substantially increased, and serum obtained from SCIT patients significantly inhibited basophil activation after 12 weeks of treatment compared to week 0. Removal of IgG4 antibodies reduced the ability of blocking basophil activation, and Der p sIgG4 and basophil activation inhibition assays had a significant relationship according to linear regression analysis.

Although it was impracticable to perform a double-blind, placebo-controlled study in this 2-year clinical observation, the current investigation is consistent with a previous study (Wang et al., 2006; Lai et al., 2013) and other studies (Keles et al., 2011; Devillier et al., 2016), which confirmed that HDM AIT is an effective treatment for allergic disease.

Induction of sIgG4 has long been regarded as a prominent immunological change induced by AIT (Sahin et al., 2016). In agreement with our previous findings and those of other studies (Wachholz et al., 2003; Shamji et al., 2012; Lai et al., 2013), we found that successful AIT could induce a substantial increase of sIgG4. The magnitude of increase in Der p sIgG4 concentrations is associated with the allergen concentration used for immunotherapy (Van Metre et al., 1980). Specific IgG4 has been proposed to block antibodies by competing with sIgE for allergens to form IgE–allergen complexes. This reduces the complex binding to sIgE receptor–expressing effector cells (Gehlhar et al., 1999; Francis et al., 2008) and prevents the allergen-dependent activation of T cells by interfering with IgE-facilitated antigen presentation (Van Neerven et al., 2006). IgG antibodies have also been shown to be associated with the inhibition of allergen-induced effector cell activation (Ball et al., 1999; Mothes et al., 2003) or with reduced allergen sensitivity (Reisinger et al., 2005). Although induction of allergen-specific serum IgG is regarded as a characteristic feature of the immunological response induced by AIT, treatment-induced changes in the levels of sIgE secretion remain a controversial issue. We found that the levels of Der p sIgE decreased significantly after 104 weeks of SCIT; this is supported by other studies (Arlian et al., 1993; Chen et al., 2017; Feng et al., 2018), However, other studies demonstrated that sIgE levels did not change after 1 year of AIT (Shamji et al., 2011; Sahin et al., 2016), and Blumberga et al. (2011) found that AIT-induced serum sIgE increased initially and then declined to baseline value after 1 year of treatment. In general, the decrease of sIgE during the late phase of AIT might be associated with the secretion of IL-10 and TGF-β by Treg cells as well as the switching of allergen-specific B cells toward IgG4 production instead of sIgE production (Frew, 2010).

We found that basophil activation triggered by submaximal allergen concentrations decreased after 16 weeks of AIT treatment. However, basophil activation was not significantly changed with maximal allergen concentrations in either the AIT or the medication group. This observation is consistent with other studies (Ebo et al., 2007; Lalek et al., 2010; Kepil Ozdemir et al., 2014), showing that basophil activation decrement was observed mainly in submaximal allergen stimulation in BAT experiments. However, in some studies, allergen-induced basophil CD203c expression did not change after 4 months of grass pollen sublingual immunotherapy (Horak et al., 2009). Erdmann et al. (2004) did not observe a change in basophil activation after 6 months of venom immunotherapy. Differences in the type of allergy, BAT markers used, time course of immunotherapy, and the allergen stimulation concentration may help explain the contradicting results in different studies.

This is the first study to assess the blocking function of serum IgG antibodies during HDM SCIT in allergic rhinitis and/or asthma patients by using the basophil activation inhibition assay. Studies have found that specific IgG4s have blocking activities by competing with sIgE for allergens to form allergen–IgE complexes. This inhibits complex binding to IgE receptor–expressing effector cells by means of the IgE-facilitated allergen binding assay (Wachholz et al., 2003; Shamji et al., 2012; Feng et al., 2018). These studies demonstrated that IgG antibodies could inhibit the allergen–IgE complex binding to the low-affinity IgE receptor (FcεRII, i.e., CD23) by use of a CD23-expressing Epstein–Barr virus-transformed B cell line and may thereby reduce allergen-specific T cell responses. We found that the serum obtained from SCIT patients significantly inhibited basophil activation and that basophils express high-affinity IgE receptor (FcεRI). Therefore, we demonstrated the effectiveness of IgG blocking activities in the high-affinity IgE receptor–expressing effector cell. We also found that the expression of CD63 in the basophil activation inhibition control (i.e., week 0) was higher than the basic basophil activation, and the Der p sIgE level significantly decreased at the late phase of SCIT. However, there was no correlation between the BAT inhibition assay and sIgE. The IgE-sensitized basophils may have already bound with IgE on the cell surface FcεRI, so that the FcεRI-bound IgE rather than serum free sIgE plays a more important role in the BAT inhibition assay.

