Engineered chimeric T cell receptor fusion construct (TRuC)-expressing T cells prevent translational shutdown in SARS-CoV-2-infected cells

  1. Ira Godbole
  2. Kevin Ciminski
  3. O. Sascha Yousefi
  4. Salma Pathan-Chhatbar
  5. Deniz Saltukoglu
  6. Niklas Vesper
  7. Pavel Salavei
  8. Juliane Strietz
  9. Nicole Gensch
  10. Michael Reth
  11. Martin Schwemmle
  12. Wolfgang W. Schamel

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Abstract

SARS-CoV-2, the causative agent of Covid-19, is known to evade the immune system by several mechanisms. This includes the shutdown of the host cellular protein synthesis, which abrogates the induction of antiviral interferon responses. The virus initiates the infection of susceptible cells by binding with its spike protein (S) to the host angiotensin-converting enzyme 2 (ACE2). Here we applied the T cell receptor fusion construct (TRuC) technology to engineer T cells against such infected cells. In our TRuCs an S-binding domain is fused to the CD3ε component of the T cell receptor (TCR) complex, enabling recognition of S-containing cells in an HLA independent manner. This domain either consists of the S-binding part of ACE2 or a single-chain variable fragment of an anti-S antibody. We show that the TRuC T cells are activated by and kill cells that express S of SARS-CoV-2 and its alpha (B.1.1.7) and beta (B.1.351) variants at the cell surface. Treatment of SARS-CoV-2 infected cells with our engineered T cells did not lead to massive cytotoxicity towards the infected cells, but resulted in a complete rescue of the translational shutdown despite ongoing viral replication. Our data show that engineered TRuC T cell products might be used against SARS-CoV-2 by exposing infected cells to the host innate immune system.

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    SciScore for 10.1101/2021.06.25.449871: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationAuthentication: Cell death was assessed by apoptotic morphology and the subsequent halt in cellular movement from the brightfield images.

