B70/B7-2 is identical to CD86 and is the major functional ligand for CD28 expressed on human dendritic cells.

Dendritic cells comprise a system of highly efficient antigen-presenting cells involved in the initiation of T cell responses. Herein, we investigated the role of the CD28 pathway during alloreactive T cell proliferation induced by dendritic-Langerhans cells (D-Lc) generated by culturing human cord blood CD34+ progenitor cells with granulocyte/macrophage colony-stimulating factor and tumor necrosis factor alpha. In addition to expressing CD80 (B7/BB1), a subset of D-Lc expressed B70/B7-2. Binding of the CTLA4-Ig fusion protein was completely inhibited by a combination of monoclonal antibodies (mAbs) against CD80 and B70/B7-2, indicating the absence of expression of a third ligand for CD28/CTLA-4. It is interesting to note that mAbs against CD86 completely prevented the binding of CTLA4-Ig in the presence of mAbs against CD80 and bound to a B70/B7-2-transfected fibroblast cell line, demonstrating that the B70/B7-2 antigen is identical to CD86. CD28 triggering was essential during D-Lc-induced alloreaction as it was inhibited by mAbs against CD28 (9 out of 11 tested). However, none of six anti-CD80 mAbs demonstrated any activity on the D-Lc-induced alloreaction, though some were previously described as inhibitory in assays using CD80-transfected cell lines. In contrast, a mAb against CD86 (IT-2) was found to suppress the D-Lc-dependent alloreaction by 70%. This inhibitory effect was enhanced to > or = 90% when a combination of anti-CD80 and anti-CD86 mAbs was used. The present results demonstrate that D-Lc express, in addition to CD80, the other ligand for CTLA-4, CD86 (B70/B7-2), which plays a primordial role during D-Lc-induced alloreaction.

Activation of human dendritic cells through CD40 cross-linking.

Dendritic 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-Lc) generated by culturing cord blood CD34+ progenitor cells with granulocyte/macrophage colony-stimulating and tumor necrosis factor alpha (TNF-alpha) express functional CD40 at a density higher than that found on B cells. Culturing D-Lc on CD40-ligand (CD40L) transfected L cells allowed D-Lc 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 levels 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-Lc maturation as illustrated by upregulation of CD25, a molecule usually expressed on interdigitating dendritic cells of secondary lymphoid organs. Finally, CD40 activated D-Lc secreted a limited set of cytokines (TNF-alpha, IL-8, and macrophage inflammatory protein 1 alpha [MIP-1 alpha]) whereas a similar activation induced elutriated monocytes to secrete IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, TNF-alpha, and MIP-1 alpha. As D-Lc activated T cells upregulated CD40L, it is likely that CD40 activation of D-Lc observed herein with a fibroblast cell line stably expressing CD40L, mimics physiological interactions between dendritic cells and T cells.

Dendritic cells enhance growth and differentiation of CD40-activated B lymphocytes.

After antigen capture, dendritic cells (DC) migrate into T cell-rich areas of secondary lymphoid organs, where they induce T cell activation, that subsequently drives B cell activation. Here, we investigate whether DC, generated in vitro, can directly modulate B cell responses, using CD40L-transfected L cells as surrogate activated T cells. DC, through the production of soluble mediators, stimulated by 3- to 6-fold the proliferation and subsequent recovery of B cells. Furthermore, after CD40 ligation, DC enhanced by 30-300-fold the secretion of IgG and IgA by sIgD- B cells (essentially memory B cells). In the presence of DC, naive sIgD+ B cells produced, in response to interleukin-2, large amounts of IgM. Thus, in addition to activating naive T cells in the extrafollicular areas of secondary lymphoid organs, DC may directly modulate B cell growth and differentiation.

CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF+TNF alpha.

