Burn injury suppresses human dermal dendritic cell and Langerhans cell function

Human skin contains epidermal Langerhans cells (LCs) and dermal dendritic cells (DCs) that are key players in induction of adaptive immunity upon infection. After major burn injury, suppressed adaptive immunity has been observed in patients. Here we demonstrate that burn injury affects adaptive immunity by altering both epidermal LC and dermal DC functions. We developed a human ex vivo burn injury model to study the function of DCs in thermally injured skin. No differences were observed in the capacity of both LCs and dermal DCs to migrate out of burned skin compared to unburned skin. Similarly, expression levels of co-stimulatory molecules were unaltered. Notably, we observed a strong reduction of T cell activation induced by antigen presenting cell (APC) subsets that migrated from burned skin through soluble burn factors. Further analyses demonstrated that both epidermal LCs and dermal DCs have a decreased T cell stimulatory capacity after burn injury. Restoring the T cell stimulatory capacity of DC subsets might improve tissue regeneration in patients with burn wounds.     

Injection of immature dendritic cells into adjuvant-treated skin obviates the need for ex vivo maturation

A key and limiting step in the process of generating human monocyte-derived dendritic cells (DC) for clinical applications is maturation. In the setting of immunotherapy, DC are matured ex vivo by culturing them with various agents that mimic the conditions encountered at a site of inflammation. This study examined whether the ex vivo DC maturation step could be replaced by maturing DC in situ by injecting immature DC into sites pre-exposed to agents that induce a microenvironment conducive to in situ maturation of the injected DC. The hypothesis was that recapitulation of the physiological conditions occurring during pathogen infection would lead to optimal conditions for DC maturation, migration, and function. Murine immature DC injected into adjuvant (Adjuprime, poly-arginine, or Imiquimod)-pretreated skin exhibited lymph node migratory capacity comparable to and immunostimulatory capacity equal to or exceeding that of ex vivo matured DC. Acquisition of migratory capacity did not always correlate with enhanced immunostimulatory capacity. Immunostimulatory capacity was not enhanced when mature DC were injected into adjuvant-pretreated sites and remained below that seen with immature DC matured in situ. Immature DC injected into adjuvant-pretreated sites were more effective than mature DC in stimulating antitumor immunity in mice. (111)Indium-labeled human monocyte-derived immature DC injected into adjuvant (Imiquimod)-pretreated sites in cancer patients acquired lymph node migratory capacity comparable to ex vivo matured DC. This study shows that in situ maturation offers a simpler and potentially superior method to generate potent immunostimulatory DC for clinical immunotherapy.     

Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity

Langerhans cells (LC) form a unique subset of dendritic cells (DC) in the epidermis but so far their in vivo functions in skin immunity and tolerance could not be determined, in particular in relation to dermal DC (dDC). Here, we exploit a novel diphtheria toxin (DT) receptor (DTR)/DT-based system to achieve inducible ablation of LC without affecting the skin environment. Within 24 h after intra-peritoneal injection of DT into Langerin-DTR mice LC are completely depleted from the epidermis and only begin to return 4 wk later. LC deletion occurs by apoptosis in the absence of inflammation and, in particular, the dDC compartment is not affected. In LC-depleted mice contact hypersensitivity (CHS) responses are significantly decreased, although ear swelling still occurs indicating that dDC can mediate CHS when necessary. Our results establish Langerin-DTR mice as a unique tool to study LC function in the steady state and to explore their relative importance compared with dDC in orchestrating skin immunity and tolerance.     

Dendritic cells,new tools for vaccination

Our rapidly expanding knowledge of the biology of the dendritic cell (DC), a major antigen-presenting cell connecting innate and adaptive immunity, suggests new possibilities for the development of vaccines and therapeutic strategies against pathogens, through the manipulation of their function in vivo, or the injection of the DC itself, once properly instructed ex vivo.     

Major subsets of human dendritic cells are efficiently transduced by self-complementary adeno-associated virus vectors 1 and 2

Dendritic cells (DC) are antigen-presenting cells pivotal for inducing immunity or tolerance. Gene transfer into DC is an important strategy for developing immunotherapeutic approaches against infectious pathogens and cancers. One of the vectors previously described for the transduction of human monocytes or DC is the recombinant adeno-associated virus (rAAV), with a genome conventionally packaged as a single-stranded (ss) molecule. Nevertheless, its use is limited by the poor and variable transduction efficiency of DC. In this study, AAV type 1 (AAV1) and AAV2 vectors, which expressed the enhanced green fluorescent protein and were packaged as ss or self-complementary (sc) duplex strands, were used to transduce different DC subsets generated ex vivo and the immunophenotypes, states of differentiation, and functions of the subsets were carefully examined. We show here for the first time that a single exposure of monocytes (M(o)) or CD34(+) progenitors (CD34) to sc rAAV1 or sc rAAV2 leads to high transduction levels (5 to 59%) of differentiated M(o)-DC, M(o)-Langerhans cells (LC), CD34-LC, or CD34-plasmacytoid DC (pDC), with no impact on their phenotypes and functional maturation of these cells, compared to those of exposure to ss rAAV. Moreover, we show that all these DC subpopulations can also be efficiently transduced after commitment to their differentiation pathways. Furthermore, these DC subsets transduced with sc rAAV1 expressing a tumor antigen were potent activators of a CD8(+)-T-cell clone. Altogether, these results show the high potential of sc AAV1 and sc AAV2 vectors to transduce ex vivo conventional DC, LC, or pDC or to directly target them in vivo for the design of new DC-based immunotherapies.     

