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

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.


Introduction
Despite significant advances in cancer therapy such as surgical techniques, radiotherapy, and systemic therapy including immune checkpoint inhibitors [1][2][3][4][5][6], it remains extremely challenging to treat advanced cancers involving organ systems and distant metastasis. Therapeutic peptide vaccines targeting tumor-associated antigens (TAAs) for cancer immunotherapy have been in development for decades [7]. The efficacy of peptide vaccines is dependent on the peptide selected for TAAs, peptide formulation,

New Approach to Manufacture a DC Vaccine
DCs were generated in compliance with Good Gene, Cellular, and Tissue-based Products Manufacturing Practice. To generate a DC vaccine, processing was validated under the clinical study approved by the Ethical Committee of Kanazawa Medical University (approval number G131). A preclinical study was taken as an accompanying study of the DC vaccination therapy performed in patients with cancer. The DC vaccination study (approval number PC4160014, 10 June 2016) was approved by the Kanazawa Medical University Certificated Committee for Regenerative Medicine (Class III technologies) (approval number of the Committee NB4150006) according to the Act on the Safety of Regenerative Medicine introduced in Japan on 25 November 2014 [37], and all investigations were performed according to the Declaration of Helsinki.
Peripheral blood mononuclear cell (PBMC)-rich fraction was collected using leukapheresis with a Spectra Optia ® cell separator (Terumo BCT, Inc., Tokyo, Japan). PBMCs were subsequently isolated using a Ficoll-Plaque Premium (GE Healthcare, Piscataway, NJ, USA) density gradient. The collection and use of blood complied with relevant guidelines and institutional practices from Ethics Committees of Kanazawa Medical University. Written informed consent was obtained from all patients.

Pinocytotic and Phagocytic Assay
To evaluate pinocytotic or phagocytic activity, 100 µg/mL FITC-dextran (Molecular Probes, Eugene, OR, USA) for pinocytotic activity or 10 µg/mL DQ-ovalbumin (Molecular Probes) for phagocytic activity was added to the maturation cocktail. After the maturation process on DCs at 37 • C for 24 h, DCs were washed twice with FACS buffer and analyzed using flow cytometry.

Measurement of Cytokine Production
Immature DCs were seeded at a density of 2 × 10 6 cells/mL with maturation cocktail onto adherent or low-attachment 24-well plates. After maturation of DCs at 37 • C for 24 h, the collected supernatants were subsequently subjected to ELISA for IL-12p70, interferon (IFN)-γ, IL-10, and transforming growth factor (TGF)-β protein expression (R&D Systems) according to the manufacturers' protocols.

CTL Induction in Vitro
PBMCs from patients compatible with HLA-A*24:02 were used to generate mDCs. For post-pulsing with peptide, cryopreserved cDCs were thawed by heat block at 37 • C for 5 min and washed with saline. Then, cDCs were pulsed with 100 µg/mL of cytomegalovirus (CMV) peptide (QYDPVAALF, GenScript, Piscataway, NJ, USA) at 4 • C for 30 min. After washing cells twice with saline, cDCs were used as a stimulator. Alternatively, DCs pre-pulsed with CMV peptide in low-adhesion culture maturation (CMV-DCs) were thawed by heat block at 37 • C for 5 min, washed twice with saline, and subsequently used as the stimulator. CD8 + T cells separated from HLA-A*24:02-autologous PBMCs using CD8 Microbeads (Miltenyi Biotec) were applied as responder cells. Stimulator (1 × 10 5 ) and responder cells were co-cultured at a ratio of 1:10 in CTL medium supplemented with IL-2 (5 ng/mL; PeproTech, Rocky Hill, NJ, USA), IL-7 (5 ng/mL; R&D Systems), IL-15 (10 ng/mL; PeproTech), and 2-mercapto-ethanol (50 µg/mL; Bio-Rad Labs, Richmond, CA, USA). AIM-V media supplemented with 10% fetal bovine serum (Biosera, Dominican Republic) was added depending on cell expansion. After five days of cultivation, a half-medium change was performed by adding cDCs post-pulsed with CMV peptide or CMV-DCs in CTL medium. After three to five days of further incubation, the cells were harvested and 1 × 10 6 cells were stained with FITC-conjugated anti-CD8 (Beckman Coulter, Inc., Brea, CA, USA) and APC-conjugated anti-CD3 (eBioscience) mAbs and T-select HLA-A*24:02 CMV pp65 Tetramer-QYDPVAALF (Medical and Biological Laboratories Co., Ltd., Nagoya, Japan) for analysis via flow cytometer. Dead cells were excluded by 7-AAD (BD Pharmingen) staining in flow cytometry analysis. were the same as a previous study [19]. The patients enrolled for DC vaccination had undergone rhG-CSF treatment 24-96 h prior to apheresis as described previously [25]. A total of seven patients with advanced cancers pathologically diagnosed as WT1 positive adenocarcinoma including stomach (three), colon/rectum (two), pancreatic (one), and salivary gland (one) cancers were enrolled; of those four patients compatible with HLA-A*24:02 were evaluated for immunological responses against WT1-CTLs.

