A nanovaccine for antigen self-presentation and immunosuppression reversal as a personalized cancer immunotherapy strategy

[ad_1]

Mice and cell lines

All the animals were cared for and treated according to the instructions and approval of the Institutional Animal Care and Use Committee of Xiamen University. Male and female C57Bl/6 mice (6–8 weeks old) were purchased from SLAC (Shanghai). B7-1/2-deficient mice with a C57BL/6 genetic background were generated by the Xiamen University Laboratory Animal Center. OT-1 mice were provided by the Chinese Academy of Medical Sciences. The mice were maintained under specific pathogen-free conditions in the animal facility at Xiamen University. 293T HEK, DC2.4, Hep1-6-OVA, B16F10, B16F10-Luc, MC-38, MC-38-OVA and LLC cells were acquired from the National Institute of Diagnostics and Vaccine Development in Infectious Diseases (Xiamen University). For the isolation of T cells from spleen or tumour tissue, the Pan T-cell Isolation Kit (Miltenyi Biotec) was used. For the isolation of CD8+ T cells, peripheral blood monocytes (PBMCs) from volunteers were isolated, or spleens from C57BL/6 or OT-1 mice were digested by Liberase (Roche Diagnostics) and DNase I (SIGMA-ALDRICH) to generate single-cell suspensions, which were then stained with R-phycoerythrin (PE)-conjugated anti-CD8 antibody and applied to anti-PE microbeads (Miltenyi Biotec) for isolation. Human PBMCs were freshly isolated from Chinese healthy adult volunteers with informed consent. All the experiments that used human PBMCs were approved by the Medical Ethics Committee of the School of Public Health of Xiamen University. Splenic lymphocytes were isolated by a splenic lymphocyte kit (Dakewe Biotech). Unless otherwise specified, DC2.4 is referred to as DC in the article. DC2.4 cells were cultured in a medium with rmGM-CSF (recombinant granulocyte-macrophage colony-stimulating factor)and rmIL-4 (all purchased from ThermoFisher). The culture medium used for DCs and T cells was RPMI-1640 (Gibco), supplemented with 10% fetal bovine serum (Gibco), 100 U ml–1 penicillin (Invitrogen) and 100 µg ml–1 streptomycin (Invitrogen). The culture medium used for 293T HEK, Hep1-6-OVA, B16F10, B16F10-Luc, MC-38, MC-38-OVA and LLC cells was Dulbecco′s Modified Eagle Medium (Gibco) supplemented with 10% fetal bovine serum, 100 U ml–1 penicillin and 100 µg ml–1 streptomycin. Throughout the studies, all the cells were used as received and tested negative for mycoplasma contamination and rodent pathogens.

Recombinant adenovirus construction

A signal sequence led by a Kozak consensus sequence was fused in a frame to the N terminus of an antigen protein/epitope (GFP/OVA, or M27-M30-TRP2/ASMTNMELM with ten copies) gene-constant region, a PDGFR transmembrane domain was fused to the C terminus and a FLAG Tag fused after the transmembrane domain to analyse the epitope expression26,27. The frame was cloned to shuttle plasmid vector pDC316. Finally, the recombinant adenovirus (rAd-GFP/OVA, rAd-MultiAgs and rAd-neo) was produced using the simple system of AdMax (Ad5) (Supplementary Fig. 1). The sequences were designed and analysed with Snapgene (version 2.3.2).

Preparation of DC-rAd-Ag

Immature DC2.4 cells were plated at 1 × 106 cells per well in 12-well plates. After 24 h, the DCs were infected with a recombinant adenovirus titre of 100 multiplicity of infection (MOI), or cells in the control groups were incubated with 20 µg of pDC316-GFP or 50 µg of antigen proteins in various formulations (that is, PBS/Ag/Liposome@Ag/Ag@IONPs), in complete media with the addition of a maturation factor combination (1,000 U ml–1 rmGM-CSF, 1,000 U ml–1 rmIL-4 and 200 U ml–1 rmTNF-α) for different lengths of time (2, 6, 24 and 48 h) at 37 °C with 5% CO2. DCs were harvested, washed with FACS buffer (1% fetal bovine serum in PBS), incubated with anti-CD16/32 (Biolegend) at room temperature and then stained on ice with anti-B7-1-APC/anti-B7-2-FITC/anti-MHC-I(H-2Kb)-PE antibodies or PE-conjugated antimouse SIINFEKL/H-2Kb monoclonal antibody 25-D1.16 (all purchased from eBioscience). Cells were analysed by flow cytometry (Cyan 5, Beckman Coulter). DCs cultured in medium with 1,000 U ml–1 rmGM-CSF and 1,000 U ml–1 rmIL-4 were stained as the background to quantify the MHC-I expression level.

