Production of lentivirus
LentiX cells were seeded in 6-well plates with 0.6 x105 cells per well in 2 mL complete DMEM (ThermoFisher). The day after seeding, for each well, 750 ng lentiviral vector, 500 ng psPAX and 260 ng pMD2G were mixed with 4.5 mL Fugene (Promega) in 100 mL Opti-MEM and incubated at room temperature for 20 minutes. After incubation, this DNA/Fugene mixture was added to the LentiX cells. The lentivirus supernatant was collected 2 or 3 days after the transfection and filtered to remove dead cells.
Cell line generation
Lentivirus encoding transfection of TCRα and TCRβ chain genes was generated as described above 1. Briefly, we cloned either the full-length TCRα or TCRβ chain gene into the pHR lentiviral vector. After lentivirus production, 2 mL of the supernatant containing TCR α and β chains with 1:1 expression was used to infect 1 million SKW3 cells. Following infection, we added 1.5 mL fresh completed RPMI to the mixture. After 2 days, we selected for SKW3 cells with surface-expressed TCRαβ using anti-TCR (Biolegend clone IP26) staining and flow cytometry (Sony SH800 sorter).
Cell culture
SKW3 cells transduced with TCR clones 55, 589 and DMF5 were cultured in RPMI 1640 GlutaMAX (Thermo Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS) (Sigma-Aldrich), 100 U/ml penicillin, and 100 U/ml streptomycin (Life technology). Prior to performing all experiments, we confirmed that cells had reached the log phase of growth (1.5 million to 2 million cells/mL), as cells in the lag phase may generate insufficient Ca2+ flux (https://osf.io/xs7zf/).
Microfluidic device design
Molding masters for both microfluidic devices used in the paper (the parallel flow focuser for high-throughput generation of the spectrally encoded ‘smart beads’ and the microwell array for on-chip loading of beads and T cells) were designed in AutoCAD (Autodesk). The microfluidic droplet generator was largely similar to our previously published design2 except that the width of orifice channels and all channel heights were changed to 25 μm to yield polymerized beads ~24 µm in diameter. All transparency masks used for fabrication of molding masters were printed at 50K dpi (Fineline Imaging). Designs of all devices are provided as Supplemental Files and in an associated OSF repository (https://osf.io/xs7zf/).
Microfluidic molding master fabrication
‘Smart bead’ generator
We fabricated the molding master for the parallel flow focuser device with a target height of 25 μm by:
(1) Spin-coating SU-8 2025 negative photoresist (MicroChem) (500 rpm for 10 s with acceleration of 133 rpm/s; 3500 rpm for 30 s with acceleration of 266 rpm/s) on a 4-inch test-grade silicon wafer (University Wafer, South Boston, MA);
(2) Soft baking the coated wafer (1 min at 65°C; 5 min at 95°C; 1 min at 65°C);
(3) Exposing the baked wafer using a UV mask aligner (Karl Suss MA6) for 26.8 s at 5.6 mW/cm2;
(4) Post-exposure baking (1 min at 65°C; 5 min at 95°C; 1 min at 65°C);
(5) Developing using SU-8 developer (Microchem Corp, Newton, MA) for ~4 min.
Microwell device for pairing beads and T cells
For the microwell device, we fabricated the microwell layer and roof layer separately on two 4-inch test-grade silicon wafers. Microwell features were fabricated using SU-8 2050 with a target height of 56 μm by:
(1) Spin-coating SU-8 2050 (MicroChem) (500 rpm for 10 s with acceleration of 133 rpm/s; 2750 rpm for 30 s with acceleration of 266 rpm/s) on a 4-inch test-grade silicon wafer;
(2) Soft baking the coated wafer (2 min at 65°C; 7 min at 95°C; 2 min at 65°C);
(3) Exposing the baked wafer using a UV mask aligner for 31.7 s at 5.6 mW/cm2;
(4) Post-exposure baking (2 min at 65°C; 6 min at 95°C; 2 min at 65°C);
(5) Developing using SU-8 developer for ~6 min.
