Whole-blood cytokine release has been described in detail elsewhere (6). Briefly, 1 ml of blood was collected into Nil Control Tubes (QuantiFERON-TB Gold In-Tube, QIAGEN, Hilden, Germany) that had 0.1 ml of PBS alone or with the mixture of three gluten peptide constituents of Nexvax2 each at 50 μg/ml added. The peptides corresponded to α-gliadin W02-E7, ω-gliadin/C-hordein W03-E7, and hordein B08-E2E7 reported by Tye-Din et al. (7). Peptides were at least 95% pure by high-performance liquid chromatography and liquid chromatography mass spectroscopy (CSBio, Menlo Park, California, USA). Tubes were incubated at 37°C for 24 hours before centrifugation and separation of plasma. Stimulation index was calculated by dividing cytokine concentration for Nexvax2 peptide incubation by the PBS control.

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