Human dendritic cells (DC) can now be generated in vitro in large numbers by culturing CD34+ hematopoietic progenitors in presence of GM-CSF+TNF alpha 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 CD1a and CD14 emerge independently. Both precursor subsets mature at day 12-14 into DC with typical morphology and phenotype (CD80, CD83, CD86, CD58, high HLA class II). CD1a+ 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 CD1a+ DC lacking Birbeck granules, E-cadherin, and Lag antigen but expressing CD2, CD9, CD68, and the coagulation factor XIIIa 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 CD1a+ precursors, represent bipotent cells that can be induced to differentiate, in response to M-CSF, into macrophage-like cells, lacking accessory function for T cells. 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.

Macrophage inflammatory protein 3alpha is expressed at inflamed epithelial surfaces and is the most potent chemokine known in attracting Langerhans cell precursors.

Dendritic cells (DCs) form a network comprising different populations that initiate and differentially regulate immune responses. Langerhans cells (LCs) represent a unique population of DCs colonizing epithelium, and we present here observations suggesting that macrophage inflammatory protein (MIP)-3alpha plays a central role in LC precursor recruitment into the epithelium during inflammation. (a) Among DC populations, MIP-3alpha was the most potent chemokine inducing the selective migration of in vitro-generated CD34(+) hematopoietic progenitor cell-derived LC precursors and skin LCs in accordance with the restricted MIP-3alpha receptor (CC chemokine receptor 6) expression to these cells. (b) MIP-3alpha was mainly produced by epithelial cells, and the migration of LC precursors induced by the supernatant of activated skin keratinocytes was completely blocked with an antibody against MIP-3alpha. (c) In vivo, MIP-3alpha was selectively produced at sites of inflammation as illustrated in tonsils and lesional psoriatic skin where MIP-3alpha upregulation appeared associated with an increase in LC turnover. (d) Finally, the secretion of MIP-3alpha was strongly upregulated by cells of epithelial origin after inflammatory stimuli (interleukin 1beta plus tumor necrosis factor alpha) or T cell signals. Results of this study suggest a major role of MIP-3alpha in epithelial colonization by LCs under inflammatory conditions and immune disorders, and might open new ways to control epithelial immunity.

Selective attraction of naive and memory B cells by dendritic cells.

In this study, we investigate whether dendritic cells (DC), known to interact directly with T and B cells, might also contribute to the recruitment of B cells through the production of chemotactic factors. We found that B cells responded to several chemokines (CXCL12, CCL19, CCL20, and CCL21), which can be produced by DC upon activation. In addition, supernatant from DC (SNDC) potently and selectively attracted naive and memory B cells but not germinal center (GC) B cells or other lymphocytes (CD4(+), CD8(+) T cells or NK cells). Production of this activity was restricted to DC and was not increased following DC activation by LPS or CD40 ligand. Surprisingly, the B-cell chemotactic response to SNDC was insensitive to pertussis toxin treatment. In addition, the chemotactic factor(s) appeared resistant to protease digestion and highly sensitive to heat. This suggested that the DC chemotactic factor(s) is different from classical chemoattractants and does not involve G(alpha(i)) proteins on the responding B lymphocytes. It is interesting that SNDC was able to synergize with several chemokines to induce massive migration of B lymphocytes. These observations show that DC spontaneously produce factors that, alone or in cooperation with chemokines, specifically regulate B-cell migration, suggesting a key role of DC in the recruitment or localization of B lymphocytes within secondary lymphoid organs.

Respective involvement of TGF-beta and IL-4 in the development of Langerhans cells and non-Langerhans dendritic cells from CD34+ progenitors.

