Journal: bioRxiv

Article Title: SARS-CoV-2 (E)-protein induces rapid TLR2-mediated T cell activation in mouse lungs revealed by intravital lung microscopy

doi: 10.64898/2026.05.03.722459

Figure Lengend Snippet: A) CD8+ T cells were isolated from spleens of WT and Tlr2-/- mice using Mouse CD8α + T Cell Isolation Kit (Miltenyi Biotec) and the autoMACS Pro separator. A representative example is shown. Cells were gated for dead cell and doublet exclusion and by FSC/SSC (upper panel). Staining of spleen cells by CD8α and TCRβ specific antibodies indicates CD8+ cell population before (middle panel) and after depletion (lower panel). Staining by I-A/E specific antibodies (mouse MHC-II) indicates potential antigen presenting cells – in spleen mainly B cells - as dominant population in the sample before purification and a minor population in purified CD8+ T cells. B) Purified WT CD8+ T cells were cultivated without any stimulation or in presence of (E)- protein (2 μg/ml,), Pam3CSK4 (10 ng/ml), interleukin-2 (IL-2, 20 ng/ml) and IL-15 (5 ng/ml) or plate-coated antibodies αCD3 and αCD28 for 16 hr. CD8+ T cell viability was determined by flow cytometry as the percentage of propidium iodide negative cells from analyzed cells. C) WT spleen-isolated CD8+ T cells were cultivated either without any stimulation (control) or stimulated as above. T cell activation was determined by detecting CD69 expression on CD8+ T cells using MACSQuant Analyzer 16 and is shown as percentage of CD69+ CD8+ T cells. Pair-wise significance was calculated using Two-way ANOVA with Tukey’s multiple comparison testing, p<0.05 (*). D) Co-stimulatory effects of (E)-protein and Pam3CSK4 on purified CD8+ T cells. T cell activation was quantified by CD69 expression on CD8+ T cells upon cytokine (IL-2/IL-15) or TCR stimulation (αCD3/αCD28). A representative flow cytometry analysis is shown with quantification of CD69 expression as fold activation over the respective control after background subtraction (WT: n=8; Tlr2 − / − : n=9). Pair-wise significance was calculated using Two-way ANOVA with Tukey’s multiple comparison testing, p<0.01 (**) and p<0.001 (***). E) Supernatants of stimulated WT and Tlr2-/- CD8+ T were analyzed for IFNγ and is shown in log scale. Significance was calculated using the ratio paired t-test with p<0.05 (*), p<0.01 (**) and p=0.0001 (***). F) Blood leukocytes were stimulated for 16 hr either with cytokines or TCR agonists in the presence or absence of (E)-protein or Pam3CSK4. Activation of CD8+ T cells was determined by CD69 surface expression after background subtraction. Starting point (cells stimulated either by cytokines or antibodies only) was the same for (E)-protein or Pam3CSK4 stimulated samples. Significance was calculated with paired t test p<0.05 (*).

Article Snippet: Cell purity was determined by staining with antibodies in staining buffer (2% FBS, 4 μM EDTA, 0.1% NaN 3 in PBS) and propidium iodide (PI, 1 μg/ml in staining buffer, Sigma) and measured in the MACSQuant Analyzer 16 (Miltenyi Biotec).

Techniques: Isolation, Cell Isolation, Staining, Purification, Flow Cytometry, Control, Activation Assay, Expressing, Comparison