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CD47 on live cells actively engages signal-regulatory protein-α (SIRP-α) on phagocytes and delivers a negative signal that prevents their elimination. We evaluated the biological consequences of SIRP-α ligation on the dendritic cell (DC) response to maturation signals and the potential interplay with the IL-10/IL-10R inhibitory pathway. At first, CD47/SIRP-α allowed the generation of mature migratory DCs not producing IL-12, IFN-γ-inducible protein-10, and CCL19. Rather, they secreted neutrophils attracting chemokine CXCL5 and IL-1β, reflecting a partial block in functional DC maturation. Afterward, semimature DCs functionally regressed in an IL-10-independent fashion toward cells that retrieved the cardinal features of immature DCs: re-expression of CCR5, loss of DC-lysosome-associated membrane protein, high endocytosis, and impaired allostimulatory functions. The global gene expression profile of IL-10 and SIRP-α-ligated DC demonstrated two distinct molecular pathways. IL-10R and SIRP-α expression were reciprocally down-regulated by CD47 and IL-10, respectively. These results emphasize that the SIRP-α pathway might be part of the molecular machinery used by the DC to dampen or resolve an inflammatory response in an IL-10-independent manner.

Receptor activator of NF-kappaB (RANK) is a recently identified member of the tumor necrosis factor receptor superfamily and is expressed on activated T cells and dendritic cells. Its cognate ligand (RANKL) plays significant roles in the activation of dendritic cell function and osteoclast differentiation. We demonstrate here the interaction of RANK with tumor necrosis factor receptor-associated factors (TRAFs) 1, 2, 3, 5, and 6 both in vitro and in cells. Mapping of the structural requirements for TRAF/RANK interaction revealed multiple TRAF binding sites clustered in two distinct domains in the RANK cytoplasmic tail. These TRAF binding domains were shown to be functionally important for the RANK-dependent induction of NF-kappaB and c-Jun NH2-terminal kinase activities. Site-directed mutagenesis demonstrated that these TRAF binding sites exhibited selective binding for different TRAF proteins. In particular, TRAF6 interacted with membrane-proximal determinants distinct from those binding TRAFs 1, 2, 3, and 5. When this membrane-proximal TRAF6 interaction domain was deleted, RANK-mediated NF-kappaB signaling was completely inhibited while c-Jun NH2-terminal kinase activation was partially inhibited. An NH2-terminal truncation mutant of TRAF6 inhibited RANKL-mediated NF-kappaB activation, but failed to affect constitutive signaling induced by receptor overexpression, revealing a selective role for TRAF6 in ligand-induced activation events.

Dendritic cells are rare haematopoietic cells that reside in a number of organs and tissues. By capturing, processing and presenting antigens to T cells, dendritic cells are essential for immune surveillance and the regulation of specific immunity. Several members of the tumour necrosis factor receptor (TNFR) superfamily are integral to the regulation of the immune response. These structurally related proteins modulate cellular functions ranging from proliferation and differentiation to inflammation and cell survival or deaths. The functional activity of dendritic cells is greatly increased by signalling through the TNFR family member CD40. Here we report the characterization of RANK (for receptor activator of NF-kappaB), a new member of the TNFR family derived from dendritic cells, and the isolation of a RANK ligand (RANKL) by direct expression screening. RANKL augments the ability of dendritic cells to stimulate naive T-cell proliferation in a mixed lymphocyte reaction, and increases the survival of RANK+ T cells generated with interleukin-4 and transforming growth factor (TGF)-beta. Thus RANK and RANKL seem to be important regulators of interactions between T cells and dendritic cells.

