Journal: ImmunoHorizons

Article Title: STAP-1-derived peptide suppresses TCR-mediated T cell activation and ameliorates immune diseases by inhibiting STAP-1–LCK binding

doi: 10.1093/immhor/vlaf015

Figure Lengend Snippet: iSP1 inhibits TCR-mediated human T cell activation. (A) Jurkat cells were treated with FITC-conjugated R8-iSP1 (FITC-iSP1) or FITC-conjugated R8-free-iSP1 (FITC-R8(-)-iSP1) peptide for 30 min, and the uptake of peptides was detected by flowcytometry analysis. (B) Jurkat cells were preincubated with iCont or iSP1 for 30 min, and spontaneous proliferation was analyzed. Data are shown as mean ± SEM of 3 independent experiments ( n = 3). * P < 0.05, by Sidak’s multiple comparisons test. (C, D)Jurkat cells were preincubated with iCont or iSP1 for 30 min and stimulated with anti-CD3/anti-CD28 mAb (C) or PMA/Ionomycin (D) for 48 h (C) or 24 h (D), and IL-2 production in the supernatant was analyzed by ELISA. Data are shown as mean ± SEM of 4 independent experiments ( n = 4). *** P < 0.001, *** P < 0.0001, by Sidak’s multiple comparisons test. (E) Jurkat cell were preincubated with iCont or iSP1 for 30 min and stimulated with anti-CD3/anti-CD28 for indicated periods, and TCR signal transduction was analyzed by Western blotting (left panel), and band intensity was quantified by Image J software (right panel). Data are shown as mean ± SEM of 5 independent experiments ( n = 5). * P < 0.05, by Dunnett’s multiple comparisons test. (F) Human PBMCs were preincubated with iCont or iSP1 for 30 min and stimulated with immobilized anti-CD3/anti-CD28 for 48 h, Cell proliferation was evaluated by CellTiter-Glo Luminescent Cell Viability Assay. Data are shown as mean ± SEM of 3 independent experiments ( n = 3). **** P < 0.0001, by Sidak’s multiple comparisons test. (G)Myc-tagged hSTAP-1-expressing Jurkat cells were preincubated with iCont or iSP1 for 30 min and stimulated with anti-CD3/anti-CD28 for indicated periods. Subsequently, association of Myc-tagged hSTAP-1 with endogenous LCK was analyzed by coimmunoprecipitation assay, followed by Western blotting. For immunoprecipitation experiment, rabbit IgG was used as isotype control.

Article Snippet: Anti-mouse CD3 (clone 145-2C11) and anti-mouse CD28 (clone PV-1) mAbs were purchased from American Type Culture Collection (Manassas, Virginia, USA) and Bio X Cell (West Lebanon, New Hampshire, USA), respectively.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Transduction, Western Blot, Software, Cell Viability Assay, Expressing, Co-Immunoprecipitation Assay, Immunoprecipitation, Control

Journal: ImmunoHorizons

Article Title: STAP-1-derived peptide suppresses TCR-mediated T cell activation and ameliorates immune diseases by inhibiting STAP-1–LCK binding

doi: 10.1093/immhor/vlaf015

Figure Lengend Snippet: iSP1 also inhibits TCR-mediated murine T cell activation. (A) EL-4 cells were preincubated with iCont or iSP1 for 30 min, and spontaneous proliferation was evaluated by WST-8. Data are shown as mean ± SEM of 3 independent experiments ( n = 3). * P < 0.001, by Sidak’s multiple comparisons test. (B)EL-4 cells were preincubated with iCont or iSP1 for 30 min and stimulated with immobilized anti-CD3/anti-CD28 mAb for 48 h, and IL-2 production in the supernatant was analyzed by ELISA. Data are shown as mean ± SEM of 4 independent experiments ( n = 4). ** P < 0.01, by Sidak’s multiple comparisons test. (C)CD4 + T cells were preincubated with iCont or iSP1 for 30 min and stimulated with immobilized anti-CD3/anti-CD28 mAb for 96 h. Cell proliferation was evaluated by WST-8. Data are shown as mean ± SEM of 4 independent experiments ( n = 4). **** P < 0.0001, by Sidak’s multiple comparisons test. (D)CD4 + T cells were preincubated with iCont or iSP1 for 30 min and stimulated with immobilized anti-CD3/anti-CD28 mAb for 48 h. IL-2 production in the supernatant was analyzed by ELISA. Data are shown as mean ± SEM of 4 independent experiments ( n = 4). *** P < 0.001, **** P < 0.0001, by Sidak’s multiple comparisons test. (E)CD4 + T cells were preincubated with iCont or iSP1 for 30 min and stimulated with anti-CD3/anti-CD28 for indicated periods, and TCR signal transduction was analyzed by western blotting (left panel), and band intensity was quantified by Image J software (right panel). Data are shown as mean ± SEM of 4 independent experiments ( n = 4). * P < 0.05, ** P < 0.01, *** P < 0.001 by Dunnett’s multiple comparisons test.

Article Snippet: Anti-mouse CD3 (clone 145-2C11) and anti-mouse CD28 (clone PV-1) mAbs were purchased from American Type Culture Collection (Manassas, Virginia, USA) and Bio X Cell (West Lebanon, New Hampshire, USA), respectively.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Transduction, Western Blot, Software