Journal: bioRxiv

Article Title: Endogenous antigen processing promotes mRNA vaccine CD4 + T cell responses

doi: 10.1101/2025.03.11.642674

Figure Lengend Snippet: a , Schematic of mRNA-miR vaccine constructs encoding influenza NP or HA, followed by miR-targeting sequences in series. b , Schematic of expected antigen expression patterns and relevant antigen processing pathways for each mRNA-miR construct. c , Representative histograms, gated on live singlets, and d , summary data, of NP expression by flow cytometry after in vitro transfection with NP-miR mRNA. e-g , NP expression in murine lymph nodes after i.m. immunization with mRNA-LNPs. e , Representative composite images (top row) and magnified insets (bottom row) of inguinal lymph nodes. ER-TR7 is a fibroblastic reticular cell marker. Scale bars indicate 100 μm. f , Surface area of positive NP staining, relative to the entire lymph node surface area. g , Integrated intensity of NP staining among all NP + pixels. f,g , Bars indicate mean of 3 biological replicates +SEM. Each symbol represents one mouse. Analyzed by 1-way ANOVA; results of Dunnett’s multiple comparison test are shown. h , Representative western blot after transfection of C2C12 myotubes with NP-miR mRNA. i , Summary data of NP expression as in h , quantified relative to GAPDH using densitometry analysis. d,i , Symbols represent n=4-8 (d) or n=10 (i) biological replicates per condition, pooled from 3 (d) or 4 (i) independent experiments and normalized to the average %NP + value (d) or average NP/GAPDH value (i) among NP-control-transfected cells, per cell type within each experiment. Analyzed by linear mixed-effects model with cell type (d) and mRNA construct (d,i) treated as fixed effects and experiment treated as a random effect (d,i) . Significance of Dunnett’s multiple comparison test within each cell type is shown. c,h , PR8 influenza-infected and untreated controls are included for comparison.

Article Snippet: Cryosections were taken at 16 µm (iLN) or 10 µm (muscle), dried, and stored at -80°C until staining. iLN sections were permeabilized in 0.5% Triton X-100 for 15 minutes, blocked in 5% goat serum in PBS for 30 minutes, and stained overnight with Influenza A NP monoclonal antibody (Clone D67J) conjugated to FITC (Invitrogen MA1-7332, diluted 1:100) and Fibroblast Marker antibody (ER-TR7) conjugated to AF546 (Santa Cruz Biotechnology, sc-73355, diluted 1:50).

Techniques: Construct, Expressing, Flow Cytometry, In Vitro, Transfection, Marker, Staining, Comparison, Western Blot, Control, Infection