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Biomimetic cell stimulation with a graphene oxide antigen-presenting platform for developing T cell-based therapies

Nature Nanotechnology (2024)Cite this article

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Abstract

Chimeric antigen receptor (CAR)-engineered T cells represent a front-line therapy for cancers. However, the current CAR T cell manufacturing protocols do not adequately reproduce immunological synapse formation. Here, in response to this limitation, we have developed a flexible graphene oxide antigen-presenting platform (GO-APP) that anchors antibodies onto graphene oxide. By decorating anti-CD3 (αCD3) and anti-CD28 (αCD28) on graphene oxide (GO-APP3/28), we achieved remarkable T cell proliferation. In vitro interactions between GO-APP3/28 and T cells closely mimic the in vivo immunological synapses between antigen-presenting cells and T cells. This immunological synapse mimicry shows a high capacity for stimulating T cell proliferation while preserving their multifunctionality and high potency. Meanwhile, it enhances CAR gene-engineering efficiency, yielding a more than fivefold increase in CAR T cell production compared with the standard protocol. Notably, GO-APP3/28 stimulated appropriate autocrine interleukin-2 (IL-2) in T cells and overcame the in vitro reliance on external IL-2 supplementation, offering an opportunity to culture T cell-based products independent of IL-2 supplementation.

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Fig. 1: Design and characterization of a cell-sized GO-APP for displaying αCD3 and αCD28 (GO-APP3/28).
Fig. 2: The applications of GO-APP3/28 in T cell culture and CAR T cell manufacture.
Fig. 3: The applications of GO-APP3/28 in CAR T cell therapy for cancer.
Fig. 4: Mechanism study of GO-APP3/28-stimulated T cell activation.

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Data availability

All deep RNA-sequencing data and single-cell RNA-sequencing data are available in GEO with accession number GSE233291. Further details regarding the data are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank the UCLA animal facility for providing animal support; the UCLA TCGB facility for providing RNA-seq services; the UCLA CFAR Virology Core for providing human cells; the UCLA Broad Stem Cell Research Centre (BSCRC) Flow Cytometry Core Facility for cell sorting support; the UCLA Electron Imaging Center of Nanomachines for TEM support; and the UCLA Materials Structure Characterization Laboratory (MSCL) for SEM support. We thank Y. Cui for the support on the negative stain of TEM samples. This work was supported by a BSCRC-CNSI Stem-cell Nanomedicine Initiative Planning Award (to L.Y. and Y.H.), an Office of Naval Research grant (grant no. N000142112285, to Y.H.), an Ablon Scholars Award (to L.Y.), and NIH/NHLBI grants (grant nos. R01HL129727 and R01HL159970, to T.H.). E.Z. is a postdoctoral fellow supported by a T32 fellowship (UCLA and Caltech integrated Cardiovascular Medicine for Bioengineers, grant no. T32HL144449). J.Y. is a predoctoral fellow supported by the UCLA BSCRC Predoctoral Fellowship. Y.-R.L. is a postdoctoral fellow supported by a UCLA MIMG M. John Pickett Postdoctoral Fellow Award and a CIRM-BSCRC Postdoctoral Fellowship. Y. Zhou is a predoctoral fellow supported by the UCLA Dissertation Year Fellowship. J.B. is a predoctoral fellow supported by the Tower Cancer Research Foundation Fellowship.

Author information

Author notes

  1. These authors contributed equally: Enbo Zhu, Jiaji Yu.

Authors and Affiliations

  1. Department of Materials Science and Engineering, University of California, Los Angeles, Los Angeles, CA, USA

    Enbo Zhu, Yang Liu & Yu Huang

  2. Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA

    Enbo Zhu, Jiaji Yu, Yan-Ruide Li, Yu-Chen Wang, Miao Li, Yu Jeong Kim, Yichen Zhu, Zoe Hahn, Yang Zhou, James Brown, Yuchong Zhang & Lili Yang

  3. Department of Bioengineering, University of California, Los Angeles, Los Angeles, CA, USA

    Enbo Zhu & Tzung Hsiai

  4. Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    Enbo Zhu & Tzung Hsiai

