IgG antibodies produced during subcutaneous allergen immunotherapy mediate inhibition of basophil activation via a mechanism involving both FcgammaRIIA and FcgammaRIIB

Abstract

The majority of human subjects who receive subcutaneous allergen immunotherapy (IT) develop decreased sensitivity to their allergens. Multiple factors may explain the efficacy of IT, some evidence support a role for allergen specific IgG antibodies. There is controversy whether such antibodies act by blocking allergen binding to IgE or initiation of active inhibitory signaling through low affinity IgG receptors (FcgammaRIIB) on mast cells and basophils. In this study, we addressed this question using peripheral blood from cat non-allergic, cat allergic, and immunotherapy-treated cat allergic subjects. Blood from subjects who received IT contain IgG antibodies that mediate inhibition of basophil activation by a mechanism that is blocked by antibodies specific for the inhibitory IgG receptor FcgammaRIIB. Surprisingly, inhibition was also blocked by aglycosylated, putatively non-FcR binding, antibodies that are specific for the FcgammaRIIA, suggesting a contribution of this receptor to the observed effect. Consistent with a cooperative effect, ex vivo basophils were found to express both IgG receptors. In other studies we found that basophils from subjects who were both chronically exposed to allergen and were producing both cat allergen specific IgE and IgG, are hyporesponsive to allergen. These studies confirm that IgG antibodies produced during IT act primarily by stimulation of inhibitory signaling, and suggest that FcgammaRIIA and FcgammaRIIB function cooperatively in activation of inhibitory signaling circuit. We suggest that under normal physiologic conditions in which only a small proportion of FcepsilonRI are occupied by IgE of a single allergen specificity, FcgammaRIIA co-aggregation may, by providing activated Lyn, be required to fuel activation of inhibitory FcgammaRIIB function.

Figure 1. Basophils from cat allergic individuals are uniquely sensitive to cat allergen induced CD203c up-regulation

A) Whole blood was stimulated with cat hair extract containing Fel d1 [0.0001 μg/ml to 1 μg/ml]. FcεRIα cross-linking antibodies and fMLP were used as positive controls. B) Cells from an allergic subject were stained with the pan-leukocyte marker CD45, IgE and CD203c to identify basophils and determine activation status. MFI of CD203c following a representative stimulation of ex vivo basophils is shown. C) A representative cat hair allergen dose response curve is shown here from a cat allergic subject. The arrow annotates the point of maximal basophil activation, or allergen “sensitivity” of the basophil. D) Average basophil CD203c expression for stimulated (as shown) non cat-allergic and cat allergic subjects. The basophil response for cat allergen extract shown was for the maximal activation; the dose required varied widely, from a fel d1 concentration of 0.001 μg/ml to the maximum does used 1 μg/ml. * p<0.001

Figure 2. IgG antibodies produced during immunotherapy acutely inhibit basophil responses to allergen by FcγRIIA and FcγRIIB dependent mechanisms

A) Serum (20% v/v) was added to whole blood from an allergic subject. “No inhibition” control serum containing no cat allergen specific IgG; “Immune serum” - serum from IT subject; “Purified IgG” and “IgG depleted serum” - see methods. Purified IgG mediated desensitization, and serum depleted of IgG lost this ability. B) Blocking with either anti-FcγRIIA (IV.3 N297Q) or anti-FcγRIIB (2B6 N297Q) reversed the effect. C) Immunofluorescence staining of ex vivo basophils with antibodies against FcγRIIA (IV.3 N297Q) (solid line) and FcγRIIB (2B6 N297Q) (heavy solid line) D) Ex vivo basophils were incubated with excess anti-FcγRIIA (IV.3 N297Q) (dashed black line) or no antibody (solid line) before addition of staining antibodies to identify basophils (CD45 and CD203) and for anti-FcγRIIB (2B6 N297Q) quantitation to assess blocking. The gray line represents isotype control. E) A murine B cell line (IIA1.6) expressing human FcγRIIB was stained by anti-FcγRIIB (2B6 N297Q) Alexa 488 (black line) but not by anti-FcγRIIA (IV.3 N297Q) Alexa 488 (gray line), or an isotype control antibody, CH4420 N297Q Alexa 488 (dotted line). F) Staining of CD20 positive peripheral blood B cell with anti-FcγRIIB (2B6 N297Q) (heavy solid line) but not anti-FcγRIIA (IV.3 N297Q) (dotted black line). The solid black line reflects isotype control staining.

