Fig. 1

Integrins mediate SARS-CoV-2 T cell entry and dysregulation of T cell response. a Expression of integrins on human primary T cells was determined by flow cytometry. Numbers within panels showed the specific mean fluorescence intensities. Gray histogram: mock control. b Precipitation of integrins from the membrane fraction of human primary T cells by protein G beads loaded with S-RBD-hFc. The binding was detected by immunoblot. Human IgG was used as isotype control. One representative result of three independent experiments is shown. c Binding of soluble S-RBD protein to T cells in different divalent cation conditions. Human primary T cells were incubated with Alexa Fluor 488-labeled S-RBD (100 μg/ml) in the presence of 5 mM EDTA, 1 mM Ca²⁺/Mg²⁺, or 1 mM Mn²⁺, respectively. The binding of soluble S-RBD protein to T cells was examined by flow cytometry and shown as the specific mean fluorescence intensity (MFI) of Alexa Fluor 488-labeled S-RBD (n = 3). d Binding of soluble S-RBD protein to T cells upon IP-10 treatment. T cells were pretreated with 2 μg/ml IP-10 and then incubated with Alexa Fluor 488-labeled S-RBD (100 μg/ml) in the presence of 1 mM Ca²⁺/Mg²⁺ (n = 3). e Expression of β1, β2, and β7 integrins was silenced in human primary T cells with the indicated sgRNA, respectively. The binding of soluble S-RBD protein (100 μg/ml) to modified T cells in the presence of 1 mM Mn²⁺ was detected by flow cytometry (n = 3). f Assessment of the binding affinity of S-RBD protein to immobilized integrin proteins containing headpiece domains (10 μg/ml) in the presence of 1 mM Mn²⁺ using ELISA. (n = 3). g Binding of soluble S-RBD protein to 293 T cells expressing WT or MIDAS mutant T cell integrins. β1-KO 293 T cells were transfected with WT or MIDAS mutant α4β1, α4β7, αLβ2, and α5β1 integrins. The binding of S-RBD (100 μg/ml) to these cells in the presence of 1 mM Mn²⁺ was measured by flow cytometry (n = 3). h Effects of integrin-binding motif mutations in S-RBD on the binding of S-RBD protein to T cells. The binding of 100 μg/ml WT or S-RBD mutant (RGE, LES, or LEI) protein to T cells in the presence or absence of 1 mM Mn²⁺ was measured by flow cytometry (n = 3). i SARS-CoV-2 pseudovirus entry into β1-KO 293 T cells ectopically expressing the indicated T cell integrins. β1-KO 293 T cells expressing the indicated integrins were cocultured with GFP-expressing SARS-CoV-2 pseudovirus in the presence or absence of 0.2 mM Mn²⁺ for 12 h. Supernatants were removed, and the cells were incubated with fresh medium for 36 h. GFP+ cells were quantified by flow cytometry (n = 3). j SARS-CoV-2 pseudovirus entry into human primary T cells before and after integrin activation. T cells were pretreated with 0.2 mM Mn²⁺ and then incubated with SARS-CoV-2 pseudovirus for 12 h. Supernatants were removed, and the cells were incubated with fresh medium for 36 h. The representative confocal images were shown (left panel). GFP+ cells were quantified by flow cytometry (n = 3) (right panel). k SARS-CoV-2 authentic virus infection of human primary T cells before and after integrin activation. T cells were pretreated with 0.2 mM Mn²⁺ and then incubated with the SARS-CoV-2 authentic virus for 1.5 h. The supernatants were removed, and the cells were incubated with fresh medium for 24 h. Relative mRNA level of the SARS-CoV-2 N gene was examined in T cells at 24 h post-infection by RT-PCR. l Activation of T cells by S-RBD protein treatment. Unactivated human primary T cells were cocultured with 0, 1, 10, or 100 μg/ml S-RBD protein, respectively, for 16 h in the presence of 0.2 mM Mn²⁺. CD25 was stained for evaluation of T cell activation capacity (n = 3). m Relative mRNA levels of proinflammatory cytokines in T cells upon S-RBD protein treatment. T cells were cocultured with 100 μg/ml S-RBD or human IgG as a control in the presence of 0.2 mM Mn²⁺ for 24 h. Cells were then lysed, and the mRNA levels of the indicated cytokines were measured by RT-PCR (n = 3). n, o Apoptosis and proliferation of T cells upon S-RBD protein stimulation. T cells were cocultured with 100 μg/ml S-RBD or human IgG in the presence of 0.2 mM Mn²⁺ for 30 min. Cells were washed and resuspended in a fresh medium for further incubation at 37 °C for 36 h. The percentage of Annexin V + and Ki67+ cells was examined by flow cytometry (n = 3). p Immunoblot analysis of phosphorylation of Src (pY416) and Akt (pS473) in T cells upon S-RBD protein stimulation. T cells were cocultured with 100 μg/ml S-RBD or human IgG for 2 h in the presence of 0.2 mM Mn²⁺. One representative result of three independent experiments is shown (left panel). The relative ratios of p-Src/Src and p-Akt/Akt were normalized to the values of the untreated group (None) (n = 3) (right panel). q The effect of integrin-blocking antibodies on S-RBD binding to T cells. Human primary T cells were pretreated with the indicated antibodies (10 μg/ml of each) followed by incubation with S-RBD protein (100 μg/ml) in the presence of 1 mM Mn²⁺. Mouse IgG was used as isotype control. The blocking efficiency of integrin-blocking antibodies was normalized to the specific MFI of the IgG group (n = 3). r SARS-CoV-2 pseudovirus entry into T cells in the absence or presence of integrin-blocking antibodies. Human primary T cells were pretreated with mouse IgG or HP2/1 + FIB504 + TS1/18 cocktail (10 μg/ml of each), followed by incubation with GFP-expressing SARS-CoV-2 pseudovirus for 12 h in the presence of 0.2 mM Mn²⁺. GFP + T cells were detected by flow cytometry after 36 h incubation (n = 3). s SARS-CoV-2 authentic infection of T cells in the absence or presence of integrin-blocking antibodies. Human primary T cells were pretreated with mouse IgG or HP2/1 + FIB504 + TS1/18 cocktail (10 μg/ml of each), followed by incubation with SARS-CoV-2 authentic virus for 1.5 h at 37 °C. Supernatants were removed, and cells were incubated with fresh medium for 48 h. Relative mRNA level of SARS-CoV-2 N gene was examined in T cells at 48 h post-infection by RT-PCR (n = 3). t The graphical abstract of the study. Data represent the mean ± s.e.m., *p < 0.05, **p < 0.01, ***p < 0.001, NS no significance