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A bead-based method for high-throughput mapping of the sequence- and force-dependence of T cell activation

Nature Methods volume 19, pages 1295–1305 (2022)Cite this article

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Abstract

Adaptive immunity relies on T lymphocytes that use αβ T cell receptors (TCRs) to discriminate among peptides presented by major histocompatibility complex molecules (pMHCs). Identifying pMHCs capable of inducing robust T cell responses will not only enable a deeper understanding of the mechanisms governing immune responses but could also have broad applications in diagnosis and treatment. T cell recognition of sparse antigenic pMHCs in vivo relies on biomechanical forces. However, in vitro screening methods test potential pMHCs without force and often at high (nonphysiological) pMHC densities and thus fail to predict potent agonists in vivo. Here, we present a technology termed BATTLES (biomechanically assisted T cell triggering for large-scale exogenous-pMHC screening) that uses biomechanical force to initiate T cell triggering for peptides and cells in parallel. BATTLES displays candidate pMHCs on spectrally encoded beads composed of a thermo-responsive polymer capable of applying shear loads to T cells, facilitating exploration of the force- and sequence-dependent landscape of T cell responses. BATTLES can be used to explore basic T cell mechanobiology and T cell-based immunotherapies.

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Fig. 1: Overview of BATTLES assay and thermo-responsive beads.
Fig. 2: Thermo-responsive beads can display 21 different peptide sequences at physiological densities.
Fig. 3: Activation responses for TCR589-transduced T cells interacting with a 21-peptide library.
Fig. 4: Activation responses for TCR55-transduced T cells interacting with 21-peptide library.
Fig. 5: Multiplexing sequence, applied ramping force, and displayed pMHC densities using BATTLES.
Fig. 6: Activation responses for the clinically tested DMF5 TCR system interacting with 11 peptide sequences from two classes.

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Data availability

All data required to reproduce all figures have been deposited to an OSF repository (https://osf.io/xs7zf/). Source data are provided with this paper.

Code availability

AutoCAD designs (parallel flow focuser and microwell device) and T cell Ca2+ flux data have been deposited to an OSF repository (https://osf.io/xs7zf/).

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Acknowledgements

This work was supported by National Institutes of Health (NIH) grant nos. 1DP2GM123641 (awarded to P.M.F.) and R01GM107132, and a Stanford Bio-X Interdisciplinary Initiatives seed grant. K.C.G. is an investigator of the Howard Hughes Medical Institute, and is also supported by grant nos. NIH-5R01AI103867, U19AI057229, Mathers Foundation and Ludwig Foundation support. P.M.F. is a Chan Zuckerberg Biohub Investigator and acknowledges the support of a Sloan Research Foundation Fellowship. Y.F. is a Cancer Research Institute Postdoctoral Fellow. X.Z. is funded by a Stanford Bio-X seed grant. A.K.W. was funded by the Natural Sciences and Engineering Research Council of Canada Postdoctoral Fellowship. Part of this work was performed at the Stanford Nano Shared Facilities, supported by the National Science Foundation under award no. ECCS-1542152. We also thank E. Appel and D. Chan for helpful discussions concerning polymers and help with shear modulus measurements and Z. Bryant and P.V. Ruijgrok for help with TIRF microscopy.

Author information

Authors and Affiliations

  1. Department of Genetics, Stanford University, Stanford, CA, USA

    Yinnian Feng, Adam K. White & Polly M. Fordyce

  2. Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA

    Xiang Zhao & K. Christopher Garcia

  3. Department of Structural Biology, Stanford University, Stanford, CA, USA

    K. Christopher Garcia

  4. Howard Hughes Medical Institute, Chevy Chase, MD, USA

    K. Christopher Garcia

  5. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Polly M. Fordyce

  6. ChEM-H Institute, Stanford University, Stanford, CA, USA

    Polly M. Fordyce

  7. Chan Zuckerberg BioHub, San Francisco, CA, USA

    Polly M. Fordyce

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  1. Yinnian Feng
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Contributions

Y.F. conceptualized the platform and validation experiments. X.Z. made all the T cell lines and generated pMHC complexes refolded with UV-labile peptides. Y.F., X.Z. and A.K.W. analyzed data. K.C.G. and P.M.F. provided funding, resources, mentorship and project supervision. Y.F., X.Z., A.K.W., K.C.G. and P.M.F. wrote the paper.

