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. 2022 Nov 15;220(2):e20220906. doi: 10.1084/jem.20220906

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© 2022 Onnis et al.

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Figure 2. — Spike suppresses signaling at an early step of IS assembly. (A–C) Left: Quantification (%) of 5-min and 15-min SAg-specific conjugates harboring PTyr (A), CD3ζ (B), or P-ZAP-70 (C) staining at the IS in CTLs (day 7) pretreated with either vehicle (PBS) or 0.05 μg/μl Spike W, then mixed with Raji cells (APCs) either unpulsed or pulsed with a combination of SEA, SEB, and SEE (SAgs), and incubated for 5 or 15 min at 37°C (≥50 cells/sample, n = 3, one-way ANOVA test; ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05). Right: Relative PTyr (A), CD3ζ (B), or P-ZAP-70 (C) fluorescence intensity at the IS (recruitment index; 10 cells/sample, n = 3, Kruskal–Wallis test; ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05). (D) Flow cytometric analysis of protein tyrosine phosphorylation in conjugates prepared as in A–C and incubated at 37°C for the indicated times. Raji cells were loaded with 1.5 μM CFSE prior to conjugate formation. Conjugates were stained with anti-PTyr mAb followed by fluorescently labeled secondary Abs. The analysis was carried out gating on CSFE⁻ cells (n = 3, one-way ANOVA test; ****, P ≤ 0.0001; ***, P ≤ 0.001; *, P ≤ 0.05). (E) Immunoblot analysis of Erk phosphorylation in CTLs activated with anti-CD3 and anti-CD28 mAbs. CTLs were incubated at 37°C for the indicated times and processed for immunoblot with Abs against the active forms of Erk1/2. Stripped filters were blotted with anti-Erk2 mAb as loading control. The migration of molecular mass markers is indicated (n = 2, one-way ANOVA test; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05). Data are expressed as mean ± SD. ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05. Nonsignificant differences are not shown. Source data are available for this figure: SourceData F2.