Naoe Michio, Ogawa Yoshio, Takeshita Kumiko, Morita Jun, Iwamoto Sanju, Miyazaki Akira, Yoshida Hideki
Department of Urology, School of Medicine, Showa University, Tokyo, Japan.
Int J Urol. 2007 Jun;14(6):532-8; discussion 538. doi: 10.1111/j.1442-2042.2006.01697.x.
Immunotherapy with bacillus Calmette-Guérin (BCG) for bladder cancer is successful, although the precise mechanism is unclear. Natural killer (NK) cells are a candidate for BCG-activated killer cells, but the roles of other T lymphocytes, such as NKT cells and gammadeltaT cells, are not fully understood. Mycobacterium tuberculosis is a potent activator of both NKT cells and gammadeltaT cells. However, it is known that the patient's prognosis is good if there are increased numbers of dendritic cells (DCs) in the urine after BCG therapy. Therefore, we investigated whether DCs are matured by BCG and whether BCG-pulsed DCs stimulate NKT cells and gammadeltaT cells.
Naïve Pan T cells were isolated form peripheral blood mononuclear cells (PBMCs) and DCs were obtained by culturing CD14(+) monocytes with granulocyte-macrophage colony-stimulating factor and interleukin-4. The DCs were pulsed with BCG and their maturation was measured by fluorescence-activated cell sorter analysis using the CD86 antibody. Naïve T lymphocytes were stimulated by coculture with BCG-pulsed DCs in vitro, followed by FACS analysis to estimate the ratio and activation of NKT cells and the ratio of gammadeltaT cells. The (51)Cr (chromium) release assay was used to estimate the cytotoxic activity of the stimulated T cells. Cytolytic proteins in the patient's PBMCs were measured during BCG therapy using semiquantitative reverse transcriptase-polymerase chain reaction.
The DCs were matured by BCG stimulation and the number of NKT cells and gammadeltaT cells increased after culturing with BCG-pulsed DCs. The BCG-pulsed DCs also activated the NKT cells and gammadeltaT cells. Also, the lymphocytes that were cocultured with the BCG-pulsed DCs showed unspecific cytotoxic activity against a bladder cancer cell line.
Sensitization of NKT cells and gammadeltaT cells by BCG-pulsed DCs might be one of the mechanisms of BCG immunotherapy.
卡介苗（BCG）用于膀胱癌的免疫治疗是成功的，尽管其确切机制尚不清楚。自然杀伤（NK）细胞是BCG激活的杀伤细胞的候选者，但其他T淋巴细胞，如NKT细胞和γδT细胞的作用尚未完全明确。结核分枝杆菌是NKT细胞和γδT细胞的有效激活剂。然而，已知BCG治疗后尿液中树突状细胞（DCs）数量增加时患者预后良好。因此，我们研究了BCG是否能使DCs成熟，以及BCG脉冲DCs是否能刺激NKT细胞和γδT细胞。
从外周血单核细胞（PBMCs）中分离出初始全T细胞，通过用粒细胞-巨噬细胞集落刺激因子和白细胞介素-4培养CD14(+)单核细胞获得DCs。用BCG对DCs进行脉冲处理，并使用CD86抗体通过荧光激活细胞分选分析来测量其成熟度。通过体外与BCG脉冲DCs共培养刺激初始T淋巴细胞，随后进行流式细胞术分析以评估NKT细胞的比例和激活情况以及γδT细胞的比例。使用（51）Cr（铬）释放试验来评估受刺激T细胞的细胞毒性活性。在BCG治疗期间，使用半定量逆转录聚合酶链反应测量患者PBMCs中的溶细胞蛋白。
BCG刺激使DCs成熟，与BCG脉冲DCs共培养后NKT细胞和γδT细胞数量增加。BCG脉冲DCs也激活了NKT细胞和γδT细胞。此外，与BCG脉冲DCs共培养的淋巴细胞对膀胱癌细胞系表现出非特异性细胞毒性活性。
BCG脉冲DCs使NKT细胞和γδT细胞致敏可能是BCG免疫治疗的机制之一。
