Fig 2: Antigen-presenting cells can readily cross-present antigens delivered by the PeptiBAC platform. Mouse dendritic cell line Jaws II was pulsed with PeptiBAC-OVA, BCG, CPP-containing SIINFEKL peptide alone or left unpulsed (cells only). Cross-presentation was determined by flow cytometry using APC-conjugated anti-H-2Kb bound to SIINFEKL. CD86 and CD40 expression (as a measure of dendritic cell maturation and activation) was determined by flow cytometry. Each bar is the mean±SEM of technical triplicates. Statistical analysis was performed with one-way analysis of variance. ****p<0.0001. APC, antigen-presenting cell; CPP, cell penetrating peptide; PeptiBAC, peptide-coated Bacillus Calmette-Guérin; PeptiBAC-OVA, OVA-targeting PeptiBAC.

Fig 3: Ppef2 is predominantly expressed by CD8+ cDC1. (A) Gene expression profiling of spleen and blood cells after sorting. Shown is the quantitative real-time PCR result for Ppef2 of three independent sort experiments (n = 3 pooled mice per sort) ± SEM analyzed by the ΔCt method with HPRT as housekeeping gene. Cells were identified and sorted as ESAMhi (CD11c+MHCII+CD11b+ESAMhi); ESAMlo (CD11c+MHCII+CD11b+ESAMlo); CD8+DC (CD11c+MHCII+CD11b−CD8+); pDC (CD11b−SiglecH+); CD4+ T cells (CD3+CD4+); CD8+ T cells (CD3+CD8+); B cells (CD19+B220+); blood monocytes NK1.1−B200−CD115+CD11b+Ly6G− cells with differential Ly6C and MHCII expression as indicated. Gating strategies are shown in Supplemental Figure 1B. (B) Ppef2 expression in GM-CSF cultured BMDCs of C57BL/6 mice after cell sorting on day 7 of culture based on the expression of CD11c, MHCII, and CD86. Error bars represent SEM of 3 independent experiments. (C) Ppef2 expression 16h after in vitro stimulation with the indicated TLR-ligands of GM-CSF- or Flt3L-cultured BMDCs of C57BL/6 mice, as well as spleen CD8+ DCs 16h after intravenous injection of 10 μg LPS as determined by qPCR. Bar graphs with SEM represent pooled data from independently performed cell cultures [GM-CSF BMDCs: unstim., LPS (n = 5); Flagellin, Poly(I:C), Pam3CSK4, CLO97, (n = 4); anti-CD40, LPS+anti-CD40 (n = 3); Flt3L BMDCs (n = 3); sorted CD8+ DCs (n = 4)]. Statistical analysis was performed using Student's t-test, with *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 4: cRNPs inhibited cGAS activation and immunostimulatory DNA‐mediated proinflammatory responses in macrophages. A) ELISA results showing that cNPs inhibited IFN‐β production in RAW264.7 cells pretreated with cGAS‐agonistic Svg3 oligonucleotide. For Svg3 + cNPs, cells were treated with 100 nM Svg3 and a series of concentrations of cNPs for 24 h. For Svg3 + washing + cNPs, cells were first treated with 100 nm Svg3 for 4 h, then washed, and further treated with cNPs for 24 h. Medium IFN‐β concentrations were measured by ELISA. B) Hydrodynamic diameters and a TEM image of cRNPs. C) Representative flow cytometry histogram (left) and mean fluorescence intensity (MFI) (right) of C6 in RAW264.7 cells after incubation with free C6 or C6/cNPs for 0.5 or 1 h. D) Confocal microscopy of the intracellular C6 delivery in RAW264.7 cells (treatment: 1 h). E) In vitro release kinetics of RU from cRNPs. F) ELISA results showing that cRNPs inhibited IFN‐β production in Svg3‐pretreated RAW264.7 cells. Cells were pre‐treated with 100 nM Svg3 for 4 h, then washed and further treated with cRNPs for 24 h (cNPs: 20 µg mL−1; RU: 2 µm). Medium IFN‐β concentrations were measured by ELISA. G) cNPs and cRNPs showed complete inhibition of IL‐6 production in RAW264.7 cells pretreated with 1 µm CpG + 100 nm Svg3 for 4 h. Pretreated cells were washed and further treated with cNPs or cRNPs for 24 h. Medium IL‐6 concentrations were measured by ELISA. H–J) qPCR results for proinflammatory cytokine genes in RAW264.7 cells treated with 1 µm CpG, 100 nm Svg3, and cRNPs or controls (RU: 2 µm) for 24 h. K) Representative flow cytometry histogram (left) and MFI (right) of CD86 on RAW264.7 cells treated as in (H–J). DNA was transfected using Lipofectamine 2000. n = 3. Data: mean ± SD. p‐Values were determined by one‐way (J) or two‐way (C, F‐I, K) ANOVA, followed by Tukey's multiple comparison test (n.s.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; *****p < 0.00001).

Fig 5: In vitro αCD137-PBNP-PTT initiates similar changes in immune-related markers on SM1 cells as PBNP-PTT after 24 h. SM1 cells were given PTT with PBNPs or αCD137-PBNPs in vitro, cultured for 24 h and then analyzed for immune marker expression levels. (A) Viability of SM1 after PTT with αCD137-PBNPs or PBNPs, or respective controls as measured by flow cytometry. (B) Intracellular expression of ATP in terms of fold change from the Untreated control. (C–I) Representative histograms of fluorescence normalized to the mode of surface (C) calreticulin, (D) PD-L1, (E) B7-H3, (F) CD80, (G) MHC-1, (H) CD86 and (I) CD137-L after PTT with αCD137-PBNPs or PBNPs, or respective controls. Values represent mean ± standard deviation. *p < 0.05; **p < 0.01 from Kruskal-Wallis test with Dunn's multiple comparisons post-hoc test against the untreated group; #p < 0.05; ##p < 0.01 from Mann-Whitney test against the untreated group.