Ex vivo depletion of alloreactive cells based on CFSE dye dilution, activation antigen selection, and dendritic cell stimulation

• Published in: Blood 2004-02, Vol.103 (3), p.1158-1165
• Main Authors: Godfrey, Wayne R., Krampf, Mark R., Taylor, Patricia A., Blazar, Bruce R.
• Format: Article
• Language: English
• Subjects: Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy; Animals; Biological and medical sciences; Bone marrow, stem cells transplantation. Graft versus host reaction; CD4-Positive T-Lymphocytes - cytology; CD4-Positive T-Lymphocytes - immunology; Cell Separation - methods; Dendritic Cells - immunology; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Graft vs Host Disease - prevention & control; Hematopoietic Stem Cell Transplantation - methods; HLA Antigens; Humans; In Vitro Techniques; Isoantigens; Lymphocyte Culture Test, Mixed; Lymphocyte Depletion - methods; Medical sciences; Mice; Mice, Inbred C57BL; Succinimides; Transfusions. Complications. Transfusion reactions. Cell and gene therapy; Transplantation, Homologous
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2003-04-1098

Eliminating alloreactive cells from T-cell populations would enable the transfer of immune function to patients who receive stem cell transplants. However, high-efficiency depletion has proved difficult to achieve. We sought to develop ex vivo approaches for the maximal depletion of alloreactive CD4+ T cells. Using a flow cytometric cell sorting approach after mixed lymphocyte reaction (MLR) culture, we have found that sorted CFSEbright (5-(and-6)-carboxyfluorescein diacetate succinmidyl ester) (nondivided) and activation antigen-negative cells are markedly depleted of alloreactivity. With HLA-mismatched peripheral blood mononuclear cell (PBMC) stimulators we have consistently attained (90%-95%) depletion of alloreactivity. Importantly, when purified matured monocyte-derived dendritic cells (DCs) are used as stimulators, a 100-fold (99%) reduction in alloreactivity was attained, resulting in abrogation of the secondary MLR. Significantly, the CFSEbright CD25- cells recovered from these cultures retained general immunoreactivity, including responses to Candida and cytomegalovirus (CMV) antigens. In addition, a CFSE-based approach was tested and found to be sufficient for graft-versus-host disease (GVHD) prevention in vivo, in a major histocompatibility complex (MHC) class II disparate murine model. This efficient approach to selectively deplete mature alloantigen-specific T cells may permit enhanced immune reconstitution without GVHD. (Blood. 2004;103:1158-1165)