Development of a local dendritic cell-therapy by using an active component of OK-432 ; Involvement of Toll-like receptor 4

脂质胆酸相关分子OK-PSA是链球菌免疫治疗剂OK-432的有效成分，是一种有效的tm型细胞因子诱导剂，在荷瘤小鼠中具有抗肿瘤活性。树突状细胞是一种有效的抗原提呈细胞，可促进对肿瘤细胞的免疫反应。在本研究中，我们首先在体外检测了OK-PSA对树突状细胞（DCs）成熟的影响。将5例健康供体和10例头颈癌患者的未成熟树突状细胞（TLR4或MiD-2 mRNA均有表达或无表达）经k - psa处理2 d后，分析其表面分子表达、细胞因子产生、T淋巴细胞刺激活性。用OK-PSA处理健康供体来源的未成熟dc可有效提高MHC II类、CD80、CD83和CD86的表面表达。ok - psa刺激的dc显著分泌可诱导th1型t细胞反应的细胞因子和趋化因子。此外，okp sa活化的dc可增强同种异体CD3+T细胞的增殖，并诱导同种异体特异性细胞毒性T淋巴细胞（ctl）。OK-PSA还激活了来自同时表达TLR4和MD-2 mrna的患者的dc。而在TLR4或MID-2 mRNA表达缺失的患者中，ok - psa刺激的dc没有诱导CTL活性。此外，转染TLR4表达载体的TLR4缺陷dc对k - psa有反应，产生IL-12并刺激同种异体T细胞。OK-PSA通过TLR4信号诱导DC成熟。OK-PSA可能是一种有用的治疗应用，作为基于dc的癌症免疫治疗的辅助剂。因此，我们接下来测试了TLR4表达对dc体内抗肿瘤作用的意义，在肿瘤内给药同源dc后再给药OK-PSA对小鼠肿瘤的抗肿瘤作用。本实验采用表达野生型TLR4的C57BL/6小鼠和C57BL/6衍生的TLR4敲除（TLR4-/-）小鼠。虽然OK-PSA显著增强了瘤内DC对野生型Lewis肺癌LL/2小鼠的抗肿瘤作用，但在TLR4-/-小鼠中，OK-PSA以及与DC和OK-PSA联合治疗的抗肿瘤作用不显著。有趣的是，即使在LL/2携带TLR4-/-的小鼠中，使用野生型小鼠来源的dc后再使用OK-PSA也显示出明显的抗肿瘤作用。在野生型小鼠中，dc和k - psa显著增加了肿瘤浸润淋巴细胞和引流淋巴结细胞对LL/2的细胞毒活性以及干扰素γ的分泌，而在TLR4-/-小鼠中则没有。在携带LL/2的TLR4-/-小鼠中，瘤内注射野生型小鼠来源的dc和OK-PSA也增强了这些活性。这些发现表明，OK-PSA可能是局部DC治疗的潜在佐剂，并且当TLR4仅在瘤内转移的DC中表达时，OK-PSA甚至能够在TLR4缺陷的宿主中引发抗肿瘤活性。少

A lipoteichoic acid-related molecule OK-PSA, an active component of a streptococcal immunotherapeutic agent OK-432, is an effective inducer of TM-type cytokines, and elicits anti-tumor activity in tumor-bearing mice. DCs are potent antigen-presenting cells which promote immune responses against tumor cells. In the current study, we first examined the effect of OK-PSA on maturation of dendritic cells (DCs) in vitro. Immature DCs derived from 5 healthy donors and 10 patients with head and neck cancer with or without the expression of Toll-like receptor 4 (TLR4) or MiD-2 mRNA were treated with OK-PSA for 2 days, then the expression of surface molecules, cytokine production, T lymphocyte-stimulating activity of the DCs were analyzed. Treatment with OK-PSA of healthy donor-derived immature DCs effectively increased the surface expression of MHC class II, CD80, CD83 and CD86. OK-PSA-stimulated DCs markedly secreted the cytokines and chemokines that can induce Th1-type T-cell response. In add … More ition, OK-P SA-activated DCs enhanced the proliferation of allogeneic CD3+T cells and induced allo-specific cytotoxic T lymphocytes (CTLs). OK-PSA also activated DCs, derived from the patients which expressed both TLR4 arid MD-2 mRNAs. while OK-PSA-stimulated DCs from the patients in whom the expression of TLR4 or MID-2 mRNA was absent, did not induce CTL activity. Furthermore, TLR4-deficient DCs transfected with TLR4 expression vector showed the response to OK-PSA to produce IL-12 as well as to stimulate allogeneic T cells. OK-PSA induced DC maturation via TLR4 signaling. OK-PSA may be a useful therapeutic application as an adjuvant for DC-based cancer immunotherapy. Thus, we next tested the significance of expression of TLR4 on the DCs in in vivo anti-tumor effect of intratumoral administration of syngeneic DCs followed by OK-PSA against established tumors in mice. C57BL/6 mice, which express wild-type TLR4, and C57BL/6-derived TLR4-knockout (TLR4-/-) mice were used for the present experiments. Although OK-PSA markedly augmented anti-tumor effect of an intratumoral DC administration in wild-type mice bearing Lewis lung carcinomas LL/2, anti-tumor effect of OK-PSA as well as of the combination therapy with DCs and OK-PSA was not significant in TLR4-/-mice. Interestingly, an administration of wild-type-mouse-derived DCs followed by OK-PSA exhibited a marked anti-tumor effect even in LL/2-bearing TLR4-/-mice. Cytotoxic activities of tumor-infiltrating lymphocytes and of draining lymph node cells against LL/2, as well as interferon-γ secretion, were significantly increased by the therapy with DCs and OK-PSA in wild-type mice but not in TLR4-/-mice. These activities in LL/2-bearing TLR4-/-mice were also enhanced by intratumoral injection of wild-type-mouse-derived DCs and OK-PSA. These findings suggest that OK-PSA may be a potential adjuvant for local DC therapy, and that DC therapy followed by OK-PSA is able to elicit anti-tumor activity even in a TLR4-deficient host when TLR4 is expressed only in intratumorally transferred DCs. Less