    Table 2: Resources

    Antibodies
    SentencesResources
    To obtain the expanded human T cells, peripheral blood mononuclear cells (PBMCs) were isolated from blood of a healthy donors by density-gradient centrifugation and grown in RPMI 1640 medium supplemented with 10% FCS and 1000 U/ml recombinant IL-2 (PeproTech) and activated with 1 μg/ml anti-CD3 and anti-CD28 antibodies.
    anti-CD3
    suggested: None
    anti-CD28
    suggested: None
    Flow cytometry: The following antibodies were used for flow cytometry staining in a 96-well format: PE-labelled anti-human CD4 (Beckman Coulter, #A07752)
    anti-human CD4
    suggested: None
    After blocking with 5% milk in PBS containing 0.1% Tween-20 the membranes were incubated with antibodies against TCRα (1:1000), TCRβ (1:100), CD3γ (1:1000), CD3δ (1:100), CD3ε (1:1000), CD3ζ (1:1000) in PBS-T followed by incubation with HRPO-conjugated secondary antibodies (1:10000).
    TCRα
    suggested: None
    TCRβ
    suggested: (Bio X Cell Cat# BE0102, RRID:AB_10950158)
    CD3γ
    suggested: None
    CD3δ
    suggested: None
    CD3ε
    suggested: None
    CD3ζ
    suggested: None
    Cells were stained with anti-CD69 antibodies and measured by flow cytometry.
    anti-CD69
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    In brief, HEK293T cells were transfected with the εTRuC-encoding lentiviral plasmids and the packaging plasmids pMD2.
    HEK293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Jukat CD3ε KO, Ramos, Vero E6, CaCo-2 and CaLu-3 cell lines were transduced with a multiplicity of infection (MOI) of 5 with the lentiviruses indicted and sorted by flow cytometry when necessary.
    Vero E6
    suggested: RRID:CVCL_XD71)
    CaLu-3
    suggested: BCRJ Cat# 0264, RRID:CVCL_0609)
    After two days, the supernatant was collected, filtered and mixed 1:1 with 300.000 Ramos-null cells that express the ecotropic receptor.
    Ramos-null
    suggested: None
    Activation assays: Ramos cells expressing or not the different S proteins were co-cultured with the different Jurkat transductant cells at a 1:3 target-to-effector ratio for 9 h.
    Jurkat
    suggested: TKG Cat# TKG 0209, RRID:CVCL_0065)
    To quantify cytokines by ELISA, εTRuC-expressing primary T cells and S-expressing Ramos cells were co-cultured for 24 h.
    Ramos
    suggested: None
    : Virus containing supernatant was harvested at the indicated time points and the viral titer was determined on VeroLucBFP cells by indirect-immunofluorescence.
    VeroLucBFP
    suggested: None
    Recombinant DNA
    SentencesResources
    pOSY120, encoding for the ACE2l-εTRuC, was generated by Gibson assembly of the XhoI-XbaI fragment of p526 anti-αCD19 (Baeuerle et al., 2019) and the PCR fragment using the primers O293 and O294 (see table 1) on the human ACE2 sequence as a template.
    pOSY120
    suggested: None
    pOSY121, encoding for the ACE2s-εTRuC, was generated by Gibson assembly of the XhoI-XbaI fragment of above and the PCR fragment using the primers O293 and O295 on the human ACE2 sequence.
    pOSY121
    suggested: None
    pOSY123, encoding the αS-εTRuC, was generated by Gibson assembly of the XhoI-XbaI fragment of above and, the PCR fragment using the primers O304 and O305 from a gBlock of CR3022 VL (Integrated DNA Technologies) and the PCR fragment using O306 and O307 from a gBlock of CR3022 VH (Integrated DNA Technologies).
    pOSY123
    suggested: None
    Molecular cloning of the luciferase-BFP and S protein-mScarlet vectors: For the molecular cloning of the pHRSIN-CS-Luc-IRES-mTagBFP2 vector, the previously described pHRSIN-CS-Luc-IRES-emGFP vector (a kind gift from A. Rodriguez, Universidad Autonoma Madrid, Spain) was digested with BstX1 and Not1 restriction enzymes to remove the emGFP.
    pHRSIN-CS-Luc-IRES-mTagBFP2
    suggested: None
    pHRSIN-CS-Luc-IRES-emGFP
    suggested: None
    mTagBFP2 was amplified from the previously described pHRSIN-CS-IRES-mTagBFP2 plasmid (Dang et al., 2020) and BstX1- and Not1-specific overhangs were added using PCR.
    pHRSIN-CS-IRES-mTagBFP2
    suggested: None
    The cDNA of the S protein was taken from the plasmid pCG1-CoV-2019-S with a codon-optimized sequence (Lapuente et al., 2021) and cloned into the pMIG vector by Gibson assembly and the cDNA of mScarlet was ligated into the vector.
    pCG1-CoV-2019-S
    suggested: None
    In brief, HEK293T cells were transfected with the εTRuC-encoding lentiviral plasmids and the packaging plasmids pMD2.
    pMD2
    suggested: None
    G (envelope) and pCMVR8.74 (gag/pol) using PEI transfection.
    pCMVR8.74
    suggested: RRID:Addgene_22036)
    To generate S-mScarlet expressing Ramos cells, the retroviral vector pMIG encoding S-mScarlet and a vector encoding the ecotropic packaging protein were co-transfected (500 ng each) into Plat-E cells with the PolyJet transfection reagent (Signagen).
    pMIG
    suggested: RRID:Addgene_9044)
    Software and Algorithms
    SentencesResources
    Cells were measured on the flow cytometer Attune NxT and the data were analyzed by FlowJo.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Multi-position videos were converted to TIFF and transferred to ImageJ.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    All p values indicated with stars (* < 0.05, ** < 0.005, *** < 0. 0005) were calculated using Prism v.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    6 software (GraphPad).
    GraphPad
    suggested: (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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 32, 33, 25, 29 and 30. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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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  2. Version published to 10.1101/2021.06.25.449871v1 on bioRxiv