DCs (dendritic cells) function as sentinels of the immune system. They traffic from the blood to the tissues where, while immature, they capture antigens. They then leave the tissues and move to the draining lymphoid organs where, converted into mature DC, they prime naive T cells. This suggestive link between DC traffic pattern and functions led us to investigate the chemokine responsiveness of DCs during their development and maturation. DCs were differentiated either from CD34+ hematopoietic progenitor cells (HPCs) cultured with granulocyte/macrophage colony–stimulating factor (GM-CSF) plus tumor necrosis factor (TNF)-α or from monocytes cultured with GM-CSF plus interleukin 4. Immature DCs derived from CD34+ HPCs migrate most vigorously in response to macrophage inflammatory protein (MIP)-3α, but also to MIP-1α and RANTES (regulated on activation, normal T cell expressed and secreted). Upon maturation, induced by either TNF-α, lipopolysaccharide, or CD40L, DCs lose their response to these three chemokines when they acquire a sustained responsiveness to a single other chemokine, MIP-3β. CC chemokine receptor (CCR)6 and CCR7 are the only known receptors for MIP-3α and MIP-3β, respectively. The observation that CCR6 mRNA expression decreases progressively as DCs mature, whereas CCR7 mRNA expression is sharply upregulated, provides a likely explanation for the changes in chemokine responsiveness. Similarly, MIP-3β responsiveness and CCR7 expression are induced upon maturation of monocyte- derived DCs. Furthermore, the chemotactic response to MIP-3β is also acquired by CD11c+ DCs isolated from blood after spontaneous maturation. Finally, detection by in situ hybridization of MIP-3α mRNA only within inflamed epithelial crypts of tonsils, and of MIP-3β mRNA specifically in T cell–rich areas, suggests a role for MIP-3α/CCR6 in recruitment of immature DCs at site of injury and for MIP-3β/CCR7 in accumulation of antigen-loaded mature DCs in T cell–rich areas.

Sllmm~'yDendritic cells, the professional antigen-presenting cells (APC) involved in T cell priming, express CD40, a molecule which triggering plays a key role in B cell growth and differentiation as well as monocyte activation. Herein we demonstrate that dendritic Langerhans cells (D-I.c) generated by culturing cord blood CD34 + progenitor cens with granulocyte/macrophage colony-stimulating and tumor necrosis factor oe (TNF-c~) express functional CD40 at a density higher than that found on B cells. Culturing D-IX on CD40-ligand (CD40L) transfected L cells allowed D-IX survival as 50 • 15% of seeded cells were recovered after 4 d while only 5% survived over control L cells. CD40 activation induced important morphological changes with a reduction of cytoplasmic content and a remarkable increase of dendrite development as well as an altered phenotype. In particular, CD40 triggering induced maintenance of high levds of major histocompatibility complex class II antigens and upregulation of accessory molecules such as CD58, CD80 (B7-1) and CD86 (B7-2). CD40 engagement also seems to turn on D-IX maturation as illustrated by upregulation of CD25, a molecule usually expressed on interdigitating dendritic cells of secondary lymphoid organs. Finally, CD40 activated D-IX secreted a limited set of cytokines (TNF-c~, IL-8, and macrophage inflammatory protein 1,', [MIP-loc]) whereas a similar activation induced dutriated monocytes to secrete IL-lot, IL-1/5, IL-6, IL-8, IL-10, TNF-~x, and MIP-lo~. As D-Ix activated T cells upregulated CD40L, it is likely that CD40 activation olD-ix observed herein with a fibroblast cell line stably expressing CD40L, mimics physiological interactiom between dendritic cells and T cells.T he CD40 antigen (1, for review) was identified by monodonal antibodies reacting with carcinomas and B cells (2) and showing costimulatory effects for B lymphocytes (3). It is a 50-kD glycoprotein which bdongs to the TNF receptor superfamily (4). Cross-linking of CD40, in conjunction with IL-4, was found to induce B cells to undergo long-term growth, as well as isotype switching, whereas addition of IL-10 results in B cell differentiation as well as isotype switch (5-8). The use of a CD40-~ fusion protein allowed the isolation of a cDNA encoding for a CD40 ligand (CD40L) 1, a new member of the TNF superfamily mainly expressed on activated T cells (9). Interaction between CD40 and CD40L has now been shown in vitro to be essential during T cell-de. pendent B cell activation (10,11). In vivo studies in mice have demonstrated that an antibody to CD40L can inhibit primary and secondary antibody production and establish- Functional CD40 molecules were found to be expressed on cells other than mature B cells. In particular, upon CD40 cross-llnklng, human progenitor B lymphocytes express CD23 and proliferate in response to . Thymic epithelial cells secrete GM-CSF in response to CD40 engagement (19). Finally, monocytes express high leveh of CD40 after eq, osure to IFN-% IL-3, and GM-CSF and CD40 cross-llnklng induces cyto...

