Journal: iScience

Article Title: SARS-CoV-2 antigen-carrying extracellular vesicles activate T cell responses in a human immunogenicity model

doi: 10.1016/j.isci.2023.108708

Figure Lengend Snippet: Characterization of 293F cell-derived EVs carrying SARS-CoV-2 spike protein (A) Schematic of EV engineering strategy using 293F cells including SARS-CoV-2 spike expression construct (see Figure S1 ) with spike protein depicted in blue. Schematic created with Biorender.com . (B) Histogram plot of particle size distribution comparing 293F EVs and Spike EVs as determined by NTA. The number of particles falling within each size bin was normalized to total particle counts and are presented as the proportion of total particles. Data represent the mean ± standard error of the mean (SEM). N = 3 independent experiments. Statistical comparisons were carried out by unpaired, two-tailed Student’s t test where p < 0.05 would be considered significant. (C) Mean and mode particle size of 293F EVs and Spike EVs as determined by NTA. Data represent the mean ± standard error of the mean (SEM). N = 3 independent experiments. Statistical comparisons were carried out by unpaired, two-tailed Student's t test where p < 0.05 would be considered significant. n.s. indicates not significant. (D) Multiplexed bead-based profiling assay of 37 surface antigens on 293F EVs and Spike EVs confirming the presence of expected EV surface markers. Data represent the median fluorescent intensity (MFI) of APC signal (reflecting CD63-APC, CD81-APC and CD9-APC counterstaining) following the subtraction of isotype controls. N = 1 independent experiment. (E) Representative western blots characterizing the presence of SARS-CoV-2 spike protein in Spike EVs and markers known to be present in EV preparations (CD63, CD9, Flotillin, TSG101, GAPDH) as well as the absence of cell contaminant markers (GM130, Calnexin). (F) Confirmation of SARS-CoV-2 spike protein presence in Spike EVs through competitive Spike-ACE2 binding assay. Spike EVs inhibit the binding of fluorescently labeled, recombinant SARS-CoV-2 spike protein competitively, in a dose-responsive manner, while 293F EVs demonstrate little inhibition. Data are mean ± SEM. N = 2 independent experiments and 2 technical replicates per sample per run. (G) Transmission electron micrographs of unstained Spike EVs confirming expected EV morphology. SARS-CoV-2 spike protein presence on 293F Spike EVs was also visually confirmed using immunogold labeling where 10 nm gold particles are seen associating with 293F Spike EVs.

Article Snippet: For immuno-gold labeling, each grid was incubated for 1 h with a drop of 1:100 Rb-SARS-CoV-2 Spike Protein (T01KHuRb) antibody (Invitrogen, 703959).

Techniques: Derivative Assay, Expressing, Construct, Two Tailed Test, Western Blot, Binding Assay, Labeling, Recombinant, Inhibition, Transmission Assay

Journal: iScience

Article Title: SARS-CoV-2 antigen-carrying extracellular vesicles activate T cell responses in a human immunogenicity model

doi: 10.1016/j.isci.2023.108708

Figure Lengend Snippet: CD4 + and CD8 + T cell Specific Activation in Response to Treatment with Spike EVs (A) Representative flow cytometric results highlighting activation-induced markers (AIM) (CD134+/CD137+) on CD4 + T cells within PBMCs derived from a single donor (Donor 962) vaccinated against SARS-CoV-2 and stimulated over 48 h with PBS (negative control), SARS-CoV-2 derived peptide covering spike protein (Spike peptide; positive control) or SARS-CoV-2 spike recombinant protein (spike protein; positive control), 293F EVs or Spike EVs. Refer to Figure S2 for full gating strategy. (B) Representative flow cytometric results highlighting AIM+ (CD69+/CD137+) CD8 + T cells within PBMCs derived from a donor (Donor 962) vaccinated against SARS-CoV-2 and stimulated over 48 h with the same conditions as described in A. Refer to Figure S2 for full gating strategy. (C) Quantification of CD4 + T cell response measured as percentage of AIM+ (CD134+/CD137+) CD4 + T Cells. Data represent the responses for nine individual vaccinated donors and 3 individual unvaccinated donors (Vaccinated N = 9; Unvaccinated N = 3). Statistical comparisons were carried out by two-way ANOVA with Tukey’s multiple comparisons test to compare all treatment groups. ∗p < 0.05; ∗∗p < 0.01. (D) Relationship between the frequency of AIM-positive CD4 + T cells in Spike EV treated PBMCs from vaccinated donors and the frequency of AIM-positive CD4 + T cells in the Spike Protein (orange) or Spike Peptide (blue) treatment conditions. N = 9. Pearson’s correlation coefficient was computed for the Spike EV and Spike Protein relationship (r = 0.979, p < 0.0001) and the Spike EV and Spike peptide relationship (r = 0.956, p < 0.0001). (E) Quantification of CD8 + T cell response measured as percentage of AIM+ (CD69+/CD137+) CD8 + T Cells. Data represent the responses for nine individual vaccinated donors and 3 individual unvaccinated donors (Vaccinated N = 9, Unvaccinated N = 3). Statistical comparisons were carried out by two-way ANOVA with Tukey’s multiple comparisons test to compare all treatment groups. ∗p < 0.05; ∗∗p < 0.01. E) Relationship between the frequency of AIM-positive CD4 + T cells in Spike EV treated PBMCs from vaccinated donors and the frequency of AIM-positive CD4 + T cells in the Spike Protein (orange) or Spike Peptide (blue) treatment conditions. N = 9. Pearson’s correlation coefficient was computed for the Spike EV and Spike Protein relationship (r = 0.979, p < 0.0001) and the Spike EV and Spike peptide relationship (r = 0.956, p < 0.0001). (F) Relationship between the frequency of AIM-positive CD8 + T cells in Spike EV treated PBMCs from vaccinated donors and the frequency of AIM-positive CD4 + T cells in the Spike Protein (orange) or Spike Peptide (blue) treatment conditions. N = 9. Pearson’s correlation coefficient was computed for the Spike EV and Spike Protein relationship (r = 0.912, p = 0.0006) and Spike peptide treatment (r = 0.929, p = 0.0003). (G) SARS-CoV-2 spike protein levels in 293F EVs and Spike EVs as determined by ELISA. Data represent the quantity of spike protein normalized to 2 x 109 particles. Data are mean ± SEM. N = 1 and N = 3 independent experiments for 293F EVs and Spike EVs, respectively and 3 technical replicates per sample per run.

