Efficient antigen cross-presentation through coating conventional aluminum adjuvant particles with PEI

Abstract

Classical aluminum adjuvant is a deficient antigen carrier for cross-presentation and cross-priming of CD8⁺ cytotoxic T cells. Our previous research has demonstrated that cross-presentation efficiency significantly increased when antigens are conjugated covalently to α-Al₂O₃ nanoparticles. Here we found that coating conventional aluminum adjuvants with polyethyleneimine (PEI) could enhance antigen cross-presentation of DCs (dendritic cells) in vitro and in vivo. PEIs exerted differential effects on antigen cross-presentation. These findings provided an alternative approach to promote the rapid translation of alumina nanoparticles adjuvants into clinical application.

Keywords: Aluminum adjuvant; PEI; antigen cross-presentation; dendritic cells.

Figure 1

Rehydragels (HPA, HS, LV) enhanced cross-presentation of murine DCs. A. Rehydragel alone, PEI alone and PEI-coated Rehydragel with different concentrations (0.03, 0.1, 0.3, 1, 3 μg/mL) were mixed with OVA protein (10 μg/mL) for 1 hour at room temperature. Mutu DCs were loaded with OVA/nanoparticles for 6 hours at 37°C and then cultured with B₃Z cells overnight. The B₃Z responses were analyzed by CPRG assay. Groups of PBS, OVA peptide (1 μg/mL) and B₃Z only were used as control in these experiments. B. The changes of DC cross-presentation by different Rehydragels were determined by CPRG assay when the concentration of PEI-coated Rehydragel was 1 μg/mL. *P<0.05, **P<0.01, ***P<0.001.

Figure 2

Different PEIs enhanced cross-presentation of murine DCs. A. HS particles were coated with bPEI0.8k or bPEI25k at different ratios and then mixed with OVA protein (10 μg/mL); B. HS particles were coated with different branched PEIs or linear PEIs and then mixed with OVA protein (10 μg/mL). Mutu DCs were loaded with HS/PEI-OVA for 6 hours at 37°C and then cultured with B₃Z cells overnight. The B₃Z responses were analyzed by CPRG assay. Groups of PBS, OVA protein (10 μg/mL), OVA peptide (1 μg/mL) and B₃Z only, B₃Z mixed with OVA were used as control in these experiments. ns, P>0.05, *P<0.05, **P<0.01.

Figure 3

Different Rehydragels and bPEIs mixtures enhanced cross-presentation of murine DCs. A. HS/bPEI0.8k (HS particles coated with bPEI0.8k), HS/bPEI25k (HS particles coated with bPEI25k), HS/bPEI0.8k+25k (HS particles coated with bPEI0.8k and bPEI25k simultaneously) and HS/bPEI0.8k+HS/bPEI25k (pure mixture of HS/bPEI0.8k and HS/bPEI25k together) were mixed with OVA protein (10 μg/mL), respectively. B. HS or LV particles were coated with bPEI0.8k or bPEI25k respectively, and then mixed with OVA protein (10 μg/mL). Mutu DCs were loaded with the Rehydragel/bPEI-OVA for 6 hours at 37°C and then cultured with B₃Z cells overnight. The B₃Z responses were analyzed by CPRG assay. Groups of PBS and OVA peptide (1 μg/mL) were used as control in these experiments. ns, P>0.05, *P<0.05, **P<0.01.

Figure 4

The mechanisms involved in enhanced DCs cross-presentation by Rehydragel/bPEI. A. Ub-OVA expression in the cytosol extract of mutu DCs. Mutu DCs cocultured with OVA protein (10 μg/mL), LV-OVA, LV/bPEI25k-OVA for 6 hours, and then the Ub-proteins from the cytosol extract were collected for Western blot analysis. The pure OVA protein (100 ng/mL) was used as control in this experiment. B. Rehydragel/bPEI increased IL-12 secretion by murine DCs. The secretion of IL-12 cytokine was detected by ELISA after mutu DCs were loaded with OVA, bPEI25k-OVA, LV-OVA or LV/bPEI25k-OVA. PBS was used as control in this experiment. *P<0.05, **P<0.01.

Figure 5

Cross-presentation of OVA protein by DCs was inhibited in the absence of 1IFNR. A. Mutu DCs, TLR3^-/- mutu DCs and IFNR^-/- mutu DCs were loaded with LV/bPEI25k-OVA for 6 hours for CPRG assay. B. Mutu DCs, murine BMDCs and CD40^-/- DCs were loaded with LV/bPEI25k-OVA for 6 hours for CPRG assay. Groups of PBS and OVA peptide (1 μg/mL) were used as control in these experiments. ns, P>0.05, *P<0.05.

Figure 6

Rehydragel/bPEI enhanced OVA cross-presentation in mutu DCs through proteasome, lysosome and TAP1 pathways. A. Mutu DCs, murine BMDCs and Tap1^-/- DCs were loaded with LV/bPEI25k-OVA for 6 hours for CPRG assay. B. Mutu DCs were treated with proteasome inhibitors and lysosome inhibitors, and then loaded with LV/bPEI25k-OVA for 6 hours for CPRG assay. Groups of PBS and OVA peptide (1 μg/mL) were used as control in these experiments. ns, P>0.05, *P<0.05, ***P<0.001.

Figure 7

Vaccination with Rehydragel/bPEI-OVA induced high frequency of OVA-specific IFN-γ producing CD8⁺ T cells in the B16-OVA tumor-bearing mice. Lymphocytes collected from the lymph nodes and spleens of B16-OVA tumor-bearing mice vaccinated with PBS, LV-OVA, LV/bPEI25k-OVA or OVA alone were co-incubated with SIINFEKL and anti-CD3. Intracellular IFN-γ staining of T cells was detected by flow cytometry. PBS and anti-CD3 were used as control in this experiment. A. Representative cytoflourograph. B. Cumulative data from three experiments. ns, P>0.05, **P<0.01, ***P<0.001.

Figure 8

Detection of the percentages of CD8⁺ T cells (Thy1.1⁺) that specifically recognize OVA_257-264 in the context of H-2K^b by Flow cytometry analysis. The B16-OVA tumor-bearing mice received adoptive transfer of OT-I spleen cells when LV-OVA, LV/bPEI25k-OVA, or OVA alone were injected into inguinal lymph nodes. The lymphocytes were stained with APC-labeled anti-mouse Thy1.1 antibody, PerCP-labeled anti-mouse CD8 antibody and MHC Dextramer H-2K^b/SIINFEKL conjugated with PE. A. Representative cytoflourograph. B. Cumulative data from three experiments. *P<0.05, **P<0.01.