WT1-DC Administration
WT1-DCs were suspended in a total volume of 1 mL of saline containing 5% albumin (Japan Blood Products Organization, Tokyo, Japan), and 1-4 × 10 7 WT1-DCs were injected at each time according to the number of DCs in each case for seven sessions (one course). The vaccine was intradermally and bilaterally administered near the axillary region and groin. DC vaccination was administered in seven sessions every two weeks following the protocol of DC vaccination [19].

Shipping of WT1-DCs
Cryopreserved WT1-DCs were thawed by heat block at 37 • C for 5 min, washed twice with saline, and suspended in saline containing 5% albumin (Japan Blood Products Organization, Tokyo, Japan) before being enclosed in a tube. After packaging the tube with BARRIA POUCH (SUGIYAMA-GEN, Tokyo, Japan), the tube was shipped by BioBoxPLUS (SUGIYAMA-GEN) to the outpatient clinic, Urata Clinic/SQOL Kanazawa. A temperature range of 2 • C to 8 • C inside the BioBoxPLUS during shipping was monitored by a temperature data logger, TEMPRETRIEVER (MadgeTech, Warner, NH, USA).

Statistical Analysis
The Wilcoxon signed-rank test was used to compare differences among groups. All statistical analyses were performed using IBM SPSS Advanced Statistics software, version 23.0 (IBM Japan, Tokyo, Japan). Differences were considered statistically significant at a p-value < 0.05.

WT1-DCs Show Remarkable Cluster and Increase in Viability and Recovery of DC/Monocyte Ratio Compared to Conventional DCs (cDCs)
In preparations of conventional DCs (cDCs) by using the adherent protocol, strong adherence to the culture dish decreases cell viability and recovery of the DC/monocyte ratio depending on the patient. For administration of cDC vaccines, cryopreserved cDC vaccines required post-pulsing with TAAs just prior to clinical use (Figure 1a, upper panel). Here, we developed a preparation of mature DCs pre-pulsed with WT1 peptides (WT1-DCs) in low-adherent conditions (Figure 1a, lower panel). After maturation stimulus with OK-432 (streptococcal preparation), PGE2, and WT1 peptides, floating cells were harvested by washing with medium, and cell morphology was observed by microscopy ( Figure 1b, upper panel). Interestingly, remarkable floating non-adherent clusters were observed in WT1-DCs. Although cDCs resided in culture dishes after harvesting, almost no WT1-DCs adhered to the low-adherent culture dish (Figure 1b, lower panel). Compared with cDCs, WT1-DCs showed higher viability and recovery of the DC/monocyte ratio (Figure 1c; viability median: cDCs, 86%; WT1-DCs, 93%; yield median: cDCs, 27%; WT1-DCs, 30%), and analysis using a flow cytometer showed purity >70% in each DC vaccine (Purity median: cDCs, 81%; WT1-DCs, 81%).

Statistical Analysis
The Wilcoxon signed-rank test was used to compare differences among groups. All statistical analyses were performed using IBM SPSS Advanced Statistics software, version 23.0 (IBM Japan, Tokyo, Japan). Differences were considered statistically significant at a p-value < 0.05.