Corresponding author

Correspondence to Polly M. Fordyce.

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Competing interests

Stanford University and Chan Zuckerberg Biohub have filed a provisional patent application (US Provisional Patent application no. 63/108,162) on the BATTLES technology described here, and Y.F., X.Z., A.K.W., K.C.G. and P.M.F. are named inventors.

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Nature Methods thanks Ellis L. Reinherz and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Madhura Mukhopadhyay, in collaboration with the Nature Methods team.

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Extended data

Extended Data Fig. 1 Generation of thermo-responsive beads using a microfluidic parallel flow focusing system.

(a) Bead generation setup that includes a PDMS parallel flow focusing device, a low-cost inverted microscope, and two syringe pumps for injection of aqueous and oil phases. Generated droplets were collected into a 24-well plate. (b) Images of droplet generation and collection at position 1 and position 2 within the PDMS device (left), with an average droplet radius of 16.1 ± 0.4 μm (right). (c) Image of thermo-responsive beads after UV polymerization of droplets and rehydration in PBST buffer (left), with an average bead radius of 23.75 ± 1.55 μm (right). Scale bars: 100 μm in b and 50 μm in c. Images from (b) to (c) represent 21 independent experiments.

Source data

Extended Data Fig. 2 Modulus of rigidity κ measurement using thermo-responsive slabs.

(a) Procedure to generate thermo-responsive slabs from an identical formulation as thermo-responsive beads followed by the measurement of κ by TA rheometer. (b) Measured storage modulus, loss modulus, and modulus of rigidity as a function of angular frequency; κ was determined from the root sum square of the storage modulus and loss modulus at each angular frequency and then averaged.

Source data

Extended Data Fig. 3 Calculated coding capacity for thermo-responsive beads.

(a) Matrix of Sm/Eu and Dy/Eu cluster variances as a function of Sm and Dy cluster medians, respectively. (b) Calculated target ratios for a thermo-responsive bead code set with 5-standard deviation spacing between cluster medians. (c) Number of calculated code clusters when requiring 3-, 4-, 5- and 6-standard deviation (STD) spacing between each code cluster.

Source data

Extended Data Fig. 4 Single molecule TIRF fluorescence imaging analysis of pMHC molecules immobilized on 12 thermo-responsive beads at the bead/glass interface.

(a) TIRF images (left) and integrated analysis (counts of bright punctae across all images, right) for a single bead from code 8 bearing biotinylated VPLTEDALL/B35 complexes. In each image, the white circle indicates the radius of the settled bead and the orange square indicates the region of interest used to estimate pMHC density. (b) Representative TIRF images (left) and integrated analysis (counts of bright punctae across all images, right) for a single negative control bead. In each experiment, biotinylated pMHC molecules were detected by incubation with PE-labeled anti-β2 mAb (clone: A17082A) immunofluorescence. Detailed bead preparation methods are found in Methods; the orange square ROI is ~840 μm2. We acquired images similar to those shown in (a) and (b) across 10 and 5 independent experiments, respectively. Scale bars: 50 µm.

Extended Data Fig. 5 Measured Ca2+-induced fluorescence as a function of time for TCR589-transduced T cells interacting with 21 different peptide sequences.

(a) All measured traces. (b) All positive traces with the indicated numbers of positive cells and measured cells and positive percentages. Light gray lines indicate traces for individual cells; dark grey lines indicate the mean signals. Light blue shade represents SEM.

Source data

Extended Data Fig. 6 Measured integrated Ca2+ fluorescence signals and estimated significance for TCR55 negative controls.

