Bone marrow-derived CD8alpha+TCR- cells that facilitate allogeneic bone marrow transplantation are a mixed population of lymphoid and myeloid progenitors.
OBJECTIVE
We have characterized a hematopoietic cell population isolated from murine bone marrow that can facilitate purified hematopoietic stem cell engraftment across fully allogeneic major histocompatibility complex barriers. These facilitating cells (FCs) are classically identified as CD8alpha(+)TCR(-) by flow cytometry. Prior work has demonstrated that FCs are comprised of a heterogeneous cell population with both lymphoid and myeloid phenotypes. The present investigation was designed to more precisely characterize these subsets in terms of both phenotype and developmental potential.


METHODS
Using fluorescence-activated cell sorting analysis, freshly isolated FCs were characterized for phenotypic expression of various lymphocyte progenitor markers. The lymphopoietic potential of FCs was evaluated by culturing freshly isolated FCs on bone marrow stroma cells overexpressing notch ligand 1 (OP9-DL1). Transcripts specific to pTalpha and TCRalpha were quantitated by employing real-time reverse transcription polymerase chain reaction. Maturation of the T-cell receptor (TCR) on FCs was biochemically analyzed by immunoprecipitation.


RESULTS
Freshly isolated FCs had significant expression of CD44(+)CD25(-) and CD44(+)CD25(+) phenotypes. A discrete subset of CD8(+)CD4(+) cells are also identified in the FC population, similar to the double-positive phase of thymocyte development. Of particular interest, FCs express pre-TCRalpha (pTalpha) mRNA and protein as demonstrated by reverse transcription polymerase chain reaction, intracellular staining and immunoprecipitation. FCs grown on OP9-DL1 with interleukin-7 and FMS-like tyrosine kinase 3 ligand can mature into CD44(-)CD25(+), CD8(+)CD4(+) and CD8(+) T cells. During this developmental process, expression of the 33-kDa pTalpha chain was replaced by a mature 40-kDa TCRalpha chain.


CONCLUSIONS
Taken together, these data demonstrate for the first time that the marrow-derived FC contains a T-cell progenitor population that closely resembles developing thymocytes.
Gangopadhyay
Nupur N
NN
Heart, Lung and Esophageal Surgery Institute, University of Pittsburgh, Pittsburgh, PA, USA. gangopadhyayn@upmc.edu
Hoffman
Rosemary A
RA
Shen
Hongmei
H
Luketich
James D
JD
Schuchert
Matthew J
MJ
D000818:Animals; D001854:Bone Marrow Cells / Q000276:immunology*; D016026:Bone Marrow Transplantation; D016827:CD8 Antigens / Q000276:immunology*; D002454:Cell Differentiation; D002469:Cell Separation; D005434:Flow Cytometry; D005453:Fluorescence; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D013234:Stem Cells / Q000166:cytology*; D014184:Transplantation, Homologous
D016827:CD8 Antigens; D011948:Receptors, Antigen, T-Cell