Fig 1: CpG immunization induces functional memory responses at day 5 post challenge with a heterosubtypic IAV infection. BALB/cByJ mice were vaccinated as indicated. Four to five weeks post immunization, mice were challenged with a 10 LD50 of PR8. (A) Lungs were harvested 5 days after infection, and the absolute number of GrB+ T and NK cells were enumerated via flow cytometry. (B) Representative FACS plots in the lung after ex vivo restimulation for 4–6 h with NP and M peptides. (C) Percentage of cytokine-producing CD4 and CD8 cells were enumerated after restimulation. Data represent two independent experiments (n = 4–5 mice per group) given in mean percentage of cytokine positive cells or absolute number of GrB+ cells + SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 2: Pregnancy affects T cell response to 3'UTR-?10-LAV vaccination in A129 mice.a Experiment scheme. Top scheme: 12-week-old female A129 mice (n = 4 or 5)were mated with male mice; at E0.5, the pregnant mice were subcutaneously immunized with 103 FFU 3'UTR-?10-LAV (?10) or PBS; at E18.5, they were sacrificed and splenocytes were harvested for T cell analysis. Bottom scheme: age-matched non-pregnant female mice (n = 3 or 6) were immunized and analyzed for T cell response to ?10 vaccination, as described above for the pregnant group. b Percentages of CD4+IFN-?+, CD8+IFN-?+, CD4+IFN-?+TNF-a+, and CD8+IFN-?+TNF-a+ cells. Spleen cells were harvested and stained for IFN-?+, TNF-a+, CD3+, and CD4+ or CD8+. Samples were acquired with BD Accuri C6 Flow Cytometer instrument. Total splenocytes were first gated to exclude, debris, cell fragments, and dead cells based on forward (FSC) and side scatter (SSC). CD3+CD4+ or CD3+CD8+ T cells were next gated for analysis of cytokine production. c Total numbers of CD4+ T cell subsets per spleen. Splenocytes were stimulated by ZIKV. d Total numbers of CD8+ T cell subsets. Splenocytes were stimulated by a ZIKV E peptide. Cytokines IL-2 (e) and IFN-? (f) in cell culture media were measured after splenocytes had been stimulated by the viral E peptide for 3 days. An unpaired t test was performed to analyze statistical significance between indicated groups. **p < 0.01, ***p < 0.001, non-significant (n.s.) p > 0.5. Triple technical replicates were performed for (c), (d), (e) and (f). Placebo-P PBS placebo pregnant mice, placebo-NP PBS placebo non-pregnant mice, vaccine-P vaccinated pregnant mice, vaccine-NP vaccinated non-pregnant mice. Error bars represent standard deviations. Source data are provided as a Source Data file.
Fig 3: CircRNA-LNP vaccine exerts synergistic efficacy with OT-I T cell transfer in late-stage B16 model. (A) Timeline of the experiment to evaluate the synergetic anti-tumor effect of vaccine (circRNAOVA-luc) and TCR-T adoptively therapy (OT-I) in late-stage orthotopic B16 tumor model, intramuscular vaccination (5 µg circRNA per mouse), intravenous administration (5×105 OT-I T cells per mouse). Average tumor growth curve (B) and the survival curve (C) of the four groups. PBS, PBS-LNP complex (n = 5); PBS+OT-I, PBS-LNP complex with OT-I transfer (n = 5), circRNAOVA-luc, circRNAOVA-luc-LNP complex (n = 5); circRNAOVA-luc+OT-1, circRNAOVA-luc-LNP with OT-I transfer (n = 6). (D-G) Individual tumor growth curves for four mouse groups. SR, survival rate. (H) Timeline of the experiment of OT-I T cell persistence evaluation. CD45.1 C57BL/6J mice were administrated with CD45.2 OT-I cells and circRNAOVA-luc-LNP (circRNAOVA-luc), intramuscular vaccination (5 µg circRNA per mouse), intravenous administration (1×106 OT-I T cells per mouse). Seven days later, spleens were collected for analysis. Representative flow dot plots (I) and statistical result (J) of the OT-I (CD45.2+) T cells in CD8+ cells. *p < 0.05, ***p < 0.001.
Fig 4: Cyclosporin A (CsA)-sensitive nuclear factors of activated T cell (NFAT) signaling is a major transcriptional pathway in Graft-Infiltrating Cells (GICs) from allogeneic heterotopic heart transplants. Syngeneic (C57BL/6, Syn) or fully allogeneic (BALB/c, Allo) heart grafts were transplanted into wild-type (WT) or NFATc1-deficient (Nfatc1flx/flxxCD4-cre) C57BL/6 recipients. A subgroup of WT recipients was treated daily with CsA (15 mg/kg, Allo CsA). GICs were purified during acute rejection phase (d5) and subjected to next-generation sequencing (NGS) assays. (A) RNA expression profiles (“heat map”) of T cell signature genes. GICs were purified from syngeneic recipients (lane 1), allogeneic recipients in the absence or presence of CsA (lanes 2 and 3, respectively), and from recipients bearing Nfatc1-/- T cells (lane 4). Their RNAs were isolated and subjected to NGS. (B) Ingenious Pathway Analyses. Expression data were normalized to the transcriptome of GICs in syngeneic transplantations. (C) RNA expression of genes encoding glycolytic enzymes. (D) Extracellular flux analyses of activated CD8+ Ts from WT and dlck-cre x Nfatc1flx/flx mice (left), and from WT and dlck-cre x Nfatc1flx/flx x caNfatc1-STOPflx/flx mice (right). Data of three independent assays are shown.
Fig 5: Rottlerin alters immunocytes compositions in IMQ-treated mice.An FACS analysis was applied to determine rottlerin activity following the oral administration of rottlerin or vehicle treatment in vivo. (A) Representative results of CD3+CD4+ T cell percentage in the DLN, spleen, and skin. The quantification of CD3+CD4+ T cell percentage is shown in the lower pannel. The data are represented as mean±SEM (n = 5 in each group). Similar results were seen in two other independent experiments. NS, P>0.05,*P<0.05,**P<0.01 vs. vehicle.(B) Representative results of the CD3+CD8+ T cell percentage in the DLN, spleen and skin. The quantification of CD3+CD8+ T cell percentage is shown in lower pannel. The data are represented as mean±SEM (n = 5 in each group). Similar results were seen in two other independent experiments. *P <0.05, ** P<0.01 vs. vehicle.
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