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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 1. — Spike suppresses TCR accumulation and phosphotyrosine signaling at the CTL IS. (A and B) RT-qPCR of human ACE2 mRNA (A) and immunoblot analysis of human ACE2 protein (B) in purified CD8⁺ T cells prior to stimulation (day 0) or after stimulation with anti-CD3/CD28 mAb–coated beads in the presence of IL-2 for the indicated times. The migration of molecular mass markers is indicated (n = 3, one-way ANOVA test for RT-qPCR analysis; *, P ≤ 0.05; n = 4, paired two-tailed Student’s t test for immunoblot analysis; **, P ≤ 0.01). (C) Immunofluorescence analysis of Spike W binding to purified CD8⁺ T cells prior to stimulation (day 0) or 7 d after stimulation with anti-CD3/CD28 mAb–coated beads in the presence of IL-2 (n = 2). Scale bar, 15 μm. (D) Immunofluorescence analysis of CTL binding of fluorescently labeled MiniVs (Spike W embedded in fluorescent NDP-labeled liposomes) or control liposomes. Representative images are shown (n = 2). Scale bar, 15 μm. (E–G) Immunofluorescence analysis of CD3ζ and PTyr in CTLs (day 7) pretreated with either vehicle (PBS) or 0.05 μg/μl Spike W (cell viability after pretreatment 91.7 ± 0.2%), mixed with Raji cells (APCs) either unpulsed or pulsed with a combination of SEA, SEB, and SEE (SAgs), and incubated for 15 min at 37°C. Representative images (medial optical sections) of the T cell:APC conjugates are shown (E). Scale bar, 5 μm. (F) Left: Quantification (%) of 15-min SAg-specific conjugates harboring PTyr staining at the IS (≥50 cells/sample, n = 3, one-way ANOVA test; ****, P ≤ 0.0001; **, P ≤ 0.01). Right: Relative PTyr fluorescence intensity at the IS (recruitment index; 10 cells/sample, n = 3, Kruskal–Wallis test; ****, P ≤ 0.0001). (G) Left: Quantification (%) of 15-min SAg-specific conjugates harboring CD3ζ staining at the IS (≥50 cells/sample, n = 3, one-way ANOVA test; ***, P ≤ 0.001; *, P ≤ 0.05). Right: Relative CD3ζ fluorescence intensity at the IS (recruitment index; 10 cells/sample, n = 3, Kruskal–Wallis test; ****, P ≤ 0.0001). (H and I) Immunofluorescence analysis of CD3ζ and PTyr in CTLs (day 7) pretreated with either 1.2 × 10⁹ control liposomes or MiniVs, mixed with Raji cells (APCs) either unpulsed or pulsed with a combination of SEA, SEB, and SEE (SAgs), and incubated for 15 min at 37°C. (H) Left: Quantification (%) of 15-min SAg-specific conjugates harboring PTyr staining at the IS (≥50 cells/sample, n = 3, one-way ANOVA test; ****, P ≤ 0.0001; **, P ≤ 0.01). Right: Relative PTyr fluorescence intensity at the IS (recruitment index; 10 cells/sample, n = 3, Kruskal–Wallis test; ****, P ≤ 0.0001). Side-by-side comparison of PTyr⁺ immune synapses formed by CTLs pretreated with the same amount of soluble and MiniV-associated Spike showed a ∼2.3-fold increase in the suppressive ability of Spike when associated with MiniVs (51 vs. 22%, n = 3). (I) Left: Quantification (%) of 15-min SAg-specific conjugates harboring CD3ζ staining at the IS (≥50 cells/sample, n = 3, one-way ANOVA test; ****, P ≤ 0.0001; **, P ≤ 0.01; *, P ≤ 0.05). Right: Relative CD3ζ fluorescence intensity at the IS (recruitment index; 10 cells/sample, n = 3, Kruskal–Wallis test; ****, P ≤ 0.0001). Nonsignificant differences are not shown. Source data are available for this figure: SourceData F1.