Semaphorins are secreted or transmembrane proteins that provide essential repulsive guidance cues to growing axons or endothelial cells through their receptors of the Plexin and Neuropilin family. Semaphorins and Plexins are also expressed in the immune system where their function remains elusive. In particular, Plexin C1 is expressed by mouse dendritic cells (DCs) and is the receptor for the poxvirus semaphorin homolog A39R. We previously found that Plexin C1 engagement by A39R inhibits integrin-mediated DC adhesion and chemokine-induced migration. Here, we show that a cellular ligand for Plexin C1 is expressed both by activated T cells and DCs, suggesting that Plexin C1 might be engaged on DCs both in cis and in trans. We used Plexin C1(-/-) mice to explore the role of Plexin C1 in DC function. DC development is unaffected in these mice. In two different in vivo assays, Plexin C1(-/-) DC migration to lymph nodes (LNs) was lower than that of wild-type (WT) DC but this difference was not statistically significant. Plexin C1(-/-) bone marrow-derived DCs induced normal in vitro T cell responses but reduced in vivo T cell responses when injected subcutaneously to WT mice. Finally, in vivo T cell responses to ovalbumin peptide and contact hypersensitivity to dinitrofluorobenzene were slightly decreased in Plexin C1(-/-) mice. These results suggest a role for Plexin C1 in DC migration or mobility within the LNs.

Dendritic cells (DCs) are a phenotypically and functionally heterogenous population of leukocytes with distinct subsets serving a different set of specialized immune functions. Here we applied an in vitro whole cell panning approach using antibody phage display technology to identify cell-surface epitopes specifically expressed on human blood BDCA3(+) DCs. A single-chain antibody fragment (anti-1F12 scFv) was isolated that recognizes a conserved surface antigen expressed on both human BDCA3(+) DCs and mouse CD8alpha(+) DCs. We demonstrate that anti-1F12 scFv binds Nectin-like protein 2 (Necl2, Tslc1, SynCaM, SgIGSF, or Igsf4), an adhesion molecule involved in tumor suppression, synapse formation, and spermatogenesis. Thus, Necl2 defines a specialized subset of DCs in both mouse and human. We further show that Necl2 binds Class-I-restricted T-cell-associated molecule (CRTAM), a receptor primarily expressed on activated cytotoxic lymphocytes. When present on antigen presenting cells, Necl2 regulates IL-22 expression by activated CD8(+) T-cells. We propose that Necl2/CRTAM molecular pair could regulate a large panel of cell/cell interactions both within and outside of the immune system.

The poxvirus A39R protein is a member of the semaphorin family that binds to Plexin C1, a molecule expressed on neutrophils and dendritic cells (DC). We previously showed that binding of A39R to Plexin C1 induces local rearrangement of the actin cytoskeleton and inhibits integrin-mediated adhesion, leading to cell retraction. As phagocytosis is dependent on both cytoskeleton integrity and integrin function, we tested the effect of A39R on DC and neutrophil phagocytosis. We found that A39R treatment strongly inhibits phagocytosis by DC and neutrophils in vitro in a Plexin C1-dependent fashion. Moreover, A39R treatment inhibited the capacity of CD8alpha+ DC to take up apoptotic bodies in vivo. As a consequence, A39R impaired the ability of CD8alpha+ DC to cross-prime CD8+ T cells ex vivo. In contrast, A39R had no effect on direct priming of CD8+ T cells by peptide-pulsed CD8alpha+ DC in vitro. These results suggest that poxviruses may use semaphorin homologs as a means to evade the immune system.

A phase I/II clinical trial was carried out to determine the safety of Flt3 ligand used as a vaccine adjuvant when administered to healthy human volunteers on two different schedules. In the first phase of this study, Flt3 ligand was administered SQ at a dose of 20 μg/kg (to a maximum of 1500 μg) every day ( N =10) or every other day ( N =10) for 1 week. The Flt3 ligand injection series was followed 1 day later by the first of three vaccinations with the licensed hepatitis B vaccine. In the second phase of the trial, 30 volunteers received either Flt3 ligand or placebo on the alternate day schedule in a randomized, double-blind design. The Flt3 ligand injections were safe and very well-tolerated. The number of lineage negative, HLA-DR hi , CD11c + , CD123 − dendritic cells (DCs) increased 23-fold, and the lineage negative, HLA-DR hi , CD11c − , CD 123bright pre-DCs increased 6-fold. There was an associated increase in monocytes and WBCs in the Flt3 ligand recipients. Despite the marked increase in peripheral circulating dendritic cells, no increase was observed in the hepatitis B antibody titers induced after vaccination.