Preparation and characterization of DCNV-rAd-Ag

Immature DC2.4 cells were infected with a recombinant adenovirus titre of 100 MOI, and cultured in the medium with the addition of a maturation factor combination for 24 h.

According to our previous work26,27,28, the cells were collected and washed in cold PBS mixed with protease inhibitor twice to remove cellular debris and culture medium. Next, the cells were suspended in saline and sonicated in a sterile 1.5 ml EP tube under a low power (22.5 W, 1 min) on ice. Then the NVs were isolated by multistep density gradient ultracentrifugation before being resuspended in saline. The product was then introduced to a Mini-Extruder (Avanti Polar Lipids) equilibrated in saline (200 nm pore-sized membrane filters) to further purify uniform NVs (DCNV-rAd-Ag).

DCNV-rAd-MultiAgs wash, DCNV-Ad was prepared from DC2.4 cells that had been infected with a blank adenovirus titre of 100 MOI for 24 h, DCNV was prepared from DC2.4 cells without any treatment, 293TNV-rAd-Ag was prepared from 293T HEK cells that had been infected with a rAd-Ag titre of 100 MOI for 24 h. All the NVs were prepared as mentioned above.

hDCNV-rAd-Ag/hDCNV-Ad/hDCNV was prepared from DCs of human PBMCs. Briefly, human PBMCs were isolated from healthy blood donors using Ficoll density gradient centrifugation (Biocoll, Biochrom AG). CD14-positive monocytes were isolated by MACS selection (Miltenyi), and cultured for 5 days in complete media with the addition of a stimulating factor combination (1,000 U ml–1 rhGM-CSF and 1,000 U ml–1 rhIL-4), and then immature DCs were obtained. Immature DCs were infected with a recombinant adenovirus titre of 100 MOI, and cultured in the medium with the addition of a maturation factor combination (1,000 U ml–1 rhGM-CSF, 1000 U ml–1 rhIL-4 and 200 U ml–1 rhTNF-α) for 24 h, and the corresponding NVs were prepared according to the DCNV-rAd-Ag preparation procedure.

The presence of NVs was verified through the morphological examination by cryo-electron microscopy, transmission electron microscopy and size measurement using dynamic light scattering (Zetasizer software 7.13). To detect the presence of major functional membrane proteins on the DCNV-rAd-Ag, a western blot assay was performed. Briefly, membrane proteins were extracted from 0.5 ml (1 mg ml–1 total protein) of DCNV-rAd-Ag by a ProteoExtract Transmembrane Protein Extraction Kit (Merck). Samples were probed with antibodies against GFP/OVA, MHC-I, B7-1, B7-2, ICAM-I or CCR7 (all purchased from Invitrogen). The major functional membrane proteins were visualized with an HRP-antimouse/rabbit immunoglobulin G antibody (Invitrogen) and ECL substrate (Thermo). To determine the content of Ag in the total amount of protein, a GFP/OVA ELISA kit or FLAG ELISA kit (Abcam) was used to directly or indirectly quantify the amount of Ag. Membrane proteins on DCs and NVs were extracted and then identified by liquid chromatography/tandem mass spectroscopy analysis. Raw data files were processed using Proteome Discoverer (PD) version 1.4 (Thermo Scientific).

