Dendritic Cells Pre-Pulsed with Wilms' Tumor 1 in Optimized Culture for Cancer Vaccination

Abstract

With recent advances in cancer vaccination therapy targeting tumor-associated antigens (TAAs), dendritic cells (DCs) are considered to play a central role as a cell-based drug delivery system in the bioactive immune environment. Ex vivo generation of monocyte-derived DCs has been conventionally applied in adherent manufacturing systems with separate loading of TAAs before clinical use. We developed DCs pre-pulsed with Wilms' tumor (WT1) peptides in low-adhesion culture maturation (WT1-DCs). Quality tests (viability, phenotype, and functions) of WT1-DCs were performed for process validation, and findings were compared with those for conventional DCs (cDCs). In comparative analyses, WT1-DCs showed an increase in viability and recovery of the DC/monocyte ratio, displaying lower levels of IL-10 (an immune suppressive cytokine) and a similar antigen-presenting ability in an in vitro cytotoxic T lymphocytes (CTLs) assay with cytomegalovirus, despite lower levels of CD80 and PD-L2. A clinical study revealed that WT1-specific CTLs (WT1-CTLs) were detected upon using the WT1-DCs vaccine in patients with cancer. A DC vaccine containing TAAs produced under an optimized manufacturing protocol is a potentially promising cell-based drug delivery system to induce acquired immunity.

Keywords: Wilms’ tumor 1; cell-based drug delivery; dendritic cells; tumor-associated antigens.

Figure 1

DCs pre-pulsed with Wilms’ tumor (WT1) peptides in low-adhesion culture maturation (WT1-DCs) form remarkable floating clusters and show higher viability and recovery of the DC/monocyte ratio. (a) In the preparation of conventional DCs (cDCs) by using the conventional adherent protocol, immature DCs were suspended with mature medium containing OK-432 and PGE2 and seeded on an adherent culture dish. After 24 h cultivation, floating and loosely attached cells were collected by washing with medium and strongly attached cells were collected by scraping. Alternatively, for the preparation of WT1-DCs, immature DCs were suspended with mature medium containing OK-432, PGE2, and WT1 peptides, seeded on a low-adherent culture dish, and harvested by washing with medium after 24 h. (b) Observation of cells using phase-contrast microscopy before and after harvesting by washing with medium. White bar indicates 400 μm. (c) Live and dead cells were measured by trypan blue staining for comparison of viability and recovery of the DC/monocyte ratio. Purity of DCs was measured by flow cytometer. PI-negative and gated cell population from FSC and SSC, excluding lymphocytes, were defined as DCs (n = 6). * p < 0.05.

Figure 2

Comparison of dendritic cell (DC) phenotypes. After harvesting cDCs and WT1-DCs prepared from the same donors, DCs were stained with antibodies for DC markers and analyzed using a flow cytometer (n = 6). The population of positive cells was determined in propidium iodide (PI)-negative and DC-gated populations excluding lymphocytes from forward and side scatter. * p < 0.05.

Figure 3

Comparison of pinocytotic and phagocytic activities. DCs were incubated with FITC-dextran for antigen pinocytotic or DQ-ovalbumin for antigen phagocytic activities with maturation cocktail. These cells were washed after a 24 h incubation, and the fluorescence intensity was examined by flow cytometer (n = 6). Δ mean fluorescence intensity (MFI) indicates a value obtained by subtracting the control incubated with DMSO. * p < 0.05.

Figure 4

Comparisons of cytokine production from cDCs or WT1-DCs. The culture supernatant after maturation of DCs were subjected to measuring of cytokine production. The amount of IL-12p70, IFN-γ, IL-10 and TGF-β were determined by ELISA (n = 8). * p < 0.05.

Figure 5

cDCs post-pulsed with cytomegalovirus (CMV) peptide and DCs pre-pulsed with CMV peptide in low-adhesion culture maturation (CMV-DCs) show equivalent antigen-presenting abilities. Representative data of CMV-specific cytotoxic T lymphocytes (CTLs) induced by cDCs post-pulsed with CMV peptide or CMV-DCs (upper panel). The cultivation of CD8⁺ T cells only was the negative control. The percentage in each panel indicates the ratio of CMV-tetramer⁺ CTLs in CD8⁺ T cells. The lower panel shows a summary of CMV-specific CTLs induction in CD8⁺ T cells (n = 6). †, post-pulsed with CMV peptide. NS, not significant.

Figure 6

Immune monitoring of WT1-specific CTLs (WT1-CTLs) in patients after WT1-DC administration. Patients No. 3 and No. 5 received one course of WT1-DC vaccination. Thereafter, the induction of WT1-CTLs was detected via WT1 tetramer analysis, and IFN-γ release from WT1-CTLs was assessed using Enzyme-linked immunospot (ELISpot) assays. Patient No. 6 received a DC vaccine post-pulsed with WT1 peptides using the previous protocol. After the second course of administration of the WT1-DC vaccine, the maintenance of WT1-CTL function was evaluated. The percentages in the dot plot panels show the ratio of WT1-CTLs in CD8⁺ T cells. The opened or closed bars indicate the numbers of spots from PBMCs stimulated with control or WT1 peptides, respectively. The mean number of spots from duplicate wells is shown.