Hematopoiesis of immature myeloid dendritic cells in stroma-dependent spleen long-term cultures occurs independently of NF-KB/RelB function.

OBJECTIVE: The nuclear factor-kappaB (NF-KB)/RelB transcription factor plays an essential role in development of some dendritic cell (DC) subsets in mice. In this laboratory, immature myeloid DC are produced in vitro in a stroma-dependent murine spleen long-term culture (LTC) system. In LTC, DC differentiate from hematopoietic progenitors maintained within the stromal cell matrix. Expression and function of RelB in development of LTC-DC has been investigated, with a view to assessing the relationship between DC produced in this system and other known subsets of DC. MATERIALS AND METHODS: RelB expression by LTC-DC was confirmed by detection of protein by Western blotting, RNA by reverse transcription polymerase chain reaction, and nuclear protein with DNA-binding function in electrophoretic mobility shift assays. The role of RelB in cell development was assessed by addition of antisense RelB oligonucleotides into LTC and colony assays established above STX3 stromal cells. RelB(-/-) mice were also examined for ability to produce LTC, and for presence of DC progenitors in spleen and bone marrow that can generate DC when overlaid on STX3 in cocultures. RESULTS: Functional RelB was detected in both LTC-DC and in STX3 stromal cells. A critical role for RelB in DC differentiation from spleen progenitors was confirmed, because antisense RelB oligonucleotides specifically and completely inhibited production of large differentiated myeloid DC in LTC. Further investigation using RelB(-/-) mice revealed that RelB expression by stromal cells rather than hematopoietic cells was required for production of LTC-DC. This was evidenced by a combination of factors, including 1) inability to generate productive LTC from RelB(-/-) mice; 2) presence of DC precursors in RelB(-/-) bone marrow and spleen, which could produce DC in stromal cocultures; and 3) increased myeloid precursor frequency among RelB(-/-) spleen cells over RelB(+/+) control cell populations. CONCLUSION: Specific development of fully differentiated, but immature myeloid CD11c(+)CD11b(+)MHC-CII(-)CD8alpha(-)CD40(-) DC in spleen LTC is dependent on expression of activated NF-kappaB/Rel-B. However, this appears to relate to stromal cell function rather than to the function of hematopoietic cells. Altogether these data confirm the importance of splenic stromal cells in myelopoiesis leading to development of immature DC as produced in LTC.

Molecular definition of an in vitro niche for dendritic cell development.

OBJECTIVE: Although dendritic cell (DC) precursors have been isolated from many lymphoid sites, the regulation and location of early DC development is still poorly understood. Here we describe a splenic microenvironment that supports DC hematopoiesis in vitro and identify gene expression specific for that niche. METHODS: The DC supportive function of the STX3 splenic stroma and the lymph node-derived 2RL22 stroma for overlaid bone marrow cells was assessed by coculture over 2 weeks. The DC supportive function of SXT3 was identified in terms of specific gene expression in STX3 and not 2RL22 using Affymetrix microchips. RESULTS: STX3 supports DC differentiation from overlaid bone marrow precursors while 2RL22 does not. A dataset of 154 genes specifically expressed in STX3 and not 2RL22 was retrieved from Affymetrix results. Functional annotation has led to selection of 26 genes as candidate regulators of the microenvironment supporting DC hematopoiesis. Specific expression of 14 of these genes in STX3 and not 2RL22 was confirmed by reverse transcription-polymerase chain reaction. CONCLUSION: Some genes specifically expressed in STX3 have been previously associated with hematopoietic stem cell niches. A high proportion of genes encode growth factors distinct from those commonly used for in vitro development of DC from precursors. Potential regulators of a DC microenvironment include genes involved in angiogenesis, hematopoiesis, and development, not previously linked to DC hematopoiesis.

Dendritic cell development in long-term spleen stromal cultures.

The cellular microenvironments in which dendritic cells (DCs) develop are not known. DCs are commonly expanded from CD34+ bone marrow precursors or blood monocytes using a cocktail of growth factors including GM-CSF. However, cytokine-supported cultures are not suitable for studying the intermediate stages of DC development, since progenitors are quickly driven to become mature DCs that undergo limited proliferation and survive for only a short period of time. This lab has developed a long-term culture (LTC) system from spleen which readily generates a high yield of DCs. Hematopoietic cells develop under more normal physiological conditions than in cultures supplemented with cytokines. A spleen stromal cell monolayer supports stem cell maintenance, renewal, and the specific differentiation of only DCs and no other hematopoietic cells. Cultures maintain continuous production of a small population of small-sized progenitors and a large population of fully developed DCs. Cell-cell interaction between stromal cells and progenitor cells is critical for DC differentiation. The progenitors maintained in LTC appear to be quite distinct from bone marrow-derived DC progenitors that respond to GM-CSF. The majority of cells produced in LTC are large-sized cells with a phenotype reflecting myeloid-like DC precursors or immature DCs. These cells are highly endocytotic and weakly immunostimulatory for T cells. This model system predicts in situ production of DCs in spleen from endogenous progenitors, as well as a central role for spleen in DC hematopoiesis.

In vitro hematopoiesis produces a distinct class of immature dendritic cells from spleen progenitors with limited T cell stimulation capacity.

The study of dendritic cells (DC) has been hampered by the difficulty of isolating rare cells for analysis of their phenotype and function. Interpretation of the DC lineage has been largely influenced by studies on cell populations which can be readily isolated and amplified in the presence of cytokines. Long term cultures (LTC) from murine spleen have been shown to support continuous in vitro hematopoiesis of DC dependent on interaction with a stromal cell monolayer. LTC-DC represent a single, stable class of DC derived by constant turnover of spleen DC progenitors maintained within stroma. They represent a resident DC population in spleen. The functional characteristics of LTC-DC have been studied in terms of capacity to stimulate T cells and response to activation by environmental stimuli. LTC-DC have many morphological, phenotypic and functional properties reflecting an immature or partially mature, marginal zone-like CD4(-)CD8(-) splenic DC subset. They are highly endocytic and can process and present protein antigen to naive hen egg lysozyme (HEL)-specific MHC-II-restricted TCR-Tg CD4(+) T cells. They do not, however, induce T cell proliferation in a mixed lymphocyte reaction. LTC-DC do not respond in a typical fashion to common DC activators like LPS and CD40L. They upregulate MHC-I and CD80/CD86 but not MHC-II and CD40. They reflect an endogenous, immature DC subset in spleen with properties distinct from immature DC located in peripheral tissues.