SARS-CoV-2 Spike protein suppresses CTL-mediated killing by inhibiting immune synapse assembly
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Abstract
CTL-mediated killing of virally infected or malignant cells is orchestrated at the immune synapse (IS). We hypothesized that SARS-CoV-2 may target lytic IS assembly to escape elimination. We show that human CD8+ T cells upregulate the expression of ACE2, the Spike receptor, during differentiation to CTLs. CTL preincubation with the Wuhan or Omicron Spike variants inhibits IS assembly and function, as shown by defective synaptic accumulation of TCRs and tyrosine phosphoproteins as well as defective centrosome and lytic granule polarization to the IS, resulting in impaired target cell killing and cytokine production. These defects were reversed by anti-Spike antibodies interfering with ACE2 binding and reproduced by ACE2 engagement by angiotensin II or anti-ACE2 antibodies, but not by the ACE2 product Ang (1-7). IS defects were also observed ex vivo in CTLs from COVID-19 patients. These results highlight a new strategy of immune evasion by SARS-CoV-2 based on the Spike-dependent, ACE2-mediated targeting of the lytic IS to prevent elimination of infected cells.
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SciScore for 10.1101/2022.05.20.492764: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Primary commercial antibodies used in this work were from SantaCruz (CD3ζ, clone 6B10.2, #sc-1239), Cell Signaling (P-ZAP-70 Y319/ Syk Y352, #2701S; PTyr, #8954S), Abcam (Pericentrin, #4448) and BD Biosciences (Granzyme B, #560211). CD3ζsuggested: (Miltenyi Biotec Cat# 130-127-939, RRID:AB_2904864)P-ZAP-70 Y319/ Syk Y352, #2701S; PTyr, #8954S), Abcam (Pericentrin, #4448)suggested: NoneAlexa Fluor 488-and 555-labeled secondary antibodies were from ThermoFisher Scientific (anti-mouse … SciScore for 10.1101/2022.05.20.492764: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Primary commercial antibodies used in this work were from SantaCruz (CD3ζ, clone 6B10.2, #sc-1239), Cell Signaling (P-ZAP-70 Y319/ Syk Y352, #2701S; PTyr, #8954S), Abcam (Pericentrin, #4448) and BD Biosciences (Granzyme B, #560211). CD3ζsuggested: (Miltenyi Biotec Cat# 130-127-939, RRID:AB_2904864)P-ZAP-70 Y319/ Syk Y352, #2701S; PTyr, #8954S), Abcam (Pericentrin, #4448)suggested: NoneAlexa Fluor 488-and 555-labeled secondary antibodies were from ThermoFisher Scientific (anti-mouse 488, #A11001; anti-rabbit 555, #A21428). anti-mousesuggested: (Thermo Fisher Scientific Cat# A-11001, RRID:AB_2534069)anti-rabbitsuggested: (Molecular Probes Cat# A-21428, RRID:AB_141784)Alternatively, CD8+ T cells (0.5×106) were incubated for 30 min at 20°C in 50 μl RPMI-HEPES in the absence of BCS with 2 μg/ml anti-ACE2 antibody (R&D Systems, #AF933) (Hoffmann et al., 2020). anti-ACE2suggested: (Thermo Fisher Scientific Cat# PA5-75453, RRID:AB_2719181)Cells were stained with primary antibodies ([1:30] CD3ζ; [1:50] P-ZAP70; [1:100] PTyr; [1:200] PCNT; [1:50] GzmB; see above catalogue #) overnight at 4°C. P-ZAP70suggested: NonePCNTsuggested: NoneExperimental Models: Cell Lines Sentences Resources The Burkitt Lymphoma derived B cell line Raji was grown at 37°C, 5% CO2, in RPMI-1640 Medium (Merck, #R8758) supplemented with 7.5% BCS. Rajisuggested: NoneSoftware and Algorithms Sentences Resources Images were processed with Zen 2009 image software (Carl Zeiss, Jena, Germany). Zensuggested: NoneRelative distances (μm) of the centrosome (marked by PCNT) from the center of the contact site with the APC, and of the lytic granules (marked by GzmB) from the centrosome, were measured using ImageJ (FigS2A). ImageJsuggested: (ImageJ, RRID:SCR_003070)3D reconstructions were obtained using Fiji (version 2.1.0). Fijisuggested: (Fiji, RRID:SCR_002285)Statistical analyses were performed with Prism software (GraphPad Software) GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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