Experimental Methods

T cell isolation and plating

Two vials of ~100 million PBMCs isolated by leukapheresis from one 28 year-old healthy male donor (StemCell) were thawed at 37°C and washed with cRPMI buffer (RPMI media with 10% Heat Inactivated FBS and Pen-Strep; GIBCO). PBMCs were then centrifuged and resuspended at 5✕10⁷ cells/mL in EasySep buffer (STEMCELL Technologies) for T cell isolation using RapidSpheres (STEMCELL Technologies) and an EasySep magnet (STEMCELL Technologies). After isolation, T cells were counted, resuspended in cRPMI, and 400,000 cells were aliquoted to each well in two 96-well Cell Culture-Treated U-bottom microplates (Fisher Scientific).

Drug treatment

JAK/STAT inhibitor drugs from the DiscoveryProbe JAK/STAT Compound Library (ApexBio) were prepared by thawing drugs from the first plate layout (L1041-01), consisting of 88 drugs in the first 11 columns of a 96-well plate, with no drug in the rightmost column. Drugs were provided at 10 mM, and were diluted to 20 μM by dilution of 1 μL of each drug in 499 μL of cRPMI. 50 μL of diluted inhibitor drugs were added to 50 μL of T cells for a final treatment concentration of 10 μM. T cells were incubated with JAK/STAT inhibitors for 1 hour at 37°C.

The list of JAK/STAT inhibitor drugs can be viewed by expanding the section below:

IL-6 stimulation

IL-6 stimulation media was prepared by dilution of 5 μL of 0.1 mg/mL Human IL-6 Recombinant Protein stock solution (Thermo Fisher) in 19,995 μL of cRPMI to a working concentration of 25 ng/mL.  After incubation with JAK/STAT inhibitors, 100 μL of cRPMI was added to each well of T cells, which were then centrifuged, and 180 μL of supernatant was removed from each well. To the remaining 20 μL of cell volume, 80 μL of IL-6 stimulation media was added to most wells for a final concentration of 20 ng/mL. Four wells, E12, F12, G12, and H12 were not stimulated and also were not treated with any JAK/STAT inhibitors. Cells were incubated with IL-6 stimulation media at 37°C.

Flow cytometry

After 15 min incubation with JAK/STAT inhibitors, one of the T cell plates was assayed using phospho-flow cytometry. 100 μL of warmed Fixation Buffer (BioLegend) was added to each well, then cells were incubated at 37°C for 15 minutes. Cells were centrifuged and resuspended in 100 μL of Cell Stain Buffer (BioLegend), then were centrifuged and resuspended in Human TruStain FcX Receptor Blocking Solution (BioLegend) and incubated at room temperature for 10 min to block. Antibodies used for subsequent staining steps are listed below:

An Extracellular Antibody Stain solution was prepared by mixing extracellular antibodies with Cell Stain Buffer and BV Staining Buffer (BioLegend). 50 μL of Extracellular Stain was added to each sample, and cells were incubated at room temperature for 30 min in the dark. After incubation, cells were washed with Cell Stain Buffer by resuspension and centrifugation. 

For intracellular staining, cells were permeabilized by resuspension in 100 μL pre-chilled Phosflow Perm Buffer III (BD Biosciences), and were incubated on ice for 45 min. Intracellular Stain Solution was prepared by mixing Intracellular antibodies with Cell Stain Buffer, BV Staining Buffer, and FcX. Cells were washed with Cell Staining Buffer, then resuspended in 100 μL of Intracellular Stain and incubated for 45 min at room temperature on a shaker. Prior to measurement, stained samples were washed 3 times with Cell Staining Buffer.

Flow cytometry was performed using a Cytek Aurora spectral cytometer, and acquisition and spectral unmixing were performed using Cytek SpectroFlo software. Analysis of flow cytometry data was performed using FlowJo (BD Biosciences).

10x Genomics Flex v2 single cell RNA-seq

After two hours of incubation with JAK/STAT inhibitors, the second plate of T cells were prepared for scRNA-seq. Cells were centrifuged and resuspended in 200 μL of PBS, then transferred to a half-skirt 96 well eppendorf plate. Sample fixation, quenching, and hybridization were carried out based on the 10x Genomics Flex v2 protocol adapted for use on a Tecan liquid handling automation system. Hybridization was performed first for gene-specific probes (L1000 and Immune panels), then with 96 well-specific Barcode probes. After hybridization, cells in each well were counted using a Cellaca MX cell counter (Nexcelom), and were pooled and washed using the Tecan system. Cell capture and GEM generation were performed using a GEM-X Flex Gene Expression Chip Kit on a 10x GEM-X system. Cells were loaded at 1.2 million per well in 4 GEM-X input wells. After GEM generation, GEM recovery, pre-amplification, SPRI cleanup, and library construction were performed according to the 10x Genomics Flex protocol. Final libraries for each well were sequenced on two Illumina NovaSeq runs at Northwest Genomics Center to target 5,000 reads per cell.