Clinical observations in patients undergoing bone marrow transplantation implicate the involvement of CD8 ؉ cells in promoting the stem-cell engraftment process. These findings are supported by mouse transplant studies, which attributed the engraftment-facilitating function to subpopulations of murine CD8 ؉ cells, but the analogous cells in humans have not been identified. Here
IntroductionClinical success of bone marrow (BM) transplantation is critically dependent on rapid hematopoietic and immune reconstitution. 1,2 Murine studies identified rare BM cells that enhance stem-cell engraftment and induce donor-specific tolerance. These facilitating cells (FCs) were defined as CD8 ϩ TCR Ϫ plasmacytoid dendritic cell precursors, 3,4 or as a subset of CD8 ϩ CD3⑀ ϩ cells, which lack conventional ␣-and ␥␦-T-cell receptor (TCR) heterodimers. 5,6 With mouse CD8 ϩ TCR Ϫ CD3 ϩ FCs, CD3 is essential for enhanced reconstitution across the allogeneic barriers. [6][7][8] Patients receiving grafts depleted of CD3 ϩ lymphocytes to avoid graft-versus-host disease (GVHD) are at high risk of graft failure. 9 The engraftment-facilitating function associated with CD8 ϩ but not CD4 ϩ T-cell subsets in both clinical transplantation 10,11 and the NOD/SCID mouse xenotransplantation model. 12 This indicates that the human CD8 ϩ population, harboring alloreactive T cells responsible for GvHD, 1 also contains cells with graft-facilitating capacity; however these immunologic properties have not yet been dissected in either clinical or experimental studies. We identified a rare population of human CD8 ϩ TCR Ϫ CD3 ϩ cells and used the NOD/SCID assay to show their graft-facilitating potential.
MethodsSample collection, cell purification, and culture Granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood (mPB), cord blood (CB), and BM collections from healthy donors were approved by the Ethical Committee of University Hospitals Basel. Informed consent was obtained in accordance with the Declaration of Helsinki. Mononuclear cells (MNCs) were isolated by Ficoll-Histopaque (Sigma-Aldrich, St Louis, MO). CB CD34 ϩ cells were isolated by magnetic-assisted cell separation (MACS; Miltenyi Biotec, Bergish Gladbach, Germany) to a purity of more than 98%. CD8 ϩ , CD8 ϩ TCR Ϫ , and CD8 ϩ TCR Ϫ CD3 ϩ cells were purified from mPB by sorting with MoFlo (Dako, Fort Collins, CO). Colony forming unit (CFU) assays of CD34 ϩ cells were performed in 1% methylcellulose containing stem-cell factor (SCF), interleukin (IL)-3, G-CSF, granulocyte-macrophage-CSF (GM-CSF), and erythropoietin (EPO) 13 after coculture with CD8 ϩ cells or subsets thereof for 2 hours at 37°C, in the presence of anti-flt3 ligand (Flt3L) monoclonal antibody (mAb; M5, Immunex, Seattle, WA) where indicated. CD8 ϩ TCR Ϫ CD3 ϩ cells were maintained in RPMI/10% fetal calf serum (FCS) and IL-7 (100 ng/mL; Novartis Basel, Basel, Switzerland) for 5 days to measure Flt3L, 14 and in IL-7 on anti-CD3 mAb-coated dishes for 3 days to isolate mRNA, or 14 days to examine cell phenotype.
Flo...