Stromal cells which direct dendritic cell development

Abstract

Development of the immune system is depicted as a hierarchical process of differentiation from hematopoietic stem cells (HSC) to lineage-committed precursors, which further develop to give mature immune cells. In the case of dendritic cell (DC) development, this linear precursor-progeny approach has led to a complex picture of relationships between various subsets of DC identified in vivo. A possible reconciliation of the diversity of DC precursors and their progeny in vivo can be achieved by assessing the role of the microenvironment in hematopoiesis. One model is that the splenic microenvironment comprises one or more populations of stromal cells, each with a unique role in DC hematopoiesis. Stromal cells derived from spleen have been studied as a model microenvironment for DC development. Cloned splenic stromal cells lines derived from a spleen long-term culture (LTC) which formerly supported production of dendritic-like cells (LTC-DC), were tested for capacity to support DC hematopoiesis in co-cultures seeded with bone marrow (BM) as a source of progenitors. In particular, the 5G3 stroma, a good supporter of DC hematopoiesis was analysed using Affymetrix microarrays for genes specifically expressed or upregulated in 5G3 over the non-supporting stroma 3B5. This resulted in identification of many genes of interest. These included genes involved in hematopoiesis, like Clcal, Sfrp2, Rspo2, Abcgl, ApoD, Aldhlal and Serpinasn, and novel genes such as Ms4a4d, Svep1, Plxdc2 and Ctla2a. The expression of these genes by 5G3 stroma was further confirmed by Realtime PCR. Cells produced in 5G3 co-cultures seeded with lineage negative (Lin') BM were characterized in terms of phenotype and function. Two populations of dendritic-like cells were characterized as CDllc.cnub+MHC-Ir L-DC and CD11c+CD11b+MHC-11+ cells resembling conventional (c)DC. The L-DC subset was shown to be phenotypically and functionally similar to LTC-DC, in terms of high endocytic capacity, ability to crosspresent antigen to CDS+ T cells and inability to activate CD4+ T cells. These features identify L-DC as distinct in comparison with other DC subsets described in spleen. The cDC-like cells showed ability to crosspresent antigen to CD8+ T cells, and were weakly endocytic. In addition, they could activate CD4+ T cells, but did not induce proliferation of CD4+ T cells, suggesting a role in tolerance or suppression. Investigation of cytokine gene 0. expression by Realtime PCR revealed many differences between the two subsets. A 10-fold increase in ILlO expression in cDC-like cells over L-DC, was also consistent with ability of cDC-like cells to be immunosuppressive. In an attempt to elucidate the stroma-dependent progenitor responsible for development of L-DC, HSC with both long-term (LT) and short-term (ST) reconstituting potential, were sorted and co-cultured over 5G3 stroma. LT-HSC required stromal contact to produce progeny CD11c+CD11b+MHC-II L-DC. In contrast, ST-HSC produced mature, myeloid CD11c"CD11b+MHC-11+ cDC-like cells in a contact-independent manner. These results identified the L-DC progenitor amongst HSC subsets, and also distinguished the lineage origin of L-DC from that of cDC-like cells. In spleen, the L-DC progenitor was identified within the LinClrl lc' c_kit10 subset which also contains HSC. These results delineate a distinct DC type in terms of phenotype and function. It can be derived by in vitro differentiation from HSC in the context of a splenic stromal niche that has a unique ability to support hematopoiesis.