| Relapse of underlying malignancies is still the major cause of failure in autologous stem cell transplantation. It has been hypothesized that relapse take place not only because of residual disease in the autograft, but because of proliferation of resistant tumor cells remaining in the patient after preparative treatment with high dose chemoradiotherapy. In that context, efforts were made to generate natural killer (NK) cells from bone marrow by incubating with interleukin-2 (IL-2) in vitro, known as IL-2 activated bone marrow (IL-2 ABM). However, the results obtained in clinical trials with IL- 2 ABM have proved disappointing in many forms of cancer. Several lines of evidence have shown that IL-2 is often not sufficiently effective because intratumoral monocytes/macrophages (MO) inhibit the IL-2-induced activation of NK cells at the site of tumor growth, an essential part of this inhibitory signal is conveged by MO-derived reactive oxygen species (ROS). Histamine, specifically acting through monocyte H2-type histamine receptors (H2R), prevents the inhibition of NK function by inhibiting the formation of ROS in monocytes. On the other hand, it has been confirmed that histamine,via H2R, triggers hematopoietic stem cell passage from the G0 state to the S-phage of the cell cycle,and promotes proliferation and differentiation of hematopoietic progenitor cells. Based on these findings, we aimed to establish a histamine/IL-2 ABM adoptive immunotherapy more effectively than IL-2 ABM. The main contents were as follows. (1) We used microcytotoxicity, CFU-Raji and CFU-GM assays to evaluate purging effect of autograft in vitro.The results showed that histamine and IL-2 synergized to activate bone marrow cells to lyse K562 cells and Raji cells in a 4h MIT reduction assay, the purging ability of IL-2 ABM can be augmented effectively by histamine or H2R agonist dimaprit, at the same time maintain hematopoietic progenitor cell activity (PCA). (2) We developed a -3- histamine/IL-2 ABM treatment protocol for B 16 melanoma bearing C57BL/6 mice that involves culturing the entire marrow autograft for 24h in the presence of histamine and IL-2, histamine/IL-2 injections immediately after marrow infusion with the goal of maintaining the in vivo cytotoxic potential of ABM cells. It was found that histamineflL-2 ABM can contribute to a more potent graft versus tumor (GVT) effect in vivo, and retain the capacity to reconstitue the long-term hematopoiesis. (3) We further characterized the mechanism of the antitumor effect generated by histamineflL-2 activated autograft. At first, monocytes recovered from peripheral blood stem cell graft were stimulated respectively by IL-2, histamine, H2R agonist ,dimaprit and H2R antagonist ranitidine. Intraceller ROS level changes were measured by laser scanning confocal microscopy (LSCM). It was found that histamine and H2R agonist dimaprit induced a significant decrease in intraceller ROS level, in contrast, IL-2 and H2R antagonist ranitidine enhanced the ROS level in monocytes significantly. Secondly, proliferation of CD34~, CD3~, CD56~CDl6~CD3 cells in the graft cultured for 1? d~y in the presence of IL-2, histamine, H2R agonist, and H2R antagonist was determined by direct immunofluoresence using flow cytometry (FCM). It was found that the relative number of NK cells remained unchanged in 1 day histaniinellL-2 activated peripbral blood graft, and a significant increase in actived NK cells (CD56~CDl6~CD3...
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