Fig 2: miR‐AB‐based multicolor‐barcoded multiplex RNAiCD8+ T cells were activated for 16 h and then cotransduced with miR‐AB retroviruses at 1:1 ratio, which carries a SplashRNA‐designed shRNAmir, targeting surface protein CD127, CD90, CD44, or CD8, with Azurite, GFP, Ametrine, or mCherry2 as fluorescent reporters, respectively, for 4 h. miR‐AB retroviruses expressing CD19‐specific shRNAmirs with same fluorescent reporters were used as controls. Surface staining was performed 72 h after transduction. FACS data were shown as overlaid histograms of control shRNAmirs (shCD19s) and target shRNAmirs (shCD127, shCD90, shCD44, or shCD8). Cells were categorized as 16 populations expressing single, double, triple, or quadruple fluorescent proteins or not (none) and fluorescent reporter‐positive plots were highlighted with thick frames and white background. Each population’s surface staining profile was shown on the right. The data are representative of two biologically independent experiments.

Fig 3: Expression of MC and Ag in cutaneous atypical APCs contribute to adjuvant-enhanced EP vaccine-mediate Ag-specific CD8+ T cell priming and is partly CD8α+/CD103+ DC-independent.C57BL/6 mice were vaccinated on days 0 and 21 with 25 μg pDNA by EP, rim was administered 1.25 mg/kg IP the day following each vaccination in MC.OVA.miR142T + rim-treated mice. (A) Splenocytes were extracted 7 days after the final vaccination (day 28) and restimulated with SIINFEKL peptide overnight. IFN-γ SFCs were quantitated by ELISpot. n = 5, *p<0.05, One-way ANOVA, Tukey correction for multiple comparisons. (B) C57BL/6 mice were injected in alternating flanks 21 days apart with 1 x 105 MPEK cells stables (neg. control, OVA, MC.OVA). Rimiducid was administered at 1.25 mg/kg IP 24 hours after each injection of cells. 7 days following the second injection, splenocytes were analyzed for SIINFEKL-specific IFN-γ-secreting CD8+ T cell by ELISpot. Error bars represent 95% confidence interval. *p<0.05 when compared to MPEK-OVA, One-way ANOVA, Dunnett’s correction for multiple comparisons. (C) Either C57BL/6 or Batf3-/- mice were vaccinated in the same manner as described for panel A. SIINFEKL-specific IFN-γ-secreting splenocytes were quantitated by ELISpot. n = 4–5, *p<0.05, One-way ANOVA, Tukey correction for multiple comparisons within mouse strain.

Fig 4: PS3D1@DMXAA inhibits breast tumor metastasis and synergizes with ICB to inhibit B16-melanoma.(A) Schematic diagram of the orthotopic breast tumor model and administration method. Mice bearing 4T1-Luci breast tumors were treated as in Fig. 5A. (B) Tumor growths are shown (n = 8). (C) Survival curves were compared using log-rank test (n = 8). (D) In vivo bioluminescence images of 4T1-Luci lung metastatic tumors. (E) Number of the lung metastatic nodules on day 25. One-way ANOVA with Tukey’s test. (F) Combined treatment scheme for mice with established B16.F10 tumors. (G) Tumor growths of B16.F10 tumor-bearing mice are shown (n = 8, means ± SEM). (H) Survival curves were compared using log-rank test (n = 8). Data are means ± SD, and statistical significance was calculated by two-tailed Student’s t test unless otherwise indicated. ***P < 0.001 and **P < 0.01. (I) Schematic illustration of the self-assembly of PS3D1@DMXAA nanoparticles with redox-responsive drug release in tumor cells. The electrostatic interaction between the tertiary amine group and DMXAA provides efficient drug loading. (1 and 2) Redox stimuli trigger SN38 release in tumor cells. SN38 induces tumor cell death and release of chemokine CCL4 that drives the infiltration of CD103+ DCs in the TME. (3) Meanwhile, PS3D1@DMXAA elicits efficient cytosolic delivery of DMXAA for STING activation in CD103+ DCs. Together, these enhance the maturation and TAA uptake of CD103+ DCs. (4 and 5) STING activation enhances the migration of mature CD103+ DCs into the tdLN and stimulates TAA cross-presentation by CD103+ DCs for cross-priming of TAA-specific effector CD8+ cytotoxic T cells. (6) PS3D1@DMXAA modulates the immunosuppressive TME and facilitates TAA-specific effector CD8+ cytotoxic T cell recruitment through CXCL9/CXCL10. All these eventually amplify the antitumor therapeutic effects.

Fig 5: LA modulates Treg-Th17 cell balance in vivo. Cells from bone marrow (BM) and spleen of sham, ovx and ovx + LA mice groups were isolated at the end of experiment, labelled and analyzed by Flow cytometry for percentage of CD4+Foxp3+, CD4+Rorγt+, CD4+IL-10+ Foxp3+ and CD4+Rorγt+IL-17+ cells. Gating was first performed for CD4+ and later analyzed for the expression of CD4+Foxp3+, CD4+Rorγt+, CD4+ IL-10+ (Tregs) and CD4+ IL-17+ (Th17) cells. A) Average percentage (%) of CD4+Foxp3+T cells. B) Average percentage (%) of CD8+Foxp3+T cells. C) Average percentage (%) of CD4+Rorγt+T cells. D) Average percentage (%) of Foxp3+ IL-10+ (Tregs) cells. E) Average percentage (%) of Rorγt+IL-17+ (Th17) cells. The results were evaluated by using ANOVA with subsequent comparisons by Student t-test for paired or nonpaired data, as appropriate. Analysis was performed using Sigma plot software (Systat Software, Inc., Germany). Values are reported as mean ± SEM (n = 10) and similar results were obtained in three independent experiments. Statistical significance was defined as p ≤ 0.05 (*p ≤ 0.05, **p ≤ 0.001) with respect to ovx mice group.