  5. Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, Los Angeles, CA, USA

    Feiyang Ma & Matteo Pelegrini

  6. Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, CA, USA

    Lili Yang

  7. Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    Lili Yang & Yu Huang

  8. Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Lili Yang

  9. California Nanosystems Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Yu Huang

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Contributions

Y.H., L.Y., E.Z., J.Y. conceived and designed the experiments. E.Z. and J.Y. led and contributed to all the experiments together. Y.-R.L. contributed to flow cell cytometry and in vivo antitumour tests. F.M., Y.-C.W., M.L. and M.P. processed the RNA-sequencing and single-cell sequencing analysis. Y.L. contributed to the GO preparation. Y.J.K., Y. Zhu and Y. Zhang contributed to cell culture. Z.H. contributed to ELISA. Y. Zhou contributed to animal experiments. J.B. and T.H. contributed to the organization of the data. E.Z., J.Y., L.Y. and Y.H. co-wrote the paper. All authors discussed the results and commented on the paper.

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Correspondence to Lili Yang or Yu Huang.

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Competing interests

Y.H., L.Y., E.Z. and J.Y. are inventors on patents relating to this study filed by the University of California, Los Angeles. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 The design and characterization of GO-APP3/28. (Related to Fig. 1).

a, An AFM image showing a representative as-prepared graphene oxide (GO) piece without any decoration. Scale bar: 5 μm. b, A representative AFM image showing a piece of modified GO decorated with antibodies (GO-APP3/28). Scale bar: 5 μm. c, UV-vis spectra analysis of the intermediate structures in the GO-APP3/28 preparation. d, Schematic of a spherical cap model for calculating the GO-APP3/28 contact area on T-cells; see Methods for details. Scale bar: 2 μm. e, A false-colored SEM image showing multiple well-dispersed T cells (blue) interacting with GO-APP3/28 (yellow). Scale bar: 10 μm. The experiment was repeated three times independently with similar results. f, Distribution of the contact area after counting n = 50 random T cells that were interacting with GO-APP3/28 and having views conducive to accurate counting. The average contact area is 75.82 ± 2.59 μm2 (mean values ± SEM).

Extended Data Fig. 2 Method development of GO-APP3/28 in T cell culture. (Related to Fig. 1).

a-d, Comparison of T-cell activations using GO-APP3/28 and the mixture of GO-APP3 and GO-APP28. GO-APP3/28 showed superior performance, indicating the importance of concurrent CD3 and CD28 stimulation in close proximity for effective T cell activation. a, Experimental design. b, T cell proliferation (number of samples n = 4) Data are presented as mean values ± SEM. c, ELISA measurements of IL-2 production in the culture on day 7 (number of samples n = 4). Data are presented as mean values ± SEM. d, ELISA measurements of IFNγ production in the culture on day 7 (number of samples n = 4). Data are presented as mean values ± SEM. e-g, SEM images showing the as-prepared GO with different sizes: < 5 µm (e), 11.8 ± 2.6 µm (f), and > 80 µm (g). Diameter of pillar reference: 5 µm. (Related to Fig. 1c) Scale bar in (e): 20 μm. Scale bar in (f): 50 μm. Scale bar in (g): 100 μm. h, A false-colored low vacuum SEM image showing oversized (>80 µm) GO-APP3/28 sheets (yellow) enveloping multiple T cells (blue). (Related to Fig. 1e) Scale bar: 2 μm. i, Comparison of T-cell proliferation using GO-APP3/28 with different sizes (number of samples n = 4). Data are presented as mean values ± SEM. j, Gradient test of GO-APP3/28 concentration in stimulating T cell expansion. Seeding cell number normalized to 1 × 106 cells (number of samples n = 4). Data are presented as mean values ± SEM. k, 0.1 µg/ml GO-APP labeled with Alexa-488 in a hemacytometer. The fluorescent (GO-APP in green) and bright-field (grids in white) images overlap. Each small square is 0.25 mm × 0.25 mm × 0.1 mm. Experiments (e-h, k) were repeated three times independently with similar results. Experiments (e-h, k) were repeated three times independently with similar results. Multiple comparisons were performed using ordinary 2-way ANOVA, followed by Tukey’s multiple comparisons test. p values less than 0.05 were considered significant. *** denotes p value < 0.001; **** denotes p value < 0.0001.