The roof features were fabricated on another wafer using SU-8 2050 with a target height of 100 μm by:
(1) Spin-coating SU-8 2050 (500 rpm for 10 s with acceleration of 133 rpm/s; 1650 rpm for 30 s with acceleration of 266 rpm/s) on a 4-inch test-grade silicon wafer;
(2) Soft baking the coated wafer (5 min at 65°C; 16 min at 95°C; 5 min at 65°C);
(3) Exposing the baked wafer using a UV mask aligner for 41.1 s at 5.6 mW/cm2;
(4) Post-exposure baking (4 min at 65°C; 9 min at 95°C; 4 min at 65°C);
(5) Developing using SU-8 developer for ~9 min.
Silane vapor deposition
To prevent sticking of PDMS during device fabrication, we treated all wafers with trichloro(1H,1H,2H,2H-perfluorooctylsilane) (PFOS, Sigma-Aldrich) by placing wafers in a vacuum chamber along with an uncapped bottle of PDMS with a few PFOS droplet in the cap, pulling vacuum for 1 min, and then maintaining vacuum (without active vacuum) for an additional 9 minutes.
Microfluidic molding master fabrication
All microfluidic devices were fabricated using standard soft lithography protocols3.
‘Smart bead’ generator
Molding masters were used to cast single-layer parallel flow-focuser devices composed of a 1:5 ratio of poly(dimethylsiloxane) crosslinker: base (PDMS, RTV 615, R.S. Hughes). After baking, devices were assembled using the ‘jumper cable’ strategy described previously 2.
Microwell pairing device
The microwell layer of the pairing device was cast using a 1:5 ratio of PDMS crosslinker (20 g crosslinker, 100 g base). After mixing in a THINKY mixer (3 min mixing followed by 3 min degassing), the PDMS mixture was poured on top of the microwell mold and this mold/PDMS assembly was placed in a vacuum chamber and subjected to vacuum for ~45 minutes to remove any air bubbles trapped inside the features. After vacuum, the PDMS-coated wafer was spun for 2 min at 200 rpm with 133 rpm/s acceleration using a spin coater (Laurell WS-650) to yield a final PDMS slab height of ~400 μm. The mold with PDMS was then baked at 80°C for 25 min in a convection oven to partially polymerize this layer. Then microwell layer was then peeled off from the mold, flipped over, and adhered to a cover glass (48×65 mm, GOLD SEAL No. 1). The roof layer was casted with a 1:10 ratio of PDMS crosslinker (10 g crosslinker, 100 g base), subjected to vacuum for 45 min, and then baked at 80°C for 30 min. The roof layer was then peeled from the wafer, inlet holes were punched using a catheter punch (SYNEO, 0.025” ID × 0.035” OD, Part No: CR0350255N20R4) to fit the outer diameters of PEEK tubing (ZEUS, 0.010” ID × 0.020” OD) and steel blunt pins (New England Small Tube, Part No: NE-1310-02), and aligned on top of the microwell layer. This two-layer device assembly was then baked at 80°C for 14 hours to fully polymerize and bond both layers. Longer baking time is not recommended as that may enhance the hydrophobicity and shrink the microwells.
Production of lanthanide-encoded ‘smart beads’
Aqueous lanthanide mixtures
For ‘smart bead’ synthesis, N-isopropylacrylamide (NIPAM), poly(ethylene glycol) diacrylate (PEG-DA, Mn=700), sodium acrylate (SAc), acrylic acid (AAc), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) were purchased from Sigma-Aldrich and used directly without further purification. SAc solution (pH=7, c[Ac]=1 M) was obtained by adding 5M NaOH into 1M AAc solution until the pH reached 7.0. The poly-acrylic acid wrapped lanthanide nanophosphors (Lns) were synthesized as described previously 2, 4. Pre-mixed Ln/polymer mixtures containing NIPAM monomer, Lns, PEG-DA, SAc and LAP were generated by varying ratios of three monomer master mixtures each containing different Lns (Table S1). The “Eu” “Dy” and “Sm” master mixtures also contained 16.3% v/v YVO4:Tm (50 mg/mL), 16.3% v/v YVO4:Dy (50 mg/mL), 16.3% v/v YVO4:Sm (50 mg/mL), respectively. A total amount of 21.3% v/v Lns maintain an equal amount of hydrophilic content across all the formulas. All master aqueous mixtures in Table S1 contained purified water with 9.2% w/v NIPAM and 5% v/v YVO4:Eu (50 mg/mL), 2.8437% v/v PEG-DA, and 5.5% v/v SAc solution. For the force multiplex experiment, 5% v/v and 6% v/v SAc solution were added into the master aqueous mixture for high and low force ramps, respectively.
