Fig 2: Gemcitabine-cisplatin chemotherapy attenuates the maturation of immunogenic CD11c + MHCII + dendritic cells.a Flow cytometric gating strategy for dendritic cells from G69-tumor tissues. b, c Flow cytometry plots and corresponding quantification of CD11c + MHCII + dendritic cells (DC) from vehicle and GC-treated G69-tumor tissues (n = 11 and n = 12 biologically independent samples respectively). Data are presented as mean values ± SEM. d Relative quantification (%CD11c + MHC2+) of H2-K, CD86, and CD40 expression in tumor-infiltrating DCs from vehicle and GC-treated G69-tumor tissues (n = 6 and n = 5 biologically independent samples, respectively). Data are presented as mean values ± SEM. (Two-tailed, unpaired t-test. ****p < 0.0001, **p = 0.008). e Mean fluorescence intensity (MFI) of IL12, IL10, and IL4 expression in CD11c + MHC2 + DCs from vehicle or GC-treated G69-tumor tissues (n = 4 and n = 6 biologically independent samples, respectively). Data are presented as mean values ± SEM. (Two-tailed, unpaired t-test. *p = 0.018, **p = 0.003). Source data are provided as a Source data file. ns not significant.

Fig 3: Gemcitabine-cisplatin chemotherapy promotes an overwhelmingly high release of inhibitory DAMP to attenuate DC maturation.a Western blot analysis of COX-2 expression in G69 cell lysates treated with either vehicle or GC for 48 h. GAPDH expression was used as loading control (n = 3 biologically independent experiments). b ELISA for PGE2 release in the culture medium of G69 cells treated with either vehicle control or GC for 48 h (n = 4 independent biological samples). Data are presented as mean values ± SEM. (Two-tailed, unpaired t-test **p = 0.0016). c COX-2 expression in G69-tumor tissue lysates treated with either vehicle or GC for two chemotherapy cycles. GAPDH expression was used as a loading control (n = 3 biologically independent experiments). d ELISA for PGE2 release in the serum of G69-tumor-bearing mice treated with either vehicle or GC for 2 cycles (n = 5 and n = 4 biologically independent samples for vehicle and GC, respectively). Data are presented as mean values ± SEM. (Two-tailed, unpaired t-test ****p < 0.0001). e Immunofluorescence analysis of COX-2 expression in G69-tumor tissues treated with either vehicle or GC for two chemotherapy cycles (Scale bar: 100 µm; Images representative of n = 3 independent experiments). f Schematic representation of BMDC treatment with conditioned media in the presence or absence of iDAMP blockade. g Flow cytometric analysis of CD40, CD86, and MHCI expression in BMDCs treated with G69-conditioned media with or without iDAMP blockade. h Geometric mean fluorescence intensity (gMFI) of CD40, CD86, and MHCI molecule expression in BMDCs upon treatment with condition media as in g (n = 2 biologically independent samples). Data are presented as mean values. i Schematic representation of footpad vaccination assay. j Flow cytometric analysis of CD40, CD86 and MHCI expression in vaccination-draining lymph node CD11c + MHC2 + DCs upon injection of G69 cells treated with GC in the presence or absence of iDAMP blockade (n = 5 biologically independent samples). Non-vaccination-draining lymph node DCs were used as control (n = 3 biologically independent samples). Data are presented as mean values ± SEM. (One-way ANOVA-Tukey’s multiple comparisons test. CD40: GC vs GC+ Celex, p = 0.045; GC vs GC+ aPGE2, p = 0.036; CD86: GC vs GC+ Celex, p = 0.046; GC vs GC+ aPGE2, p = 0.0007; H2-K: GC vs GC+ Celex, p = 0.0004; GC vs GC+ aPGE2, p = 0.005). Source data are provided as a Source data file. ns not significant.

Fig 4: 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 5: iDAMP blockade inhibits tumor growth by promoting pro-inflammatory DC maturation.a Percent growth curves (top) and tumor weight (bottom) of G69 tumors from vehicle (n = 7 biologically independent animals), Celecoxib (n = 7 biologically independent animals), GC (n = 8 biologically independent animals), and GC+ Celecoxib-treated mice (n = 7 biologically independent animals) upon two cycles of chemotherapy. Percent tumor growth was obtained by normalizing to starting tumor volumes. Doted gray rectangles denote the starting point and the end point of each GC cycle. Celecoxib was injected ip 48 h before GC treatment initiation and continued daily to endpoint. Data are presented as mean values ± SEM. (Top: One-way ANOVA-Tukey’s multiple comparisons test. GC+ Celex vs Vehicle p = 0.006; vs Celex p = 0.029; vs GC p = 0.001; Bottom: One-way ANOVA-Tukey’s multiple comparisons test. GC+ Celex vs Vehicle p = 0.0006; vs Celex p = 0.007; vs GC p = 0.019). b Percent growth curves (top) and tumor weight (bottom) of G7 tumors from vehicle (n = 5 biologically independent animals), Celecoxib (n = 6 biologically independent animals), GC (n = 8 biologically independent animals), and GC+ celecoxib-treated mice (n = 8 biologically independent animals) upon two cycles of chemotherapy. Data are presented as mean values ± SEM. (One-way ANOVA-Tukey’s multiple comparisons test. GC+ Celex vs Vehicle; vs Celex p = 0.0002; vs Celex p = 0.0003; vs GC p = 0.028; Bottom: One-way ANOVA-Tukey’s multiple comparisons test. GC+ Celex vs Vehicle p < 0.0001; vs Celex p = 0.002; vs GC p = 0.004). c, d Flow cytometry plots and corresponding quantification of CD11c + MHCII + dendritic cells (DC) from vehicle (n = 11 biologically independent samples), Celecoxib (n = 5 biologically independent samples), GC (n = 12 biologically independent samples), and GC+ Celecoxib-treated (n = 12 biologically independent samples) G69-tumor tissues. Data are presented as mean values ± SEM. e Relative quantification (%CD11c + MHC2+) of H2-K, CD86, and CD40 expression in tumor-infiltrating DCs from vehicle (n = 6 biologically independent samples), celecoxib (n = 5 biologically independent samples), GC (n = 5 biologically independent samples), and GC+ Celecoxib-treated (n = 4 biologically independent samples) G69-tumor tissues. Data are presented as mean values ± SEM. (One-way ANOVA-Tukey’s multiple comparisons test. H2-k: Vehicle vs GC p < 0.0001, Celex vs GC p = <0.0001, GC vs GC+ Celex p = 0.004; CD86:Vehicle vs GC p = 0.047, Celex vs GC p = 0.038). f Mean fluorescence intensity (MFI) of IL12, IL10, and IL4 expression in CD11c + MHC2 + DCs from vehicle (n = 4 biologically independent samples), celecoxib (n = 5 biologically independent samples), GC (n = 6 biologically independent samples), and GC+ Celecoxib-treated (n = 5 biologically independent samples) G69-tumor tissues. Data are presented as mean values ± SEM. (One-way ANOVA-Tukey’s multiple comparisons test. IL10: Vehicle vs Celex p = 0.004, vs GC p = 0.006, Celex vs GC p = <0.0001, vs GC+ Celex p = 0.002, GC vs GC+ Celex p = 0.005; IL12:Vehicle vs Celex p = 0.02; vs GC p = 0.009). g Representative images of immunofluorescence analysis of CD8+ T cells (red) interaction with CD11c + DCs (green) and CRT + G69 cells (white) upon treatment with either GC or GC+ Celecoxib for 2 cycles (Scale bar: 100 µm; Images representative of n = 3 independent experiments). Source data are provided as a Source data file.