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Multimodal probing of T-cell recognition with hexapod heterostructures

Nature Methods (2024)Cite this article

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Abstract

Studies using antigen-presenting systems at the single-cell and ensemble levels can provide complementary insights into T-cell signaling and activation. Although crucial for advancing basic immunology and immunotherapy, there is a notable absence of synthetic material toolkits that examine T cells at both levels, and especially those capable of single-molecule-level manipulation. Here we devise a biomimetic antigen-presenting system (bAPS) for single-cell stimulation and ensemble modulation of T-cell recognition. Our bAPS uses hexapod heterostructures composed of a submicrometer cubic hematite core (α-Fe2O3) and nanostructured silica branches with diverse surface modifications. At single-molecule resolution, we show T-cell activation by a single agonist peptide-loaded major histocompatibility complex; distinct T-cell receptor (TCR) responses to structurally similar peptides that differ by only one amino acid; and the superior antigen recognition sensitivity of TCRs compared with that of chimeric antigen receptors (CARs). We also demonstrate how the magnetic field-induced rotation of hexapods amplifies the immune responses in suspended T and CAR-T cells. In addition, we establish our bAPS as a precise and scalable method for identifying stimulatory antigen-specific TCRs at the single-cell level. Thus, our multimodal bAPS represents a unique biointerface tool for investigating T-cell recognition, signaling and function.

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Fig. 1: Hexapod-enabled molecular level interrogation of T-cell recognition.
Fig. 2: Hexapod-based biochemical investigation of T-cell calcium signaling.
Fig. 3: Magnetic hexapod-enabled torque on floating T cells.
Fig. 4: Hexapod-bAPS biophysical stimulation of T cells and CAR-T cells.
Fig. 5: Hexapod system enables precise and scalable identification of stimulatory CD8+ antigen-specific TCRs at the single-cell level.

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Data availability

Source data are also provided at https://osf.io/c5y3q/. Sequence data generated in this study are deposited in the National Center for Biotechnology Information Gene Expression Omnibus at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229249. Source data are provided with this paper.

Code availability

The source code for the customized magnet rotator used in this study can be accessed at https://github.com/MagneticRotor/rotating-magnetic-field. Sample codes for analyzing calcium signals using MATLAB and rotation magnetic field simulation using Wolfram Mathematica are available at https://osf.io/c5y3q/. All sequencing analysis was done using standard R packages.

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Acknowledgements

We thank M. Swartz for the generous gift of the OT-1 mice, J. Rowley for the generous gift of the NALM-6 cells, and H. Schreiber for the generous gift of the E6-1 Jurkat cells. We also thank L. Pulido and N. Ankenbruck for general technical assistance, M. Berthoud for technical assistance with the rotating magnetic field device, C. Labno for technical assistance with the FRET experiments, and T.-X. Zheng for providing support on magnetic field calculations. K.M. Watters is thanked for editing the manuscript. We acknowledge start-up grants from the University of Chicago (to B.T. and J.H.), and partial support from the University of Chicago Materials Research Science and Engineering Center (DMR-2011854; B.T.) and Chicago Immunoengineering Innovation Center (J.H.).

Author information

Author notes

  1. These authors contributed equally: Xiaodan Huang, Lingyuan Meng.

Authors and Affiliations

  1. Pritzker School of Molecular Engineering, University of Chicago, Chicago, IL, USA

    Xiaodan Huang, Lingyuan Meng, Guoshuai Cao, Yifei Hu, Min Chen, Thao Cao & Jun Huang

  2. Department of Chemistry, University of Chicago, Chicago, IL, USA

    Aleksander Prominski, Jiuyun Shi, Jiping Yue & Bozhi Tian

  3. Pritzker School of Medicine, University of Chicago, Chicago, IL, USA

    Yifei Hu

  4. The James Franck Institute, University of Chicago, Chicago, IL, USA

    Chuanwang Yang & Bozhi Tian

  5. Department of Physics, University of Chicago, Chicago, IL, USA

    Charles Gallagher

  6. The Institute for Biophysical Dynamics, University of Chicago, Chicago, IL, USA

    Bozhi Tian

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Contributions

Hexapod synthesis, quantification and characterization: L.M., X.H. and J.S. with input from B.T.; cell culture and cell rotation with hexapods: X.H., L.M., J.Y. and Y.H. with input from J.H.; CAR-T cell construction: Y.H.; T-cell calcium, flow cytometry and ELISA: X.H., M.C. and Y.H.; single-molecule imaging and analysis: X.H. and G.C.; rotation analysis, particle tracking and force estimation: L.M., X.H., A.P. and C.G.; magnetic field calculation and measurement: A.P., L.M. and C.Y.; FRET and 3D imaging: M.C., T.C. and X.H.; identification of antigen-specific TCRs with the hexapod system: X.H. and Y.H.; single-cell sequencing data analysis: G.C.; statistical analysis: L.M. and X.H.; figure preparation and manuscript writing: L.M., X.H., A.P., G.C., M.C., Y.H., T.C. and J.Y. with input from all authors; funding acquisition and supervision: B.T. and J.H. Conception and design of T-cell assays: J.H.

