Generation of dendritic cells from lymphoid precursors

Dendritic cells (DC) are "professional" antigen presenting cells (APC) that are pivotal for the initiation of T-cell-dependent immune responses (1). DC are widely distributed cells in the body, but are scarce and thus difficult to purify. Methods have become available to grow DC in culture, facilitating the study of their biological and developmental properties. One such method is described in this chapter to generate DC in vitro from CD34(+) Lineage(-) (Lin(-)) CD10(+) bone marrow (BM) progenitor cells that are easily committed to the lymphoid lineages. This source of DC precursors thus distinguishes itself from mature monocytes or from total CD34(+) cells, which have also been employed to generate DC in vitro (2-5). It is unclear at this point if DC produced from lymphoid progenitor cells or from monocytes or total CD34(+) cells are equivalent. Lymphoid progenitor cells displaying the cell-surface phenotype CD34(+) Lin(-) CD10(+) rapidly lose CD34 cell-surface expression in culture and quickly differentiate into T-, B-, or natural-killer (NK) lymphocytes in the appropriate SCID-hu assay or culture systems (6,7; Galy, unpublished observations). This population of CD34(+) BM progenitor cells contains cells expressing the IL-7Rα chain, TdT, RAG-1, the transcription factors Pax5 and Ikaros, and represent the earliest recognizable human B-cell precursor identified in human BM (7,8). BM cells of CD34(+) Lin(-) CD10(+) phenotype can differentiate into antigen-presenting DC under the appropriate culture conditions, generating "lymphoidrelated DC" (6). Limiting dilution analyses demonstrated that single clones of CD34(+) Lin(-) CD10(+) progenitor cells can generate B cells, NK cells, and DC suggesting that these three lineages of cells share a common developmental stage in the BM and thus have close developmental relationships (6). In spite of being multipotent cells, CD34(+) Lin- CD10(+) cells do not differentiate into myeloid or erythroid cells in the presence of cytokines permissive for the production of such lineages from CD34(+) cells (6,7). In contrast, cytokine combinations such as c-kit-ligand+GM-CSF+flt3-ligand+IL-1+IL-7, which cause total CD34(+) cells to become myeloid cells, induce CD34(+) Lin(-) CD10(+)to differentiate into DC. Such lymphoid-related DC display characteristic long membrane processes, have immunostimulatory properties, and express on the cell surface high levels of MHC class II molecules, CD 1a, CD40 with little or no CD14, CD19, or CD15 antigens. Owing to their distinct hematopoietic origin, lymphoid-related DC may represent a separate population of DC. It is tempting to speculate that lymphoid-related DC may develop in lymphoid organs such as the thymus where progenitor cells with similar dual lymphoid/DC potential have been described (9). The method described here consists of preparing mononuclear cells (MNC) from BM that are depleted by negative selection of cells expressing lineage markers (CD2, CD3, CD19, CD20, CD14, CD15, CD32, glycophorin A), found on T cells, B cells, monocytes, and erythrocytes. Lineage-depleted cells are labeled with fluorochrome-conjugated antibodies and CD34(+) Lin(-) CD10(+)cells are purified by flow cytometry. Sorted cells are placed in culture in the presence of Flt3-ligand, c-kit-ligand, GM-CSF, IL-1, and IL-7 for 2 wk to generate lymphoid-related DC.