Characterisation of Dendritic Cells Arising from Progenitors Endogenous to Murine Spleen

Abstract

Heterogeneity amongst dendritic cell (DC) subsets leads to a spectrum of immune response capacity against pathogens. Several DC subsets in spleen have been described which differ in terms of phenotype and function. We have previously reported a distinct population of CD11cloCD11bhiMHC-II−CD8− dendritic-like “L-DC” in murine spleen, which can also be generated in splenic stromal longterm cultures. Here, the ontogeny of L-DC development in perinatal mice has been compared with other known splenic DC subsets. Flow cytometric analysis has revealed the presence of L-DC at embryonic age (E)18.5 spleen, while plasmacytoid (p)DC and conventional (c)DC appear at 2 and 4 days following birth. Co-cultures of E18.5 spleen above splenic stroma also showed production of only L-DC, while spleen cells from D0 through D5 neonates showed production of both L-DC and cDC-like cells. Addition of an M-CSFR inhibitor to co-cultures revealed that while the development of cDC-like cells depended on M-CSF, many L-DC developed independently of M-CSF. Furthermore, purified hematopoietic stem cells (HSC) and multipotential progenitors (MPP) isolated from neonatal D1 spleen are capable of developing into L-DC in co-cultures. These studies reveal a lineage of dendritic-like cells developing in the spleen microenvironment, and which appear to arise from endogenous progenitors laid down in spleen during embryogenesis.

Redefining Myeloid Cell Subsets in Murine Spleen

“…Antibody staining followed by flow cytometry was performed to analyze cell surface marker expression as described previously ( 32 ). Non-specific antibody binding via Fc receptors was blocked by incubating cells (≤ 10 6 ) with anti-CD16/32 (FcBlock) (Biolegend: San Diego, CA, USA) at 5 μg/mL.…”

“…Novel dendritic-like cells termed L-DC, were recently discovered in murine and human spleen on the basis of their resemblance with dendritic-like cells produced in vitro in splenic long-term cultures (LTC-DC) ( 28 – 30 ), or in co-cultures of bone marrow precursors over selected splenic stroma ( 31 , 32 ). L-DC have a distinct CD11b hi CD11c lo MHCII − CD8 − phenotype and dendritic-like appearance ( 33 ).…”

Spleen as a Site for Hematopoiesis

“…Indeed, it has become clear that L-DC progenitors in vivo reflect HSC endogenous to spleen, and that the process of L-DC development reflects extramedullary hematopoiesis. L-DC progenitors first appear in murine spleen at E18.5, while progenitors of cDC appear after 4 days (Petvises and O'Neill, 2014a). This raises the possibility that hematopoietic progenitors in spleen are laid down during ontogeny, and that hematopoiesis in spleen giving rise to differentiation of dendritic-like L-DC occurs as an active process, independent of inflammatory signalling.…”

Delineation of a novel dendritic-like subset in human spleen

“…Previous studies have demonstrated the ability of the murine 5G3 splenic stroma to support myelopoiesis from hematopoietic stem/progenitor cells present in both murine bone marrow and spleen. 12,14,15,30,31 Cell growth was monitored, and non-adherent cells were collected and flow cytometrically assessed at 7-day intervals. Live cells were gated, and HLA-DR versus hCD11c staining was used to delineate any dendritic-like cells produced in the cocultures.…”