Assay for monitoring in vitro selective depletion strategies in allogeneic stem cell transplantation

• Published in: Cytotherapy (Oxford, England) Vol. 9; no. 6; pp. 600 - 610
• Main Authors: Villa, I, Kvale, E.O, Lund-Johansen, F, Olweus, J., MD, PhD
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 2007; Informa UK Ltd
• Subjects: Advanced Basic Science; Allodepletion; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Biological Assay - methods; Biomarkers; Cell Proliferation; Cell Separation; Humans; immunotherapy; Interleukin-2 Receptor alpha Subunit; Kinetics; Lectins, C-Type; Lymphocyte Activation; Other; Stem Cell Transplantation; T-Lymphocytes - cytology; Transplantation, Homologous; Allodepletion; stem cell transplantation; immunotherapy
• ISSN: 1465-3249; 1477-2566
• DOI: 10.1080/14653240701510573

Background GvHD is a serious and potentially life-threatening side-effect of allogeneic BMT, caused by alloreactive cells attacking normal host cells. A number of different approaches have been attempted to remove allo-activated cells from the graft prior to transplantation. When developing such assays, there is a need to control for unwanted removal of cells, as well as depletion efficiency related to activation kinetics. Methods The specific activation induced by the superantigens SEB and TSST-1 of T cells with defined Vβ chains was utilized to follow activation of bystander cells and the kinetics of specific cellular activation by flow cytometry. Results The activation marker CD69 was up-regulated on bystander T cells, and was only transiently highly expressed on the specific T cells, making this marker unreliable for removal of alloreactive cells. In contrast, CD25 was found only on specifically activated T cells and was stably expressed over several days. However, it was not detected on all specific cells until day 6. Likewise, proliferation occurred only in T cells expressing the expected Vβ chains, with all activated cells having undergone at least one cell cycle by day 4. Discussion In conclusion, our assay demonstrates that only temporary bystander activation occurs when polyclonally activating T cells by SEB or TSST-1, and that CD25, but not CD69, can be used for removal of specifically activated cells. Furthermore, this assay is useful for monitoring methods aiming at specific removal of cycling cells.