Droplet generation
2.5% v/v LAP (39.2 mg/mL in DI water) was added to each solution right before its injection into the droplet generator. Pre-mixed Ln/polymer mixtures and HFE7500 with Ionic Krytox (IK) and 0.05% v/v AAc were flowed into the aqueous inlet and oil inlets of the droplet generator device, respectively, yielding high-throughput production of pre-gel droplets at the flow-focusing nozzle. Droplets (radius=16.1 ± 0.4 μm) were generated with aqueous and oil flow rates of 500 μL/h and 3200 μL/h and then collected through Tygon tubing into a 24-well plate (Thermo Fisher Scientific). ~80 μL running oil was added into the each well prior to the collection of the droplets to prevent evaporation of HFE7500 and resultant droplet breakage.
Bead polymerization and functionalization
During droplet generation, the AAc in the oil phase gradually diffuses into the aqueous phase to form a carboxy shell to allow subsequent covalent coupling of streptavidin to the bead surface. For 2 formulas at a time, we applied flood UV light (IntelliRay, UV0338) at 100% amplitude (7” away from the lamp, power= ~50-60 mW/cm2) for 2 minutes to induce polymerization and crosslinking of carboxyl groups at interfacial surfaces. After polymerization, beads were transferred into a 2 mL fritz column with ~20 μm pore size (Biotage) and then washed with: 2 mL dimethylformamide (DMF, Thermo Fisher Scientific) for 20 s; 2 mL dichloromethane (DCM, Thermo Fisher Scientific) for 10 s; and 2 mL methanol (Thermo Fisher Scientific) for 20 s. After washing, beads were resuspended in 1 mL PBST buffer for the aqueous EDC chemistry.
EDC chemistry for streptavidin surface conjugation
To functionalize ‘smart’ beads with streptavidin, 150 μL of carboxy ‘smart’ beads (~200,000 beads) were washed with 200 μL 0.1 M MES buffer (pH = 4.5) supplemented with 0.01% (v/v) Tween-20 (activating buffer) three times prior to resuspension in 200 μL of activating buffer. Next, 200 μL of a freshly made 2%w/v 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, Sigma-Aldrich) in activating buffer was added to the bead solution. The entire reaction was then incubated for 3.5 hours at room temperature on a rotator with end-over-end mixing (10 rpm). The bead slurry was then washed with 1 mL 0.1 M borate buffer (pH = 8.5) supplemented with 0.01% (v/v) Tween-20 (conjugating buffer) and subsequently resuspended in 400 μL of conjugating buffer. Conjugation of streptavidin (Sigma-Aldrich) was carried out by adding 16 μL streptavidin solution (dissolved in 1X PBS at 1 mg/mL) into the mixture and rotated the whole slurry overnight at 4°C. The reaction was quenched by adding 10 μL of 0.25 M ethanolamine in conjugating buffer to the mixture and rotating for 30 minutes at 4°C. The final product was washed 3 times with PBST buffer and resuspended in 200 μL of the same buffer for subsequent bright field and fluorescence imaging. The efficiency and consistency of streptavidin coupling was checked by incubating 0.5 µL 1 mg/mL Biotin-Atto647 and ~5 µL bead slurry (~500 beads, either streptavidin-functionalized or without streptavidin as a negative control) for 1 h at room temperature and imaging in the Cy5 channel.
