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ASNase hypersensitivities are IgG/IgE dependent
depleted to below 5%) is sufficient to completely protect mice from the development of anti-ASNase IgE antibodies and the onset of ASNase hypersensitivity (Figure 5C). The development of ASNase hypersensitivity after B-cell depletion is consistent with previous clinical data indicat- ing that patients are depleted of B cells but not T cells dur- ing leukemia treatment, but yet develop hypersensitivity reactions to ASNase.31 The presence of anti-ASNase IgE may explain why some patients develop ASNase hyper- sensitivity in the absence of detectable anti-ASNase IgG,2 suggesting that ASNase hypersensitivity reactions proba- bly also occur via multiple pathways in humans. Regarding the prevention of ASNase-mediated immune responses, our results suggest that although treatment with rituximab during ALL induction therapy may protect against the inactivation and accelerated clearance of ASNase by antigen-specific IgG antibodies, rituximab-pre- treated patients may not necessarily be protected against ASNase hypersensitivity. It is possible that pretreating patients before ASNase administration with rituximab and antihistamine or an agent that can attenuate T-cell activation may mask or mitigate hypersensitivity reac- tions while allowing ASNase therapeutic drug levels to be achieved.
It is not routine practice to evaluate anti-ASNase IgE antibody levels during ASNase therapy,2,32,33 and while some studies have suggested a role of anti-ASNase IgE during hypersensitivity reactions to ASNase,14-16 the pres- ence or absence of plasma or serum antigen-specific IgE may not necessarily be predictive of FcεRI receptor-bound antigen-specific IgE.34 Likewise, performing skin prick tests,35 basophil activation tests that solely incorporate markers of IgE-mediated hypersensitivity,36 or anti-
ASNase IgG antibody measurements2 cannot accurately explain or predict clinical reactions to ASNase. Therefore, investigating receptors or immune cells that are directly involved in hypersensitivity reactions, rather than relying on biomarkers of sensitization for prediction, may be more useful. We hypothesize that a cell-based approach will distinguish when antigen and antibody levels are suf- ficient to form immune complexes capable of binding the Fcγ receptor, and also be able to detect antigen binding to cell-associated IgE. Consistent with our hypothesis, the ASNase basophil activation test can detect activation via both the classical and alternative pathway of anaphylaxis (Figure 6C-D). Although the changes in CD200R3 meas- ured were modest (but statistically significant), they are similar to those we observed using a positive control (i.e., 2.4G2) and to those that have been previously described.22,23
ASNase ex vivo binding was simultaneously detected in an FcγRIII- and IgE-dependent mechanism of hypersensi- tivity (Figure 2), and due to the simultaneous upregulation of CD200R1 and downregulation of CD200R3 by the BAT, we hypothesized that ASNase hypersensitivity was both FcγRIII- and IgE-dependent. Supporting our hypoth- esis, blocking IgE-mediated hypersensitivity using EM-95 or antihistamine only partially mitigated ASNase hyper- sensitivity (Figure 7). Similar results were obtained when blocking IgG-mediated hypersensitivity reactions using 2.4G2 or PAF receptor antagonist (Figure 7). However, ASNase hypersensitivity reactions were completely inhib- ited when both pathways of anaphylaxis were simultane- ously blocked using EM-95 and PAF receptor antagonist, 2.4G2 and antihistamine, or PAF receptor antagonist and antihistamine. It is not yet known whether both path-
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 chal- lenged with ASNase to induce hyper- sensitivities. The AUC of the tempera- ture vs. time curve was estimated. All mice receiving any pretreatment med- ication had a significant reduction in the severity of ASNase hypersensitivi- ties relative to sensitized, non-pretreat- ed mice (P<1x10-4). A total of 5 mice were included in each analysis, as indi- cated, and P value significance is indi- cated as * for P<0.05, ** for P<0.01, *** for P<1x10-3, and **** for P<1x10-4.
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