In 1999, we reported seven cases of an unusual hematologic malignancy with primary cutaneous presentation that appeared as a distinct clinicopathologic entity characterized by medium-sized tumor cells with a peculiar CD3- CD4+ CD56+ CD43+ HLA-DR+ cell surface phenotype. Because the origin of tumor cells was not clear and they exhibited a nonlineage-specific phenotype, we hypothesized that such tumors likely originated from hematologic-myeloid precursor cells and were tentatively assigned the designation "agranular CD4+ CD56+ hematodermic neoplasms." In the present study we report 14 cases (seven already reported and seven additional cases) of these tumors, and simultaneously we present now a rare population of cells that we have identified in the peripheral blood of healthy volunteers treated with Flt3 ligand. These cells express all the characteristic markers of CD4+ CD56+ hematodermic neoplasms. This population appears to be related to plasmacytoid monocytes because they also expressed CD68 and bright levels of CD123. To confirm the relationship between these normal cells and CD4+ CD56+ hematodermic neoplasms, we conducted an extensive comparative phenotypic study. Results show that these two cell types are indeed related because they share many phenotypic features, including the presence of CD4, CD56, CD43, CD68, and HLA-DR and the absence of other T, B, NK, or myelomonocytic markers. More importantly, we found that the bright expression of CD123 by immunohistochemistry is a distinctive characteristic of CD4+ CD56+ hematodermic neoplasms because all (n = 14) cases expressed this marker, whereas only two specimens in a control panel comprising 30 samples of related tumors expressed comparable levels of CD123. We therefore propose that oncogenic transformation of NCAM-expressing plasmacytoid monocyte-like cells may lead to "agranular CD4+ CD56+ hematodermic neoplasm."

CD123bright plasmacytoid cells (PC) and CD1c+ peripheral blood myeloid dendritic cells (DC) are two human DC precursors that can be expanded in vivo by Fms-like tyrosine kinase 3 ligand (FL). It has been proposed that PC and myeloid CD1c+ DC may represent two distinct lineages of DC. However, the phylogenetic affiliation of PC and its relationship with myeloid DC remain controversial. Here we show that CD123brightHLA-DR+ PC from FL-treated healthy volunteers can be divided into mutually exclusive subsets that harbor either lymphoid or myeloid features. Lymphoid-like PC represent the majority of PC and include pTα-, CD3ε-, and CD7-expressing cells. They exhibit TCR-β gene loci in germline configuration and show low allostimulatory capacity, but produce type I IFN upon virus infection and can be differentiated in vitro into potent APC. Myeloid-like PC represent a minor fraction of the total PC population. They exhibit a striking PC/myeloid DC intermediate phenotype (CD5+CD11clowCD45RAlowCD45RO−CD101+), produce proinflammatory cytokines, and do not require in vitro maturation to act as potent APCs. We propose that, rather than forming a lineage, PC might represent a population of lymphoid cells undergoing an in vivo cell fate conversion from a lymphoid to a myeloid cell type.

The poxvirus A39R protein is a member of the semaphorin family previously reported to bind plexin C1. We show that, in the mouse, plexin C1 is expressed on dendritic cells (DCs) and neutrophils and is the only receptor for A39R on these cells. The biological effects of a recombinant form of A39R were examined in vitro on mouse DCs derived from wild-type or plexin C1−/− mice. A39R binding to plexin C1 on DCs inhibited integrin-mediated adhesion and spreading in vitro. This phenomenon was accompanied by a decrease in integrin signaling, measured by focal adhesion kinase phosphorylation, and a rearrangement of the actin cytoskeleton, without inducing DC maturation or affecting their viability. The A39R effect on DC adhesion was blocked by a specific inhibitor of cofilin phosphorylation, suggesting that the regulation of F-actin turnover by plexin C1 was essential to induce cellular retraction. Furthermore, A39R binding to plexin C1 inhibited chemokine-induced migration of DCs in vitro, suggesting that plexins and semaphorins could be involved in the regulation of leukocyte movement.