5A). In Pt #2, while specific

CD4+ T cells were not observed before vaccination, NY-ESO-1119–141–specific CD4+ T cells were elicited after vaccination. The vaccine-induced NY-ESO-1119–141–specific CD4+ T cells were also detected in the CD4+CD25−CD45RO+ (effector/memory) T-cell population, as observed in Pt #1 (Fig. 5B). We then asked whether vaccine-induced T cells had a high-affinity TCR that recognized naturally processed antigens [21, 28]. We established NY-ESO-1–specific CD4+ T-cell clones. Four clones and a single clone that recognized different epitopes were generated from Pt #1 and Pt #2, respectively. Four minimal epitopes (NY-ESO-183–96, HDAC inhibitor 94–109, 119–130,121–134) were defined from CD4+ T-cell 5-Fluoracil clones derived from Pt #1 (Fig. 6A and data not shown). Both spontaneously induced (#2–11) and vaccine-induced (#3–1) CD4+ T-cell clones recognized naturally processed NY-ESO-1 protein and as little as 0.1 nM of peptide (Fig. 6A). One minimal epitope defined from Pt #2 was NY-ESO-1122–133 and the vaccine-induced CD4+ T-cell clone (#1–1) again recognized both the naturally processed NY-ESO-1 protein and as little as 0.1 nM of peptide (Fig. 6B), indicating that these T-cell clones had high-affinity TCRs

against NY-ESO-1. Together, OK-432 as an adjuvant could overcome Treg-cell suppression and activate high-affinity preexisting NY-ESO-1–specific CD4+ T-cell precursors. While a subset of patients treated with immunotherapy has been shown to experience objective and durable clinical responses, it is becoming increasingly clear that several mechanisms downregulate antitumor immunity during the course of the immune response and play a major role in limiting the effectiveness of cancer immunity [6, 35, 36]. A plethora of cell types, cell surface molecules, and soluble factors mediate this suppressive activity [3, 6, 35, 36]. Among them, CD4+CD25+Foxp3+ Treg cells play a crucial role by suppressing a wide variety of immune responses, and finding ways to control Treg-cell suppression is a major priority

in this field [6, 7]. In this study, we showed the potential of OK-432 (a penicillin-inactivated and lyophilized preparation of Streptococcus Tangeritin pyrogenes) which stimulates TLR signals [30, 33, 34] to control Treg-cell suppression, supporting the idea that OK-432 may be a promising adjuvant for cancer vaccines by inhibiting Treg-cell suppression and by augmenting induction of tumor-specific T cells against coadministered protein antigens. Appropriate adjuvant combinations, such as those that are MyD88-dependent or MyD88-independent, or those that are TRIF-coupled and include endosomal signals, are known to synergistically activate DCs with regard to the production of inflammatory cytokines [37, 38]. As OK-432 is derived from bacterial components, its capacity to bind a combination of various TLRs makes it attractive.