Analysis of Clonal Contributions to T Lymphoid and Myeloid Lineages during Early Hematopoiesis Following Autologous Transplantation in the Rhesus Macaque.

Hematopoietic stem cells are defined by their ability both to self-renew and to differentiate into cells that repopulate all hematopoietic lineages. A common lymphoid progenitor (CLP) from mouse marrow has been phenotypically purified and shown to only transiently repopulate all lymphoid lineages (T, B, and NK) but not myeloid lineages when transplanted into lethally irradiated recipients (Kondo et al, 1997). Using the rhesus macaque as a model for human hematopoiesis, we asked whether lineage-restricted repopulating cells contribute to hematopoietic output and for how long following engraftment. In order to follow the progeny of progenitor or stem cell clones in vivo, retroviral vectors were used to mark primitive hematopoietic cells before reinfusion into lethally irradiated autologous recipients. The conditions utilized result in high level of stable retroviral marking in all lineages. We have previously reported use of the sensitive Linear Amplification Mediated - Polymerase Chain Reaction (LAM-PCR) showing that a highly polyclonal set of stable clones accounts for myelopoiesis (Schmidt et al, 2002). In the current study, detailed characterization of the full array of clones contributing to granulocytes, T-lymphocytes, and B-lymphocytes post-engraftment was performed, via sequence analysis of individual clones delineated by individual insertion site sequences at various time points following transplantation of CD34+ cells. Thus far, we have identified 59 granulocyte and 148 T-lymphocyte clones at 1 month following transplantation. Most clones detected at this time point uniquely contribute to only the lineage from which they were identified, only three clones were common to both lineages (1.5% overlap, 95%CI=(0–3.1%)), suggesting independent cells are responsible for hematopoietic reconstitution at this early time point. Six-months following transplantation fewer clones were detected to contribute to hematopoiesis. Interestingly, of the 22 granulocyte and 46 T-lymphocyte clones identified, only two were shared between both lineages (3.0% overlap, 95%CI= (0–7.2%)), further supporting the hypothesis that lineage committed progenitors are responsible for both early and potentially more stable hematopoiesis in this model. To our knowledge, no other study has provided such an extensive characterization of clones contributing to early or more stable in vivo hematopoiesis in the primate model. Preliminary sequence specific tracking experiments reveal that the clones contributing at six-months remain lineage specific over time. Further tracking of clones is being conducted to fully assess the role of such lineage specific progenitor cells in B-lymphopoiesis and long-term hematopoiesis.