Broadly recognized, cross-reactive SARS-CoV-2 CD4 T cell epitopes are highly conserved across human coronaviruses and presented by common HLA alleles

Version published to 10.1016/j.celrep.2022.110952

Jun 1, 2022

SciScore for 10.1101/2022.01.20.477107: (What is this?)

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

Table 1: Rigor

Ethics	IRB: EXPERIMENTAL MODEL AND SUBJECT DETAILS: Blood, PBMCs and HLA typing: Whole blood from COVID-19 convalescent donors, healthy donors, or vaccine recipients was collected under protocol approved by the UMass Chan Medical School Institutional Review Board of the University of Massachusetts and informed consent was obtained from all subjects. Consent: EXPERIMENTAL MODEL AND SUBJECT DETAILS: Blood, PBMCs and HLA typing: Whole blood from COVID-19 convalescent donors, healthy donors, or vaccine recipients was collected under protocol approved by the UMass Chan Medical School Institutional Review Board of the University of Massachusetts and informed consent was obtained from all subjects.
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
For blocking of antigen-stimulation assays, in-house produced antibodies to HLA-DR (LB3.1), HLA-DQ (SPVL-3), HLA-DP (B7/21), or HLA-ABC (w6/32), were added at a final concentration of 10 μg/mL.	HLA-DR suggested: None HLA-DP suggested: None HLA-ABC suggested: None
Cells were collected, washed, and stained using a standard protocol which included: staining for dead cells with Live/Dead Fixable Aqua Dead Cell Stain Kit™ (Life Technologies, Thermo Fisher Scientific, Waltham, MA); blocking of Fc receptors with human Ig (Sigma-Aldrich, St. Louis, MO); staining with the mix of DP4-PE and APC tetramers (final concentration of 2-4 μg/mL each) at 37°C for 2 hours; surface staining antibodies CD3-APC-H7, CD4-PerCP-Cy5.5, CD8-APC-R700, CD14-BV510, CD19-BV510, CD56-BV510 were added for the last 20 minutes of incubation, followed by washes and resuspension in buffer for data acquisition.	CD19-BV510 suggested: None CD56-BV510 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Partially-match HLA cell lines: EBV-transformed LG2 cell line (10984, IPD-IMGT/HLA), 9068 cell line (BM9, IHWG), and mouse DP4-transfected MN605 cell line (M12C3-DPA10103/DPB10401; (Williams et al., 2018); kindly provided by Dr. S. Kent, UMMS), were maintained in RPMI 1640 medium supplemented with L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and 10% FBS at 37°C/5% CO2.	MN605 suggested: None
Software and Algorithms
Sentences	Resources
After 6 hours incubation, cells were collected, washed, and stained using a standard protocol, which included: staining for dead cells with Live/Dead Fixable Aqua Dead Cell Stain Kit™ (Life Technologies, Thermo Fisher Scientific, Waltham, MA); blocking of Fc receptors with human Ig (Sigma-Aldrich, St. Louis, MO); surface staining with mouse anti-human CD3-APC-H7 (SK7), CD4-PerCPCy5.5 (RPA-T4), CD8-APC-R700 (RPA-T8), CD14-BV510 (MϕP9), CD19-BV510 (SJ25C1), CD56-BV510 (NCAM16.2); fixation and permeabilization using BD Cytofix/Cytoperm™; and intracellular staining with mouse anti-human IFN-γ-V450 (B27), TNF-α-PE-Cy7 (MAb11), IL-2-BV650 (5344.111), (all from BD Biosciences, San Jose, CA).	BD Cytofix/Cytoperm™ suggested: None
Data were acquired using a BD LRSII flow cytometer equipped with BD FACSDiva software (BD Biosciences, San Jose, CA) and analyzed using FlowJo v.	BD FACSDiva suggested: (BD FACSDiva Software, RRID:SCR_001456)
Polyfunctional analysis was performed in FlowJo, defining Boolean combinatorial gates for all the markers in the CD3+/CD4+/CD8-population.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
These results were visualized in SPICE software v6.0 (Roederer et al., 2011). t-SNE analysis was done in concatenated samples (control, SARS-CoV-2, peptide 163 and peptide 164) from 3 donors using the available plugin in FlowJo.	SPICE suggested: (SPICE, RRID:SCR_016603)
All primers sequences shown in STAR Methods.	STAR suggested: (STAR, RRID:SCR_004463)
These files were processed using MIGEC v1.2.9 pipeline: Checkout-batch for de-multiplexing and UMI tag extraction, Histogram for MIG (molecular identifier groups) statistics, and Assemble-batch to perform UMI-guided assembly (Shugay et al., 2014); followed by MiXCR v3.0.13: analyze amplicon pipeline, to align, assemble and export clonotypes (Bolotin et al., 2015).	MIGEC suggested: (migec, RRID:SCR_016337)
HLA-peptide binding prediction was performed with NetMHCIIpan v4.0 server (Reynisson et al., 2020).	NetMHCIIpan suggested: None

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: We detected the following sentences addressing limitations in the study:

There are several limitations of our study. We mapped the specificity of the cross-reactive response by following IFN-γ-secreting cells, but non-IFN-γ-secreting populations could also contribute to the response. In expanded T cell lines we observed higher frequencies of T cells staining with DP4-163/164 tetramer than responding to the same peptide in IFN-γ ELISPot essays, indicating that some T cells can recognize the epitope but not secrete IFN-γ. We observed the cross-reactive T cell response to involve mostly CD4+ T cells. This might be due to in vitro culture conditions that favor CD4+ over CD8+ T cell populations, or an intrinsic bias of cross-reactive T cells because of the different patterns of pMHC-TCR interaction for MHC-I and MHC-II proteins. We studied a relatively small group of 27 individuals exposed to SARS-CoV-2 antigens by infection or vaccination, mostly over 40 years of age. Younger individuals with more frequent previous exposures to HCoVs might show a different pattern of response. Our initial screen for immunodominant epitopes involved only three donors, all of whom recognized 163/164, but other immudominant cross-reactive epitopes might have escaped our attention, including those recognized by other MHC proteins. For all of the donors, previous HCoV infection was inferred but not observed, and we did not attempt to determine which donors were exposed previously to which of the HCoVs. In conclusion, we identified a pan-coronavirus epitope that dominates t...

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 and 36. 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:

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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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Broadly recognized, cross-reactive SARS-CoV-2 CD4 T cell epitopes are highly conserved across human coronaviruses and presented by common HLA alleles

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