Asparaginase immune complexes induce Fc-γRIII-dependent hypersensitivity in naive mice

Abstract

Asparaginase (ASNase) is an important drug for the treatment of leukemias. However, hypersensitivity to ASNase can increase the risk of leukemia relapse. Two mechanisms of ASNase hypersensitivity have been identified in mice. The existence of a pathway involving anti-ASNase IgG and Fc-γ receptor III (Fc-γRIII) implies that IgG and ASNase immune complexes (ICs) could directly induce hypersensitivity. The aim of this study was to detect ASNase ICs in mice after hypersensitivity reactions and determine their role in hypersensitivity. Protein G beads were used to detect plasma ASNase ICs by flow cytometry. Anti-ASNase IgG was purified from the plasma of sensitized mice, and ASNase ICs were prepared ex vivo at various ratios of ASNase to anti-ASNase IgG. The levels of ASNase ICs detected after hypersensitivity reactions correlated with reaction severity (R² = 0.796; P = 0.0005). ASNase ICs prepared ex vivo required high levels of anti-ASNase IgG for formation, and binding to naive and sensitized immune cells depended on soluble anti-ASNase IgG, antigen:antibody ratio, and Fc-γRIII. Similarly, basophil activation by ASNase ICs depended on the antigen:antibody ratio and Fc-γRIII. Consistent with the ex vivo results, naive mice receiving ASNase ICs developed hypersensitivity reactions. Our data demonstrate that ASNase ICs can directly contribute to the onset and severity of ASNase hypersensitivity.-Rathod, S., Ramsey, M., DiGiorgio, D., Berrios, R., Finkelman, F. D., Fernandez, C. A. Asparaginase immune complexes induce Fc-γRIII-dependent hypersensitivity in naive mice.

Keywords: anaphylaxis; antidrug antibodies; basophil activation test; immunogenicity; leukemia.

Figure 1

ASNase ICs are detected after an ASNase hypersensitivity reaction, and their levels correlate with the severity of the reaction. A) ASNase ICs were detected in plasma samples collected 2 h after the onset of an ASNase hypersensitivity reaction. B) The percentage of ASNase ICs positively correlates with the severity of ASNase hypersensitivity, as quantified by rectal temperature AUC. C, D) No correlation was detected between anti-ASNase IgG antibody levels measured before (C) or after (D) the onset of ASNase hypersensitivity. OD, optical density.

Figure 2

ASNase IC binding to immune cells is Fc-γRIII–dependent. A) ASNase ICs were prepared ex vivo using either commercially available mouse or rabbit anti-ASNase IgG, or ASNase-sensitized mouse plasma that contained anti-ASNase IgG and detected by flow cytometry. B) ASNase ICs prepared ex vivo at an ASNase to anti-ASNase IgG weight ratio of 1:10 show strong binding to naive and ASNase-sensitized total leukocytes (CD45⁺). C) Blocking Fc-γRIII using an mAb inhibits ASNase IC binding to naive and ASNase-sensitized leukocytes. D) The Fc-γRIII expression of leukocytes was determined by flow cytometry and indicates that naive mice have lower Fc-γRIII expression than ASNase-sensitized mice. E, F) Blocking anti-ASNase IgE with anti-IgE mAb (EM-95) (E), or removing soluble anti-ASNase IgG through lysis and washing (F) shows that ASNase IC binding to leukocytes is not dependent on IgE and Fc-ɛRI or soluble anti-ASNase IgG.

Figure 3

Binding of ASNase ICs to immune cells requires high anti-ASNase IgG antibody levels and correlates with Fc-γRIII expression. A) Ex vivo ASNase ICs were prepared at weight ratios of ASNase to anti-ASNase IgG of 1:1, 1:2.5, 1:5, 1:10, and 1:20 and their binding to total leukocytes (CD45⁺) was determined. B–F) ASNase IC binding to naive and sensitized macrophages (B), neutrophils (C), basophils (D), B cells (E), and T cells (F) was determined at a 1:1, 1:10, and 1:20 weight ratio of ASNase to anti-ASNase IgG (n = 15). G) The Fc-γRIII expression of ASNase-naive and -sensitized macrophages, neutrophils, basophils, B cells, and T cells was determined, and found to positively correlate with the ASNase ICs binding of macrophages, neutrophils, and basophils (H–J). MFI, mean fluorescence intensity.

Figure 4

ASNase ICs activate naive and sensitized basophils in an Fc-γR–dependent manner. A) ASNase exposure induces ASNase-sensitized basophils to up-regulate CD200R1 and down-regulate CD200R3 comparably with their respective modification by anti-IgE mAb (EM-95) and anti–Fc-γRIIB/III mAb (2.4G2), respectively, which were used as positive controls. In contrast, ASNase ICs do not affect CD200R1 expression, suggesting that they activate basophils through an Fc-γR–dependent mechanism. B) The down-regulation of CD200R3 by ASNase ICs only occurs at higher weight ratios of ASNase to anti-ASNase IgG antibody.

Figure 5

ASNase ICs prepared ex vivo can trigger anaphylaxis in ASNase-naive mice. A) Naive C57BL/6 mice received intravenous vehicle control (PBS), ASNase (1 µg), or ASNase ICs prepared ex vivo at a 1:10 ratio of ASNase (1 µg) to anti-ASNase IgG (10 µg). The core body temperatures of mice were monitored for the development of hypothermia and the onset of hypersensitivity. B, C) The data indicate that mMCP-1 (B) and anti-ASNase IgG (C) levels were lower in naive mice receiving ASNase ICs compared with ASNase-sensitized mice challenged with ASNase. D) Low ASNase activity levels were detected in naive mice receiving ASNase ICs and ASNase-sensitized mice challenged with ASNase. OD, optical density.