Lymphocyte development involves several stages beyond the commitment to the T-cells, B-cells, or NK lineages. Last year we isolated the clonal common lymphocyte progenitor (CLP), a rare cell in mouse bone marrow (~.02 percent) which in vitro or in vivo can give rise to T Iymphocytes, B lymphocytes, and NK cells, but not any myeloerythroid cells, including dendritic APC. This application represents an attempt to define stages in development between hematopoietic stem cells (HSC), multipotent progenitors (MPP) and CLP, as well as define the genes express in CLP that may have committed HSC/MPP to become CLP, or govern the differentiation of CLP to each of their lymphoid lineage outcomes. In addition, the ontogeny of CLP from embryogenesis to old age is another major goal of investigation, as well as attempts to use CLP in addition to HSC to accomplishment rapid immune reconstitution in lethally irradiated mice representing models for both autologous and folly allogeneic human transplantation. The approach to lineage analysis from HSC/MPP to CLP is to place purified HSC or MPP onto clonal stromal monolayers that permit the development of B lymphocytes and myelopoiesis, either at the bulk level to measure population effects or at the single cell level, to determine whether the dividing CLP progeny from these multipotent progenitors results from a symmetric or asymmetric division. The pathway between CLP and defined lymphoid outcomes will use almost exactly the same assay, except that thymic stromal cells will be used alone or in addition to the cloned bone marrow stromal cells in order to test all 3 outcomes from bulk or single CLP put in culture T-cells, B-cells, and NK cells. Again, the single cell experiments are designed to determine whether asymmetric or symmetric outcomes occur when single CLP divide. The expressed gene approaches will involve establishing full cDNA libraries that are normalized and placed on microchips, and then screened using HSC/MPP probes, CLP probes, and probes of the downstream T lineage, B lineage, and NK lineage cells. Interesting discovered genes selectively expressed in CLP, or carrying motifs indicating receptor function and/or relationship to other genes involved in lineage commitments will be isolated. Retroviral vectors will be used to transfect either HSC/MPP for CLP specific genes, or CLP for genes that might specify the particular lymphoid lineage they could become. CLP will be isolated from fetal liver, young adult mice, and quite old mice, and will be tested for their homogeneity and clonal activity for lymphoid lineages at least. In addition, fetal CLP will be investigated for the possibility of giving rise, in addition to T-, B-, and NK cells, to other lymphoid related cells such as dendritic APC, allograft facilitator cells, and cells in the mast cell/basophil lineage. Finally, graded doses of CLP will be used to reconstitute Ly5 congenic and folly allogeneic mice along with radioprotective doses of stem cells in order to test whether a) lymphocyte recovery can be hastened, b) resistance to CMV infection can be established, and c) immunological transplant tolerance to CLP genetic donor heart grafts can be established.
| Ooi, A G Lisa; Karsunky, Holger; Majeti, Ravindra et al. (2009) The adhesion molecule esam1 is a novel hematopoietic stem cell marker. Stem Cells 27:653-61 |