Production of inclusion bodies of B35 MHC heavy chain and human b-2-microglobulin
The codon-optimized B35 MHC heavy chain gene or human β-2-microglobulin gene was cloned into a pET28a vector. The construct was transformed into BL21 (DE3) E. coli and grown over night on LB agar plates supplemented with kanamycin. Single colonies were then picked, used to inoculate 10 mL LB broth containing kanamycin, and then grown overnight at 37°C with shaking at 250 rpm. The 10 mL overnight culture was added into 1 L of LB broth containing kanamycin and grown until OD= 0.5. IPTG was added into the same culture at a final concentration of 1 mM and shaken for another 3 hours. The bacteria were pelleted down by centrifuge at 6,000g for 30 min and then resuspended in 50 mL Buffer 1 (50 mM Tris-HCl, pH=8, 100 mM NaCl, 1mM DTT, 5% Triton-X-100, 1 mM EDTA). The bacteria were then sonicated with 2-second sonication and 2-second rest for 2 min and rested for another 2 min on ice. The sonication was repeated for 4 times. The lysed bacteria were then pelleted down at 6,000 g for 15 min, washed in 50 mL Buffer 1, and subjected to the same sonication procedure two more times. The pellet was then washed in 50 mL Buffer 2 (50 mM Tris-HCl, pH=8, 100 mM NaCl, 1 mM EDTA) and subjected to the same sonication procedure 2 more times. Finally, the pellet was solubilized in 25 mL Buffer 3 (8 M urea, 50 mM Tris-HCl pH=8, 10 mM EDTA, 10 mM DTT) by rotating overnight. The inclusion body was run on SDS-PAGE to check the protein molecular weight.
Refolding of peptide-MHC
We prepared 1 L refolding buffer containing 5 M urea, 400 mM L-Arginine, 100 mM Tris-HCl (pH=8), 2 mM EDTA, 0.5 mM oxidized glutathione, and 5 mM reduced glutathione. We then dissolved 30 mg UV-cleavable peptide in 1 mL DMSO and added it into the refolding buffer. Next, we mixed 30 mg B35 MHC heavy chain inclusion body and 30 mg human β-2-microglobulin inclusion body and added this into refolding buffer through a 23G needle by gravity. The refolding buffer was then transferred to dialysis tubing and dialyzed against 10 L 10 mM Tris-HCl (pH=8). Every 12 hours, the 10 mM Tris-HCl (pH=8) buffer was changed to fresh buffer; the dialysis was performed for a total of 60 hours. After dialysis, the refolding buffer was purified by flowing through diethylaminoethyl (DEAE-) cellulose which was equilibrated using 10 mM Tris-HCl (pH=8). The refolded pMHC was eluted from the DEAE-cellulose using 0.5 mM NaCl in 10 mM Tris-HCl (pH=8), buffer exchanged to 10 mM Tris-HCl (pH=8), and concentrated by Amicon concentrator. The protein was then biotinylated overnight at 4 °C. The biotinylated pMHC protein was then purified via size-exclusion chromatography (GE Superdex 200 increase 10/300 GL) and fractions were analyzed by running on SDS-PAGE. Fractions with refolded pMHC protein were further purified by running on a Mono-Q column. Fractions were again analyzed by SDS-PAGE and fractions with refolded pMHC protein were then flash frozen in liquid nitrogen and stored in -80 °C.
Peptide production
21 peptide ligands (length = 9 aa) were produced by Fmoc-based solid-phase peptide synthesis (GenScript). The UV-labile peptide used here was KPIVVLJGY, where "J" is Fmoc-(S)-3-Amino-3-(2-Nitro-Phenly)-Propionic Acid (also produced by GenScript). All peptides were dissolved in dimethyl sulfoxide (20 mM) and stored at -20 °C until further use.
Peptide exchange reaction
UV-facilitated peptide exchange was performed as described previously 5. Briefly, we first prepared peptide exchange buffer containing 20 mM Tris-HCl (pH 7.0) and 150 mM NaCl. Next, for each exchange reaction, we combined 40.4 μL exchange buffer, 5.6 μL of the peptide to exchange (500 μM, diluted from 20 mM stocks in DMSO in exchange buffer) and 10 μL UV-labile pMHC (2.8 μM in PBST) in one well of a 96-well plate. The plate was placed on a thermo-mixer whose temperature was pre-equilibrized to 4°C. A UVP compact and handheld UV Lamp (365 nm, UVP95000505, 6W) was placed over the sample for 2 h, with the distance between the lamp and sample set to ~3 cm. After that, the sample was withdrawn from the wells and purified and concentrated using 3K spin columns (ThermoFisher) three times at 4°C. The final concentration was estimated using a NanoDrop 2000 spectrophotometer (Thermo Scientific).
