T cell analysis by flow cytometry

To generate Fab-K_D-Fabs, Fab-X and Fab-Y molecules were mixed in TexMACs cell culture medium at an equimolar ratio and incubated for 1 h at 37°C. PBMC (1.00 × 10⁵) were added to 384-well tissue culture plates, followed by addition of the Fab-K_D-Fab proteins. The cells were then activated via T cell receptor stimulation either by addition of 250 ng/mL (final assay concentration) mouse anti-human CD3 (eBioscience, UCHT1) or 1 µg/mL (final assay concentration) SEB (Sigma-Aldrich). Medium alone was added to unstimulated cells. Cells were incubated at 37°C, 5% CO₂ for 48 h and then centrifuged at 500 × g for 5 min and the conditioned medium transferred to a storage plate and placed at −80°C. Cell plates were then washed twice in flow buffer using an ELX automated plate washer (BioTek). Cells were resuspended by vortexing prior to antibody staining. Antibody details are shown in Table S3. Cells were stained at 4°C in the dark for 45 min and washed twice with flow buffer before fixing in BD CellFix overnight. The plates were then centrifuged at 500 × g for 5 min, the supernatant was removed, and the pellet resuspended in FACS buffer before acquiring on the iQue® Screener Plus flow cytometer (IntelliCyt®). ForeCyt® software (version 6.3, IntelliCyt®) was used to gate on CD14+ monocytes, CD56+ NK cells, CD19+ B-cells, CD4+ and CD8+ memory and naïve T cells. For each cell population, the cellular expression of CD69, CD25, CD71 and CD137 was measured as reported median fluorescence intensity values. The data were then used to calculate the log2 fold changes in expression relative to control well values and exported into Spotfire® (version 7.11.1, TIBCO®) for analysis and visualization.