Profiling the Immune Response to Peanut Using Mass Cytometry
Saturday, March 5, 2016
South Exhibit Hall H (Convention Center)
Leticia Tordesillas, PhD, Adeeb H. Rahman, PhD, Hugh A. Sampson, MD FAAAAI, M. Cecilia Berin, PhD
Rationale: CyTOF (mass cytometry) improves multidimensional single cell analysis, with greater multiplexing capability, lower background, and higher sensitivity for detection of phosphorylation compared to flow cytometry. We developed a CyTOF panel to study acute responses to food allergens in whole blood.

Methods: Fresh blood samples from 6 peanut-allergic and 3 healthy subjects were activated with peanut extract, anti-IgE, or media for 15 and 30 minutes, all in presence of IL3. Samples were combinatorially barcoded with palladium isotopes to reduce sample-specific batch effects and data collection variation. Pooled samples were stained with a panel of 29 metal-conjugated antibodies including markers for cell identification and activation. Cells were acquired on a CyTOF2, and SPADE analysis performed using Cytobank.

Results: Basophils from patients but not healthy controls upregulated known activation markers (CD63) after peanut stimulation. When activated, basophils increased phospho-ERK, -p38, and -S6, and downregulated CD38 and the Fcγ receptor CD32. Aggregation of basophils and platelets was observed upon activation with peanut, measured by co-expression of CD61. In addition to basophils, we confirmed that other cells types showed evidence of activation after exposure to peanut in allergic but not healthy control subjects. cDCs, pDCs and monocytes increased phosphorylation of S6 upon peanut exposure. We did not detect activation in neutrophils, eosinophils, T cells or B cells.

Conclusions: We have established methods for profiling antigen-responsive cells in whole blood using mass cytometry. This provides insight into novel cell types activated by allergen, and elucidation of signaling pathways activated in allergic effector cells.