Fig 2: Replication-defective BAdv85C5 vaccine enhances galectin and cathepsin-dependent antigen presentation in mouse DCs and human macrophagesC57BL/6 mouse-derived DCs were infected with vaccines or control vectors, washed, and processed.(A) At 18 h post-infection, lysates collected in Trizol were used for qPCR using primers (Method details) for endosome trafficking genes enriched during transcriptomics (Figure 2) (∗≥1.4-fold increase in mRNA expression, n = 2, 2 experiments).(B) At 18 h post-infection, lysates collected in anti-protease buffer analyzed using specific antibodies and Wes Simple blot protocol (Method details). Densitometry panels shown.(C and D) DCs and macrophages were subjected to siRNA versus Lgals-3 and Lgals-8 or scrambled controls and at 18 h, infected using vaccines or vectors for 4 h. Washed DCs were overlaid using BB7 CD4 T cells, and 18 h later, supernatants assayed for IL-2 using sandwich ELISA (triplicate wells of DCs per group and 2 independent experiments).(E) Lysates collected at 18 h post-infection were used for qPCR using primers (Method details) for cathepsins enriched during transcriptomics (Figure 2) (∗≥1.4-fold increase in mRNA expression, n = 2, 2 experiments).(F) Lysates collected at 18 h post-infection were analyzed using specific antibodies and Wes Simple blot protocol (Method details).(G) WT-DCs or ATG7KO-DCs DCs were left untreated or treated with cathepsin inhibitors as indicated, followed by vaccine or vector infection and antigen presentation. NALLM, N-acetyl-l-leucyl-l-leucyl-l-methional; calpeptin; E64, each at 30 μM; triplicate wells of DCs per group and 2 independent experiments.∗p < 0.01, ∗∗p < 0.006, 1-way ANOVA with Tukey’s post-test.

Fig 3: BAdv85C5 booster induces stronger cytokine-positive T cells and effector (TEM) and memory T cells (TCM) in BCG-vaccinated mice following Mtb challenge(A–D) Lungs and spleens of mice vaccinated and challenged as in Figure 6B were stained for CD4 and CD8 T cells expressing IFN-γ and IL-2 and analyzed using flow cytometry (∗∗∗∗p < 0.001; ordinary 1-way ANOVA). Arrowheads indicate BAdv85C5-induced T cell expansion. Flow cytometry data presented for 1 of 2 similar experiments integrated in Figure 6C.(E–H) The lungs and spleen of mice vaccinated and challenged as in Figure 6B were stained for CD4 and CD8 TEM and TCM followed by flow cytometric analysis (∗p < 0.01, ∗∗∗,∗∗∗∗p < 0.001, ordinary 1-way ANOVA). Arrows indicate BAdv85C5-induced T cell expansion. TRMs (CD4+CD103+CD69+) on day 21 for post-vaccination (vax) and day 60 post-Mtb challenge are shown in Figure S7.

Fig 4: Immune responses post P@aPDL1 and Vadimezan treatment in the 4T1 tumour model.a Relative quantification of the CD3+ T cells in the PB, n = 5 biologically independent animals. b Representative flow cytometry analysis of CD4+ T cells, CD8+ T cells gating on CD3+ T cells. Relative quantification of the c CD4+ T cells and d CD8+ T cells, n = 5 biologically independent animals. e Representative immunofluorescence images of metastatic tumour in the lung showing CD4+ T cells (green) and CD8+ T cells (red) after treatment, scale bar: 100 µm. Representative flow cytometry analysis of f IFN-γ+CD8+ T cells and g Ki-67+CD8+ T cells gating on CD3+ T cells, n = 5 biologically independent animals. h Cytokine levels in the serum of mice, n = 5 biologically independent animals. G1, PBS; G2, aPDL1; G3, Vadimezan; G4, P@aPDL1; G5, P@aPDL1 + Vadimezan; G6, P@IgG + Vadimezan. Data are presented as mean ± SD, statistical significance was analysed via ANOVA (one-way, Tukey post-hoc test). P-value: **P < 0.01, ***P < 0.001. a G1 vs. G5: P < 0.0001, G2 vs. G5: P < 0.0001, G3 vs. G5: P < 0.0001, G4 vs. G5: P < 0.0001, G6 vs. G5: P < 0.0001. c G1 vs. G5: P < 0.0001, G2 vs. G5: P = 0.0007, G3 vs. G5: P = 0.0018, G4 vs. G5: P = 0.0005, G6 vs. G5: P < 0.0001. d G1 vs. G5: P < 0.0001, G2 vs. G5: P < 0.0001, G3 vs. G5: P < 0.0001, G4 vs. G5: P < 0.0001, G6 vs. G5: P < 0.0001. f G1 vs. G5: P < 0.0001, G2 vs. G5: P < 0.0001, G3 vs. G5: P < 0.0001, G4 vs. G5: P < 0.0001, G6 vs. G5: P < 0.0001. g G1 vs. G5: P < 0.0001, G2 vs. G5: P < 0.0001, G3 vs. G5: P < 0.0001, G4 vs. G5: P < 0.0001, G6 vs. G5: P < 0.0001. h TNF-α: G1 vs. G5: P < 0.0001, G2 vs. G5: P = 0.0009, G3 vs. G5: P < 0.0001, G4 vs. G5: P < 0.0001, G6 vs. G5: P < 0.0001; IFN-γ: G1 vs. G5: P < 0.0001, G2 vs. G5: P < 0.0001, G3 vs. G5: P < 0.0001, G4 vs. G5: P < 0.0001, G6 vs. G5: P < 0.0001; IL-2: G1 vs. G5: P < 0.0001, G2 vs. G5: P < 0.0001, G3 vs. G5: P < 0.0001, G4 vs. G5: P = 0.0007, G6 vs. G5: P < 0.0001; IL-6: G1 vs. G5: P < 0.0001, G2 vs. G5: P < 0.0001, G3 vs. G5: P < 0.0001, G4 vs. G5: P < 0.0001, G6 vs. G5: P < 0.0001; IL-12: G1 vs. G5: P < 0.0001, G2 vs. G5: P < 0.0001, G3 vs. G5: P < 0.0001, G4 vs. G5: P < 0.0001, G6 vs. G5: P < 0.0001.

Fig 5: mtDNA transfection activates Influenza A-specific CD8+ T cells Scheme of the immunoproteasome-specific T cell assay detecting the HLA-A2 restricted GILGFVFT peptide in phLF.Flow cytometric detection of Influenza-M1/HLA-A2 complex in phLF upon transfection with mtDNA, mtDNA pretreated with DNase I, or treatment with IFNß (n = 3 independent experiments).Scheme of M1/HLA-A2 restricted CD8+ T cells (4VA1 T cell clone) activated by mtDNA stressed phLF that had been loaded with Influenza-M1 peptide.CD8+ T-cell activation assay of Influenza-M1/HLA-A2 restricted CD8+ T cells upon co-culture with mtDNA-transfected phLFs loaded with M1 peptide or upon treatment with IFNß or IFN? displaying secretion of IL-2 from three different biological experiments. Data information: All data are shown as mean ± SEM and analyzed with one-way ANOVA test. Asterisks indicate significance as *P < 0.05, **P < 0.01. Source data are available online for this figure.