pMHC-functionalized ‘smart’ bead
The streptavidin (SA)-coupled ‘smart’ beads were washed with PBST buffer three times. To create sample beads with pMHC at physiological density (for normal BATTLES and force multiplex experiments), we mixed 0.5 µL of 10 nM pMHCs with ~20,000 streptavidin ‘smart’ beads in 50 µL PBST buffer. The mixture was gently rotated for 1 h at room temperature, washed with 200 µL PBST four times, and then resuspended in 200 µL PBST buffer. Bead surfaces were blocked by adding 5 µL of 20 mg/mL biotin-BSA (Thermo Scientific) to the pMHC coated bead slurry for 1 h at room temperature, washed three times in PBST, and then resuspended in 50 µL PBST for further use. The BSA-coated surface eliminates potential non-specific binding of the bead to the T cell6. For the concentration multiplex experiment, we mixed 0.5 µL of 100 nM (7X) or 500 nM (27 X) pMHCs with ~20,000 streptavidin ‘smart’ beads in 50 µL PBST buffer and followed identical bead-coating procedures. For quantifying the concentration difference, we added 1 μL of alexa-647 labeled anti-beta-2 microglobulin monoclonal antibody (Thermo Fisher, clone: B2M-01, 1 mg/mL) to 5 µL beads solution containing ~2,000 pMHC-coated beads and gently rotated for 1 h. We then washed beads with PBST five times, resuspended in 20 µL 1X PBS buffer, and imaged on an inverted microscope (Nikon Eclipse Ti, Nikon) with a SOLA light engine (380 nm -680 nm, Lumencor, Beaverton, OR) and a Cy5 filter cube set.
pMHC quantification by single molecule TIRF microscopy
To determine the actual density of pMHC molecules on ‘smart bead’ surfaces, we imaged beads via single-molecule total internal reflection fluorescence (smTIRF) microscopy (evanescent wave penetration depth of ~400 nm and a ~50 µm× 50 µm field of view). To detect bead-bound pMHCs, we added 1 µL of PE anti-human β2-microglobulin antibody (BioLegend, clone: A17082A, 0.2 mg/ml) to 5 µL beads solution containing either ~2,000 pMHC- or 2,000 SA-coated beads (for control experiment) and gently rotated for 1 h. After staining, we washed beads with PBST five times, resuspended in 50 µL 1X PBS buffer, and placed samples on ice prior to imaging. Stained beads were imaged by smTIRF microscopy using a simple flow cell created using Scotch double-sided tape, a cover glass (VMR, 22 × 50 mm, #1.5), and a glass slide (Corning, 75 × 25 mm). After loading ~200 beads into the flow cell, the channel was washed with 1X PBS five times and then sealed with nail polish to prevent evaporation during the imaging. For one bead in the field of view, we recorded 10 sequential images at 100 ms intervals. To quantify individual pHMC molecules, we counted the number of bright spots using “Find maxima” in ImageJ with an intensity prominence of 40 and spots with different coordinates were summed across all 10 images.
Shear modulus measurement
To access the modulus of rigidity/shear modulus of each code, we made NIPAm slabs containing identical ingredients as the 21 codes and then added 50 µL of each code to the ~15 mm diameter wells in the lid of 24-well plate. After 2-min UV polymerization, we washed the slab three times and rehydrated in colorless RPMI. We then measured the shear modulus (modulus of rigidity) using the TA rheometer (HR 10, Discovery Hybrid Rheometer) with a 20 mm parallel plate associated with Peltier plate Steel plus Solvent Well. The strain and angular frequency were set to 0.015% and 0.1 rad/s to 100 rad/s with 5 points per decade, respectively. The shear modulus is the root sum square of the storage modulus and loss modulus.
