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S. Rathod et al.
However, the binding of ASNase to immune cells might have depended on anti-ASNase IgG, primarily the anti- ASNase IgG1 subclass (Online Supplementary Figure S4), because CD45+ASNase+ cells were solely detected on Day 23 of our protocol (Figure 1C, P<1x10-4), which is when plasma anti-ASNase IgG antibody levels became elevated relative to naïve mice (Figure 1D, P<1x10-4).
Basophils express both the high-affinity IgE receptor, FcεRI, and the low-affinity IgG receptor, FcγRIII, and, therefore, can bind antigen via cell-associated IgE or anti- gen-specific IgG (i.e., after immune complex formation). Suggesting that anti-ASNase IgE may play a role in the detected binding to basophils, plasma anti-ASNase IgE antibodies were elevated in the plasma samples of sensi- tized mice on Day 23 relative to controls by ELISA (Figure 1E). Due to the possible interference of anti-ASNase IgG during the detection of anti-ASNase IgE by ELISA,26 the presence of anti-ASNase IgE in plasma samples was con- firmed by capturing IgE using anti-IgE-coated polystyrene beads and staining with labeled ASNase (Figure 1F). Based on the antigen-specific antibody isotypes detected, it is possible that recognition or binding of free ASNase to immune cells after sensitization can occur through cell associated-IgE among cells expressing FcεRI (e.g., basophils, Figure 1B) or via the binding of ASNase-IgG immune complexes to cells expressing FcγRIII (e.g., neu- trophils, macrophages/monocytes, basophils, Figure 1B). Supporting this proposed mechanism of recognition, the binding of ASNase to the basophils of sensitized mice is decreased by blocking antibodies targeting the FcγRIII receptor (2.4G2, Figure 2, P< 1x10-4) or IgE (EM-95, Figure 2, P<1x10-4). The binding of ASNase to the basophils of
sensitized mice is further decreased when both antibodies are used in combination rather than singly (Figure 2, P<1x10-3). Thus, basophils can bind ASNase immune com- plexes and free ASNase, and both can occur simultaneous- ly. In contrast, the binding of ASNase to IgE- leukocytes of sensitized mice is decreased after blocking with 2.4G2 (Figure 2, P<1x10-3) but binding is not affected by EM-95 (Figure 2, P>0.05). Consistent with requiring immune complex formation prior to ASNase binding to cells expressing the FcγRIII receptor, if soluble IgG is removed during erythrocyte lysing before staining cells, then the binding of ASNase to basophils is decreased relative to non-lysed samples (43% decrease, Figure 2, P<1x10-4). Blocking lysed cells with 2.4G2 has little to no effect on the binding of ASNase to basophils; in contrast, blocking with EM-95 strongly inhibited binding (Figure 2, P<1x10-4). The results suggest that ASNase binding to immune cells depends on the surface expression of FcγRIII and an IgE receptor, most likely, FcεRI.
The frequencies of blood basophils, B cells, and CD4+ Tregs increase during sensitization to ASNase
Upon challenge with ASNase, immune cell binding to ASNase in sensitized mice may lead to a hypersensitivity reaction, yet other cells that do not recognize or bind to ASNase likely play a role during sensitization to the drug. To identify other cells that may be involved in sensitiza- tion, we measured the frequency of B cells, CD4+/8+ T cells, CD4+/8+ Tregs, neutrophils, macrophages/mono- cytes, and basophils in blood within the CD45+ leukocytes population at five different time points during sensitiza- tion (Figure 3A-D; Online Supplementary Figure S5A-D).
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<1x10-4). 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<1x10-3, and **** for P<1x10-4.
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haematologica | 2019; 104(2)