Their eponymous morphology and unique ability to activate naive T cells are hallmark features of dendritic cells (DCs). Specific properties of the actin cytoskeleton may define both characteristics. In search for regulators that coordinate DC phenotype and function, we observed strongly increased expression of the actin-remodeling GTPases Cdc42 and Rac1 during DC development from human stem cells. Cdc42 and Rac1 are constitutively active in immature DCs, and their activity is further up-regulated by maturational stimuli such as LPS or CD40L. Activation of Rac1 is associated with its rapid recruitment into lipid rafts. Cdc42 is not recruited into rafts, but readily activated by raft-associated moieties. The functional interplay of rafts, GTPases, and cortical actin is further shown by GTPase activation and actin remodeling after pharmacological disruption of lipid rafts and by the loss of the actin-based DC morphology by transfection of dominant-negative Cdc42 and Rac1. Both Cdc42 and Rac1 also control the transport of essential immunostimulatory molecules to the DC surface. Transfection with dominant-negative GTPases led to reduced surface expression of MHC class I and CD86. Consecutively, DCs display a reduced stimulatory capacity for CD8+ T cells, whereas MHC class II-dependent stimulation of CD4+ T cells remains unperturbed. We conclude that Cdc42 and Rac1 signaling controls DC morphology and conditions DCs for efficient CD8+ T cell stimulation.
Langerhans cells (LC) are CD1a+E-cadherin (E-cad)+Birbeck granule+ but CD11b−CD36−factor XIIIa (FXIIIa)− members of the dendritic cell (DC) family. Evidence holds that LC originate from CD1a+CD14− rather than CD14+CD1a− progenitors, both of which arise from GM-CSF/TNF-α-stimulated CD34+ stem cells. The CD14+CD1a− progenitors, on the other hand, can give rise to a separate DC type characterized by its CD1a+CD11b+CD36+FXIIIa+E-cad−BG− phenotype (non-LC DC). Although GM-CSF/TNF-α are important for both LC and non-LC DC differentiation, TGF-β1 is thought to preferentially promote LC development in vitro and in vivo. However, the hemopoietic biology of this process and the nature of TGF-β1-responsive LC precursors (LCp) are not well understood. Here we show that CD14+ precursors in the presence, but not in the absence, of TGF-β1 give rise to a progeny that fulfills all major criteria of LC. In contrast, LC development from CD1a+ progenitors was TGF-β1 independent. Further studies revealed that CD14+ precursors contain a CD11b+ and a CD11b− subpopulation. When either subset was stimulated with GM-CSF/TNF-α and TGF-β1, only CD14+CD11b− cells differentiated into LC. The CD11b+ cells, on the other hand, acquired non-LC DC features only. The higher doubling rates of cells entering the CD14+ LCp rather than the CD1a+ LCp pathway add to the importance of TGF-β1 for LC development. Because CD14+CD11b− precursors are multipotent cells that can enter LC or macrophage differentiation, it is suggested that these cells, if present at the tissue level, endow a given organ with the property to generate diverse cell types in response to the local cytokine milieu.
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