Preparation and characterization of ASPIRE

A signal sequence was fused in the N-terminus of the anti-PD1 scFv gene region, and a HIS-Tag was fused before the scFv gene region. To facilitate the analyses of epitope expression, a flexible peptide linker (GGGGS)3 and a PDGFR transmembrane domain were fused to the C terminus. The gene encoding anti-PD1 ScFv was finally cloned into plasmid vector pcDNA3.1(+) to express membrane-localized anti-PD1 antibodies in DCs (αPD1-DC) (Supplementary Fig. 16). Immature DCs transfected with pcDNA3.1(+)-αPD1 (105 cells µg–1) were cultured in the culture medium with the addition of growth factor combination (that is, 1,000 U ml–1 rmGM-CSF and 1,000 U ml–1 rmIL-4). After 48 h of culture, the cells were infected with the recombinant adenovirus mentioned previously (rAd-GFP/OVA, rAd-MultiAgs, rAd-neo), and cultured in the culture medium with the addition of maturation factor combination (1,000 U ml–1 rmGM-CSF, 1,000 U ml–1 rmIL-4 and 200 U ml–1 rmTNF-α) for another 12 h. The cells were collected and NVs were extracted in a similar procedure to that in Preparation and characterization of DCNV-rAd-Ag.

αPD1-DCNV/αPD1-DCNVB7–/– were prepared after αPD1-DC/αPD1-DCB7–/– were induced by maturation factor combination with 12 h of incubation. The cells were collected and NVs were extracted in a similar procedure to that in Preparation and characterization of DCNV-rAd-Ag.

To detect the orientation of the αPD-1 antibody on ASPIRE or αPD1-DCNVs, an immunoprecipitation assay was performed. Briefly, 0.5 ml (1 mg ml–1 total protein) of ASPIRE or αPD1-DCNVs were incubated with beads (Santa Cruz Biotechnology) conjugated to protein A/G for 1 h at room temperature after the addition of 2 μg of anti-His antibody specific for the fusion protein HIS-αPD1. The beads were washed gently thrice with PBS. Sample mixtures were resolved and subjected to 10% SDS–PAGE and analysed by western immunoblot assay.

Antigen self-presentation to activate naive CD8+ T cells

To assess the ability of DCNV-rAd-Ag for antigen self-presentation and to directly activate naive T cells, CD8+ T cells (2 × 105 per well) selected from the spleen of C57BL/6 mice were co-cultured with different doses of DCNV-rAd-GFP/293TNV-rAd-GFP/DCNV-Ad/DCNV formulations (0.5, 5 and 20 µg ml–1 NVs) in RPMI 1640 culture medium. After 7 days of incubation, the cells were transferred to ELISPOT plates (Merck), which were pretreated with 95% ethanol and washed with sterile water and PBS before coating with 15 µg ml–1 anti-IFN-γ or anti-TNF-α (Mabtech) overnight at 4 °C. Unbound antibodies were removed by washing with sterile filtered PBS. After 24 h of incubation, the cells were washed away and the plates were incubated with 1 µg ml–1 biotinylated anti-IFN-γ or anti-TNF-α antibody (Mabtech) for 2 h at room temperature. Thereafter, the plates were washed with PBS and incubated for 90 min at room temperature with streptavidin–alkaline phosphatase (Mabtech) (1/1,000 dilution) followed by washing with PBS. The plates were finally incubated with a BCIP/NBT substrate solution (CALBIOCHEM) at room temperature until spots emerged, which took approximately 1 h. The colour development was stopped by repeated washings with tap water. After drying, the spots were counted with an ELISPOT reader using AID ELISPOT software. To detect the activation of primary human T cells, human PBMCs were isolated from healthy blood donors using Ficoll density gradient centrifugation. CD8-positive T cells were isolated by MACS selection (Miltenyi). CD8+ T cells (2 × 105 per well) were co-cultured with different doses of hDCNV-rAd-GFP/293TNV-rAd-GFP/hDCNV-Ad/hDCNV formulations prepared in PBMCs isolated from the same volunteer (0.5, 5 and 20 µg ml–1 NVs) in RPMI 1640 culture medium. After 7 days of incubation, the cells were transferred to the ELISPOT plates (Merck), and T-cell activation was detected by ELISPOT assay as mentioned above.

To further verify that DCNV-rAd-Ag directly activates T cells and stimulates T-cell specific proliferation, 105 OT-I CD8+ T cells per well were co-cultured with different doses of DCNV-rAd-OVA and 293TNV-rAd-OVA formulations (0.5, 5 and 20 µg ml–1 NVs) in RPMI 1640 culture medium. After 24 h of incubation, the media were aspirated and 150 µl of CPRG/lysis buffer was added and incubated for 90 min, measured at 570 nm using a microplate reader. The IL-2 and TNF-α in the culture supernatant were determined by a Mouse IL-2 ELISA kit or a TNF-α ELISA kit (both purchased from Dakewe Biotech).