Figure 3. Elevation in cat allergen specific IgG antibodies is associated with environmental exposure or immunotherapy

Levels of allergen specific IgG Abs were determined by ELISA (see methods). Non cat allergic (◇) and cat allergic (△) individuals were separated into those with a cat in the home > 3 months (Cat Keeping) vs. those never living with a cat. Immunotherapy subjects are represented by circles (o). Dunnett’s method with control (either no cat keeping or yes cat keeping as indicated) was used for statistical analysis.

Figure 4. Both FcγRIIA and FcγRIIB are expressed on human basophils and immunotherapy modulates FcγRIIB expression

Grouping was based on self-reported allergy symptoms and immunotherapy treatment. Diamonds (◇)represent subjects denying any allergy symptoms, triangles (△) represent subjects with allergies, circles (o) represent subjects on IT and x’s are the isotype control for each antibody. A) Basophils from all subjects expressed FcγRIIA (IV.3 N297Q), no difference was seen between groups B) The MFI of FcγRIIB (2B6 N297Q) was significantly decreased on basophils from subjects on IT compared to non allergic controls (MFI 96 vs. 152).

Figure 5. Reduced basophil sensitivity to allergen is observed in subjects with elevated cat hair allergen specific IgG1

To assess the association of between basophil sensitivity and allergen-specific IgG, we analyzed the relationship between cat allergen specific IgG levels and basophil sensitivity (y-axis), dividing subjects into those whose sera contained high vs. low cat specific IgG. The analysis was done for cat hair allergen specific IgG1 (high > 400 relative units (RU)), IgG2 (high > 100 RU), and IgG4 (high > 50 RU). The relationship between high vs. low allergen-specific IgG and basophil sensitivity reached significance for IgG1 (*p=0.04), but not IgG2 (p=0.24) or IgG4 (p=0.21). Mean cat allergen specific IgE were as follows: IgG1 low and high, 5.3 and 6.45 kU/dl respectively, IgG2 low and high 5.2 and 6.6 kU/dl respectively and IgG4 low and high 5.7 and 5.5 kU/dl respectively.

Figure 6. IT reduces basophil sensitivity to cat hair allergen

Data was collected for one individual pre- and post-immunotherapy. A). After seven months of maintenance IT, basophil sensitivity had decreased 10 fold. Pre-IT cat hair allergen specific IgG was 0.2 RU, post-IT cat hair allergen specific IgG was 5.5 × 10³ RU. Expression levels of low affinity IgG receptors were minimally changed (B). FcγRIIA: pre IT △ MFI 4.7; post △ MFI 4.1; FcγRIIB: pre IT △ MFI 109; post △ MFI 93.

Figure 7. Current exposure to environmental cat allergen decreases acute IgG mediated effects on basophil sensitivity

Serum from the IT subject used in Figure 2 (20% v/v), containing high amounts of cat specific IgG, was added to whole blood of allergic subjects prior to cat allergen stimulation. The immune serum mediated a greater change in basophil responsiveness in subjects not exposed to cat in the home (left panel). Baseline cat hair allergen specific IgG is documented in each of the plots by “IgG”. The filled square is the geometric mean fluorescence intensity of CD203c expression following 10 minutes of stimulation with fMLP (see methods).

Figure 8. Hyporesponsiveness of “IT” basophils does not reflect acute FcγR signaling

Blocking antibodies against either FcγRIIA (IV.3 N297Q) or FcγRIIB (2B6 N297Q) were added to samples 30 minutes before allergen was added. Subject B recorded a negative skin prick test to cat allergen and reported efficacy of his treatment (he currently keeps a cat). In this individual, blocking FcγRIIA had a greater effect than blocking FcγRIIB, but both resulted in enhanced basophil sensitivity. Despite high levels of cat hair allergen specific IgG Abs in all other subjects, blocking FcγR did not decrease basophil sensitivity. Cat hair allergen specific IgG levels: A - 4.0 × 10³, B - 6.1 × 10⁵, C - 5.0 × 10³, D - 5.8 × 10⁴, E - 1.5 × 10³ and F - 5.5 × 10³.