Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Immunology

Language

English (en)

Date of Award

1-1-2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Wayne Yokoyama

Abstract

While there has been much progress in defining the specificity and function of natural killer: NK) cells, their differentiation has not been fully elucidated. Previous studies of thymocyte development in vitro indicate that double negative: CD4&minusCD8&minus, DN) thymocytes can develop into cells with NK cell markers, but these cells have not been well characterized. Moreover, a subpopulation of NK cells which requires an intact thymus, i.e, thymic NK cells, has been described with selective expression of CD127 although their origin and differentiation are also poorly understood. Herein, we generated and characterized NK cells differentiating from thymic DN precursors. We enriched potential progenitors by sorting DN1: CD44&plusCD25&minus) CD122&minusNK1.1&minus thymocytes from Rag1 deficient mice for adoptive transfer into Rag1&minus/&minusLy5.1 congenic mice. Following intrathymic injection, donor-derived cells phenotypically resembling thymic CD127&plus NK cells were found in thymus and spleen. To further characterize these cells, we seeded sorted DN1 CD122&minusNK1.1&minus thymocytes on a confluent monolayer of irradiated OP9 bone marrow stromal cells in the presence of IL15, IL7, FMS-like tyrosine kinase 3 ligand: Flt3L) and stem cell factor: SCF). Flow cytometry results showed NK1.1&plus cells emerged after at least 7 days in culture. By using limiting dilution analysis, we demonstrated a cell frequency of 0.24%: 1 out of 414 sorted thymocytes were able to generate an NK1.1&plus cell population). In vitro differentiated NK cells acquired markers associated with the development of conventional bone marrow&ndash derived splenic NK cells, but also expressed CD127, which is typically found on thymic NK cells. In-depth studies using gene chip microarrays further confirmed in vitro differentiated NK cells more closely resembled thymic NK cells as both expressed novel markers such as CD25 and CD103, which were not expressed by splenic NK cells. Finally, we found that in vitro cells generated from thymic precursors secreted cytokines when stimulated and degranulated upon target exposure, indicating that they were functional. Together, these data indicate that thymic NK cells can develop from a DN1 progenitor cell and may perhaps have a specific role that sets them apart from their splenic counterparts.

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Permanent URL: http://dx.doi.org/10.7936/K7X63JZ0

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