During the current funding period, they have made substantial progress in understanding in vitro lymphopoiesis. Perhaps the most important finding for the success of investigations proposed here is that a murine fetal liver cell line, AFT024, can efficiently support differentiation of putative human stem cells at the single cell level into NK cells and myeloid cells while expanding CD34+ progenitors. The role of IL-2, IL-7, c-kit ligand (KL), Flt3 ligand (FL) and IL-3 has been established and they have defined molecular events which occur during this process. This assay will be optimal to address several important questions in NK cell differentiation. There are two related themes as the focus of these investigations. They hypothesize that early differentiation of stem cells into an NK cell restricted progenitor (loss of myeloid capacity) and further NK cell commitment is governed by factor provided by the microenvironment, and factors inherent to the stem cell source. They will use the differentiating of AFT024 to study NK cell differentiation from stem cells using a sequential """"""""switch culture assay"""""""" which allows separation of early and late differentiation stages. The second line of investigation is based on the discovery of class I binding receptors, which are comprised of immunoglobulin-type or lectin-type domains with genes clustered to regions of human chromosomes 19 and 12, respectively. In addition to KIR, other related receptors (LIR-1, LAIR-1) of the immunoglobulin family will also be studied. They hypothesize that inhibitory or activating receptor repertoires are determined late in development and that receptor ligation during development may signal the cell to stop acquiring new receptors.
In Specific Aim 1, the role of cytokines, chemokines, proteoglycans, and the complex microenvironment will be explored in NK cell development using single cell differentiation of stem cell populations from adult marrow and cord blood. The role of these factors in determining the balance between progenitor maintenance and NK cell differentiation will be assessed. These studies integrate well with goals in Specific Aim 2 to study the ontogeny of class I binding receptors and receptors of related families. They will determine how and what developmental stage receptor acquisition occurs in developing NK cells.
In Specific Aim 3, they will evaluate transcription factors within developing cells to determine their role in lineage restriction. Gene manipulation (to over-express or block function) by retroviral transduction into single primitive stem cell populations will verify their role in the differentiation process. Preliminary data suggests a role for targeting TCF-1 as an initial focus using this approach.
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