(a) Left: Integrated Ca2+ signals for each positive cell (light grey markers) and the mean integrated Ca2+ signal (light grey square) at 34 °C. Right: Estimated p-value vs. log2-transformed mean fold change for TCR55-tranduced T cells interacting with 21 different peptide sequences at 34 °C. Grey box indicates Bonferroni-corrected p-value at a significance of 0.05 (p = 0.0024). Correspondence between thermo-responsive bead spectral code and displayed peptide sequence is shown at right. n = 1320 cells in total. (b) Left: Integrated Ca2+ signals for each positive cell (light grey markers) and the mean integrated Ca2+ signal (light grey square) at 37 °C. Right: Estimated p-value vs. log2-transformed mean fold change for TCR55-tranduced T cells interacting with 21 different peptide sequences at 37 °C. Grey box indicates Bonferroni-corrected p-value at a significance of 0.05 (p = 0.0024). Correspondence between thermo-responsive bead spectral code and displayed peptide sequence is shown at right. n = 845 cells in total. The box width in (a) and (b) defines the interquartile range (IQR) and whiskers extend to 1.5 × IQR in either direction. p-values were calculated via two-sided bootstrapping as detailed in Methods. (c) Integrated Ca2+ signals for all positive cells (dark grey markers) at 34 °C, 37 °C and with expansion force applied (bars and error bars indicate mean and standard deviation, respectively; positive cell numbers and percentages are indicated above each bar.

Source data

Extended Data Fig. 7 Measured integrated Ca2+ fluorescence signals and estimated significance for TCR55 interacting with either: (1) PEG-DA beads (which do not change size upon a small temperature change) bearing the stimulatory peptide VPLTEDALL; (2) thermo-responsive NIPAM beads bearing the non-stimulatory peptide VPIVEDSFL; and (3) thermo-responsive NIPAM beads bearing the stimulatory peptide VPLTEDALL.

(a) Results at 34 °C alone, (b) results at 37 °C alone, and (c) results for heating to 37 °C and then cooling to 34 °C. Each panel shows: (left) integrated Ca2+ signals for each positive cell (light grey markers) and the mean integrated Ca2+ signal (light grey square) and (right) estimated p-value vs. log2-transformed mean fold change for TCR55-tranduced T cells interacting with each peptide-bead combination. Positive cell percentages are indicated above each bar. Grey box indicates Bonferroni-corrected p-value at a significance of 0.05 (p = 0.0167). Correspondence between embedded spectral code and displayed peptide sequence is shown at right. In all box plots, the box width defines the interquartile range (IQR) and whiskers extend to 1.5 × IQR in either direction. p-values were calculated via two-sided bootstrapping as detailed in Methods.

Source data

Extended Data Fig. 8 Changes in bead radii as a function of temperature for bead codes containing different SAc concentrations.

(a) Measured radii as a function of time for thermo-responsive beads per code upon changing the temperature to 37 °C at time t = 0 s and to 34 °C at time t = 60 s; all traces are offset such that initial bead radii are set to the mean radius for each code at time t = 0 s. The red portion of each trace indicates bead heating from RT to 37 °C; the blue portion of each trace indicates bead cooling from 37 °C to 34 °C. (b) Mean (marker) and standard deviation (error bar) for change in radius upon cooling for beads from each code. n = 5-9 beads for each code; the full dataset is available for download in the OSF repository.

Source data

Extended Data Fig. 9 Force multiplex for two TCR55 replicates.

(a) Measured integrated Ca2+ fluorescence signals for each positive cell (light grey markers) and the mean integrated Ca2+ signal (light grey square) for two TCR55 replicates interacting with 4 different peptides at three force ramps for Replicate 1 (top) and Replicate 2 (bottom). The box width defines the interquartile range (IQR) and whiskers extend to 1.5 × IQR in either direction. n = 1498 cells and 1256 cells for rep. #1 and rep. #2, respectively. (b) Correlation plot of the means of integrated Ca2+ signals between two replicates. Dashed black line indicates the line with a slope equal to 1; red dashed line indicates a linear regression. Error bars indicate SEM. (c) Integrated Ca2+ signal as a function of applied force for 4 peptides for Replicate 2. Zero force data is from a TCR55 control experiment where the temperature was maintained at 34 °C; error bars indicate SEM. n = 1256 cells for force ramps experiments and 1320 cells at zero force ramp. Error bars indicate SEM.

Source data

Extended Data Fig. 10 Sequence and concentration multiplexed measurements for two TCR55 replicates.

