Fig 1: TAC-OVA suppresses endogenous immune responses to OVA challenge irrespective of the spleen. (A-G) C57BL/6 mice were untreated or treated with TAC squeezed with PBS (Empty TAC) or OVA (TAC-OVA) on days -7 and -4. Mice were immunized on day 0 with CFA and OVA. 7 days later, immune responses were assessed in the draining LNs. (B) Number of IL-2 and (C) IFNγ producing cells were assayed by ELISpot after restimulation with (B) OVA protein or (C) SIINFEKL peptide. B-C are n = 3 for naïve and n=5 for other groups. Secretion of (D, E) Th1 and (F, G) Th17 associated cytokines after OVA protein restimulation. (D–G) show n=4 pools of 2 mice each per group. (H-J) C57BL/6 mice underwent sham surgery or splenectomy. Mice were either untreated or treated with TAC-OVA and challenged with OVA in CFA. OVA-specific immune responses were assessed in the draining LNs after 7 days by ELISpot. (I) IFNγ and (J) IL-2 producing cells after restimulation with (I) SIINFEKL peptide or (J) OVA protein. (I-J) n = 4-5 per group. (A-J) TAC dose was 1x109 and OVA squeeze concentration was 100μM. Mean +/- SD. Mann-Whitney U Test. Representative of at least 2 independent experiments.
Fig 2: Gamma and ancestral RBD formulated with Alum induce T cell responses in mice.BALB/c mice were immunized at day 0 and day 14 via i.m. with: Gamma RBD + Alum (blue circles) or Ancestral RBD + Alum (red triangles) and 28 days after T cell responses were evaluated. a Splenocytes were stimulated with complete medium or a peptide pool derived from RBD (Gamma and Ancestral) and then brefeldin A was added. Afterward, cells were harvested and stained with specific Abs anti-CD8, and anti-CD4, fixed, permeabilized, and stained intracellularly with anti–IFN-γ, TNF-α and anti-IL-2. Results are presented as percentage of cytokine-producing T cells. Bars are means ± SEM. n = 6 mice per group. Kruskal Wallis test. *p < 0.05, **p < 0.01, ***p < 0.001. Exact P values are shown. Representative of two experiments. b Evaluation of secreted cytokines in the supernatants from stimulated splenocytes by flow cytometry. Bars are means ± SEM. n = 5 mice per group. Kruskal Wallis test. *p < 0.05, **p < 0.01. Exact P values are shown. Representative of two experiments. Source data are provided as a Source Data file.
Fig 3: T-cellular immune response to the rNA-N1-MPP vaccine adjuvanted with BDX100 or BDX301 in C57BL/6 mice.a Flow cytometry plots showing the percentage of IL-2- and TNFα-producing CD4+ EM T-lymphocytes in mouse lungs and spleens 10 days after the booster immunization (gated on live CD3+CD19‒CD4+CD62L‒CD44+ cells). Cells were incubated with overlapping peptides, covering the whole sequence of N1 NA-protein for 6 h in the presence of Brefeldin A and co-stimulatory anti-CD28 antibodies. Plots were produced by combining data from each separate sample into one file for each group. b, c Percentage of different cytokine-producing cell populations within the total CD4+ EM T-cell subset after background subtraction in the lungs (b) and spleen (c). Average and standard error (SE) values are shown. Statistical analyses were performed using one-way ANOVA with Tukey posthoc test. P-values for the groups with statistically significant differences are shown on the plots.
Fig 4: Protection and T-cellular immune response after the heterologous challenge.a Body weight dynamics and survival of the BALB/c mice after the heterologous challenge with 5 × LD50 (1 × 104 PFU/mouse) of A/bald eagle/Florida/W22-134-OP/2022 (H5N1 reassortant with A/Puerto Rico/8/1934 vaccine backbone) or 5 × LD50 (7.5 × 103 PFU/mouse) A/New Caledonia/20/1999 (H1N1) (n = 8). b Viral titers in lungs and nasal turbinates of the BALB/c mice on day 4 after the challenge (n = 4). c Flow cytometry plots showing the percentage of IL-2- and TNFα-producing CD4+ EM T-lymphocytes (gated on live CD3+CD19‒CD4+CD62L‒CD44+ cells) in C57BL/6 mouse lungs and spleens 5 days after the challenge with 0.1 × LD50 (2 × 102 PFU/mouse) of A/bald eagle/Florida/W22-134-OP/2022 (H5N1). Cells were incubated with overlapping peptides, covering the whole sequence of N1 NA-protein for 6 h in the presence of Brefeldin A and co-stimulatory anti-CD28 antibodies. Plots were produced by combining data from each separate sample into one file for each group. d, e Percentage of different cytokine-producing cell populations within the total CD4+ EM T-cell subset after background subtraction in the lungs (d) and spleen (e). Average and standard error (SE) values are shown. Statistical analyses were performed using one-way ANOVA with Tukey posthoc test. P-values for the groups with statistically significant differences are shown on the plots.
Fig 5: gRb1 promotes Treg over Th17 cell identity in mouse splenocytes. (A–G) Splenocytes isolated from naïve mice were stimulated with anti‐CD3/CD28 antibody‐coupled beads in the presence of gRb1 (100 μM) or vehicle control for 3 days. After 3 days, the cells were treated with a protein transport inhibitor for 3 h and then harvested. (A‐E) Representative plots illustrating the proportion of CD25+Foxp3+ (A), CTLA‐4+Foxp3+ and GITR+Foxp3+ (B), IL‐10+Foxp3+ (C), TGF‐β+Foxp3+ (D), and IL‐17+ROR‐γt+ T cells (E), among the CD4+ T cells. Mann–Whitney U test. (F) Representative plots of ROR‐γt expression in CD4+ T cells. Mann–Whitney U test. (G) Ratio of Tregs to Th17 cells. Mann–Whitney U test. *p < 0.05, **p < 0.01. The data are representative of at least three separate experiments. The box in a box‐and‐whisker plot extends from the 25th to the 75th percentile, and the whiskers are plotted from the minimum to the maximum. It also plots each individual value as a point overlaid on the graph; n = 5–15 animals in each group. gRb1, ginsenoside Rb1; Treg, Regulatory T; Foxp3, Forkhead Box P3; CD, Cluster of Differentiation; CTLA‐4, Cytotoxic T‐Lymphocyte Antigen 4; GITR, Glucocorticoid‐Induced Tumor Necrosis Factor Receptor; IL, Interleukin; TGF‐β, Transforming Growth Factor β; Tr1, Type 1 Regulatory T; Th3, T Helper 3; ROR‐γt, Retinoic Acid Receptor‐Related Orphan Receptor γ t; gMFI, geometric mean fluorescence intensity.
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