Streptococcal Preparation OK-432 Promotes Fusion Efficiency and Enhances Induction of Antigen-Specific CTL by Fusions of Dendritic Cells and Colorectal Cancer Cells

Abstract

Dendritic/tumor fusion cell (FC) vaccine is an effective approach for various types of cancer but has not yet been standardized. Antitumor activity can be modulated by different mechanisms such as dendritic cell (DC) maturation state. This study addressed optimal strategies for FC preparations to enhance Ag-specific CTL activity. We have created three types of FC preparations by alternating fusion cell partners: 1) immature DCs fused with autologous colorectal carcinoma cells (Imm-FCs); 2) Imm-FCs followed by stimulation with penicillin-inactivated Streptococcus pyogenes (OK-432) (Imm-FCs/OK); and 3) OK-432-stimulated DCs directly fused to autologous colorectal carcinoma cells (OK-FCs). Both OK-FCs and Imm-FCs/OK coexpressed the CEA, MUC1, and significantly higher levels of CD86, CD83, and IL-12 than those obtained with Imm-FCs. Short-term culture of fusion cell preparations promoted the fusion efficiency. Interestingly, OK-FCs were more efficient in stimulating CD4+ and CD8+ T cells capable of high levels of IFN-γ production and cytolysis of autologous tumor or semiallogeneic targets. Moreover, OK-FCs are more effective inducer of CTL activation compared with Imm-FCs/OK on a per fusion cell basis. The pentameric assay confirmed that CEA- and MUC1-specific CTL was induced simultaneously by OK-FCs at high frequency. Furthermore, the cryopreserved OK-FCs retained stimulatory capacity for inducing antitumor immunity. These results suggest that OK-432 promotes fusion efficiency and induction of Ag-specific CTL by fusion cells. We conclude that DCs fused after stimulation by OK-432 may have the potential applicability to the field of antitumor immunotherapy and may provide a platform for adoptive immunotherapy in the clinical setting.

Synergistic Induction of Antigen-Specific CTL by Fusions of TLR-Stimulated Dendritic Cells and Heat-Stressed Tumor Cells

“…CEA-and MUC1-specific CTLs were induced simultaneously by OK-FCs at higher frequency, than by Imm-FCs or Imm/HS-FCs. These results indicate that the fusion process (41) and heat-treatment, in the absence of stimuli such as OK-432, induce insufficient FC activation and initiate TAA-specific CTL responses. Heat-treated tumor cells are an effective source of TAAs for a FC-based vaccine.…”

“…Ag presentation by DCs, in the absence of proper costimulation, induces tolerance (1). In particular, recent studies suggest that TLR-agonist CpG ODNs or conserved pathogen-associated molecular patterns, such as OK-432, start the DC maturation process, which is a critical event in the induction of full effector function in T cells (20,31,32,35,41,57,58). Therefore, we next investigated whether DCs activated by a combination of TLR ligation and up-regulated HSPs enhance the immunogenicity of FCs.…”

“…Both OK-432 and HSPs are putative ligands of the TLR, stimulate DCs rapidly, and elicit a Th1-promoting phenotype (61,62), all of which are essential for exhibiting capacities to prime naive T cells and develop antitumor CTL responses. Our previous study, which found that depletion of CD4 ϩ T cells significantly impairs CTL induction by DCs/tumor FCs (11,41,63,64), indicates that efficient CTL generation requires the help of CD4 ϩ T cells. Our present study has shown that OK/HS-FCs are superior to other FC preparations for expanding the population of both CD4 ϩ and CD8 ϩ T cells that produce high levels of IFN-␥.…”

“…Moreover, OK-432 promotes functional maturation of DCs through the TLR4 and ␤ 2 integrin system to enhance Ag-specific CTL responses to a greater extent than does a previously reported mixture (consisting of TNF-␣, IL-1␤, IL-6, and PGE 2 ) or LPS (34 -40). We have also reported that OK-432 promotes fusion efficiency and the induction of tumor-specific CTL by FCs (41).…”

Dendritic and tumor cell fusions transduced with adenovirus encoding CD40L eradicate B-cell lymphoma and induce a Th17-type response

“…8,9 In addition, as the FC approach delivers not only TA epitopes but also the genes encoding those TAs, they continuously produce TA proteins for several days after fusion. 10 FCs function in a manner similar to APCs with the expression of costimulatory molecules and the ability to migrate to draining lymph nodes, in which they interact with CD4 + and CD8 + T cells and induce potent antitumor immunity. In addition, the use of several TAs may be simultaneously advantageous, as policlonal activation of T cells against different TAs could be a better approach to eradicate tumor cells.…”