Extended Data Fig. 3 The applications of GO-APP3/28 in T cell culture. (Related to Fig. 2).

a-f, Study of GO-APP3/28-activated T cell culture from healthy donor PBMCs. a, ELISA measurements of IFN-γ production on day 7 (number of samples n = 4). Data are presented as mean values ± SEM. (Related to Fig. 2d) b, Flow cytometry analysis of surface CD25 (IL-2 receptor) expression on T cells stimulated with the indicated methods on day 7 (number of samples n = 4). Data are presented as mean values ± SEM. (Related to Fig. 2d) c, Flow cytometry analysis of CD4+ and CD8+ populations in cultured T cells on day 12. (Related to Fig. 2c) d-f, Flow cytometry analysis showing the IL-2 intracellular expression levels in T cells stimulated by the indicated methods on day 7 with gating on CD4+ (d) and CD8+ (e) populations. f, Quantification of (d) and (e) (number of samples n = 3). Data are presented as mean values ± SEM. g-h, Study of T cell culture from PBMCs with a titration of external IL-2. g, Experimental design. h, T cell proliferation on day 12 (number of samples n = 4). Data are presented as mean values ± SEM. Multiple comparisons were performed using ordinary 2-way ANOVA, followed by Tukey’s multiple comparisons test. p values less than 0.05 were considered significant. ‘ns’ denotes not significant; ** denotes p value < 0.01; **** denotes p value < 0.0001.

Extended Data Fig. 4 The in vivo persistency and biodistribution of GO-APP3/28 CAR-T cells. (Related to Fig. 3).

a-b, Study of the in vivo persistency and biodistribution of GO-APP3/28 CAR-T cells in a human Raji-FG xenograft mouse model (see Fig. 3i for study design). a, Representative flow plots of Fig. 3j showing biodistribution of GO-APP3/28 CAR-T cells across tissues collected at the terminal analysis. b, Representative flow plots of Fig. 3k showing phenotyping of GO-APP3/28 CAR-T cells liver at the terminal analysis. c, Study of in vivo persistency and biodistribution of GO-APP3/28 CAR-T cells in a human AsPC-1-FG xenograft mouse model (see Fig. 3l for study design). Representative flow plots of Fig. 3p showing persistence of GO-APP3/28 CAR-T cells in tumor and peripheral blood at the terminal analysis.

Extended Data Fig. 5 Single-cell gene profiling of GO-APP3/28-stimulated T cell activation. (Related to Fig. 4).

a, Gene set enrichment analysis (GSEA) of CD8+ T cells for CD8+ T cell activation. (Related to Fig. 3i) b, GSEA of CD8+ T cells for cytokine production. (Related to Fig. 3j) c, GSEA of CD4+ T cells for cytokine production. (Related to Fig. 3m) d, Violin plot showing the expression distribution of the indicated genes encoding the master transcription factors of different Th subtypes in CD4+ T cells. (TBX21: TH1; GATA3: TH2; Foxp3: Treg; RORC: TH17).

Extended Data Fig. 6 Mechanism study of GO-APP3/28-stimulated T cell activation at the protein expression level. (Related to Fig. 4).

a-b, Flow cytometry analysis of GO-APP3/28 T cells and Beads3/28 T cells. a, Flow plots showing the indicated intracellular protein expression. b, Mean fluorescence intensity (MFI) measurement from flow cytometry showing the TCR and IL-2 signaling pathway molecules in GO-APP3/28 T cells and Beads3/28 T cells at the protein level (number of samples n = 3). Data are presented as mean values ± SEM. (Related to Fig. 4o) Pairwise comparisons were made using a 2-tailed Student’s t test. p values less than 0.05 were considered significant. ‘ns’ denotes not significant; * denotes p value < 0.05; ** denotes p value < 0.01; *** denotes p value < 0.001; **** denotes p value < 0.0001.

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Western blot source data, including molecular weight markers.

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Zhu, E., Yu, J., Li, YR. et al. Biomimetic cell stimulation with a graphene oxide antigen-presenting platform for developing T cell-based therapies. Nat. Nanotechnol. (2024). https://doi.org/10.1038/s41565-024-01781-4

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