Quantifying bead shrinking and swelling with changes in temperature
All bead imaging used a small dissection scope (AmScope) equipped with a 20X objective with either a 0.5X relay lens or a 1X relay lens (for the force multiplexing beads) and a Thorlabs camera (DCC3240M). Prior to imaging, we washed SA-coated ‘smart’ beads with PBST three times, resuspended in colorless RPMI buffer, and then loaded them into a flow cell. After bead loading, we sealed the flow cell with nail polish (to prevent evaporation) and placed the flow cell on an indium tin oxide (ITO) glass slide (Bioscience Tools, TC-1-100s). While imaging continuously, we: (1) heated the sample from room temperature (25°C) to 37°C for 1 min (the heating ramp took ~8 sec), (2) cooled the sample to 34°C (the cooling ramp took ~30 sec) and then maintained the sample at 34°C for 2 min. Finally, we quantified bead edges from bright field images by tracing bead edges for every frame using ImageJ and plotting the Feret diameter over time.
‘Smart’ bead loading
Prior to loading ‘smart beads’, we treated microwell devices with air plasma for 4 min at 150 W plasma (Femto, Diener). Using a syringe pump, we then immediately filled the device with PBST 100 µL/h to expel the air trapped inside each well (for ~10 min) and subsequently flowed PBST at 20 µL/h for 1 h to block the surface. To load beads, we pooled ~10 µL of resuspended solution from each code, washed with PBST one time, and then resuspended in 20 µL PBST. This bead mixture was loaded into the microwell device at 50 µL/h to fill the chamber and then at 10 µL/h for ~ 2h in order to fill most of the wells with beads. We then rinsed the device with PBST at 100 µL/h to flush away extra beads before introducing cells.
T cell staining
To monitor TCR signaling, Cal-520 AM (AAT Bioquest, Inc.) was used to stain T cells and observe intracellular calcium flux associated with early T cell activation. Briefly, cells were washed, resuspended at 2 million cells/mL in 600 µL of colorless RPMI 1640 (Life technology), 3% (v/v) FBS, 0.02% (w/v) Pluronic F-127 (Sigma-Aldrich, 3% w/v in PBS buffer) and 2 µM Cal-520 (AAT Bioquest, 2 mM in DMSO), and incubated for 40 min at 37°C. During this incubation, cells were resuspended by gently pipetting the solution every 10 min. T cells were then washed with 1 mL colorless RPMI 1640 at RT and resuspended in 60 µL colorless RPMI 1640 with 3% (v/v) FBS at RT prior to loading into the device.
Cell loading
During cell staining, the microwell device was washed with colorless RPMI 1640 at 50 µL/h for 40 min at RT. The stained cells were then loaded into the microwell chip at 30 µL/h for 5 min and then incubated for 10 min to allow stained T cells to settle on top of the beads within the microwells. To ensure even cell loading, we then removed the syringe containing the stained cells from the device input, resuspended cells again via repetitive cycles of withdrawing and injection, and then repeated cell loading from the opposite inlet via an identical procedure. Repeating this injection twice allowed most of the beads to be associated with at least one cell. After loading, we washed away unbound cells using colorless RPMI 1640 supplemented with 3% (v/v) FBS at a flow rate of 20 μL/h for 30 min and then incubated at RT for another 30 min. Based on device microwell dimensions, flow rates <50 μL/h only generated drag forces <4 pN, preventing potential force-induced T cell activation during the loading and washing steps; cell loading was also carried out at room temperature to avoid generating any thermo-responsive forces during cell loading.
Ca2+ flux imaging
Time-lapse imaging experiments were performed on an automated inverted microscope (Nikon Eclipse Ti, Nikon) with a motorized filter turret. For 10-min Ca2+ imaging, exposure times were kept 300 ms during all experiments to prevent pixel intensity saturation. Images were acquired at × 4 magnification (S Plan Fluor ELWD 20x Ph1 ADM; Nikon) with 2×2 binning on a sCMOS Andor camera (Zyla 4.2, Andor Technology plc., Belfast, Northern Ireland) using µManager Software. To heat and cool beads, the entire microwell chip assembly was placed on an ITO glass slide mounted on the ASI stage. To exert force on bead-associated T cells, the chip was heated to and maintained at 37°C for 1 min and then cooled to and kept at 34°C for 2 min. Immediately after cooling, we acquired a total of 150 Ca2+ fluorescence images at 4 s intervals.
