Fig. 7: TLR2 expression by the hematopoietic lineage is required for optimal Spike-specific antibody and CD4+ T-cell responses in the lung following mucosal vaccination and boosting with Pam2Cys Spike.

Tlr2−/− (KO) or wild type (WT) mice were lethally irradiated then received transfer of Tlr2−/− or WT bone marrow (BM) cells i.v. Mice were rested for 12 weeks to allow hematopoietic reconstitution and replacement, then immunized with Pam₂Cys Spike i.n three times, two weeks apart. Immune responses were characterized at one week post final vaccination. Control mice were unvaccinated Tlr2−/− (UNV). a Anti-spike IgG and IgA titres (log₁₀ scale) in serum and bronchoalveolar lavage fluid (BALF) determined by ELISA. b Neutralizing antibody (nAb) titres (log₁₀ scale) in serum and BALF of vaccinated mice were determined as the titre needed for 50% inhibition of SARS-CoV-2 Spike-expressing lentivirus in HEK293-ACE2 cells. Limits of detection are indicated by the dotted lines. c Proportion of CD4⁺ T-cells and Bcl6⁺ CD4⁺ T-cells in mediastinal lymph nodes. Frequency of antigen-specific cytokine-producing CD4⁺ T-cells in the d lungs and e spleen, detected by intra-cellular staining and flow cytometry following recall with Spike protein in the presence of Brefeldin A. KO host KO BM, black; KO host WT BM, red; WT host KO BM, blue; WT host WT BM, purple. Statistics: p-values indicated, means + /− SEM are shown. Box and whiskers plots show median (centre), 25th and 75th percentile (box), lowest and highest value (whiskers). a–c Data are for individual mice from two independent biological experiments (n = 6 each), Kruskal–Wallis test, Dunn’s multiple comparisons. d, e Representative of two independent biological experiments (n = 6 each), two-way ANOVA, Tukey’s multiple comparisons test. Source data are provided as a Source Data file.