Ex vivo depletion of alloreactive cells based on CFSE dye dilution, activation antigen selection, and dendritic cell stimulation.
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 CFSE(bright) (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 CFSE(bright) 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.
Godfrey
Wayne R
WR
Department of Pediatrics, University of Minnesota Cancer Center, Minneapolis, MN 55455, USA. godfr007@umn.edu
Krampf
Mark R
MR
Taylor
Patricia A
PA
Blazar
Bruce R
BR
D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D002469:Cell Separation / Q000379:methods*; D003713:Dendritic Cells / Q000276:immunology; D005434:Flow Cytometry; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006086:Graft vs Host Disease / Q000517:prevention & control; D006680:HLA Antigens; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D007519:Isoantigens; D007959:Lymphocyte Culture Test, Mixed; D008212:Lymphocyte Depletion / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D013388:Succinimides; D014184:Transplantation, Homologous
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006680:HLA Antigens; D007519:Isoantigens; D013388:Succinimides