Journal: Nature Communications
Article Title: Optimal murine CD4 + T cell priming by mRNA-lipid nanoparticle vaccines requires endogenous antigen processing
doi: 10.1038/s41467-025-68080-x
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, d Representative histograms, gated on live singlets ( c ), and summary data ( d ) of %NP-expressing cells 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 Bars represent mean + SEM of biological replicates 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. Sample sizes: fibroblasts n = 7 (NP-control) or n = 8 (NP-miR142t, NP-miR206t) ( d ); DC2.4 n = 5 per condition ( d ); BMDC n = 4 per condition ( d ); C2C12 n = 10 per condition ( i ). d , i 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 pairwise comparisons to control mRNA, determined by likelihood ratio test followed by Dunnett’s multiple comparison test within each cell type, are shown. d Among DC2.4, NP-control vs. NP-miR142t p < 1 × 10 -10 and NP-control vs. NP-miR206t p = 3.51 × 10 -7 ; among BMDC, NP-control vs. NP-miR142t p < 1 × 10 -10 and NP-control vs. NP-miR206t p = 1.1 × 10 -8 . i NP-control vs. NP-miR206t p < 1 × 10 -10 . c , h PR8 influenza-infected and untreated controls are included for comparison. d , f – i Source data are provided as a Source Data file.
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 min, 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
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