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. 2021 May 18;12:2935. doi: 10.1038/s41467-021-23244-3

Fig. 4. PLGA-MP containing co-encapsulated tumor antigens and Riboxxim efficiently induce CD8+ T-cell proliferation, increased tumor-specific T-cell responses, and peptide-specific in vivo cytotoxicity.

Fig. 4

Analysis of immune responses against PLGA-microparticles (MP) containing OVA (a–f) or tumor-associated antigen (TAA)-epitopes (g) and polyI:C (pIC, blue circles) or Riboxxim (Rib, red squares). a, b For adjuvant and particle dose titration, C57BL/6J mice (n = 4) were immunized s.c. with 5 mg MP charged with 250 µg OVA and either different amounts of dsRNA analogs per MP (a) or varying amounts of MP-OVA-polyI:C (OVA/pIC, blue circles, n = 4) or MP-OVA/Riboxxim (OVA/Rib, red squares, n = 4) per mouse (b). Six days post-immunization, CTL activation was monitored by ICS for IFNγ+ of CD8+ splenocytes. Statistics: two-way ANOVA followed by Šídák’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. c Improved CTL-mediated cytotoxicity after vaccination of C57BL/6J mice with MP loaded with 250 µg OVA and different amounts of polyI:C (OVA/pIC, blue circles, n = 6) compared to different amounts of Riboxxim (OVA/Rib, red squares, n = 6) was monitored in vivo by specific lysis of CFSE-labeled, SIINFEKL peptide-loaded target cells. Mice immunized with MP-Empty (Ø, black, n = 3) served as a control for antigen-specific killing. Representative flow cytometry histograms of peptide-pulsed target cells and unpulsed reference cells are shown on the right. Statistics: two-way ANOVA followed by Šídák’s multiple comparisons test. *P < 0.05; ***P < 0.001. d Kinetics of antigen-specific immune responses after vaccination of C57BL/6J mice (n = 5–8 for OVA/pIC, n = 5–7 for OVA/Rib) with 5 mg MP charged with 250 µg OVA and 2.5 µg dsRNA analogs was monitored by ICS for IFNγ+ of CD8+ splenocytes at different time points after immunization. PLGA-MP containing dsRNA adjuvants alone served as control (Adj ctrl, adjuvant control, n = 8). Statistics: two-way ANOVA followed by Šídák’s multiple comparisons test. **P < 0.01; ****P < 0.0001. e Clonal expansion of CFSE-labeled OT-1 cells was analyzed in vivo after i.v. injection into PLGA-MP-vaccinated C57BL/6J mice (n = 3) (e) or in vitro by T-cell count after co-culture with PLGA-MP-treated BMDCs (n = 6, MP Empty, black diamonds; MP OVA/pIC, blue circles, MP OVA/Rib, red squares) or unpulsed BMDCs (Ø, black diamonds, n = 3) (f) by CFSE dye dilution using flow cytometry. Statistical significance was analyzed compared to the MP Empty group by one-way ANOVA followed by Tukey’s multiple comparisons test. ****P < 0.0001. g Immune responses against indicated tumor peptide antigens were monitored by ICS for IFNγ+ of CD8+ splenocytes 6 days after vaccination of AAD mice (n = 3–20) with 50 µg of different TAA and 2.5 µg Riboxxim encapsulated into PLGA-MP. Mice immunized with MP Empty served as a control (Empty). All data are presented as means ± SD. Graphs represent pooled data derived from at least two independent experiments with a similar outcome.