(a) Measured integrated Ca2+ fluorescence signals for each positive cell (light grey markers) and the mean integrated Ca2+ signal (light grey square) for two TCR55 replicates interacting with 4 different peptides at three pMHC concentrations. The box width defines the interquartile range (IQR) and whiskers extend to 1.5 × IQR in either direction. n = 2535 cells and 1699 cells for rep. #1 and rep. #2, respectively. (b) Top row: mean integrated Ca2+ signals for positive cells interacting with VPLTEDALL, VPITEDSQL, VPLTEDAEL and IPLTEEAEL peptides at 1X, 7X and 27X pMHC concentrations and the summarized integrated Ca2+ signals for four selected peptides under three pMHC concentrations for replicate 2. Error bars indicate SEM. (c) Estimated p-value (calculated via two-sided bootstrapping, see Methods) vs. log2-transformed mean fold change of integrated Ca2+ signals for 4 peptides at 3 different pMHC concentrations; grey area represents p-value>0.013 (Bonferroni-corrected p-value at a significance of 0.05). Correspondence between thermo-responsive beads spectral code and displayed peptide sequence combined its concentration is shown at right. (d) Correlation plot of the means of integrated Ca2+ signals between two replicates. Dashed black line indicates the line with a slope equal to 1; red dashed line indicates a linear regression. Error bars indicate SEM.

Source data

Supplementary information

Supplementary Information

Supplementary Methods, Discussion, Figs. 1–24 and Tables 1–7.

Reporting Summary.

Peer Review File.

Supplementary Video 1

Parallel flow focuser for production of thermo-responsive beads with linear amplification (four flow-focusing channels).

Supplementary Video 2

Thermo-response of the thermo-responsive beads with 55 mM sodium acrylate during the heating and cooling process. Code 9 was used in this video.

Supplementary Video 3

Initial bead loading followed by cell loading before on-chip imaging.

Supplementary Video 4

T cells remained in contact with bead surfaces throughout the heating and cooling process. Four representative wells are presented.

Supplementary Video 5

Representative Ca2+ flux within single TCR589 T cells interacting with pMHC-coated thermo-responsive beads bearing the stimulatory HIV-pol peptide (IPLTEEAEL) and the nonstimulatory peptide (VPLTEDAEL).

Supplementary Video 6

Thermo-response of the thermo-responsive beads with 50 mM (code 1), 55 mM (code 15) and 60 mM (code 9) sodium acrylate during the heating and cooling process.

Source data

Source Data Fig. 2

Thermo-responsive beads can display 21 different peptide sequences at physiological densities.

Source Data Fig. 3

Activation responses for TCR589-transduced T cells interacting with a 21-peptide library.

Source Data Fig. 4

Activation responses for TCR55-transduced T cells interacting with 21-peptide library.

Source Data Fig. 5

Multiplexing sequence, applied ramping force and displayed pMHC densities using BATTLES.

Source Data Fig. 6

Activation responses for the clinically tested DMF5 TCR system interacting with 11 peptide sequences from two classes.

Source Data Extended Data Fig. 1

Droplet radius and bead radius.

Source Data Extended Data Fig. 2

Measured storage modulus, loss modulus and modulus of rigidity as a function of angular frequency.

Source Data Extended Data Fig. 3

Calculated coding capacity for thermo-responsive beads.

Source Data Extended Data Fig. 5

Activation responses for TCR589-transduced T cells interacting with a 21-peptide library.

Source Data Extended Data Fig. 6

Measured integrated Ca2+ fluorescence signals for TCR55 negative controls.

Source Data Extended Data Fig. 7

Measured integrated Ca2+ fluorescence signals for TCR55 interacting with PEG-DA beads and thermo-responsive NIPAM beads bearing different stimulatory peptides at different conditions.

Source Data Extended Data Fig. 8

Changes in bead radii as a function of temperature for bead codes containing different SAc concentrations.

Source Data Extended Data Fig. 9

Multiplexing sequence and applied ramping force using BATTLES.

Source Data Extended Data Fig. 10

Multiplexing sequence and displayed pMHC densities using BATTLES.

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Feng, Y., Zhao, X., White, A.K. et al. A bead-based method for high-throughput mapping of the sequence- and force-dependence of T cell activation. Nat Methods 19, 1295–1305 (2022). https://doi.org/10.1038/s41592-022-01592-2

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