Antigenically Modified Human Pluripotent Stem Cells Generate Antigen-Presenting Dendritic Cells

Abstract

Human pluripotent stem cells (hPSCs) provide a promising platform to produce dendritic cell (DC) vaccine. To streamline the production process, we investigated a unique antigen-loading strategy that suits this novel platform. Specifically, we stably modified hPSCs using tumour antigen genes in the form of a full-length tumour antigen gene or an artificial tumour antigen epitope-coding minigene. Such antigenically modified hPSCs were able to differentiate into tumour antigen-presenting DCs. Without conventional antigen-loading, DCs derived from the minigene-modified hPSCs were ready to prime a tumour antigen-specific T cell response and further expand these specific T cells in restimulation processes. These expanded tumour antigen-specific T cells were potent effectors with central memory or effector memory phenotype. Thus, we demonstrated that immunocompetent tumour antigen-loaded DCs can be directly generated from antigenically modified hPSCs. Using such strategy, we can completely eliminate the conventional antigen-loading step and significantly simplify the production of DC vaccine from hPSCs.

Figure 1. Tumour antigen gene-modified hPSCs produce tumour antigen-expressing DCs.

(a) Structure of lentivector LV.MP carrying a tumour antigen gene MART-1. (b) GPF expression in H1.MP cells, a H1 cell line generated by LV.MP transduction and G418 selection, as detected by flow cytometry. (c,d) MART-1 expression in H1.MP cells as measured by RT-PCR (c) and immunostaining (d). (e) GFP expression in H1.MP-derived DCs (H1.MP-DCs) as detected by flow cytometry. (f) MART-1 expression in H1.MP-DCs as measured by RT-PCR.

Figure 2. DCs derived from tumour antigen gene-modified hPSCs present tumour antigen.

(a) Proliferation of GFP^high H1.MP cells after sorting. (b) GFP expression in sorted GFP^high H1.MP cells as detected by flow cytometry. (c,d) MART-1 expression in GFP^high H1.MP cells as measured by RT-PCR (c) and immunostaining (d). (e) Expansion of primed MART-1-specific CD8+ T cells by GFP^high H1.MP-derived DCs (GFP^high H1.MP-DCs) as detected by pentamer staining and flow cytometry.

Figure 3. Modification of hPSCs with tumour antigen epitope-coding minigene.

(a) Structure of lentivector LV.ME carrying MART-1 epitope-coding minigene. (b) GPF expression in H1.ME cells, a H1 cell line generated by LV.ME transduction and G418 selection, as detected by flow cytometry. (c) Minigene expression in H1.ME cells as measured by RT-PCR. (d) SSEA-4 expression in H1.ME as detected by immunostaining.

Figure 4. Tumour antigen epitope-coding minigene is expressed in DCs derived from minigene-modified hPSCs.

(a–c) Morphology (a), yield (b) and phenotype (c) of DCs derived from minigene-modified hPSCs (H1.ME-DCs). The statistical significance of difference was determined by two-sided Student’s t-test (mean ± SD, n = 10) in (b). (d) GFP expression in H1.ME-DCs as detected by flow cytometry. (e) Expression of MART-1 epitope-coding minigene in H1.ME-DCs as measured by RT-PCR. (f) CD83 expression on H1.ME-DCs after treatment with TNF. (g) Allostimulatory function of H1.ME-DCs on CD4+ T cells after treatment with TNF. The percentages of divided CD4+ T cells are indicated.

Figure 5. DCs derived from minigene-modified hPSCs efficiently prime tumour antigen-specific T cell response.

(a) Induction of MART-1-specific CD8+ T cell response by H1-DCs pulsed with MART-1 peptide of various concentrations. (b,c) Induction of MART-1-specific CD8+ T cell response by H1.ME-DCs in low-responsive PBLs. The antigen-specific T cells were stained by pentamer and detected by flow cytometry nine days after DC/PBL coculture. (b) Contour plots of a representative experiment. The numbers in plots indicate the percentage of pentamer+ CD8+ cells in total T cells. (c) Quantitative analysis of the experiments. The statistical significance of differences were determined by two-sided Student’s t-test (mean ± SD, n = 6). (d,e) Induction of MART-1-specific CD8+ T cell response by H1.ME-DCs in high-responsive PBLs. (f) Comparing T cell priming ability of H1.ME-DCs and MART-1 peptide-pulsed moDCs. The statistical significance of difference was determined by two-sided Student’s t-test (mean ± SD, n = 5). (g) Comparing T cell priming ability of MART-1 peptide-pulsed H1-DCs and H1.ME-DCs after prolonged culture. H1-DCs were pulsed with MART-1 peptide, washed and further cultured for seven days before applying for priming. Unpulsed H1-DCs and H1.ME-DCs were employed as controls. (h) Induction of MART-1-specific CD8+ T cell response by H1.ME-DCs using different DC:PBL ratios.

Figure 6. CTLs expanded by DCs derived from minigene-modified hPSCs are immunocompetent.

(a) Expansion of MART-1-specific CD8+ T cells by H1.ME-DCs in bulk culture. HLA-A2+ PBLs were primed and then restimulated twice with H1.ME-DCs. MART-1-specific T cell expansion during this process was monitored by flow cytometry at the indicated time points. The percentages of pentamer+ CD8+ cells in total T cells are shown in the representative contour plots. (b) Phenotype of MART-1-specific T cells expanded by H1.ME-DCs. (c) GrB secretion by MART-1-specific T cells expanded by H1.ME-DCs as measured by ELISPOT. The statistical significance of difference was determined by two-sided Student’s t-test (mean ± SD, n = 3). (d) Specific cytotoxicity of MART-1-specific T cells expanded by H1.ME-DCs. The statistical significance of difference was determined by two-sided Student’s t-test (mean ± SD, n = 3, *p < 0.002).

Figure 7. Schematic summary of a novel antigen-loading strategy for DC vaccine production from hPSCs.

In a traditional patient blood cell-dependent platform, antigen-loading is limited to DCs, wherein antigen payloads in various forms are delivered into DCs by conventional antigen-loading approaches. In an hPSC-DC platform, antigen-loading can be done in hPSCs other than DCs by antigenically modifying the hPSCs. From such antigenically modified hPSCs, antigen-loaded DCs can be generated without a conventional antigen-loading step. Using this novel antigen-loading strategy, there are no more requirements of clinical-grade payload production and additional DC manipulation. Thus, DC vaccine production from hPSCs is significantly simplified.