| ObjectiveAlong with chemotherapy regimen modified and transplantation technique matured, complete remission of leukemia have been further improved, but recurrence is the current difficulties and that was cause for minimal residual disease(MRD). CIK/DC-CIK cells have been widely applied in clinic due to their high antitumor activity and easy proliferation in vitro, MHC-unrestriction and mild adverse effects on normal tissues. However, its clinical efficacy have been mixed, we think Trafficking of the transferred immunocytes toword minimal residual leukemia lesions may be the key mechanism behind the low effects in vivo. Here, we investigate the distribution of DC-CIK cells in leukemia host and exploit and determine whether Tim-3repector expression on DC-CIK cells and blocking Tim-3might affect homing to tumor sites, influencing its efficacy in vivo; seek the method to improve the clinical effects of DC-CIK in MRD.MethodsCIK cells and DC-CIK cells were cultured for2weeks from the peripheral blood mononuclear cells of patients. The immune molecules on CIK/DC-CIK cells and PBMCs were detected by FACS in order to determine CIK cells and DC-CIK cells. Subcutaneous tumor model was established by K562leukemia cells in nude mice. DC-CIK cells were labeled with CFSE derectly and then inoculated into nude mice with subcutaneous tumor by intravenous injection. Distribution of DC-CIK cells in the organ and subcutaneous tumor sites and bone marrow was observed by automatic microplate reader and fluorescence microscope. Tim-3expression on CIK/DC-CIK cells and PBMCs were detected by FACS and find the difference among three groups, doing further work to confirm Tim-3expression on CIK/DC-CIK cells by RT-PCR. We combine transwell and fluorescence spectrophotometer and tecan genios to determine whether blocking Tim-3receptor on DC-CIK cells would affect their ability to migrate across tumor endothelial cells in vitro; trafficking towards tumor sites and bone marrow in tumor-bearing nude mice were detected by automatic microplate reader and fluorescence microscope after blocking Tim-3, observing the variation of their efficacy in vivo.ResultsFresh PBMCs only include4.31%CD3+CD56+cells, but the absolute number of immune cells increased markedly cultured with anti-CD3antibodies, IFN-γ and IL-2for2weeks. When we examined the phenotypes of the cultured cell population with fluorescence-activated cell sorting analyses, the cell population was composed of85.7%CD3+,22.7%CD3+CD56+,74.5%CD3+CD4-,76.5%CD8+,3.7%CD4+,31.6%CD3+CD8+, coinciding with CIK cells. Also the proportion of CD3+CD56+and CD3+CD8+/CD3+subsets of DC-CIK cells co-cultured with DC have been raised. Subcutaneous K562tumor model was established successfully. DC-CIK cells in subcutaneous K562tumor and bone marrow at24h point after injected was rare and the accumulation of DC-CIK cells in organs of tumor-bearing nude mice from the highest to the lowest were as follows:kidney, liver, intestine, spleen, lung and tumor by automatic microplate reader. It was confirmed that Tim-3receptor was expressed highly on CIK cells by FACS and RT-PCR,(83.26±5.69)%on CIK cells and even (95.07±3.69)%on CD3+CD56+subset. DC-CIK cells also expressed (64.93±14.77)%Tim-3recepter, less than CIK cells, however, the expression rate is the same with CIK cells on CD3+CD56+subset. The positive rate of Tim-3on fresh PBMCs was quite low, only (0.39±0.32)%, and only (6.22±3.61)%on CD3+CD56+subset. Migration test in vitro comfirmed thtat transmigrated DC-CIK cells across tumor endothelial cells in a period of1h increased up to52.66%from38.16%after blocking Tim-3receptor(P<0.001). Biodistribution test in vivo showed that the DC-CIK cells homing to tumor sites and bone marrow were increased(P<0.01), blocking Tim-3with anti-human Tim-3antibody. Compared with the RPMI1640-treated group, subcutaneous tumor of K562cells growth was inhibited greatly by injected intravenously at does of10million DC-CIK cells once a week. And median survival time of DC-CIK cells by Tim-3blocked treated tumor-bearing nude mice was longer than the other two groups(P<0.05).ConclusionDistribution of DC-CIK cells in subcutaneous tumor sites and bone marrow were rare by injected intravenously. Blocking Tim-3could promote DC-CIK cells migrating across tumor endothelial cells and trafficking toward tumor sites and bone marrow and finally prolong survival time of tumor-bearing nude mice. Tim-3maybe a novel effective target to improve adoptive immunotherapy of DC-CIK/CIK cells and blocking Tim-3could eliminate MRD and reduce the recurrence of leukemia in future. |
