Multiplex array

This coculture model to study mucosal immunity was previously described in more detail [19 (link)]. In brief, healthy donor PBMC from volunteers, who had given written informed consent for research purposes, were obtained from the Dutch Blood bank (Amsterdam, The Netherlands). Monocytes and naïve Th cells were isolated using negative selection by magnetic beads. Monocytes were differentiated into immature moDC for 6 days using GM-CSF and interleukin (IL)4, and Th cells were stored in liquid nitrogen. After HT-29 cells reached confluence in the transwells, 5 × 10⁵ moDC were added to the basolateral compartment for 48h. The epithelial cells in the HT-29/moDC coculture were apically exposed to chicken TM or shrimp TM. Simultaneously, wells containing moDC without IEC, were also exposed to chicken TM or shrimp TM for 48h, allowing direct interaction with the moDC. Subsequently, moDC were collected for analysis by flow cytometry (FACS CantoII, BD Biosciences, Franklin Lakes, NJ, USA) and coculture with allogenic naïve T cells (stimulated with αCD3 and IL2). moDC and T cells (in a 1:10 ratio) were cocultured for 96 h. Cells were collected for flow cytometric analysis and supernatants were collected to measure cytokines using ELISA (R&D systems, Minneapolis, MN, USA) or multiplex array (Meso Scale Discovery, Rockville, MD, USA).

Blood collection for plasma angiogenic biomarkers was mandatory for all participants. Samples were collected at various time points: baseline (prior to starting therapy on Day 1), on Day 2 (prior to the second dose of ponatinib), on Day 1 of subsequent cycles, and off‐treatment. Plasma protein measurements were performed using multiplex array (Meso‐Scale Discovery) or standard ELISA kits (R&D Systems) in the Clinical Laboratory Improvement Amendments (CLIA)‐certified facility of the Steele Laboratories at Massachusetts General Hospital as previously described.11Tumor genotyping was performed as part of routine clinical care in the CLIA‐certified facilities at Dana‐Farber Cancer Institute and/or Massachusetts General Hospital using methods as previously described.12, 13