Pacific orange

PBMCs were thawed in warm media, washed twice and resuspended at 0.5×10⁶ viable cells/microliter. 200 microliter of cells were plated per well in 96-well deep-well plates. After resting for 1 hour at 37° C, cells were, stimulated by addition of 50 microliter solutions of cytokines: IFNα, IFNγ, IL-6, IL-7, IL-10, IL-2, or IL-21 (Data S2D) respectively and incubated at 37°C for 15 minutes. Cells were then immediately fixed in 1.6% paraformaldehyde, permeabilized with 100% cold methanol, and kept at −80° C overnight. Each well was bar-coded by a unique combination of Pacific Orange and Alexa-750 dye concentrations (Invitrogen/Life Technologies, Inc.), the cells were washed with FACS buffer (PBS supplemented with 2% FBS and 0.1% sodium azide), and stained with our phospho-Flow antibody panel (Data S2E). Finally cells were washed and resuspended in FACS buffer and 100,000 cells per stimulation condition were collected using DIVA 6.0 software on an LSRII flow cytometer (BD Biosciences). Data analysis was performed using FlowJo v9.3 (TreeStar, Inc.) as shown in Figure S5 and the Mean Fluorescence Intensity (MFI) of the 90^th percentile was used for downstream analysis. All stimulated samples were compared to unstimulated control (baseline) samples and fold-changes calculated. Responses above 1.5-fold change as well as baseline MFI values were used for heritability estimates.

To determine tumor burden and the percentage of live cells, PBMCs and tissue-derived, single-cell suspensions were stained with LIVE/DEAD fixable violet solution (VIVID; Invitrogen, Grand Island, NY, USA) and Annexin-V (BD Biosciences, Franklin Lakes, NJ, USA) according to manufacturer’s instructions. Mouse anti- CD45R/B220 and CD5 were used to identify TCL1-192 cells. Determination of absolute cell counts was done using AccuCount blank particles (Spherotech, Lake Forest, IL, USA). Intracellular staining was done as previously described.(3 (link)) Briefly, cells were fixed in 4% paraformaldehyde, permeabilized in 90% methanol or 80% ethanol at −20°C and stained with surface antibodies and intracellular stains. Intracellular stains included anti- Ki67, phospho-PLCγ2(Y759), phospho-ERK1/2(T202/Y204) and phospho-p65(S529) (BD Biosciences). Cut-off for positive cells was determined by isotype controls. Cells were analyzed on a FACS Canto II flow cytometer using FACS-DIVA (BD Biosciences) and FlowJo software (version 9; TreeStar, Ashland, OR, USA). For some experiments, cells were barcoded with different concentrations of pacific orange and pacific blue (Invitrogen) to allow for analysis of multiple samples in one tube.(21 (link))

This assay was performed by the HIMC at Stanford University. PBMCs were thawed in warm medium, washed twice and resuspended at 0.5 × 10⁶ viable cells ml⁻¹. Then, 200 μl of cells were plated per well in 96-well deep-well plates. After resting for 1 h at 37 °C, cells were stimulated by adding 50 μl of cytokine (IFN-α, IL-6, IL-10 or IL-2) and incubated at 37 °C for 15 min. PBMCs were then fixed with paraformaldehyde (PFA), permeabilized with methanol and stored at −80 °C overnight. Each well was barcoded using a combination of Pacific Orange and Alexa-750 dyes (Invitrogen) and pooled in tubes. Cells were washed with FACS buffer (PBS supplemented with 2% FBS and 0.1% sodium azide) and stained with the following antibodies (all from BD Biosciences): CD3 Pacific blue, CD4 PerCP-Cy5.5, CD20 PerCp-Cy5.5, CD33 PE-Cy7, CD45RA Qdot 605, pSTAT-1 AlexaFluor488, pSTAT-3 AlexaFluor647 and pSTAT-5 PE. The samples were then washed and resuspended in FACS buffer. Then, 100,000 cells per stimulation condition were collected using DIVA 6.0 software on an LSRII flow cytometer (BD Biosciences). Data analysis was performed using FlowJo v.9.3 by gating on live cells based on forward versus side-scatter profiles, then on singlets using forward scatter area versus height, followed by cell subset-specific gating.