SummaryHuman dendritic cells (DC) can now be generated in vitro in large numbers by culturing CD34 + hematopoietic progenitors in presence of GM-CSF+TNFet for 12 d. The present study demonstrates that cord blood CD34 + HPC indeed differentiate along two independent DC pathways. At early time points (day 5-7) during the culture, two subsets of DC precursors identified by the exclusive expression of CDla and CD14 emerge independently. Both precursor subsets mature at day 12-14 into DC with typical morphology and phenotype (CDS0, CD83, CD86, CD58, high HLA class II). CDla + precursors give rise to cells characterized by the expression of Birbeck granules, the Lag antigen and E-cadherin, three markers specifically expressed on Langerhans cells in the epidermis. In contrast, the CD14 + progenitors mature into CDla + DC lacking Birbeck granules, E-cadherin, and Lag antigen but expressing CD2, CD9, CD68, and the coagulation factor XIlla described in dermal dendritic cells. The two mature DC were equally potent in stimulating allogeneic CD45RA + naive T cells. Interestingly, the CD14 + precursors, but not the CDla + precursors, represent bipotent cells that can be induced to differentiate, in response to M-CSF, into macrophage-like cells, lacking accessory function for T ceils.Altogether, these results demonstrate that different pathways of DC development exist: the Langerhans cells and the CD14+-derived DC related to dermal DC or circulating blood DC. The physiological relevance of these two pathways of DC development is discussed with regard to their potential in vivo counterparts.

SummaryIn the present report, we have investigated the in vitro differentiation of surface(s) slgD + and slgD-human B cells into Ig-secreting calls in response to various stimuli, slgD + B cells homogeneously expressed some of the antigens identifying mantle zone B cells, but lacked expression of germinal center markers, thus confirming that the B cell populations positively selected on the basis of slgD expression were highly enriched for naive B lymphocytes. Conversely, slgD-B cells expressed some of the antigens spedfically associated with germinal center B cells. T cell-independent differentiation of slgD + and sIgD-B cells could be achieved by simultaneous crosslinking of sIgs and CD40 in the presence of a mouse Ltk-cell line stably expressing human CDw32/Fc'yPdI (CDw32 L cells). In this experimental system, sIgD + B cells were exclusively proned for IgM synthesis, whereas sIgD-B ceils produced IgG, IgM, and IgA. Both the human and viral forms of interleukin 10 (ILd0) strongly increased the Ig secretion by sIgD + and slgD-B cells simultaneously activated through slgs and CD40. IgM and IgG constituted the predominant Ig isotype produced by slgD + and slgD-B cells, respectively, in response to IL-10. slgD + B cells could be induced for IgA synthesis upon co-culturing with transforming growth factor (TGF-~) and II.-10, in the presence of an anti-CD40 monodonal antibody presented by the CDw32 L cells. In contrast, TGF-~ suppressed the IL-10-mediated IgG, IgM, and IgA secretions by slgD-B cells, slgD + B cells could not be induced for IgA synthesis by TGF-~ and Ibl0 after crosslinking of their slgs, suggesting that ligation of CD40 was one of the obligatory signals required for commitment of naive B cells to IgA secretion. Limiting dilution experiments indicated that the IgA-potentiating effect of TGF-B was due to its capacity to increase the frequency of IgA-producing cells, most likely as a consequence of class switching. Taken together, our data strongly suggest that TGF-B is involved in the regulation of IgA isotype selection in humans.

We have identified a novel lysosome-associated membrane glycoprotein localized on chromosome 3q26.3-q27, DC-LAMP, which is homologous to CD68. DC-LAMP mRNA is present only in lymphoid organs and DC. A specific MAb detects the protein exclusively in interdigitating dendritic cells. Expression of DC-LAMP increases progressively during in vitro DC differentiation, but sharply upon activation with LPS, TNFalpha, or CD40L. Confocal microscopy confirmed the lysosomal distribution of the protein. Furthermore, DC-LAMP was found in the MHC class II compartment immediately before the translocation of MHC class II molecules to the cell surface, after which it concentrates into perinuclear lysosomes. This suggests that DC-LAMP might change the lysosome function after the transfer of peptide-MHC class II molecules to the surface of DC.