Rainbow calibration particles

Stimulations were stopped by incubation in 2mM EDTA for 5 min. Surface staining was performed for 15 min in the presence of 1 mg/ml of Beriglobin (CSL Behring) with the following fluorochrome-conjugated antibodies titrated to their optimal concentrations as specified in Supplementary Table 2: anti-CD3-FITC (Miltenyi), anti-CD4-VioGreen (Miltenyi), anti-CD8-VioBlue (Miltenyi), anti-CD38-APC (Miltenyi), and anti-HLA-DR-PerCpVio700 (Miltenyi). During the last 10 min of incubation, Zombie Yellow fixable viability staining (Biolegend) was added. Fixation and permeabilization were performed with eBioscience™ FoxP3 fixation and PermBuffer (Invitrogen) according to the manufacturer’s protocol. Intracellular staining was carried out for 30 min in the dark at room temperature with anti-4-1BB-PE (Miltenyi), anti-CD40L-PEVio770 (Miltenyi) and anti-CD40L-PECy7 (Biolegend), anti-IFN-γ-A700 (Biolegend) and anti-TNF-α-BV605 (Biolegend). All samples were measured on a MACSQuant^®Analyzer 16 (Miltenyi). Instrument performance was monitored prior to every measurement with Rainbow Calibration Particles (BD Biosciences).

All samples were analyzed using a BD LSRFortessa cell analyzer. Each sample set in the study was analyzed using a common configuration established for the flow cytometer at the beginning of the study. For each sample run, eight peaked Rainbow Calibration Particles (BD Biosciences) were run to track cytometer performance and compensation was calculated using single antibody stained beads (BD CompBead) to eliminate spectral overlap associated with simultaneous usage of multiple fluorochrome-labeled antibodies. In addition, Fluorescence Minus One controls were periodically performed to ensure antibody binding specificity as well as to demarcate gating areas for positive cell populations. This required the preparation of multiple cocktails, each one missing one specific antibody from our panel and staining pooled conjunctival IC samples (from one individual) separately with each of these minus-one antibody cocktails. Flow cytometer outputs were imported and analyzed using the FCS Express 6 data analysis software (De Novo Software). All analysis was performed using a hierarchical gating strategy developed at Ocular Biomarker Laboratory. To reduce user-based variations in gating of cell populations, the two personnel conducting the analysis periodically analyzed and compared data with a common set of samples.

At time points indicated, 10⁴ beads (Rainbow calibration particles; BD Biosciences) were added to tissue culture wells immediately before analysis, and the ratio of beads to live cells was used to calculate the absolute cell number in each sample. Propidium iodide (0.2 μg mL⁻¹, Sigma) was used for dead‐cell exclusion.
To distinguish between naïve (CD45.2) and memory (CD45.1) OT‐I T cells in the coculture experiment, an anti‐CD45.1‐APC antibody (BD Biosciences) was added to the bead suspension.
Flow cytometry was performed on FACSCanto II or Fortessa X‐20 cytometer (both BD Biosciences). Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

To standardize the analysis, we used Rainbow Calibration Particles (6 peaks; BD Biosciences), which contain a mixture of particles of similar size with different fluorescence intensities. The particles were used according to the manufacturer’s protocol and were reconstituted with phosphate-buffered saline (BD Biosciences). Equipment voltages and the mean fluorescence intensity (MFI) were adjusted for each fluorochrome on a daily basis in order to standardize the protocol, reducing the existing inter-test variability.
Calibration was performed at the start of the experiments (both baseline and follow-ups). All included patients were analyzed with the same voltage setting of the cytometer lasers.

Leftover satellite vials from mobilized PBSC grafts from patients undergoing ASCT for MM or healthy donors were analyzed from the Fred Hutchinson Cancer Center (Seattle). A healthy donor cohort from the QIMR Berghofer Medical Research Institute (Brisbane) was used as a comparator. Healthy volunteer PB samples were collected and PBMCs isolated using Ficoll-Paque PLUS (Cytiva) prior to cryopreservation in IMDM (GIBCO by Life Technologies) media containing 20% FBS (GIBCO by Life Technologies) and 10% DMSO. Healthy sibling transplant donors were administered G-CSF for 4 consecutive days and PB collected before and after G-CSF mobilization, together with a sample of the apheresis product. Spherotech Rainbow Calibration Particles (8 peak) from the same lot number were employed to calibrate the voltages across BD FACSymphony instruments used to acquire human data from the Seattle and Brisbane cohorts (cohorts 1 and 2, respectively).

All flow cytometric analyses were performed using a BD FACS Fortessa (BD Biosciences, Franklin Lakes, NJ, USA). To ensure comparable mean fluorescence intensities (MFIs) over time of the analyses, Cytometer Setup and Tracking beads (CST beads, BD Biosciences, Franklin Lakes, NJ, USA) and Rainbow Calibration Particles (BD Biosciences, Franklin Lakes, NJ, USA) were used. For flow cytometric analysis, the following fluorochrome-labeled antibodies were used: BUV737 anti-CD11c (BD, clone B-ly6), BUV395 anti-CD14 (BD, clone M5E2), BUV395 anti-CD3 (BD, clone UCHT1), BV786 anti-CD27 (BD, clone L128), BV711 anti-CD19 (BD, clone SJ25C1), BV605 anti-CD24 (BD, clone ML5), BV510 anti-CD10 (BD, clone HI10A), BV421 anti-CXCR5 (BD, clone RF8B2), PE-CF594 anti-IgD (Biolegend, San Diego, CA, USA, clone IA6-2), APC-Cy7 anti-CD38 (Biolegend, clone HIT2), PE-Cy7 anti-IgG (BD, clone G18-145), anti-IgA-Biotin (BD, clone G20-359), BV650 anti-IgM (BD, clone MHM-88), FITC anti-TNFα (Biolegend, clone Mab11), BV650 anti-IFNγ (BD, clone 4S.B3), BV786 anti-CD40L (Biolegend, clone 24-31), PE-CF594 anti-CD137 (Biolegend, clone 4B4-1). Numbers of absolute B and T cells were measured with Trucount (BD) and samples were processed according to the manufacturer’s instruction.