Corresponding authors

Correspondence to Jun Huang or Bozhi Tian.

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Competing interests

The University of Chicago has filed a provisional patent for the synthesis and application of hexapods for T-cell studies. B.T., J.H., X.H. and L.M. are co-inventors. All other authors have no competing interests.

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Nature Methods thanks Cheng Zhu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Madhura Mukhopadhyay, in collaboration with the Nature Methods team.

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Extended data

Extended Data Fig. 1 Hexapod synthesis and heterostructure protein modifications.

a, The synthesis and modification of hexapods: i) hematite cores are dispersed in PVP pentanol solution. The viscous solution prevents particle precipitation and hematite aggregation. Water forms emulsion droplets on hematite surface, while PVP and citrate help the oil/water phase separation. TEOS is the silica precursor, NH4OH is the catalyst for TEOS condensation, and the addition of C2H5OH forms a homogeneous solution in the droplets. Silica branches grow via tetraethoxysilane (TEOS) condensation, forming one branch on each facet of the cubic hematite core. ii) Schematic showing individual surface modification steps. b, 3D reconstructed fluorescent image of CD19-hexapods labeled with AF488-anti-CD19 fluorescent dye.

Extended Data Fig. 2 Determination of the ligand number on a hexapod tip by single-molecule imaging.

a, Cartoon illustration of ligand presentation by tetrameric-SA(A4). b, Representative image of PE-conjugated-hexapod and single PE molecules on the coverslip. Left to right: DIC image, fluorescent (PE) channel, segmentation of single PE molecules on the coverslip (arrow: single PE; square: hexapod), overlay of PE and DIC. c, Fluorescence intensity comparison between single PE molecules (n = 64 independent measurements) and hexapod tips (n = 23 independent measurements). Box boundaries span the 25th to 75th percentiles with the median marked, and all the data points from min to max presented. Data were examined from independent measurements and statistical analysis was performed using by Wilcoxon signed-rank test. d, Fluorescence signal along a line scan drawn through the position of a single molecule, either on the hexapod tip or the coverslip. The single peak confirms the presence of a single molecule and indicates its location on the hexapod tip or the coverslip. The experiment was repeated independently three times with similar results.

Source data

Extended Data Fig. 3 Induced calcium fluxes in CAR T cells.

a, Representative time-lapse images of the induced calcium fluxes in 1st generation CAR T cells using anti-CD3-hexapod or CD19-hexapod, respectively. Scale bar, 5 µm. The experiment was repeated independently five times with similar results. b, Calcium profiles (mean F/F0 ± SEM,) show that anti-CD3-hexapod (n = 5 independent experiments) induced stronger calcium than CD19-hexapod (n = 5 independent experiments) in 1st generation CAR T cells, indicating that TCR transduction has higher efficiency than CAR transduction. c, NALM-6 cell induced saturated calcium signals in CAR T cells. Scale bar, 5 µm. The experiment was repeated independently for six times with similar results. d, Calcium profile (mean F/F0 ± SEM) of NALM-6 cell-induced calcium (n = 6 independent experiments). NALM-6 cells serve as a positive control. e, Calcium intensity at each time point (1 frame per two seconds or n = 300 data points) of stimulated CAR T cells. Lines represent mean F/F0 ± SD. Statistical analysis was performed using one-way ANOVA test with Tukey’s multiple comparisons.

Source data

Extended Data Fig. 4 CD19-hexapod strongly colocalized with anti-CD19 CAR T cell.

a, Representative confocal images of a CD19-hexapod with an anti-CD19 CAR T cell. CAR T cells were transduced with anti-CD19-mGFP (monomeric green fluorescent protein), and the CA19-hexapod was labeled with an Alexa Fluor555-conjugated streptavidin. Scale bars, 5 μm. b, Histograms from the respective line scan quantifications. Fold intensities were calculated from the raw fluorescence intensities along the linear ROI (yellow) in the confocal images. c, The Pearson’s correlation coefficient (PCC) values for the anti-CD19 CAR T cell and CD19-hexapod overlay (0.79 ± 0.05, mean ± SEM, n = 3). Experiment was repeated independently for five times with similar results.

Source data

Extended Data Fig. 5 FRET assay using confocal microscopy.

a, Photobleaching of CD19-conjugated AF555-hexapod substantially increases the fluorescence of CAR conjugated GFP. Shown from left to right are excited donor (GFP-anti-CD19 CAR) images before bleaching, excited acceptor (AF555-CD19-hexapod) images before bleaching, excited donor images after bleaching, excited acceptor images after bleaching, calculated FRET efficiency images (pseudo-color, where a yellow color spectrum represents strong to weak FRET efficiency), and differential interference contrast (DIC) images. b, Substituting CD19 with anti-CD45, an antibody that links the hexapod to CAR-T cell by binding with CD45 on the cell surface (instead of CD19 CAR binding activity), significantly decreased the FRET efficiency. The experiments were conducted in the same manner as in a, except AF555-CD19-hexapod was replaced with AF555-anti-CD45-hexapod. Scale bar, 5 μm. The experiment was repeated independently for five times with similar results.

