Fig 2: CpG immunization induces APC maturation in the lung. BALB/cByJ mice were immunized intranasally. (A) Representative FACS plots from the lung at day 1 show (i) interstitial macrophages (F4/80+), (ii) alveolar macrophages (F4/80+ CD11c+), and (iii) conventional DCs (CD11c+). DCs, interstitial macrophages, and alveolar macrophage populations were enumerated at day 1 (B) and day 3 (F) in the lungs. Innate immune cell populations were assessed for their surface expression of the costimulatory molecule CD86 by measuring the median fluorescence intensity (MFI) (C,G). The influx of CD103+ dendritic cells (D,H) and TLR9+ (E,I) cells was also enumerated after immunization. CD69 upregulation on CD4 and CD8 T cells in the lungs 3 days after immunization (J). Graphs represent two separate experiments (n = 3–5 mice per group) with data given in mean percentage ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 3: Therapeutic effect of intratumoral gene electrotransfer (GET) with plasmid interleukin 15 (IL-15)/IL-15Ra in breast tumor. Breast tumors were established by injections of 1 × 106 4T1-luc cells in the left posterior mammary fat pad of female Balb/c mice. Tumors (30–80 mm3) were treated with intratumoral saline (Ctrl, n = 10) or plasmid DNA IL-15/IL-15Ra (GET, n = 8) with electrical pulse deliveries at days 0, 4, and 7 (indicated by arrows). (A) Growth curves of 4T1-luc breast tumors and survival curves of tumor bearing animals. *s p = 0.008–0.038 between Ctrl and GET groups during days 10–24 by t-test. *p < 0.05 between Ctrl and GET treatment by Log-Rank test. (B) Percentages of memory CD8/CD4 T cells in PBMCs. Flow cytometric analysis was performed at day 15 for tumor mice (Ctrl, n = 3) or day 107 for long-term tumor free mice after GET treatment (GET, n = 2). (C) Systemic and regional changes of immune cells after GET. Two days after the completion of GET treatment, animals (n = 5) were euthanized to analyze MDSCs in blood and spleens, and CD103+CD8+ tissue resident memory cells and Nkp46+CD3+ NKT cells in draining lymph nodes. Error bars represent standard deviations. $p = 0.074, #p = 0.058, *p < 0.05 and ***p < 0.001 by t-test.

Fig 4: PSA protection from HSE is independent of induced Tregs. a % FoxP3+ CD4 Tregs and b CD69+ CD4 T cells in spleen and CLN of PSA or PBS-treated WT mice at day 6 pi. c CD25 expression within FoxP3+ CD4 Tregs in WT mice at day 6 pi, % and mean fluorescence intensity (MFI) in () shown in right top quadrant. d % CD25 within FoxP3+ Tregs (left plot) and FoxP3- CD4+ T cells (right plot), e CD103 expression within FoxP3+ Tregs in WT mice at day 6 pi; % and MFI in () shown in right top quadrant. f CD103 within FoxP3+ Tregs (left plot) and FoxP3- CD4+ T cells (right plot) in the spleen or CLN of WT mice at day 6 pi. Data from three experiments shown. g PSA-treated Treg depleted and control WT mice were monitored for survival after HSV infection and ACV treatment as in Fig. 1a, ns: not significant determined by log rank Mantel–Cox test (n = 11–12 mice). After administration of three (1 week) or six doses (2 weeks) of PSA, MLN in uninfected WT mice were monitored for h cellularity, i % CD4 and CD8 T cells, and j # ICOS+, CD39+, and CD73+ CD4 and CD8 T cells (n = 3 mice); ****p < 0.0001, **p < 0.01 as determined by two-way ANOVA or one-way ANOVA with Sidaks or Turkeys correction, respectively, for multiple comparisons tests. All data show mean ± SEM

Fig 5: Antigen presentation and activation of bone marrow-derived dendritic cells after infection with viRNA-mOVA.Mouse bone marrow-derived dendritic cells were infected with sucrose purified viRNA-mOVA WR, sucrose purified mOVA complexed with cationic liposomes as a control, virus alone or non-treated cells as a mock. A) Antigen presentation was determined by flow cytometry using APC-conjugated anti-H-2Kb bound to SIINFEKL. B) Dendritic cell activation and C) migration marker expression was determined by flow cytometry using antibodies against murine CD86 and murine CD103, respectively. Each bar is the mean ± SEM of technical triplicates.