We describe a new B220 ؉ subpopulation of immaturelike dendritic cells (B220 ؉ DCs) with low levels of expression of major histocompatibility complex (MHC) and costimulatory molecules and markedly reduced T-cell stimulatory potential, located in the thymus, bone marrow, spleen, and lymph nodes. B220 ؉ DCs display ultrastructural characteristics resembling those of human plasmacytoid cells and accordingly produce interferon-␣ after virus stimulation. B220 ؉ DCs acquired a strong antigen-presenting cell capacity on incubation with CpG oligodeoxynucleotides, concomitant with a remarkable up-regulation of MHC and costimulatory molecules and the production of interleukin-12 (IL-12) and IL-10. Importantly, our data suggest that nonstimulated B220 ؉ DCs represent a subset of physiological tolerogenic DCs endowed with the capacity to induce a nonanergic state of T-cell unresponsiveness, involving the differentiation of T regulatory cells capable of suppressing antigen-specific T-cell proliferation. In conclusion, our data support the hypothesis that B220 ؉ DCs represent a lymphoid organ subset of immature DCs with a dual role in the immune system-exerting a tolerogenic function in steady state but differentiating on microbial stimulation into potent antigen-presenting cells with type 1 interferon production capacity. IntroductionMaintenance of immunologic self-tolerance is an essential process directed at preventing harmful autoimmune diseases caused by autoreactive T cells capable of responding to self-antigens. Avoidance of pathologic reactivity of self-reactive T cells may occur as a consequence of T-cell deletion, T-cell unresponsiveness, or, in some instances, T helper cell type 2 (TH2) skewing (reviewed in Hackstein et al 1 ). Deletion of autoreactive T-cell clones, resulting in T-cell-negative selection, takes place essentially in the thymus under the control of thymic dendritic cells (DCs) and epithelial cells (reviewed in Ardavín 2 ). In contrast, the molecular mechanisms controlling T-cell unresponsiveness or anergy, which is the basis of peripheral tolerance, are not fully understood. However, increasing evidence supports that T regulatory (T reg ) cells play an essential role in the control of autoreactive T-cell clones and, therefore, in the maintenance of T-cell peripheral tolerance because of their capacity to suppress antigen-specific T-cell responses (reviewed in Roncarolo and Levings 3 ). Interestingly immature DCs have been demonstrated to participate in the differentiation of T reg cells (reviewed in Jonuleit et al 4 ). In this sense, human and mouse interleukin-10 (IL-10)-treated immature DCs have been reported to induce antigen-specific T-cell anergy. [5][6][7][8][9] In addition, in vitrogenerated human immature DCs have been demonstrated to induce the differentiation of T reg cells in vitro and in vivo. 9,10 Therefore, on the basis of these data, the tolerogenic potential of DCs has been proposed to be correlated with an immature DC state. 1 On the other hand, DC-mediated induction of murine T-ce...

Dendritic cells (DCs) are essential for the establishment of immune responses against pathogens and tumour cells, and thus have great potential as tools for vaccination and cancer immunotherapy trials. Experimental evidence has led to a dual DC differentiation model, which involves the existence of both myeloid- and lymphoid-derived DCs. But this concept has been challenged by recent reports demonstrating that both CD8- and CD8+ DCs, considered in mice as archetypes of myeloid and lymphoid DCs respectively, can be generated from either lymphoid or myeloid progenitors. The issue of DC physiological derivation therefore remains an open question. Here we report the characterization of a DC-committed precursor population, which has the capacity to generate all the DC subpopulations present in mouse lymphoid organs---including CD8- and CD8+ DCs, as well as the B220+ DC subset---but which is devoid of myeloid or lymphoid differentiation potential. These data support an alternative model of DC development, in which there is an independent, common DC differentiation pathway.

