Research ArticleCELL BIOLOGY

Promoting the activation of T cells with glycopolymer-modified dendritic cells by enhancing cell interactions

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Science Advances  20 Nov 2020:
Vol. 6, no. 47, eabb6595
DOI: 10.1126/sciadv.abb6595
  • Fig. 1 Schematic process of DC modification by glycopolymers.

    (A) Strategy for DC surface modification with glycopolymers. DCs were first matured and then transfected by HTP using a photoporation delivery system. Then, the synthetic glycopolymers were attached to the cell surface through the reaction between HTP anchors and the polymer end groups. (B) DC stably modified with glycopolymers on the cell surface promotes the efficiency of T cell activation. (C) Molecular structures of synthetic glycopolymers and graphic notes.

  • Fig. 2 DC cell surface modification with glycopolymers.

    (A) Schematic of HTP transfection with a photo perforation transfection system (PTS) was used. (B) Representative images showing HTP transfection. Nuclei were stained by DAPI (blue), and the HA-tagged HTP was stained by FITC-avidin (green). Scale bar, 100 um. (C) Quantification of transfection efficiency compared with Lip2000. ***P < 0.001 compared with Lip2000. Data are means ± SEM (n = 3). (D) Schematic of DC modification with glycopolymers. (E) Representative images showing green fluorescence on the DC cell surface. Nuclei were stained by DAPI (blue) and biotin-labeled poly-(MAG) (pMB) by FITC-avidin (green). (F) Representative images showing the modified DCs incubated in complete medium for specified times (1, 3, and 7 days). (G) Viability of engineered DC over the 7-day period. Data are means ± SEM (n = 3). N.D., not determined.

  • Fig. 3 Promotion of T cell activation by glycopolymer-engineered DCs.

    (A) Schematic of experiment on T cell proliferation and the release of cytokines. (B) Mean fluorescence intensity distribution of CFSE-stained T cells. (C) Mean fluorescence intensity of T cells induced by engineered and native DC. Data are means ± SEM (n = 3). “CON” represents T cells without induction by DCs, DC-T represents T cells induced by native DCs, “DC-pMAG-T” represents T cells induced by pMAG-modified DCs, and “DC-pMAM-T” represents T cells induced by pMAM-modified DCs. ***P < 0.001. (D and E) Cytokine release from T cells induced by different DCs. Data are means ± SEM (n = 3). **P < 0.01 and ***P < 0.001.

  • Fig. 4 Representation that T cells activated by glycopolymer-modified DCs have increased cancer cytotoxicity and the T cell specificity is not affected.

    (A) Schematic showing T cell induction process. B16 antigens were used to stimulate DC to present antigens to T cells, making the T cells specific to B16 (TB16), similarly for CT26 antigen making T cells specific to CT26 (TCT26). (B) Representative cell images after coculturing B16 with T cells: DC-T represents native DC-induced T cells, “pMAG-DC-T” represents T cells induced by pMAG-modified DC, and “pMAM-DC-T” represents T cells induced by pMAM-modified DC. (C) Representative data on LDH release from B16 after treatment with T cells induced by different kinds of DC. Data are means ± SEM (n = 3). ***P < 0.001. (D) Representative cell images after coculturing CT26 with T cells. (E) Representative data on LDH release from B16 after treatment with T cells induced by different kinds of DCs. Data are means ± SEM (n = 3). ***P < 0.001. (F) Showing that glycopolymer-modified DCs had no impact on T cell specificity. Representative cell images show TB16 and TCT26 cells cross-linked to B16 and CT26 cells, respectively. (G) Representative data on LDH release from B16 and CT26 after cross-treatment with specific T cells. Data are means ± SEM (n = 3). ***P < 0.001. OD, optical density.

  • Fig. 5 Glycopolymers modified on the DC cell surface were necessary for enhancing the interaction with T cells.

    (A) Image tracking of DC and T cell migration over time. (B) Frequency of contact between DC and T cell. Scale bar, 10 μm. (C) Duration of contact between DC and T cell. Data analysis using Tukey’s multiple comparison test, with T cell number n = 70. Data are means ± SEM (n = 3).*P < 0.05, **P < 0.01, and ***P < 0.001. (D) QCM measurements of interactions between pMAG with proteins PD-1 and CD40L on the T cell surface. (E) Quantitative analysis of protein adsorption difference. Data shown are means ± SEM (n = 3). ***P < 0.001 (t test). (F to H) Free glycopolymers that were added to the medium at concentrations of 0.1 mg/ml did not help to improve the interactions between DCs and T cells. (F) Image tracking of DCs and T cells migration over time. Scale bar, 10 μm. (G) The frequency of contact between DCs and T cells. (H) Duration of contact between DCs and T cells. Data analysis using Tukey’s multiple comparison test, with T cell number n = 70. Data are means ± SEM (n = 3). ***P < 0.001. (I to K) d-(+)-mannose (1 mg/ml) blocked the interactions between glycopolymer-modified DCs and T cells. (I) Image tracking of DC and T cell migration over time. Scale bar, 10 μm. (J) The frequency of contact between DCs and T cells. (K) Duration of contact between DCs and T cells. Data analysis using Tukey’s multiple comparison test, with T cell number n = 70. Data are means ± SEM (n = 3). **P < 0.01, and ***P < 0.001.

  • Fig. 6 Glycopolymer-modified BMDCs enhanced T cell viability that was inhibited by blocking sugar receptors on the T cell surface.

    (A) Representative images of BMDCs modified with pMAG. (B) TNF-α expression of T cells induced by pMAG-modified BMDCs. ***P < 0.001 compared with mDC. (C) IFN-γ expression of T cells induced by pMAG-modified BMDCs. (D) Representative image of B16 cancer cells treated by unmodified DC-induced T cells. (E) Representative images of B16 cancer cells treated by glycopolymer-engineered T cells. pMAG-DC-T represents T cells induced by MAG-modified DC, and pMAM-DC-T represents T cells induced by MAM-modified DC. (F) Representative images of B16 cancer cells treated by sugar receptor–blocking T cells. “IG-T” represents T cells incubated with glucose and then induced by pMAG-modified matured DCs. “IM-T” represents T cells incubated with mannose and then induced by pMAG-modified matured DCs. Scale bar, 10 μm. (G) B16 cancer cell viabilities treated by different BMDC-induced T cells. Data analysis using Tukey’s multiple comparison test. Data are means ± SEM (n = 3). *P < 0.05 compared with pMAG-DC-T, #P < 0.05 compared with pMAM-DC-T.

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