Brandau S, Suttmann H, Riemensberger J, Seitzer U, Arnold J, Durek C, Jocham D, Flad H D, Böhle A
Department of Immunology and Cell Biology, Research Center Borstel, Germany.
Clin Cancer Res. 2000 Sep;6(9):3729-38.
Immunotherapy with Bacillus Calmette-Guérin (BCG) is clinically established in the treatment of superficial bladder cancer. In our attempt to clarify the underlying immunological mechanism, we could previously show that stimulation of PBMC with BCG leads to the generation of cytotoxic BCG-activated killer (BAK) cells. Among others, these BAK cells as well as lymphokine-activated killer (LAK) cells have been suggested as possible effector cells during BCG therapy. To understand BCG-induced activation of effector lymphocytes more precisely, we investigated the lytic pathways of human BAK cells and compared BAK cell cytotoxicity with LAK cell cytotoxicity. Perforin and Fas ligand (FasL) are the major cytolytic molecules of cytotoxic lymphocytes. Our results demonstrate that BAK and LAK cells showed an increased expression of perforin and FasL as compared with unstimulated controls. Killing of T-24 bladder tumor as well as Jurkat cells by BAK and LAK cells was predominantly mediated via perforin as demonstrated by a drastically reduced lysis in the presence of concanamycin A and EGTA/MgCl2, respectively. In contrast, lysis (radioactive release assay) and membrane disintegration (Annexin V binding) of both targets by BAK and LAK cells could not be blocked with an inhibitory anti-FasL monoclonal antibody (NOK-1). Nevertheless, T-24 and Jurkat were susceptible to killing by recombinant soluble FasL and by Chinese hamster ovary cells expressing membrane-bound FasL. We conclude that cellular mediators of BCG effector mechanisms, such as BAK and LAK cells, kill their targets via perforin and independent of the FasL pathway. Because we also found increased numbers of perforin-expressing lymphocytes in patients after BCG therapy, our findings have potential clinical relevance because BCG therapy would not be impaired by FasL resistance of target cells, which recently has been described for some tumors.
卡介苗（BCG）免疫疗法在浅表性膀胱癌的治疗中已得到临床确立。在我们试图阐明其潜在免疫机制的过程中，我们之前能够证明用卡介苗刺激外周血单核细胞（PBMC）会导致细胞毒性卡介苗激活杀伤（BAK）细胞的产生。其中，这些BAK细胞以及淋巴因子激活杀伤（LAK）细胞被认为是卡介苗治疗期间可能的效应细胞。为了更精确地了解卡介苗诱导的效应淋巴细胞激活，我们研究了人BAK细胞的裂解途径，并将BAK细胞的细胞毒性与LAK细胞的细胞毒性进行了比较。穿孔素和Fas配体（FasL）是细胞毒性淋巴细胞的主要溶细胞分子。我们的结果表明，与未刺激的对照相比，BAK细胞和LAK细胞中穿孔素和FasL的表达增加。如在存在 concanamycin A和EGTA/MgCl2时裂解显著减少所证明的，BAK细胞和LAK细胞对T-24膀胱肿瘤以及Jurkat细胞的杀伤主要通过穿孔素介导。相比之下，BAK细胞和LAK细胞对两个靶标的裂解（放射性释放测定）和膜崩解（膜联蛋白V结合）不能被抑制性抗FasL单克隆抗体（NOK-1）阻断。然而，T-24和Jurkat细胞对重组可溶性FasL以及表达膜结合FasL的中国仓鼠卵巢细胞的杀伤敏感。我们得出结论，卡介苗效应机制的细胞介质，如BAK细胞和LAK细胞，通过穿孔素并独立于FasL途径杀死其靶标。因为我们还发现卡介苗治疗后患者中表达穿孔素的淋巴细胞数量增加，我们的发现具有潜在的临床相关性，因为卡介苗治疗不会受到靶细胞FasL抗性的影响，而最近已在一些肿瘤中描述了这种抗性。