In vivo, dendritic cells (DC) form a network comprising different populations. In particular, Langerhans cells (LC) appear as a unique population of cells dependent on transforming growth factor beta(TGF-beta) for its development. In this study, we show that endogenous TGF-beta is required for the development of both LC and non-LC DC from CD34+ hematopoietic progenitor cells (HPC) through induction of DC progenitor proliferation and of CD1a+ and CD14+ DC precursor differentiation. We further demonstrate that addition of exogenous TGF-beta polarized the differentiation of CD34+ HPC toward LC through induction of differentiation of CD14+ DC precursors into E-cadherin+, Lag+CD68-, and Factor XIIIa-LC, displaying typical Birbeck granules. LC generated from CD34+ HPC in the presence of exogenous TGF-beta displayed overlapping functions with CD1a+ precursor-derived DC. In particular, unlike CD14(+)-derived DC obtained in the absence of TGF-beta, they neither secreted interleukin-10 (IL-10) on CD40 triggering nor stimulated the differentiation of CD40-activated naive B cells. Finally, IL-4, when combined with granulocyte-macrophage colony-stimulating factor (GM-CSF), induced TGF-beta-independent development of non-LC DC from CD34+ HPC. Similarly, the development of DC from monocytes with GM-CSF and IL-4 was TGF-beta independent. Collectively these results show that TGF-beta polarized CD34+ HPC differentiation toward LC, whereas IL-4 induced non-LC DC development independently of TGF-beta.

Polymerase chain reaction-based identification of a novel serpin from human dendritic cells.

A subtraction library of CD40-stimulated human tonsil dendritic cells has been constructed using a polymerase chain reaction approach adapted for low numbers of cells. From this library we identified a cDNA for a serine protease inhibitor, a serpin, which is absent from monocytes, B cells and T cells but expressed in CD40-activated monocyte- and progenitor cell-generated dendritic cells. In addition, the serpin is expressed in a lung fibroblast cell line and keratinocytes. Its mRNA is detected only in tonsil and thymus. The serpin described here reportedly functions as a megakaryocyte maturation factor in the presence of interleukin (IL)-3 and IL-11. This suggests that dendritic cells may promote the immune response by protecting IL-3 and IL-11 or other essential proteins from degradation.

Interleukin-3 cooperates with tumor necrosis factor alpha for the development of human dendritic/Langerhans cells from cord blood CD34+ hematopoietic progenitor cells.

We have previously shown that tumor necrosis factor (TNF)alpha strongly potentiates the granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin (IL)-3-dependent proliferation of CD34+ hematopoietic progenitor cells (HPC) through the recruitment of early progenitors with high proliferative potential. Furthermore, the combination of GM-CSF and TNFalpha allows the generation of large numbers of dendritic/Langerhans cells (D-Lc). Herein, we analyzed whether IL-3, when combined to TNFalpha would, as does GM-CSF, allow the generation of CD1a+ D-Lc. Accordingly, cultures of cord blood CD34+ HPC with IL-3 + TNFalpha yielded 20% to 60% CD14+ cells and 11% to 17% CD1a+ cells, while IL-3 alone did not generate significant numbers of CD1a+ cells. Although the percentage of CD1a+ cells detected in IL3 + TNFalpha was lower than that observed in GM-CSF + TNFalpha (42% to 78%), the strong growth induced by IL-3 + TNFalpha generated as many CD1a+ cells as did GM-CSF + TNFalpha. The CD14+ and CD1a+ cells generated with IL-3 + TNFalpha are similar to CD14+ and CD1a+ cells generated in GM-CSF alone and GM-CSF + TNFalpha, respectively. CD1a+ cells differed from CD14+ cells by (1) dendritic morphology, (2) higher expression of CD1a, CD1c, CD4, CD40, adhesion molecules (CD11c, CD54, CD58), major histocompatibility complex (MHC) class II molecules and CD28 ligands (CD80 and CD86), (3) lack of Fc receptor FcgammaRI (CD64) and complement receptor CR1 (CD35) expression, and (4) stronger induction of allogeneic T-cell proliferation. Thus, in combination with TNFalpha, IL-3 is as potent as GM-CSF for the generation of CD1a+ D-Lc from cord blood CD34+ HPC. The dendritic cell inducing ability of IL-3 may explain why mice with inactivated GM-CSF gene display dendritic cells.

IL-10 induces CCR6 expression during Langerhans cell development while IL-4 and IFN-gamma suppress it.