WT1-DCs Show Remarkable Cluster and Increase in Viability and Recovery of DC/Monocyte Ratio Compared to Conventional DCs (cDCs)
In preparations of conventional DCs (cDCs) by using the adherent protocol, strong adherence to the culture dish decreases cell viability and recovery of the DC/monocyte ratio depending on the patient. For administration of cDC vaccines, cryopreserved cDC vaccines required post-pulsing with TAAs just prior to clinical use (Figure 1a, upper panel). Here, we developed a preparation of mature DCs pre-pulsed with WT1 peptides (WT1-DCs) in low-adherent conditions (Figure 1a, lower panel). After maturation stimulus with OK-432 (streptococcal preparation), PGE2, and WT1 peptides, floating cells were harvested by washing with medium, and cell morphology was observed by microscopy ( Figure 1b, upper panel). Interestingly, remarkable floating non-adherent clusters were observed in WT1-DCs. Although cDCs resided in culture dishes after harvesting, almost no WT1-DCs adhered to the low-adherent culture dish (Figure 1b, lower panel). Compared with cDCs, WT1-DCs showed higher viability and recovery of the DC/monocyte ratio ( Figure 1C; viability median: cDCs, 86%; WT1-DCs, 93%; yield median: cDCs, 27%; WT1-DCs, 30%), and analysis using a flow cytometer showed purity >70% in each DC vaccine (Purity median: cDCs, 81%; WT1-DCs, 81%).  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.

WT1-DCs Have Abilities of Lower Pinocytosis and IL-10 Production Compared with cDCs
To validate the potency of pre-pulsing of antigen and processing during maturation, pinocytosis and phagocytosis activities were examined during the maturation of DCs. Pinocytosis was observed

WT1-DCs Have Abilities of Lower Pinocytosis and IL-10 Production Compared with cDCs
To validate the potency of pre-pulsing of antigen and processing during maturation, pinocytosis and phagocytosis activities were examined during the maturation of DCs. Pinocytosis was observed by using FITC-dextran. Compared with cDCs, WT1-DCs showed slightly lower FITC ∆ mean fluorescence intensity (∆MFI) (median of ∆MFI: cDCs, 58; WT1-DCs, 54) (Figure 3, left panel). A lower pinocytosis activity was observed in WT1-DCs. In addition, analysis using DQ-ovalbumin, a self-quenched albumin that fluoresces upon proteolytic degradation, revealed that the fluorescence intensity generated from each DC was equivalent. These results indicated that the ability of cDCs and WT1-DCs to phagocytose was equivalent (Figure 3, right panel). Furthermore, the production of cytokines involved in the induction of CTLs was measured (Figure 4). Production of IL-12p70 and IFN-γ, which promote CTL induction, were equivalent. Despite a varying level based on each patient, WT1-DCs generated from three of seven showed higher IL-12p70 production than cDCs. The WT1-DCs also produced higher IFN-γ than cDCs. No change was observed in TGF-β secretion; however, a lower production of IL-10 was observed in WT1-DCs compared with cDCs (cDCs, 293 pg/mL; WT1-DCs, 39 pg/mL). Thus, compared with cDCs, WT1-DCs exhibited lower phagocytosis and IL-10 production. DCs also produced higher IFN-γ than cDCs. No change was observed in TGF-β secretion; however, a lower production of IL-10 was observed in WT1-DCs compared with cDCs (cDCs, 293 pg/mL; WT1-DCs, 39 pg/mL). Thus, compared with cDCs, WT1-DCs exhibited lower phagocytosis and IL-10 production.

Antigen-Presentation Ability of DCs Pre-Pulsed with CMV Peptide in Low-Adhesion Culture Maturation (CMV-DCs) are Similar to cDCs Post-Pulsed with CMV Peptide
To evaluate the antigen-presenting ability to activate CTLs, we prepared DCs pre-pulsed with CMV peptide in low-adhesion culture maturation (CMV-DCs). Compared with a culture of CD8 + T DCs also produced higher IFN-γ than cDCs. No change was observed in TGF-β secretion; however, a lower production of IL-10 was observed in WT1-DCs compared with cDCs (cDCs, 293 pg/mL; WT1-DCs, 39 pg/mL). Thus, compared with cDCs, WT1-DCs exhibited lower phagocytosis and IL-10 production.