For the detection of T-cell proliferation dilution, an OT-I CD8+ T-cell suspension was stained with a carboxyfluorescein succinimidyl ester solution, and co-cultured with 20 µg ml–1 NVs in RPMI 1640 culture medium for 48 h, followed by flow cytometric analysis.

In vivo imaging of DCNVs

For LN draining studies, C57BL/6 mice were treated with DCNV-rAd-OVA@ICG or 293TNV-rAd-OVA@ICG by s.c. injection at the tail base24,26. Under isoflurane anaesthesia, in vivo near-infrared fluorescence imaging was performed using an IVIS Lumina II imaging system at 12 h postinjection. C57BL/6 mice were injected with DCNV-rAd-GFP or DCNV-rAd-GFP. After 12 h, inguinal LNs were harvested, and after a 10% formaldehyde solution fixation, paraffin embedding and freezing section, the GFP fluorescence signal was measured.

In vivo immunization and cancer immunotherapy studies

All the mice used for immunological studies were 6–8-week-old females with a C57Bl/6 background. All the animals were cared for and treated according to the instructions and approval of the Institutional Animal Care and Use Committee of Xiamen University (no. XMULAC20190146), and it was defined that the tumour load of mice must not exceed 1.7 cm (diameter). The tumour volume throughout this study was calculated by the equation: tumour volume = length × width2 × 0.5. Animals were euthanized when the tumour masses reached 1.5 cm in diameter or when the animals became moribund with severe weight loss or ulceration. C57BL/6 mice were immunized with different formulations: NVs (60 µg per mouse), recombinant adenovirus (2 × 107 vector particles per mouse) or complete dendritic cell vaccine (2 × 105 cells per mouse) in 100 µl by s.c. injection at the tail base at the indicated time points. In some studies, antigen emulsified in AlumOH served as a positive control. Briefly, antigen protein (2 nmol) in 0.5 ml of PBS was thoroughly emulsified in 0.5 ml of AlumOH until the mixture was homogeneous, and then administered subcutaneously in a 100-µl-injection volume.

For prophylactic tumour challenge studies, vaccinated animals were challenged on day 14 after the final immunization by the s.c. injection of 2 × 105 Hep1-6-OVA cells per mouse on the right flank, and tumour growth was monitored every other day. Livers were excised on day 18, followed by enumeration of the Hep1-6-OVA lung tumour nodules. For the CD4+/CD8+ cell depletion, 250 µg of GK1.5 antibody or 53-6.7 antibody (BioXcell) were administered to the vaccinated tumour-bearing mice by intraperitoneal injection every 2 weeks. For B7-1/2–/– mice, vaccinated gene knockout animals were challenged after the final immunization by Hep1-6-OVA cells.

For the therapeutic tumour vaccination studies, C57BL/6 mice were inoculated with 1 × 105 B16F10 or MC-38 cells per mouse on the right flank by s.c. injection on day 0 and vaccinated on the indicated days. For the combinatorial therapy groups, the antimouse PD-1 antibody (10.3 µg per mouse; clone, J43 (BioXcell)) was administered intraperitoneally after each vaccination. For the lung metastasis model, the surviving mice after ASPIRE treatment were rechallenged by the intravenous injection of 5 × 104 B16F10-Luc cells per mouse, and a group of untreated mice were inoculated as a control. Under isoflurane anaesthesia, the fluorescence signal of lungs metastasis was measured every day using an IVIS Lumina II imaging system.

For the co-stimulatory study of the anti-PD1 antibody and B7-1/2, C57BL/6 mice were subcutaneously inoculated with 2 × 105 LLC cells and CD4 depletion was maintained for the duration of the experiment by repeated injections of 250 µg of GK1.5 antibody every 7 days. αPD1-DCNVs were administered by s.c. injection at the tail base every 3 days. For the co-stimulatory study in KO mice, B7-1/2–/– mice were inoculated with 1 × 105 MC-38 per mouse by s.c. injection on day 0, and MC-38-specific CTLs were intraperitoneally administered after each vaccination on the indicated days.

For the therapeutic tumour vaccination studies, C57BL/6 mice were inoculated with 1 × 105 MC-38 cells per mouse on the right flank by s.c. injection on day 0, and vaccinated on days 10 and 13 with αPD1-DCNV or DCNV-rAd-neo separately in the order indicated, or vaccinated on day 13 with αPD1-DCNV, DCNV-rAd-neo or ASPIRE (60 µg per dose).

