Extramedullary hematopoiesis leading to the production of a novel antigen-presenting cell type in murine spleen.

The concept of extramedullary hematopoiesis for production of organ-specific antigen presenting cells has importance in immunity in terms of the compartmentalisation of the immune response in different tissue sites. A new and distinct dendritic-like antigen presenting cell subtype is described which is dependent on the spleen microenvironment for development. Cells arise by a unique developmental pathway distinct from other dendritic cells (DC). In particular, a self-renewing progenitor of these cells has been identified in spleen upstream of the earliest DC progenitor currently identified in bone marrow. This progenitor depends on the splenic microenvironment for maintenance and proliferation, adding further support for spleen as a site for hematopoiesis.

Spleen stroma maintains progenitors and supports long-term hematopoiesis.

Hematopoietic stem/progenitor cells (HSPC) differentiate in the context of stromal niches producing cells of multiple lineages. Limited success has been achieved in the past with induction of hematopoiesis in vitro. Previously, spleen long-term stromal cultures (LTC) were shown to continuously support restricted hematopoiesis for production of novel dendritic-like cells (LTC-DC). An in vivo equivalent dendritic cell type was then described which is specific for spleen. The in vivo counterpart cell was termed 'L-DC' and represents a dendritic-like CD11c(lo)CD11b(hi)CD8α-MHC-II- cell which differs phenotypically and functionally from monocytes/macrophages and conventional and plasmacytoid DC. Splenic stroma is now shown to maintain HSPC and to support their restricted in vitro differentiation to give this 'L-DC' subset. In order to characterise progenitors of this distinct cell type, LTC were analysed for cell subsets produced, and these subsets sorted and assessed for hematopoietic potential in subsequent co-cultures over STX3 stroma. Progenitors were defined as a lineage (Lin)(-)ckit(lo) subset reflecting HSPC. Furthermore, when Lin(-)ckit(hi)Sca1(+)Flt3(-) HSPC were sorted from bone marrow, they colonised splenic stroma with long-term production of L-DC. The maintenance of HSPC by splenic stroma was confirmed when non-adherent cells collected from LTC showed oligopotent reconstitution of the hematopoietic compartment of lethally irradiated mice. All data support a model whereby spleen houses a niche for HSPC in the resting state, with production of progenitors, and their differentiation to give tissue-specific antigen presenting cells.

Technical advance: in vitro production of distinct dendritic-like antigen-presenting cells from self-renewing hematopoietic stem cells.

A novel CD11c(lo)CD11b(hi)MHC-II⁻CD8α⁻ dendritic-like cell (L-DC) develops in cocultures of bone marrow over splenic stroma. L-DCs are distinct from other DC subsets and have potential importance in spleen for immunity to blood-borne antigens. As production is maintained in cultures for >12 months, L-DC development evidently depends on self-renewing progenitors. To improve this culture system, highly purified HSCs were sorted from bone marrow and used to establish cocultures. Nonadherent cells produced were analyzed for surface marker expression and capacity to activate/inhibit T cells. Cocultures produced a pure population of L-DCs for up to 12 months, which were strong activators of CD8⁺ T cells. The in vitro production of a pure population of L-DCs from HSCs--in numbers amenable to in vitro assays of function and development--therefore represents an important advance.

Spleen as a site for hematopoiesis of a distinct antigen presenting cell type.

While spleen and other secondary tissue sites contribute to hematopoiesis, the nature of cells produced and the environment under which this happens are not fully defined. Evidence is reviewed here for hematopoiesis occurring in the spleen microenvironment leading to the production of tissue-specific antigen presenting cells. The novel dendritic-like cell identified in spleen is phenotypically and functionally distinct from other described antigen presenting cells. In order to identify these cells as distinct, it has been necessary to show that their lineage origin and progenitors differ from that of other known dendritic and myeloid cell types. The spleen therefore represents a distinct microenvironment for hematopoiesis of a novel myeloid cell arising from self-renewing hematopoietic stem cells (HSC) or progenitors endogenous to spleen.