Antigen-Presentation Ability of DCs Pre-Pulsed with CMV Peptide in Low-Adhesion Culture Maturation (CMV-DCs) are Similar to cDCs Post-Pulsed with CMV Peptide
To evaluate the antigen-presenting ability to activate CTLs, we prepared DCs pre-pulsed with CMV peptide in low-adhesion culture maturation (CMV-DCs). Compared with a culture of CD8 + T cells alone, co-culture of CD8 + T cells with cDCs post-pulsed with CMV peptide or CMV-DCs resulted in a marked increase in CMV-specific CTLs ( Figure 5, upper panel; CD8 + T cells, 0.1%; CD8 + T cells +

Antigen-Presentation Ability of DCs Pre-Pulsed with CMV Peptide in Low-Adhesion Culture Maturation (CMV-DCs) are Similar to cDCs Post-Pulsed with CMV Peptide
To evaluate the antigen-presenting ability to activate CTLs, we prepared DCs pre-pulsed with CMV peptide in low-adhesion culture maturation (CMV-DCs). Compared with a culture of CD8 + T cells alone, co-culture of CD8 + T cells with cDCs post-pulsed with CMV peptide or CMV-DCs resulted in a marked increase in CMV-specific CTLs ( Figure 5, upper panel; CD8 + T cells, 0.1%; CD8 + T cells + cDCs post-pulsed with CMV, 9.9%; CD8 + T cells + CMV-DCs, 9.1%). There was no significant difference in the ratio of CMV-specific CTLs induced by cDCs post-pulsed with CMV and CMV-DCs (median: cDCs post-pulsed with CMV, 5.9%; CMV-DCs, 6.3%) ( Figure 5, lower panel).

Administration of WT1-DCs Induces WT1-Specific CTLs in Patients with Cancer
As an interim analysis, four patients having HLA-A*24:02 received the WT1-DCs vaccine were evaluated, which had been shipped to the neighboring clinic within 1 h after release. Immunohistochemistry was also performed for WT1 antigens on paraffin embedded tissues before enrolling the study (data not shown). The condition of all vaccines met the administration criteria. Immune monitoring using tetramer analysis and ELISpot assays were performed after one course of the DC vaccination. Of the four patients completing one course of WT1-DCs vaccination three had gastric cancer and one had salivary gland cancer. Pre-DC vaccination status, post-DC vaccination status, and immunological responses are shown in Table 1; Table 2. WT1-CTLs from three male patients with gastric cancer were detected using WT1-tetramer analysis ( Figure 6). The detection of IFN-γ-producing cells showed elevation after WT1-DCs vaccination in three patients using ELISpot assays ( Figure 6). Conversely, in patient No.4 with salivary gland cancer, the immunological responses failed as negative following to the criteria of immune monitoring for WT1-CTLs [38].
In patient No. 3 (upper panel in Figure 6), the induction of WT1-CTLs was observed via tetramer analysis (Before Vac., 0.01%; After Vac., 0.10%). Despite the increased level of WT1 peptides according to ELISpot assays, the non-specific elevation of IFN-γ-producing cells was found in the control stimulation after one course of WT1-DC vaccination. Therefore, the specificity for detecting WT1-

Administration of WT1-DCs Induces WT1-Specific CTLs in Patients with Cancer
As an interim analysis, four patients having HLA-A*24:02 received the WT1-DCs vaccine were evaluated, which had been shipped to the neighboring clinic within 1 h after release. Immunohistochemistry was also performed for WT1 antigens on paraffin embedded tissues before enrolling the study (data not shown). The condition of all vaccines met the administration criteria. Immune monitoring using tetramer analysis and ELISpot assays were performed after one course of the DC vaccination. Of the four patients completing one course of WT1-DCs vaccination three had gastric cancer and one had salivary gland cancer. Pre-DC vaccination status, post-DC vaccination status, and immunological responses are shown in Table 1; Table 2. WT1-CTLs from three male patients with gastric cancer were detected using WT1-tetramer analysis ( Figure 6). The detection of IFN-γ-producing cells showed elevation after WT1-DCs vaccination in three patients using ELISpot assays ( Figure 6). Conversely, in patient No.4 with salivary gland cancer, the immunological responses failed as negative following to the criteria of immune monitoring for WT1-CTLs [38]. Table 1. Clinical characteristics of patients treated with the WT1-pulsed DC vaccine.

