Development of Two Distinct Dendritic-Like APCs in the Context of Splenic Stroma

Abstract

Murine splenic stroma has been found to provide an in vitro niche for hematopoiesis of dendritic-like APC. Two distinct cell types have been characterized. The novel “L-DC” subset has cross-presenting capacity, leading to activation of CD8+ T cells, but not activating CD4+ T cells, which is consistent with their CD11cloCD11bhiMHC-II− phenotype. For L-DC, an equivalent tissue-specific APC has been found only in spleen. A second population of CD11chiCD11bloMHC-II+ cells resembling conventional dendritic cells (cDC) can activate both CD4 and CD8 T cells. Production of L-DC but not cDC-like cells is now shown to be dependent on contact between the L-DC progenitor and stroma such that the presence of a Transwell membrane can prevent L-DC development. Since L-DC can be produced continuously in vitro in stromal co-cultures overlaid with bone marrow (BM) progenitors, it was hypothesized that L-DC progenitors are self-renewing. The L-DC progenitor is shown here to be defined by the Flt3−c-kit+Lin−Sca-1+ (F−KLS) subset of adult BM which contains primitive HSC. Since the less primitive F+KLS HSC subset also contains L-DC progenitors, Flt3 does not appear to be a defining marker for this progenitor. Precursors of the cDC-like subset are found only within the F+KLS subset and seed production of a transient population of APC. All data identify differentiation of L-DC from HSC, and of cDC-like cells from DC precursors, which occurs independently of inflammatory signals and is dependent on a splenic stromal microenvironment.

Distinct Progenitor Origin Distinguishes a Lineage of Dendritic-Like Cells in Spleen

“…Co-cultures initiated with known HSPC subsets sorted from BM show differential capacity to produce the distinct L-DC and cDC-like subsets which are produced together in co-cultures established with a heterogeneous Lin − BM subset (7, 10). Both MDP, as progenitors of monocytes and DC, as well as CDP, the more restricted progenitor of cDC and pDC, both produce only cDC-like cells in co-cultures.…”

Impact of the c-MybE308G mutation on mouse myelopoiesis and dendritic cell development

“…This procedure has been described in detail previously [27, 28, 34, 35]. Medium change was performed every 3–4 days by replacement of 2.5 ml medium.…”

Delineation of a novel dendritic-like subset in human spleen

“…L-DCs reflect a novel type of APC that is distinguishable by stromadependent development, lack of MHC-II expression, and high endocytic activity, which is consistent with their ability to efficiently cross-present antigens to CD8 T lymphocytes. 10,11,38,39 The in vivo equivalent of L-DCs was later characterized in mouse spleens and shown to have similar phenotype and function. 9 In cocultures of human splenocytes with the 5G3 murine stromal line, several subsets of DC-like cells are produced, and they differ in their expression of HLA-DR and hCD11c.…”

“…On the basis of high CD11c and MHC-II expression, these cells resemble cDC-like cells, previously reported as a transient early population produced in murine splenic cocultures. 10,11 These cells disappear after 21-28 days of coculture. The hCD11c lo HLA-DR 2 subset was consistently produced and represented a maximum of 17% of cells over time.…”