Hypersensitivity reactions to asparaginase in mice are mediated by anti-asparaginase IgE and IgG and the immunoglobulin receptors FcεRI and FcγRIII

Abstract

Asparaginase is an important drug for the treatment of leukemias. However, anti-asparaginase antibodies often develop, which can decrease asparaginase drug levels and increase the risk of relapse. The aim of this study is to identify the immunoglobulin isotypes and receptors responsible for asparaginase hypersensitivities. Mice immunized with asparaginase developed anti-asparaginase IgG1 and IgE antibodies, and challenging the sensitized mice with asparaginase induced severe hypersensitivity reactions. Flow cytometry analysis indicated that macrophages/monocytes, neutrophils, and basophils bind asparaginase ex vivo through FcγRIII. In contrast, asparaginase binding to basophils was dependent on FcγRIII and IgE. Consistent with the asparaginase binding data, basophil activation by asparaginase occurred via both IgG/FcγRIII and IgE/FcεRI. Depleting >95% of B cells suppressed IgG but not IgE-dependent hypersensitivity, while depleting CD4⁺ T cells provided complete protection. Combined treatment with either anti-IgE mAb plus a platelet-activating factor receptor antagonist or anti-FcγRIII mAb plus a H1 receptor antagonist suppressed asparaginase hypersensitivity. The observations indicate that asparaginase hypersensitivity is mediated by antigen-specific IgG and/or IgE through the immunoglobulin receptors FcγRIII and FcεRI, respectively. Provided that these results apply to humans, they emphasize the importance of monitoring both IgE- and IgG-mediated asparaginase hypersensitivities in patients receiving this agent.

Figure 1.

ASNase is recognized ex vivo by B cells, neutrophils, macrophages/monocytes, and basophils after sensitization. (A) Peripheral blood cells were collected from naïve mice, cultured with labeled ASNase for 30 minutes, and analyzed for ASNase positive immune cells by flow cytometry. (B) The ex vivo ASNase-specific recognition by B cells, neutrophils, macrophages/monocytes, basophils, and T cells of the blood were analyzed within CD45⁺ populations by flow cytometry of sensitized (red data points) and non-sensitized (green data points) mice on Day 23 of the sensitization protocol. (C) ASNase binding to total leukocytes (CD45⁺) and (D) anti-ASNase IgG antibodies were measured throughout the sensitization protocol. Anti-ASNase IgE antibodies were measured by (E) ELISA and (F) flow cytometry using plasma samples collected on Day 23 of the sensitization protocol. A total of 5 to 20 mice were included in each analysis, as indicated, and P value significance is indicated as * for P<0.05, ** for P<0.01, *** for P<1×10⁻³, and **** for P<1×10⁻⁴.

Figure 2.

Ex vivo ASNase binding to basophils is dependent on FcγRIII and FcεRI. Ex vivo ASNase binding to basophils after immunoglobulin receptor blocking with 2.4G2 (anti-FcγRIIB/III mAB) and/or EM-95 (anti-IgE mAB) suggests that the binding is dependent on both FcγRIII and FcεRI (n = 10). Furthermore, lysing cells and removing soluble IgG antibodies before measuring ASNase binding reduces the frequency of ASNase⁺ cells relative to non-lysed cells (P<1×10⁻⁴). The binding of ASNase to basophils is attenuated by EM-95 but not 2.4G2 after removing soluble IgG. In contrast, ASNase binding to CD45⁺ IgE- cells decreases after blocking with 2.4G2 but not after blocking with EM-95 (n=10). P value significance is indicated as * for P<0.05, ** for P<0.01, *** for P<1×10⁻³, and **** for P<1×10⁻⁴.

Figure 3.

Frequencies of CD4⁺ Tregs, B cells, and basophils increase in blood after ASNase sensitization. The frequency of (A) macrophages/monocytes, (B) CD4⁺ Treg cells, (C) B cells, and (D) basophils was measured in the blood of mice at various time points during sensitization. A total of 5 or 10 mice were included in each analysis, as indicated, and P value significance is indicated as * for P<0.05, ** for P<0.01, *** for P<1×10⁻³, and **** for P<1×10⁻⁴.

Figure 4.

ASNase-sensitized mice develop hypersensitivity reactions when challenged. The rectal temperature of sensitized and non-sensitized mice was monitored for 2 hours after the ASNase challenge on Day 24. (A) Sensitized mice experienced a drop in rectal temperature that was quantified by estimating the AUC of the rectal temperature versus time curve. (B) Sensitized mice had elevated levels of mMCP-1 and (C) low ASNase drug levels compared to non-sensitized mice. (D) ASNase binds to B cells, neutrophils, macrophages/monocytes, basophils, and T cells after challenging with ASNase on Day 24. A total of 5 or 20 mice were included in each analysis, as indicated, and P value significance is indicated as * for P<0.05, ** for P<0.01, *** for P<1×10⁻³, and **** for P<1×10⁻⁴.

Figure 5.

The in vivo depletion of CD4⁺ T cells but not B cells prevents ASNase-mediated hypersensitivity reactions. (A) B cells or (B) CD4⁺ T cells were depleted in vivo using anti-CD19 or anti-CD4 mAb three days prior to each ASNase sensitization dose. Less than 5% of B or CD4⁺ T cells remained after depletion in the blood. (C) The rectal temperature AUC after B or CD4⁺ T-cell depletion was determined after challenging with ASNase. (D) Anti-ASNase IgG levels, (E) ASNase activity, (F) mMCP-1 concentrations, and anti-ASNase IgE levels, as assessed by (G) ELISA and (H) flow cytometry, were measured from the plasma samples of mice collected after the challenge. A total of 5 or 10 mice were included in each analysis, as indicated, and P value significance is indicated as * for P<0.05, ** for P<0.01, *** for P<1×10⁻³, and **** for P<1×10⁻⁴.

Figure 6.

Ex vivo ASNase activates basophils in an IgG- and IgE-dependent manner. (A) IgE-mediated basophil activation by EM-95 (anti-IgE mAb) upregulates CD200R1 basophil expression relative to media control. (B) IgG-mediated basophil activation by 2.4G2 (anti-FcγRIIB/III mAB) downregulates CD200R3 basophil expression relative to media. (C) ASNase exposure among sensitized peripheral blood cells upregulates the CD200R1 expression of basophils in the presence and absence of anti-ASNase IgG (no IgG). (D) Downregulation of basophil CD200R3 expression in response to ASNase in ASNase-sensitized mice requires anti-ASNase IgG. A total of 10 mice were included in each analysis, as indicated, and P value significance is indicated as * for P<0.05, ** for P<0.01, *** for P<1×10⁻³, and **** for P<1×10⁻⁴.

Figure 7.

FcεRI and FcγRIII play a role in ASNase hypersensitivity. Sensitized mice were pretreated with EM-95 (anti-IgE mAB), 2.4G2 (anti-FcγRIIB/III mAB), CV-6209 (PAF receptor antagonist), triprolidine (antihistamine), EM-95 and CV-6209, 2.4G2 and triprolidine, or triprolidine and CV-6209 and challenged with ASNase to induce hypersensitivities. The AUC of the temperature vs. time curve was estimated. All mice receiving any pretreatment medication had a significant reduction in the severity of ASNase hypersensitivities relative to sensitized, non-pretreated mice (P<1×10⁻⁴). A total of 5 mice were included in each analysis, as indicated, and P value significance is indicated as * for P<0.05, ** for P<0.01, *** for P<1×10⁻³, and **** for P<1×10⁻⁴.