The monocyte capacity to differentiate into dendritic cells (DCs) was originally demonstrated by human in vitro DC differentiation assays that have subsequently become the essential methodologic approach for the production of DCs to be used in DC-mediated cancer immunotherapy protocols. In addition, in vitro DC generation from monocytes is a powerful tool to study DC differentiation and maturation. However, whether DC differentiation from monocytes occurs in vivo remains controversial, and the physiologic counterparts of in vitro monocyte-derived DCs are unknown. In addition, information on murine monocytes and monocyte-derived DCs is scarce. Here we show that mouse bone marrow monocytes can be differentiated in vitro into DCs using similar conditions as those defined in humans, including in vitro cultures with granulocyte-macrophage colony-stimulating factor and interleukin 4 and reverse transendothelial migration assays. Importantly, we demonstrate that after in vivo transfer monocytes generate CD8 ؊ and CD8 ؉ DCs in the spleen, but differentiate into macrophages on migration to the thoracic cavity. In conclusion, we support the hypothesis that monocytes generate DCs not only on entry into the lymph and migration to the lymph nodes as proposed, but also on extravasation from blood and homing to the spleen, suggesting that monocytes represent immediate precursors of lymphoid organ DCs.

IntroductionDendritic cells (DCs) play a central role in the immune system due to their main function as initiators and regulators of antigenspecific antiviral T-cell responses and in the pathogenesis of a variety of viruses, such as human immunodeficiency virus (HIV), cytomegalovirus, measles virus, herpes virus, influenza virus, and respiratory syncytial virus. 1 However, little is known about the subpopulations of DCs involved in antiviral responses, the kinetics of the variations of DC subpopulations, and, importantly, the mechanisms of recruitment of DCs to the lymph nodes (LNs) during inflammatory responses driven by viral infections. On the other hand, despite the available information dealing with the phenotype and function of the 2 main mouse DC subsets, namely, CD8 Ϫ and CD8 ϩ DCs, their involvement in antiviral immune responses in vivo as well as their functional relationships and origin remain largely unknown.Over the last years CD8 Ϫ and CD8 ϩ DCs have been ascribed to the myeloid and lymphoid lineages, respectively, and consequently considered as independent DC categories. 1 However, more recently 2 reports from our group and Dr Weissman's laboratory (Stanford University) have described the generation of both CD8 Ϫ and CD8 ϩ DCs from a common lymphoid precursor 2 or from either myeloidor lymphoid-committed precursors. 3 These results suggest that in fact CD8 Ϫ and CD8 ϩ DCs might not represent independent cell types, and in this sense we have previously reported that migrationor CD40L ligation-induced Langerhans cell (LC) maturation involved CD8 up-regulation. 4,5 In these reports, which have been confirmed by a recent article by Merad et al, 6 we showed that the LN-restricted CD8 int DC population derived from LCs. 4,5 On the basis of these previous data on LN DCs and using the dramatic changes occurring in the draining popliteal LNs (PO-LNs) after infection by the Swiss (SW) strain of the mouse mammary tumor virus (MMTV), 7 we have extensively analyzed the mechanisms of DC recruitment during the inflammatory responses induced by a viral infection as well as the relationships between the different DC subsets involved. The results presented here indicate that MMTV(SW) induces a strong migration of blood-borne CD8 Ϫ DCs to the PO-LNs via high endothelial venules (HEVs), providing direct evidence of DC recruitment to the LNs during an in vivo viral infection-driven inflammatory process. Moreover, our data strongly suggest that LN CD8 ϩ DCs originate as the result of CD8 up-regulation by LN CD8 Ϫ DCs, supporting the view that these subsets represent different physiologic states of the same DC population. Finally, the fact that DCs become infected by MMTV(SW) suggest their participation in the immune response against this virus. Materials and methods Experimental infection with MMTV(SW)The MMTV(SW) was purified from milk and titered as described previously. 8 Eight-to 10-week-old BALB/c mice were given a 10-L injection Supported by the European Commission (grant no. QLRT-1999-00276) For personal use on...

1. Shih, I. H. & Been, M. D. Catalytic strategies of the hepatitis delta virus ribozymes. Annu. Rev. Biochem. 71, 887-917 (2002). 2. Rosenstein, S. P. & Been, M. D. Self-cleavage of hepatitis delta virus genomic strand RNA is enhanced under partially denaturing conditions. Biochemistry 29, 8011-8016 (1990). 3. Reid, C. E. & Lazinski, D. W. A host-specific function is required for ligation of a wide variety of ribozyme-processed RNAs.