For the antitumour study of ASPIRE in heterogeneous tumours, C57BL/6 mice and antigen-presentation-deficient mice were inoculated subcutaneously with 3 × 105 heterogeneous tumour cells (MC-38-OVA cells mixed with MC-38 cells in various proportions) and vaccinated with the indicated formulations (60 µg of NVs, 2 × 105 DCs or 0.2 nmol OVA) on days 10, 17 and 24. The mature DCs (CD11c+CD80+MHCII+) in tumour-infiltrating DCs were detected on day 20. Intracellular protein staining and flow cytometric analysis were used for the analysis of OVA in heterogeneous tumour cells at the indicated time points.

Phenotypic and functional assessment of T cells

For the analysis of the activation of peripheral blood lymphocytes, at 2 weeks after the final immunization peripheral blood was harvested from the animals in all the treatment groups. The peripheral blood lymphocytes were prepared and incubated in RPMI 1640 media with 1 μM of the SIINFEKL peptides added. After 24 h, the IL-2/TNF-α/IFN-γ production was measured with the ELISA assay system (purchased from Dakewe Biotech) according to the manufacturer’s instructions.

Immunized mice were analysed for the percentages of tumour antigen-specific CD8+ T cells using the tetramer staining assay with a peptide–MHC tetramer tagged with PE (H-2Kb-restricted SIINFEKL, MBL Beijing Biotech). Briefly, the peripheral blood lymphocytes were resuspended in a mouse CD16/32 antibody (0.025 mg ml–1) solution to block non-specific and FcR-mediated antibody binding. The suspension was incubate for 10 min at room temperature and washed five times with FACS buffer. Then H-2Kb OVA Tetramer-SIINFEKL-PE solution was added to each sample and incubated for 30 min on ice. Anti-CD8-APC was added to each experimental sample and incubated for 20 min on ice. The samples were washed twice with FACS buffer, fixed and the cells resuspended for flow cytometry analysis. To assess the functionality of the primed CD8+ T cells, PBMCs were stimulated ex vivo with the peptide pool (1 μM of each antigen peptide, M27, M30 and TRP2) for 6 h, fixed, permeabilized, stained with anti-IFN-γ-eFluor 660/TNF-α-FITC and anti-CD8-APC, and analysed by flow cytometry.

For the analysis of tumour cells or tumour-infiltrating T cells, tumour tissues were excised at the indicated time points, cut into small pieces of 2–4 mm and then placed in a dissociation buffer (1 mg ml–1 collagenase type IV and 0.1 mg ml–1 DNase I in RPMI) for 30 min at 37 °C with gentle shaking. The cell suspension was passed through a 70 µm strainer, washed with FACS buffer and stained with the indicated antibodies or its isotype control, followed by flow cytometric analysis. The intracellular cytokine staining assays on tumour-infiltrating T cells were performed with anti-IFN-γ-eFluor 660 and anti-TNF-α-FITC.

For the analysis of CD8+ T cells in the draining LNs, the draining LNs were excised at the indicated time points. Cell suspensions were prepared and stained with anti-CD8-APC, followed by flow cytometric analysis.

Immunized MC-38 mice were analysed for the percentages of tumour-infiltrating antigen-specific CD8+ T cells using a tetramer staining assay with the peptide–MHC tetramer tagged with PE (H-2Kb-restricted ASMTNMELM, MBL Beijing Biotech), functional cell subtypes were analysed by staining with FITC-anti- PD-1/eFluor450-anti-CD38/PerCP-eFluor710-anti-Granzyme B/FITC-anti-CD44/PE-antimouse CD62L for flow cytometry analysis.

Statistical analysis

All the animal studies were performed after randomization. Experiments were not performed in a blinded fashion. Data were analysed by one- or two-way ANOVA, followed by Bonferroni post hoc test for comparison of multiple groups with Prism (v5.0 and v7.0) (GraphPad Software). Data were normally distributed and the variance between groups was similar. P values less than 0.05 were considered statistically significant. All the values were reported as mean ± s.d. with the indicated sample size. No sample in any representative experiment was excluded from the analysis.

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

[ad_2]

Source link

Leave a Reply

Your email address will not be published.