Figure 1.

Design of anti-SARS-CoV-2 S1 CAR-Ts and effect on CD69 activation marker expression

(A) Design of anti-SARS-CoV-2 S1 CAR-Ts. The CR3022 scFv region was coupled to a Flag-Tag followed by different hinge regions and then the CD28 transmembrane (TM) domain and intracellular domain and a CD3ζ intracellular domain. The hinge regions included a 39 amino acid CD28 hinge (CR3022-28Z), a 47 aa CD8α chain (CR3022-8a-28Z), a 119 aa CH3 region hinge (CR3022-CH3-28Z), or a 229 aa IgG4 hinge (CR3022-IgG4-28Z). As a control, a CAR lacking a scFv region (Flag-28Z) and another with the scFv region of monoclonal antibody CR3014 (CR3014-28Z).

(B) Flow cytometric profiles of the CAR construct expression in T cells. CAR expression was detected by staining the Flag-tag, and the EGFP was used for cell sorting (n = 5) (also see Figures S1 and S2).

(C) Expression of surface activation antigen CD69 on CAR-Ts. (Left panel) Histogram showing CD69 expression on different CAR-Ts (Flag-28Z, CR3014-28Z, CR3022-28Z, CR3022-8a-28Z, CR3022-CH3-28Z, and CR3022-IgG4-28Z). (Right panel) FACS profile showing CD69 expression on the different CARs after incubation with 293-ACE2 or 293-ACE2-RBD cells for 20 h. Data are shown as mean ± SD of triplicate samples. ∗∗∗∗p < 0.0001 (compared with Flag-28Z by one-way ANOVA, n = 5) (also see Figure S3).

(D) Increased CD69 expression on CR3022-28Z CAR-T in response to 293-ACE2 cells preincubated with different concentrations of the RBD peptide. CAR-Ts were co-cultured for 20 h with 293-ACE2 cells preincubated with various concentrations of the RBD peptide for 1 h prior to co-culture. Data are shown as mean ± SD of triplicate samples. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (two-tailed Student’s t test, n = 2).

(E) viSNE profiles of CAR and CD69 expression on CAR-Ts. (Left panel) Anti-Flag staining shows the surface expression of the Flag-tagged CARs in islands i, ii, and iii after 20 h of incubation with 293-ACE2 and 293-ACE2-RBD cells, whereas anti-CD69 staining shows the expression of CD69 in CAR-Ts (CR3022-28Z, CR3022-8a-28Z, CR3022-CH3-28Z, or CR3022-IgG4-28Z CAR-Ts) (n = 3). (Right panels) Histograms showing changes in the percent representation of CAR-Ts in island i relative to total CAR-Ts (upper right panel) and in the relative mean fluorescent intensity (MFI) values in island i (lower right panel) (also see Figure S4).

(F) Induction of S6 phosphorylation in response to 293-ACE2-RBD cells. CR3022-8a-28Z CAR-Ts were incubated with 293-ACE2 or 293-ACE2-RBD cells for 20 min prior to the detection of S6 phosphorylation by flow cytometry. In parallel, CR3022-8a-28Z CAR-Ts were incubated with PMA and ionomycin for the same period. Data are shown as mean ± SD of triplicate samples. ∗p < 0.05, ∗∗p < 0.01 (compared with CAR-T only group by one-way ANOVA, n = 5).

(G) CR3022-28Z downregulation in response to 293-ACE2 cells preincubated with different concentrations of the RBD peptide. CAR-Ts were co-cultured for 20 h with 293-ACE2 cells preincubated with various concentrations of the RBD peptide for 1 h prior to the co-culture. Data are shown as mean ± SD of triplicate samples. ∗p < 0.05, ∗∗∗∗ p < 0.0001 (two-tailed Student’s t test, n = 2).