Age (Years)
In patient No. 3 (upper panel in Figure 6), the induction of WT1-CTLs was observed via tetramer analysis (Before Vac., 0.01%; After Vac., 0.10%). Despite the increased level of WT1 peptides according to ELISpot assays, the non-specific elevation of IFN-γ-producing cells was found in the control stimulation after one course of WT1-DC vaccination. Therefore, the specificity for detecting WT1-CTLs could not be confirmed according to the previously reported criteria [39]. In patient No. 5 (middle panel in Figure 6), an increased number of WT1-CTLs was detected after WT1-DC vaccination (Before Vac., 0.00%; After Vac., 0.05%). Conversely, a slight increase in the number of spots containing WT1 peptides was observed after one course of vaccination, the number of spots containing control peptides also increased similarly. Patient No. 6 (lower panel in Figure 6) received the WT1-DCs vaccine under this protocol but had also undergone cDCs vaccination study using the previous protocol with post-pulsed WT1 peptides [approval number PC4160014, June 10, 2016]. In this case, the number of WT1-CTLs increased after the second course of DC vaccination (before second Vac., 0.16%; after second Vac., 0.19%) on the positive baseline of both tetramer analysis and ELISpot assays. The number of IFN-γ spots further increased after the second course of DC vaccination pulsed with WT1-235 killer and WT1-34 helper peptides compared with that after the first session without additional chemotherapy.
Pharmaceutics 2020, 12, x; doi: FOR PEER REVIEW www.mdpi.com/journal/pharmaceutics previous protocol with post-pulsed WT1 peptides [approval number PC4160014, June 10, 2016]. In this case, the number of WT1-CTLs increased after the second course of DC vaccination (before second Vac., 0.16%; after second Vac., 0.19%) on the positive baseline of both tetramer analysis and ELISpot assays. The number of IFN-γ spots further increased after the second course of DC vaccination pulsed with WT1-235 killer and WT1-34 helper peptides compared with that after the first session without additional chemotherapy.

