It has been demonstrated an anti-CD25 immunotoxin (IT) can specifically kill mature activated T cells which respond in a mixed lymphocyte reaction (MLR), while preserving third party reactivity. The objective of our study was to optimize specific killing using IT enhancers. We tested the ricin-A chain ontaining anti-CD25 immunotoxin, RTF5-SMPTdgA, using the one-way MLR to monitor depletion of alloreactive cells. To increase immunotoxin activity, we studied the effect of ammonium chloride, brefeldin A, monensin and perhexiline in this setting. The one-way MLR was performed in three steps: ( 1 ) Irradiated stimulators (peripheral blood mononuclear cells [PBMC]) were incubated with responders (PBMC from HLA-mismatched individual) for 24 hours. (2) RTF5-SMPT-dgA ±enhancers were added for 24 hours. (3) The IT and the enhancers were removed and cells were cultured for 5 days. During the last 18 hours, 3H-thymidine was added to the cultures to measure the proliferative activity of remaining cells. All three steps were carried out with negative controls and in (3), irradiated third party PBMCs were added to culture to demostrate preservation of third party activity. Both ammonium chloride and monensin enhanced IT-mediated depletion of alloreactive responder cells (>95% and >98%, respectively) while preserving third party reactivity (>79% and >47%, respectively). Brefeldin A did not enhance specific depletion and perhexiline was toxic at 1 -100|iM. In conclusion, both ammonium chloride and monensin enhance the ability of RTF5-SMPT-dgA to specifically deplete cells which proliferate in a one-way MLR.
|Original language||English (US)|
|Issue number||11 PART II|
|State||Published - Dec 1 2000|
ASJC Scopus subject areas
- Cell Biology