Immune responses are initiated by dendritic cells (DC) that form a network comprising different populations. In particular, Langerhans cells (LC) appear as a unique population of cells colonizing epithelial surfaces. We have recently shown that macrophage-inflammatory protein-3alpha/CCL20, a chemokine secreted by epithelial cells, induces the selective migration of LC among DC populations. In this study, we investigated the effects of cytokines on the expression of the CCL20 receptor, CCR6, during differentiation of LC. We found that both IL-4 and IFN-gamma blocked the expression of CCR6 and CCL20 responsiveness at different stages of LC development. The effect of IL-4 was reversible and most likely due to the transient blockade of LC differentiation. In contrast, IFN-gamma-induced CCR6 loss was irreversible and was concomitant to the induction of DC maturation. When other cytokines involved in DC and T cell differentiation were tested, we found that IL-10, unlike IL-4 and IFN-gamma, maintained CCR6 expression. The effect of IL-10 was reversible and upon IL-10 withdrawn, CCR6 was lost concomitantly to final LC differentiation. In addition, IL-10 induced the expression of CCR6 and responsiveness to CCL20 in differentiated monocytes that preserve their ability to differentiate into mature DC. Finally, TGF-beta, which induces LC differentiation, did not alter early CCR6 expression, but triggered its irreversible down-regulation, in parallel to terminal LC differentiation. Taken together, these results suggest that the recruitment of LC at epithelial surface might be suppressed during Th1 and Th2 immune responses, and amplified during regulatory immune responses involving IL-10 and TGF-beta.

Critical role of IL-12 in dendritic cell-induced differentiation of naive B lymphocytes.

Dendritic cells (DC) are potent APCs initiating immune responses. In a previous report, we demonstrated that DC directly enhance both proliferation and differentiation of CD40-activated naive and memory B cells. The present study deciphers the molecular mechanisms involved in DC-dependent regulation of B cell responses. Herein, we have identified IL-12 as the mandatory molecule secreted by CD40-activated DC that promote the differentiation of naive B cells into plasma cells secreting high levels of IgM. In fact, IL-12 synergizes with soluble IL-6R alpha-chain (sgp80), produced by DC, to drive naive B cell differentiation. IL-12 is critical for the differentiation of naive B cells into IgM plasma cells, whereas IL-6R signaling mainly promotes Ig secretion by already differentiated B cells. The differentiation of naive B cells in cocultures of B cells, T cells, and DC is IL-12 dependent, definitely demonstrating that the role of DC in humoral responses is not confined to the activation of T cells and further extending the physiologic relevance of DC/B cell interaction. Finally, this study also identifies differential requirements for DC-dependent naive and memory B cell differentiation, the latter being IL-12 independent. Altogether these results indicate that, in addition to prime T cells toward Thl development, DC, through the production of IL-12, may also directly signal naive B cell during the initiation of the immune response.

Interleukin 10 inhibits T cell alloreaction induced by human dendritic cells.

Human dendritic cells (DC) generated from CD34+ hematopoietic progenitors cultured in the presence of granulocyte macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-alpha are related to Langerhans cells (DLC) and have been shown to induce a strong proliferation of allogeneic CD4+ T cells. The present study shows that recombinant human IL-10 (h-IL-10) inhibits the primary and secondary proliferative responses of both CD4+ and CD8+ T cells induced by allogeneic CD1a+ DLC. The alloreaction induced by DLC generated after 5-18 days of culture of CD34+ HPC was equally inhibited by h-IL-10, thus indicating that DLC were sensitive to h-IL-10 at all stages of differentiation. This is further indicated by the h-IL-10-induced inhibition of the T cell alloreaction mediated by interdigitating DC freshly isolated from tonsils. h-IL-10 specifically acted on DLC as it did not affect the proliferation induced by Epstein-Barr virus lymphoblastoid cell lines (EBV-LCL) nor that induced by immobilized anti-CD3. The inhibitory effect of h-IL-10 was not due to the production of suppressive factors by the DLC, as the addition of DLC and IL-10 did not inhibit EBV-LCL-induced T cell proliferation. Rather, the inhibition of cytokine production (IL-2, GM-CSF, TNF, IFN-gamma) observed after 24 h of co-culture may explain the inhibition of T cell DNA synthesis detected 3 days later. The h-IL-10-induced inhibition of human DC mediated alloreaction advocates considering the use of h-IL-10 in the prevention of transplant rejection and graft versus host disease, phenomena initiated by DC.

CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to granulocyte-macrophage colony-stimulating factor plus tumor necrosis factor alpha: II. Functional analysis.

In response to granulocyte-macrophage colony-stimulating factor plus tumor necrosis factor alpha, cord blood CD34+ hematopoietic progenitor cells differentiate along two unrelated dendritic cell (DC) pathways: (1) the Langerhans cells (LCs), which are characterized by the expression of CD1a, Birbeck granules, the Lag antigen, and E cadherin; and (2) CD14+ cell-derived DCs, characterized by the expression of CD1a, CD9, CD68, CD2, and factor XIIIa (Caux et al, J Exp Med 184:695, 1996). The present study investigates the functions of each population. Although the two populations are equally potent in stimulating naive CD45RA cord blood T cells through apparently identical mechanisms, each also displays specific activities. In particular CD14-derived DCs show a potent and long-lasting (from day 8 to day 13) antigen uptake activity (fluorescein isothiocyanate dextran or peroxidase) that is about 10-fold higher than that of CD1a+ cells, which is restricted to the immature stage (day 6). The antigen capture is exclusively mediated by receptors for mannose polymers. The high efficiency of antigen capture of CD14-derived cells is coregulated with the expression of nonspecific esterase activity, a tracer of lysosomial compartment. In contrast, the CD1a+ population never expresses nonspecific esterase activity. The most striking difference is the unique capacity of CD14-derived DCs to induce naive B cells to differentiate into IgM-secreting cells, in response to CD40 triggering and interleukin-2. Thus, although the two populations can allow T-cell priming, initiation of humoral responses might be preferentially regulated by the CD14-derived DCs. Altogether, those results show that different pathways of DC development might exist in vivo: (1) the LC type, which might be mainly involved in cellular immune responses, and (2) the CD14-derived DC related to dermal DCs or circulating blood DCs, which could be involved in humoral immune responses.

Human dendritic Langerhans cells generated in vitro from CD34+ progenitors can prime naive CD4+ T cells and process soluble antigen.

Earlier studies have concluded that fresh Langerhans cells (LC) are able to capture and process native Ags, whereas cultured LC have lost these functions while acquiring the capacity to prime naive T cells. Herein we studied the functions of human dendritic/Langerhans cells (d-Lc) generated in vitro by culturing CD34+ hemopoietic progenitor cells in the presence of granulocyte-macrophage CSF (GM-CSF) + TNF-alpha. Less than 50 d-Lc were found to strongly stimulate the proliferation of 2.5 x 10(4) allogeneic naive CD4+ T cells. Furthermore, six to 50 d-Lc induced half-maximal proliferation of naive syngeneic CD4+ cord blood T cells, in the presence of picomolar concentrations of superantigens. During the alloreaction, the CD4+ T cells were expanded up to 100-fold within two successive stimulation cycles with the same d-Lc, and the recovered T cells were specific for the d-Lc alloantigen. HLA-matched tetanus toxoid (TT)-specific T cell clones were found to proliferate in response to TT presented by CD1a+ d-Lc. Finally, electron microscopy demonstrated that CD1a+ d-Lc were able to capture an Ag (gold-labeled Igs) through receptor-mediated endocytosis. Thus, in vitro generated d-Lc can prime naive T cells and process native Ags, a property that might eventually prove useful for priming Ag-specific naive T cells for cellular immunotherapy.

Dendritic cell biology and regulation of dendritic cell trafficking by chemokines.

