Developing a nonhuman primate basophil activation assay by flow cytometry

Abstract

Abstract The Human Basophil Activation test (BAT) has been validated for many disease conditions, used for the diagnosis of allergy to drugs and foods, and for monitoring the role of basophils in immunotherapy. While several BAT kits are commercially available and widely used for humans, the BAT assay in nonhuman primate (NHP) is scarcely present in the literature. Given that NHPs are used as a nonclinical model in drug development and safety assessment when other animal species cannot be used, we developed a BAT using peripheral blood of cynomolgus macaques (Cyno). For human, the mAb panels 1) CCR3+SSClow,2) CD203c+SSClow, 3) CRTH2+CD3−, and 4) CD66−CD3−CD123+HLA-DR− have been used to identify total basophils. Following stimulation with allergens or a positive control (eg. anti-human FcɛRIα or IgE), the activated basophils can be gated as CD63+ and/or CD203+. For the Cyno BAT development, first, the anti-human mAbs noted above were tested for their cross-reactivity with Cyno. Second, the anti-human FcɛRIα and IgE mAbs were tested for their ability to crosslink and activate the Cyno basophils. Third, the basophil stimulation buffer (BSB) was formulated with calcium and heparin with or without IL-3 for blood collected in EDTA tubes. The FcɛRIα but not the IgE mAb, consistently activated basophils and upregulated CD63 expression. IL-3 added to BSB resulted in high background CD63 expression on unstimulated basophils and therefore was not used in the finalized method. Our data indicates that the only panel with all mAbs cross-reactive to Cyno was 4) CD66−CD3−CD123+HLA−. This panel identified the Cyno basophils that were SSCMed not SSCLow. In summary, a BAT has been successfully developed for use in exploratory NHP studies.