Since the reduction of BAT appeared associated with the concentration of the triggering allergen, we considered the likely involvement of inhibiting antibodies. The inhibition experiments showed that serum from SCIT patients reduced basophil allergen threshold sensitivity but had no effect on basophil reactivity. We also depleted IgG4 from the serum of patients after completion of 2 years of SCIT and found that the IgG4-deficient serum demonstrated reduced inhibitory effects on basophil activation. Linear regression showed significant correlations between sIgG4 and the basophil activation inhibition assay. Thus, IgG4 antibodies appear to play an important role in reducing allergen sensitivity rather than reducing reactivity during AIT. IgG4 depletion could not remove the inhibition ability completely; the existence of other antibody isotypes such as IgA and IgG1 may also have inhibition ability (Santos et al., 2015). Further investigation on these antibody isotypes may help explain this phenomenon.

IgG4 is a unique antibody with a half-antibody exchange; it is also referred to as “Fab-arm exchange,” which results in monovalent and non-crosslinking antibodies (Aalberse et al., 2009). IgG4 antibodies can bind to allergens and reduce the free allergen concentration but not induce effector cell activation. Our results are consistent with other studies. For example, Nopp et al. (2009) observed negative correlations between basophil activation and specific IgG antibodies (20-fold median increase of sIgG4 after 9 months of SCIT). Lalek et al. (2010) showed that birch-specific IgG antibodies are responsible for the reduction of basophil allergen threshold sensitivity (fivefold median increase of sIgG4 after 2–4 months of SCIT). We reported 10-fold increases of sIgG4 after 4 months of SCIT. However, Horak et al. (2009) demonstrated that basophil responsiveness did not change after 4 months of grass sublingual immunotherapy and only found a twofold median increase in sIgG4 antibodies. These results may also explain why AIT had no or less effect on basophil reactivity (Lalek et al., 2010), as there were not sufficient antibodies to compete with IgE binding to the allergen.

Previous studies demonstrated that AIT could induce a substantial increase of sIgG4 antibodies and had blocking activity, but allergen-specific IgG does not always correlate with therapeutic effects (Bodtger et al., 2005). This could be explained by AIT not only increasing the level of serum IgG4 antibodies but also altering their specificity and/or affinity. Many studies have demonstrated that AIT not only increases the level of serum IgG4 antibodies but also alters their specificity (Michils et al., 1997, 1998). In venom immunotherapy, Michils et al. (1997) found that specificity change can occur at the beginning of treatment and long before the change of antibody titers could be detected. Kolbe et al. (1995) showed that administration of high concentrations of allergen in murine models alter not only antibody quantity but also affinity and specificity. However, Wachholz et al. (2003) showed that the binding affinities of allergen-specific IgG or IgE did not change after immunotherapy. Thus, the relationship between IgG4 and clinical efficacy requires further investigation.

We demonstrated the time course of both the cellular and humoral immune responses during HDM immunotherapy, including the induction of sIgG4 antibodies and reduction of basophil sensitivity to the allergen. We also demonstrated that an increase of Der p sIgG4 rather than a reduction of Der p sIgE correlates with the reduction of basophil activation. SCIT reduced the allergen basophil threshold sensitivity in Der p allergic rhinitis and/or asthma patients through induction of sIgG4.

All datasets generated for this study are included in the article/Supplementary Material.

The studies involving human participants were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

JL designed the study, collected the data, and performed the statistical analysis. MF had responsibility for the experiments, performed the statistical analysis, and drafted the manuscript. XZ and RQ were involved in statistical analysis. QS, MX, and XS were involved in data collection.

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.