In vitro selective depletion of CD4(+)CD25(+) regulatory T-cells from PBMC using anti-tac-SAP.

It has been shown that naturally occurring regulatory T-cells (CD4(+)CD25(+) Foxp3(+) T-cells) have critical roles in tumor invasion and down-regulation of immune response against established tumors. High expression of CD25 (IL-2Rα) by regulatory T (T(reg)) cells may cause an inefficient response when using IL-2-based cancer vaccines. It seems that selective elimination of T(reg) cells before treatment of tumor-bearing T-cells can strongly increase the efficacy of a vaccine. The aim of this study was to set up an efficient cost-effective protocol to eliminate CD4(+)CD25(+) T-cells-using the immunotoxin anti-tac-SAP. Peripheral blood mononuclear cells (PBMC) taken from colon cancer patients were treated with different concentrations (i.e., 0-100 µg/dl) of the immunotoxin. Flow cytometric analyses were then preformed to analyze expression of CD4, CD25, CD3, CD8, and CD45 surface markers; semi-quantitative fluorescent-PCR was used for the detection of Foxp3 expression before and after anti-tac-SAP treatment. The results indicated that anti-tac-SAP effectively eliminated CD4(+)CD25(+) T(reg) cells and that 25 µg/dl was the optimal concentration of anti-tac-SAP for selective depletion of these cells. These outcomes were verified by analyses of Foxp3 expression. The results also indicated that this immunotoxin had no non-specific effects on other T-cells, including CD4(+)CD25(-) and CD8(+)CD45(+) T-cells. Building on the work here, ongoing/future studies with the anti-tac-SAP will focus on functional assessments of the remaining (i.e., non-eliminated) T-cells (i.e., CD8, CD4; using proliferation and peptide sensitization assays) to ascertain if the immunotoxin inadvertently alters the functions of these cells-an untoward outcome.