IL-6–targeted therapies to block the cytokine or its receptor drive distinct alterations in T cell function

a 96-well plate (referred to hereafter as 8- or 9-assay plates) were used to assess day 0 and day 14 T cell responses in 4-hour con-ditioned media from CD3/CD28 Dynabead-activated CD3 + T cells. Eight-assay plate analytes included IL-2, IL-6, IL-7, IL-10, IL-15, IL-21, IL-22, and IL-27. Nine-assay plate analytes included IL-6, IL-7, IL-10, IL-15, IL-21, IL-22, IL-27, TNF- α , and TNF- β . Activated cell culture supernatants were collected prior to processing cells for phospho-flow and stored at –80°C. Thawed samples were tested undi-luted in duplicate, where unstimulated samples served as negative controls, and 24-hour T cell–condi-tioned media from a healthy control individual served as positive control. Calibrators and test samples were incubated overnight on coated plates and assayed according to the manufacturer’s instructions for U-PLEX Custom Biomarker Group 1 assays (human) (Meso Scale Discovery). Meso Scale Discovery assay plates were read on a Sector SQ120MM instrument. Data were analyzed using Meso Scale Discovery Workbench v4.0 software. Mean calculated concentration coefficient of variation was 4.10% and 2.95% for the 8-assay and 9-assay plate calibrators, respectively, demonstrating the high reproduc-ibility of the U-PLEX assays, with limits of detection within the low to sub pg/mL range. Of note, IL-2 levels were above the calibrator curve fit range for nearly all siltuximab study samples, whereas IL-7 and IL-15 were below calibrator curve fit range; therefore, these analytes were not included in the comparison of the effect of siltuximab and tocilizumab on T cell production of cytokines.