Article Snippet: For immuno-gold labeling, each grid was incubated for 1 h with a drop of 1:100 Rb-SARS-CoV-2 Spike Protein (T01KHuRb) antibody (Invitrogen, 703959).

Techniques: Activation Assay, Derivative Assay, Negative Control, Positive Control, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: SARS-CoV-2 antigen-carrying extracellular vesicles activate T cell responses in a human immunogenicity model

doi: 10.1016/j.isci.2023.108708

Figure Lengend Snippet: Preferential Uptake of CFSE-Stained Spike EVs in Antigen-Presenting Cells within Mixed Peripheral Blood Mononuclear Cell Population (A) Representative flow cytometric results of PBMCs derived from a single donor (Donor 965) vaccinated against SARS-CoV-2 and incubated with Spike EVs pre-stained with CFSE as well as PBS only and PBS with CFSE as controls. Flow cytometric plots highlight the frequency of total CFSE-positive cells from a single run following the exclusion of dead cells and doublets (Refer to Figure S3 for gating strategy). (B) Graphical representation of CFSE signal in PBMCs collected following the conditions outlined in (A). N = 3 independent experiments; Data represent the mean ± SEM. (C) Representative flow cytometric results of PBMCs following 16 h of incubation with Spike EVs pre-stained with CFSE following manual gating for cell subpopulation analysis. Refer to Figure S3 for full gating strategy. (D) The percentage of each cell subtype that is CFSE-positive following incubation with CFSE-stained Spike EVs. N = 3 independent experiments; Data represent the mean ± SEM. (E) t-distributed Stochastic Neighbor Embedding (t-SNE) analysis of live PBMCs after PBS treatment (left plot) or CFSE-stained Spike EV treatment (right plot) to cluster cell subpopulations based on flow cytometric measurements of CD3, CD4, CD8, CD19, CD14, CD16 and HLA-DR. Manual gating of cell populations and CFSE-positivity (fluorescent green) were overlayed to visually depict EV uptake across cell populations. (F) Percentage of antigen-presenting cells (dendritic cells, B cells and monocytes) displaying the surface expression of CD80 and CD86 as determined by flow cytometry following 24 h stimulation with PBS, SARS-CoV-2 derived peptide pool covering spike protein (Spike peptide) or SARS-CoV-2 spike recombinant protein (Spike protein), 293F EVs or Spike EVs in PBMCs from vaccinated donors. N = 8 independent donors; Data represent the mean ± SEM; Statistical comparisons were carried out by two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: For immuno-gold labeling, each grid was incubated for 1 h with a drop of 1:100 Rb-SARS-CoV-2 Spike Protein (T01KHuRb) antibody (Invitrogen, 703959).

Techniques: Staining, Derivative Assay, Incubation, Expressing, Flow Cytometry, Recombinant

Journal: iScience

Article Title: SARS-CoV-2 antigen-carrying extracellular vesicles activate T cell responses in a human immunogenicity model

doi: 10.1016/j.isci.2023.108708

Figure Lengend Snippet:

Article Snippet: For immuno-gold labeling, each grid was incubated for 1 h with a drop of 1:100 Rb-SARS-CoV-2 Spike Protein (T01KHuRb) antibody (Invitrogen, 703959).

Techniques: Recombinant, Transduction, Transfection, Expressing, Electron Microscopy, Membrane, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Plasmid Preparation, Software, Sequencing

Journal: The FEBS journal

Article Title: Cardiomyocyte external mechanical unloading activates modifications of α-actinin differently from sarcomere-originated unloading

doi: 10.1111/febs.16925

Figure Lengend Snippet: List of antibodies for western blot analysis.

Article Snippet: RB Acetylated-Lysine MultiMab (Cell Signaling Tech #9814) , 1 : 1000.

Techniques: Western Blot