Extended Data Fig. 6 Hexapod-bAPS amplified 5C.C7 CD4+ T cell activation.

a, Representative histograms from sextuplicate independent staining experiments of primary naïve 5C.C7 CD4+ T cells. Staining of the unstained, unstimulated cells is shown in gray and is used for gating. Filled plots indicate co-cultured cells with (red) or without (blue) rotation. Left, CD69; right, CD25. b, Bar graphs of sextuplicate data depicting the mean fluorescence intensity (MFI) for CD69 or CD25 and the percentage of CD69 or CD25 positive cells. The expression levels of CD69 and CD25 were significantly amplified by magnetic rotation (that is, biophysical forces) in naïve 5C.C7 CD4+ T cells. c, Secretion levels of TNF-α and IL-2 in day 6-10 CD4+ T cell blasts were measured using an enzyme-linked immunosorbent assay (ELISA). Rotation significantly boosted the cytokine production compared to static hexapod (no rotation) groups. All statistical analyses were performed using one-way ANOVA tests with Bonferroni’s multiple comparisons. Results show individual replicates alongside the mean value ± SEM of multiple independent measurements (n = 6 cultures for b and n = 3 independent repeats for c).

Source data

Extended Data Fig. 7 Streptavidin hexapods do not stimulate T cells in the presence and absence of shear stresses.

Intracellular staining and ELISA were used to measure IFN-γ (a), TNF-α (b), and IL-2 (c) expressions. Additionally, surface staining measured CD25 (d) and CD69 (e) expressions in OT-1 T cells after a 12-hour incubation under various experimental conditions, as indicated by colors. Histograms show the expression of primary OT-1 CD8+ T cells, with sextuplicate independent experiments performed. Bar graphs were generated to depict the percentage of positive cells (left), mean fluorescence intensity (MFI) (middle), and cytokine level (right). These graphs were based on sextuplicate data obtained from staining primary CD8+ T cells. All statistical analyses were performed using one-way ANOVA tests paired with Bonferroni’s multiple comparisons. Data are represented as the mean value ± SEM with individual replicates (n = 6 independent repeats).

Source data

Extended Data Fig. 8 The hexapod system facilitates precise and scalable identification of stimulatory CD4+ antigen-specific TCRs at the single-cell level.

a, Distribution of clonotype annotations within the repertoire, computed across the entire repertoire (left) and only among those with a productive TCR (right). When compared to the known 5C.C7 CDR3 amino acid (aa) sequence (alpha: AAEASNTNKVV; beta: ASSLNNANSDYT), each cell was classified as either a ‘5C.C7’ T cell (with the corresponding TCR alpha and/or TCR beta CDR3 aa sequence), an ‘other’ T cell (with a different productive CDR3 aa sequence), or ‘NA’ if a cell appears in the scRNA-seq data without the corresponding TCR information (potentially due to limited depth or dropout). b, Uniform Manifold Approximation and Projection (UMAP) showing single cells with the respective TCR clonotype annotations color-coded. c, Gene Set Enrichment Analyses (GSEA) showing selected pathways enriched in 5C.C7 cells compared to other cells. A positive enrichment score indicates enrichment in 5C.C7 cells over other cells, and a negative enrichment score implies enrichment in other cells compared to 5C.C7 cells. P-value was generated from a permutation test and adjusted for multiple testing using the Benjamini–Hochberg procedure. The false discovery rate (FDR) was estimated via the FDR q-values. d, Normalized gene expression of selected genes that are differentially expressed between 5C.C7 and other T cells. Group-wise mean expression was calculated as the average scaled gene expression of all cells within that group, and the final values were capped to maximize the dynamic range of the color scale. Genes were grouped into functional categories related to 5C.C7 cell TCR sequence or function.

Supplementary information

Supplementary Information

Supplementary Methods, Figs. 1–21, Tables 1–3 and description of supplementary videos.

Reporting Summary

Supplementary Video 1

Calculated rotating magnetic fields in trajectory tracking studies.

Supplementary Video 2

Rotating hexapods in a rotating magnetic field.

Supplementary Video 3

A hexapod-bound cell in the rotating magnetic field.

Supplementary Video 4

3D imaging of a hexapod.

Source data

Source Data

Statistical source data with named tabs for each figure/extended data figure item. Also available at https://osf.io/c5y3q/.

Image Source Data

Unprocessed image source data for figures and extended data figures. Also available at https://osf.io/c5y3q/.

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Huang, X., Meng, L., Cao, G. et al. Multimodal probing of T-cell recognition with hexapod heterostructures. Nat Methods (2024). https://doi.org/10.1038/s41592-023-02165-7

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