‘Smart bead’ imaging
To identify cells within each well, we first imaged the device via bright-field imaging with 2x2 binning using a 4x objective. To identify embedded spectral codes within each bead, we then illuminated the device from above using 292 nm excitation via a Xenon arc lamp (Lambda LS, Sutter Instruments, Novato, CA) equipped with an automated filter wheel (Lambda 10-2, Sutter Instruments, Novato, CA) containing a 292/27 bandpass excitation filter (Semrock, Rochester, NY) paired with UG11 absorptive glass (Newport, Irvine, CA). Emitted light was passed through an additional UV blocking filter mounted within a custom 3D printed holder mounted over the objective and then collected within Ln-channels using nine emission filters (435/40, 474/10, 536/40, 546/6, 572/15, 620/14, 630/92, 650/13, and 780/20 nm). For each image, we then identified all beads and determined the Ln ratios most likely to have produced the observed spectra associated with each pixel via linear unmixing relative to a series of Ln reference spectra as described previously 7. Finally, we created a matrix associating each microwell (indexed by row and column) with the spectral code of the bead within it.
Image analyses
Ca2+ fluxes for individual cells were analyzed in ImageJ. First, we duplicated the full stack of the fluorescence images. For one replicate, we segmented individual cells by finding all local maxima using “Find maxima” in ImageJ with an intensity prominence of 350. For the other replicate, we generated thresholded images using the triangle method and converted to binary images. We then combined the segmented images with the thresholded images using the “AND” operator under image calculator to identify individual cells. For Ca2+ measurements, we: (1) selected a region of interest associated with each cell using the particle analysis tool in ImageJ with a size from 4-pixel units to infinity, and then (2) recorded fluorescence signals and centroids across 150 images. We verified that cells do not move out of ROIs during the course of the experiment for Ca2+ analyses by tracking the centroid of each individual cell using the MultiTracker plugin. We then calculated time-lapse fluorescent intensities for selected ROIs and assigned each to a cell number with unique x and y coordinates. Signals were analyzed in Matlab (MathWorks) by custom written scripts and can be provided upon request. Briefly, we: (1) extracted the spatial coordinates of centers of individual microwells from the bright-field image, (2) converted spatial coordinates to microwell row and column numbers, (3) assigned each cell to a specific microwell by comparing cell and microwell centroids and assuming a 7.5 µm well diameter, and then (4) associated traces for each cell with the embedded code (and thus the peptide sequence) of the bead present within that microwell.
Ca2+ traces were normalized for each cell by plotting the fluorescence ratio at each time point divided by the measured intensity at time zero. Integrated Ca2+ signals were calculated by subtracting 1 from each timepoint and integrating Ca2+ traces over 10 min; all cells with an integrated Ca2+ signal > 0 were considered ‘positive’ cells.
Bootstrap Hypothesis Testing
To calculate bootstrapped p-values for each peptide tested using BATTLES, we iteratively: (1) pooled integrated Ca2+ signals of all peptides, (2) calculated the number of measurements associated with the peptide of interest (n) and for this entire pool (m), (3) sampled n and m observations with replacement from the merged pool, and (4) calculated the difference (t*) between the mean of the first n observations and mean of the second m observations. In each case, each observation was the fold-change of a particular integrated Ca2+ signal relative to the mean Ca2+ signal across all measurements. We repeated this procedure 100,000 times and then estimated the probability that an observed distribution was statistically significantly different from the pooled distribution as follows:
For TCR589 and TCR55 cells interacting with 21 peptides, we applied a Bonferroni correction for multiple hypothesis testing at a significance of 0.05 (0.05/21 = 0.0024). For concentration multiplex experiments, we pooled specific peptides at a given concentration with the same peptide at other concentrations for bootstrap hypothesis testing and therefore determined a Bonferroni-corrected significance threshold of p = 0.013 (0.05/4). For DMF5 T cell interacting with two different peptide classes, we pooled all 5 peptides within each class plus the Tax peptide and therefore determined a Bonferroni-corrected significance threshold of p = 0.008 (0.05/6).