Fig 2: pAntp CPP conjugates exhibit enhanced LN accumulation and uptake by APCs in dLNs relative to free peptide antigen. Groups of C57BL/6 mice (n = 3 per group) were immunized subcutaneously with 25 nmol cy5-labeled gp100 or cy5-gp100-pAntp with 25 µg c-di-GMP, and then at selected times inguinal dLNs were isolated for histology or flow cytometry analysis. (A and B) Immunofluorescence of dLNs 48 h after immunization with cy5-gp100 (A) or cy5-gp100-pAntp (B). (Scale bars, 100 µm for main images and 50 µm for Insets.) (C–F) Representative dot plots from day 2 and quantification of percentage of peptide+ cells at 2- or 6-d postvaccination with cy5-gp100 and cy5-gp100-pAntp for (C) CD19+ cells; (D) F4/80+ macrophages; (E) cDC1 cells; and (F) cDC2 cells. **P < 0.01, *P < 0.05, by unpaired Student’s t test. (G) Representative flow cytometry plots showing up-regulation of MHCII and CD86 on CD11c+ DCs in response to the vaccination with the gp100 peptides with or without addition of c-di-GMP adjuvant. (H) Quantification (n = 3 animals per group) of percentage of MHCIIhi/CD86hi DCs in each condition. n.s., not significant by two-way ANOVA with a Bonferroni posttest.

Fig 3: 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 4: 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 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.