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. 2018 Jan 29;9:78. doi: 10.3389/fimmu.2018.00078

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Copyright © 2018 Liang, Mao, Sun, Li, Li, Yu, Ma, Gu, Zhang, Taniguchi and Li.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Lack of core fucosulation reduced interaction ability between T cell receptor (TCR) and pMHC-II and impaired the signaling via TCR. (A) ZAP70 phosphorylation of Fut8^+/+OT-II T–B cells. (T) B cell conjugate formation was initiated by centrifuging together CD4⁺ T cells with or without 1 µg/mL OVA_323–339-loaded B cells. The blots were probed by anti-pZAP70 Ab and anti-ZAP70 Ab. Data are representative of three independent experiments. Densitometric analysis of the bands of pZAP70 normalized against ZAP70. Data are shown as the mean ± SD (**p < 0.01). (B) ZAP70 phosphorylation of Fut8^+/+OT-II CD4⁺ T cells with or without fucosidase treatment. Purified CD4⁺ T cells were treated with or without 100 mU Bovine Kidney Fucosidase, and then coincubated with 1 µg/mL OVA_323–339 loaded Fut8^+/+OT-II B cells for 30 min. The blots were probed by anti-pZAP70 Ab and anti-ZAP70 Ab. Data are representative of three independent experiments. Densitometric analysis of the bands of pZAP70 normalized against ZAP70. Data are shown as the mean ± SD (**p < 0.01; ***p < 0.001). (C) Compare of activation of Fut8^+/+OT-II T + B cells and Fut8^−/−OT-II T + B cells. CD4⁺ T cells were conjugated with OVA_323–339 loaded B cell. After 30 min, the T–B cells were fixed and stained with anti-TCRβ and anti-CD69 Abs. Data are shown as mean values ± SD (**p < 0.01). (D,E) Distribution of MHC on the T and B cells. The T–B cells were fixed, permeabilized, and stained with Abs to MHC-II (green). Data are from three separate experiments. The ratio of MHC II intensity at the T–B cell conjugate site relative to non-conjugate areas. Data are shown as the mean values ± SD (**p < 0.01). (F) FACS analysis of the conjugates between B cells and CD4⁺ T cells. B cells (OVA_323–339-loaded or not loaded) were labeled with anti-MHC-II (FITC) and CD4⁺ T cells were labeled with anti-TCRβ (PE-Cy5). T and B cells were quickly mixed and conjugated by a brief centrifugation step. They were then incubated at 37°C for 30 min. A representative staining profile with anti-MHC-II and anti-TCRβ mAb is shown. The conjugate cells are double positive (MHC-II⁺TCRβ⁺) cells. (G) Percentage of T–B cell conjugates (both MHC-II and TCRβ positive cells) was calculated. Data are reported as the mean ± SD (**p < 0.01) in four independent experiments. (H) The secrition of IL-2 was downregulated in the culture media of Fut8^−/−OT-II T–B cells. OD values were measured at 492 nm using a microplate reader. Data are reported as the mean ± SD (**p < 0.01) in three independent experiments. (I) Proliferation of Fut8^+/+OT-II CD4⁺ T cells with OVA_323–339 loaded Fut8^+/+OT-II B cells. Fut8^+/+OT-II CD4⁺ T cells were purified and labeled with carboxyfluorescein diacetatesuccinimidyl ester (CFSE). Fut8^+/+OT-II CD4⁺ T cells were cocultivated with 1 µg/mL OVA_323–339 loaded Fut8^+/+OT-II B cells for 48 h, the divided cells were analyzed by FACS. One representative experiment is shown. M1–M4 indicates daughter cell populations which have subsequently lost half of their CFSE signal.

Lack of core fucosulation reduced interaction ability between T cell receptor (TCR) and pMHC-II and impaired the signaling via TCR. (A) ZAP70 phosphorylation of Fut8^+/+OT-II T–B cells. (T) B cell conjugate formation was initiated by centrifuging together CD4⁺ T cells with or without 1 µg/mL OVA_323–339-loaded B cells. The blots were probed by anti-pZAP70 Ab and anti-ZAP70 Ab. Data are representative of three independent experiments. Densitometric analysis of the bands of pZAP70 normalized against ZAP70. Data are shown as the mean ± SD (**p < 0.01). (B) ZAP70 phosphorylation of Fut8^+/+OT-II CD4⁺ T cells with or without fucosidase treatment. Purified CD4⁺ T cells were treated with or without 100 mU Bovine Kidney Fucosidase, and then coincubated with 1 µg/mL OVA_323–339 loaded Fut8^+/+OT-II B cells for 30 min. The blots were probed by anti-pZAP70 Ab and anti-ZAP70 Ab. Data are representative of three independent experiments. Densitometric analysis of the bands of pZAP70 normalized against ZAP70. Data are shown as the mean ± SD (**p < 0.01; ***p < 0.001). (C) Compare of activation of Fut8^+/+OT-II T + B cells and Fut8^−/−OT-II T + B cells. CD4⁺ T cells were conjugated with OVA_323–339 loaded B cell. After 30 min, the T–B cells were fixed and stained with anti-TCRβ and anti-CD69 Abs. Data are shown as mean values ± SD (**p < 0.01). (D,E) Distribution of MHC on the T and B cells. The T–B cells were fixed, permeabilized, and stained with Abs to MHC-II (green). Data are from three separate experiments. The ratio of MHC II intensity at the T–B cell conjugate site relative to non-conjugate areas. Data are shown as the mean values ± SD (**p < 0.01). (F) FACS analysis of the conjugates between B cells and CD4⁺ T cells. B cells (OVA_323–339-loaded or not loaded) were labeled with anti-MHC-II (FITC) and CD4⁺ T cells were labeled with anti-TCRβ (PE-Cy5). T and B cells were quickly mixed and conjugated by a brief centrifugation step. They were then incubated at 37°C for 30 min. A representative staining profile with anti-MHC-II and anti-TCRβ mAb is shown. The conjugate cells are double positive (MHC-II⁺TCRβ⁺) cells. (G) Percentage of T–B cell conjugates (both MHC-II and TCRβ positive cells) was calculated. Data are reported as the mean ± SD (**p < 0.01) in four independent experiments. (H) The secrition of IL-2 was downregulated in the culture media of Fut8^−/−OT-II T–B cells. OD values were measured at 492 nm using a microplate reader. Data are reported as the mean ± SD (**p < 0.01) in three independent experiments. (I) Proliferation of Fut8^+/+OT-II CD4⁺ T cells with OVA_323–339 loaded Fut8^+/+OT-II B cells. Fut8^+/+OT-II CD4⁺ T cells were purified and labeled with carboxyfluorescein diacetatesuccinimidyl ester (CFSE). Fut8^+/+OT-II CD4⁺ T cells were cocultivated with 1 µg/mL OVA_323–339 loaded Fut8^+/+OT-II B cells for 48 h, the divided cells were analyzed by FACS. One representative experiment is shown. M1–M4 indicates daughter cell populations which have subsequently lost half of their CFSE signal.