Discussion
In this study, we performed phenotypic and functional analyses on WT1-DCs pre-pulsed with WT1 peptides in low-adhesion culture maturation, and we evaluated active WT1-CTLs after WT1-DC administration in patients with cancer. Compared with cDCs, WT1-DCs formed floating clusters and increased in viability and recovery of the DC/monocyte ratio. By contrast, co-stimulatory molecule CD80 and the immune checkpoint factor PD-L2 on WT1-DCs expressed lower levels than those on cDCs. In addition, the production of immune suppressive cytokine IL-10 from WT1-DCs was extremely low. Nevertheless, different DC maturation protocols (cDC vs. WT1-DC) did not affect antigen-presentation ability. Furthermore, immune monitoring of WT1-CTLs as practical application of the total process including shipping of the WT1-DC vaccine after WT1-DC-administration demonstrated that WT1-DCs induced WT1-CTLs in patients with cancer.
In the conventional adherent protocol for monocyte-derived DC generation, adherence of cells to the culture plate was dependent on the patient from whom the cells originated (Figure 1b). Recovery of adherent cells by scraping causes a decrease in cell viability and recovery of the DC/monocyte ratio yield. WT1-DCs generated from a low-adhesion dish were easy to recover by washing with medium and showed a high viability and recovery. Low expression of DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN, also called CD209) and PD-L2 has been reported for these populations [33]. DC-SIGN is involved in antigen uptake [39]; thus, it is speculated that the reduction of pinocytosis might have been caused by low DC-SIGN expression on WT1-DCs. However, WT1-DCs have an equivalent capacity for antigen presentation as cDCs ( Figure 5). The difference of pinocytosis between cDCs and WT1-DCs did not affect CTL induction in vitro.
Our phenotypic analysis of WT1-DCs also showed that CD80 and PD-L2 expression were significantly reduced compared to cDCs. The streptococcal preparation OK-432 engages TLR2 or TLR4 [29] and promotes maturation of human monocyte-derived DCs correlated with increased expression of CD80, CD83, and CD86 [29][30][31]. The downregulation of CD80 and PD-L2 together with the remarkable cluster formation that occurs with WT-DCs might therefore reduce the signaling of OK-432 via TLRs and affect the maturation. We expected to produce non-adherent DCs equipped with homogeneous in phenotype and function by using the optimized manufacturing protocol of WT-DCs. WT1-DCs exhibited heterogeneous phenotype and function, the control of cluster formation may be an important issue for the progress of homogeneous WT1-DCs. Size-dependent hepatic differentiation of human induced pluripotent stem (iPS) cells has been reported [40]. The control of cell mass size is important for the efficiency and reproducibility of differentiation of functional cells from iPS cells. The regulation of cluster size of DCs could contribute to generating homogenous DC vaccines equipped with the ability to induce high acquired immunity.
Compared with cDCs, WT1-DCs showed an equivalent production of IL-12p70, IFN-γ, and TGF-β but a low production of the immunosuppressive cytokine IL-10. IL-10 production from antigen-presenting cells is specific for TLR2 agonists [41][42][43][44]. We speculate that insufficient OK-432 signaling through TLR2 might led to a decrease in the IL-10 production of WT1-DCs. Mycobacterium avium induces PD-L2 expression on mouse bone marrow-derived dendritic cells in an IL-10-dependent manner via the TLR2-p38-MAPK signaling pathway [45]. Therefore, the low PD-L2 expression observed in WT1-DCs may be due to a reduction in IL-10 production, but further study is needed to test this.
Knockdown of PD-L1 and PD-L2 in monocyte-derived DCs enhances CTL induction [46], and IL-10 has known immunosuppressive effects [47]. Therefore, reduced PD-L2 and IL-10 in WT1-DCs were expected to enhance their induction of CTLs. However, in vitro CTL induction revealed that antigen-presentation abilities were equivalent in cDCs and WT1-DCs ( Figure 5). Nevertheless, some suppression of PD-L1 and PD-L2 expression is needed to enhance CTL induction. DC vaccines with siRNA silencing of PD-L1 and PD-L2 augment the expansion and function of CD8 + T cells specific for minor histocompatibility antigens [48]. Indeed, clinical trials for hematological malignancies using DCs with siRNAs against PD-L1 and PD-L2 (NCT02528682) are expected to lead to the development of promising DC vaccines. The acquisition of WT1-CTLs as a proof-of-concept drug delivery in vivo was observed in patients with cancer who received WT1-DCs vaccination. The effectiveness of WT1-DCs to induce acquired immunity was confirmed. Specifically, IFN-γ production was negative in two of three cases treated with WT1-DCs (Table 1, Table 2 and Figure 6). It is important to deliberate the possible reason why WT1-CTLs showed IFN-γ negativity to understand its antitumor activity. WT1-DCs vaccination was conducted without any adjuvants in this study, which may have led to a failure in the induction function in vivo. Administration of WT1-DCs with OK-432 might be essential for achieving sufficient induction of functional WT1-CTLs in patients with cancer. In fact, the induction of IFN-γ producing WT1-CTLs was observed after the vaccination with WT1-post-pulsed DCs in combination with OK-432 in vivo [19,49]. It has been reported that OK-432 induces IL-12 production from human PBMCs and promotes a Th1 dominant state that is suitable for inducing antitumor immunity [50,51]. Moreover, OK-432 significantly enhanced in vitro proliferation of CD4 + effector T cells by regulatory T (Treg)-cell suppression, and this blocking effect depended on IL-12 derived from antigen-presenting cells [52]. The induction of IFN-γ producing WT1-CTLs without an increase in Treg cells was observed after the administration of WT1-post-pulsed DCs with OK-432 [53]. Several preclinical and clinical studies suggest that Treg cells prevent the development of effective antitumor immunity in tumor-bearing patients and promote tumor progression [54]. The activity of OK-432 to Treg-cell suppression could be beneficial for the induction of functional WT1-CTLs in vivo. Further clinical studies using WT1-pre-pulsed DCs with OK-432 for patients with cancer would be needed to monitor the induction of IFN-γ producing WT1-CTLs as wells as to improve the immune environment in vivo.

Conclusions
In conclusion, we established a protocol for the preparation of WT1-DCs pre-pulsed with WT1 peptides in optimized culture maturation. WT1-DCs exhibit high viability, recovery, and equivalence in in vitro CTL induction compared with cDCs. After the administration of WT1-DCs, immune monitoring demonstrated that WT1-DCs induce acquired immunity in patients with cancer. DCs function as adjuvants in vivo and are expected to be applied to cancer treatments that promote long-lasting effects with few side effects. WT1-DC vaccination for patients with cancer demonstrated the safety and immunogenicity in vivo. Prospective clinical trials are required to evaluate the efficacy of acquired immunity in response to WT1-DC vaccination in large number of cancer patients.

Patents
S.S. and T.K. are inventors of the patent for the manufacturing of a DC vaccine using G-CSF (PCT/JP/2014/053676). H.S. is the inventor of the WT1 patent (PCT/JP2010/057149 and PCT/JP2006/323827).