DC (dendritic cells) represent an heterogeneous family of cells which function as sentinels of the immune system. They traffic from the blood to the tissues where, while immature, they capture antigens. Then, following inflammatory stimuli, they leave the tissues and move to the draining lymphoid organs where, converted into mature DC, they prime naive T cells. The key role of DC migration in their sentinel function led to the investigation of the chemokine responsiveness of DC populations during their development and maturation. These studies have shown that immature DC respond to many CC and CXC chemokines (MIP-1 alpha, MIP-1 beta, MIP-3 alpha, MIP-5, MCP-3, MCP-4, RANTES, TECK and SDF-1) which are inducible upon inflammatory stimuli. Importantly, each immature DC population displays a unique spectrum of chemokine responsiveness. For examples, Langerhans cells migrate selectively to MIP-3 alpha (via CCR6), blood CD11c+ DC to MCP chemokines (via CCR2), monocytes derived-DC respond to MIP-1 alpha/beta (via CCR1 and CCR5), while blood CD11c- DC precursors do not respond to any of these chemokines. All these chemokines are inducible upon inflammatory stimuli, in particular MIP-3 alpha, which is only detected within inflamed epithelium, a site of antigen entry known to be infiltrated by immature DC. In contrast to immature DC, mature DC lose their responsiveness to most of these inflammatory chemokines through receptor down-regulation or desensitization, but acquire responsiveness to ELC/MIP-3 beta and SLC/6Ckine as a consequence of CCR7 up-regulation. ELC/MIP-3 beta and SLC/6Ckine are specifically expressed in the T-cell-rich areas where mature DC home to become interdigitating DC. Altogether, these observations suggest that the inflammatory chemokines secreted at the site of pathogen invasion will determine the DC subset recruited and will influence the class of the immune response initiated. In contrast, MIP-3 beta/6Ckine have a determinant role in the accumulation of antigenloaded mature DC in T cell-rich areas of the draining lymph node, as illustrated by recent observations in mice deficient for CCR7 or SLC/6Ckine. A better understanding of the regulation of DC trafficking might offer new opportunities of therapeutic interventions to suppress, stimulate or deviate the immune response.

In vitro HIV1 infection of CD34+ progenitor-derived dendritic/Langerhans cells at different stages of their differentiation in the presence of GM-CSF/TNF alpha.

Langerhans cells (LC) are antigen-presenting cells which are found in areas at risk of inoculation by the human immunodeficiency virus (HIV). LC were shown to be sensitive to in vitro infection by HIV1. They could be generated in vitro by culturing CD34+ haematopoietic progenitors with GM-CSF+TNF alpha. In this study, we tested the sensitivity to HIV1 infection of in vitro generated LC throughout their differentiation and we investigated the effect of such an infection on in vitro differentiation. Phenotypic controls were performed using FACS analysis on day 6 for the presence of a CD1a+ cell population, and differentiation was assessed by transmission electron microscopy on day 13 for the presence of Birbeck granules. CD34+ cells were purified from cord blood mononuclear cells by magnetic separation. Cell suspensions were infected with either a T-lymphotropic, syncytium-inducing isolate (HXB2) or a macrophage-tropic, non-syncytium-inducing isolate (Ba-L). Viral particle release was measured by p24 antigen production in the culture supernatant. A high level of p24 production was noted on day 13 of postinfection only when infection was carried out with Ba-L isolate on cells generated after 6 days in culture with GM/CSF+TNF alpha. No infection of CD34+ progenitor cells was obtained either with Ba-L isolate or HXB2. The sensitivity of Langerhans cell/dendritic cell (LC/DC) precursors to NSI isolate (Ba-L) seemed to coincide with the early stage of differentiation (CD1a antigen appearance). The infection did not alter the differentiation of in vitro generated LC, which presented their specific ultrastructural marker of epidermal environment, i.e. Birbeck granules from day 15 of the culture as compared to control culture. These results highlight the HIV infectibility of a differentiated population of LC/DC generated in vitro from CD34+ progenitors.