title: In vitro selective depletion of CD4(+)CD25(+) regulatory T-cells from PBMC using anti-tac-SAP.
abstract: 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.
authors.affiliation: Department of Immunology, Isfahan University of Medical Sciences, Isfsahan, Iran. akbari@resident.mui.ac.ir
authors.forename: Akbar
authors.initials: A
authors.lastname: Akbari
authors.forename: Abbas
authors.initials: A
authors.lastname: Rezaei
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000627:therapeutic use*; D001427:Bacterial Toxins / Q000235:genetics; D015704:CD4 Antigens / Q000276:immunology; D002478:Cells, Cultured; D003110:Colonic Neoplasms / Q000628:therapy*; D005098:Exotoxins / Q000235:genetics; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000276:immunology; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000627:therapeutic use*; D053645:Interleukin-2 Receptor alpha Subunit / Q000276:immunology*; D008212:Lymphocyte Depletion / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000627:therapeutic use*; D054789:Ribosome Inactivating Proteins, Type 1 / Q000627:therapeutic use*; D012503:Saponins / Q000235:genetics / Q000627:therapeutic use*; D016176:T-Lymphocyte Subsets / Q000187:drug effects / Q000276:immunology*; D050378:T-Lymphocytes, Regulatory / Q000187:drug effects / Q000276:immunology*; D037521:Virulence Factors / Q000235:genetics; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D015704:CD4 Antigens; D005098:Exotoxins; D051858:Forkhead Transcription Factors; D000922:Immunotoxins; D053645:Interleukin-2 Receptor alpha Subunit; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D012503:Saponins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C418974:FOXP3 protein, human; C581146:anti-tac-SAP