Efficient transduction of monocyte- and CD34+-derived Langerhans cells with lentiviral vectors in the absence of phenotypic and functional maturation

BACKGROUND: Gene delivery in dendritic cells (DC) has raised considerable interest to modulate DC functions and induce therapeutic immunity or tolerance in an antigen-specific fashion. Among immature DC, Langerhans cells (LC) are attractive candidates for antigen delivery using lentiviral vectors (LV). METHODS: LC derived from monocytes (Mo-LC), or derived from CD34+ cells (CD34-LC) in the presence of cytokine cocktail, were transduced with LV expressing enhanced green fluorescent protein (E-GFP) under the control of the ubiquitous phosphoglycerate kinase (PGK) promoter at a multiplicity of infection of 18, at days 0 to 3 for Mo-LC, or at days 0 to 12 for CD34-LC. We assessed gene transfer levels from the percentage of E-GFP+ cells in the final cultures, and examined the morphology, immunophenotype, state of differentiation and function of transduced LC. RESULTS: Day 0 transduction of monocytes or CD34+ progenitors before cytokine pre-activation and LC differentiation resulted in stable gene expression in 7.8% of Mo-LC and 24% of CD34-LC. Monocyte-derived DC (Mo-DC) differentiated in serum-free medium were also efficiently transduced up to 13.2%. Interestingly, Mo-LC cells committed towards LC phenotype were permissive for transduction up to day 3. Transduction levels of CD34-LC peaked at day 6 to 44% and decreased thereafter. LV transduction did not perturb viability, phenotype and function of E-GFP-expressing LC. CONCLUSIONS: LC generated ex vivo can serve as vaccine vehicles in humans through efficient transduction by LV. These LC will be helpful to assess in vitro the immunogenicity of gene therapy vectors, from the characterization of their phenotypic and functional maturation.     

Dendritic cells in distinct oral mucosal tissues engage different mechanisms to prime CD8+ T cells

Although oral dendritic cells (DCs) were shown to induce cell-mediated immunity, the identity and function of the various oral DC subsets involved in this process is unclear. In this study, we examined the mechanisms used by DCs of the buccal mucosa and of the lining mucosa to elicit immunity. After plasmid DNA immunization, buccally immunized mice generated robust local and systemic CD8(+) T cell responses, whereas lower responses were seen by lining immunization. A delayed Ag presentation was monitored in vivo in both groups; yet, a more efficient presentation was mediated by buccal-derived DCs. Restricting transgene expression to CD11c(+) cells resulted in diminished CD8(+) T cell responses in both oral tissues, suggesting that immune induction is mediated mainly by cross-presentation. We then identified, in addition to the previously characterized Langerhans cells (LCs) and interstitial dendritic cells (iDCs), a third DC subset expressing the CD103(+) molecule, which represents an uncharacterized subset of oral iDCs expressing the langerin receptor (Ln(+)iDCs). Using Langerin-DTR mice, we demonstrated that whereas LCs and Ln(+)iDCs were dispensable for T cell induction in lining-immunized mice, LCs were essential for optimal CD8(+) T cell priming in the buccal mucosa. Buccal LCs, however, failed to directly present Ag to CD8(+) T cells, an activity that was mediated by buccal iDCs and Ln(+)iDCs. Taken together, our findings suggest that the mechanisms engaged by oral DCs to prime T cells vary between oral mucosal tissues, thus emphasizing the complexity of the oral immune network. Furthermore, we found a novel regulatory role for buccal LCs in potentiating CD8(+) T cell responses.     

CD4 T cell-induced, bid-dependent apoptosis of cutaneous dendritic cells regulates T cell expansion and immune responses

The fate of dendritic cells (DCs) after Ag presentation may be DC subset-specific and controlled by many factors. The role of activation-induced apoptosis in regulating DC function is not clear. We investigated the fate of cutaneous DCs (cDCs), specifically Langerhans cells (LCs), and observed that they undergo apoptosis after successful Ag presentation to CD4 T cells. Caspase-specific inhibitors revealed that LC lines use a type II apoptosis pathway in response to CD4 T cells. In support of this, BH3-interacting domain (Bid) protein was present at high levels and specifically cleaved in the presence of Ag-specific T cells. Significant resistance to apoptosis by OT-2 CD4 cells was also observed for Bid knockout (KO) LCs in vitro. To test whether Bid was required to regulate LC function in vivo, we measured contact sensitization and topical immunization responses in Bid KO mice and observed markedly enhanced ear swelling and proliferation responses compared with wild-type mice. Furthermore, when Ag-pulsed Bid KO migratory cDCs were inoculated into wild-type recipients, an increase in both the rate and percentage of expanded OT-2 T cells expressing IFN-gamma was observed. Thus, enhanced Ag presentation function was intrinsic to Bid KO cDCs. Therefore, Bid is an important regulator of LC viability and Ag presentation function.     

Langerhans cells are required for efficient presentation of topically applied hapten to T cells

Dendritic cells (DC) play a pivotal role in the control of T cell immunity due to their ability to stimulate naive T cells and direct effector function. Murine and human DC are composed of a number of phenotypically, and probably developmentally, distinct subsets, which may play unique roles in the initiation and regulation of T cell responses. The skin is populated by at least two subsets of DC: Langerhans cells (LC), which form a contiguous network throughout the epidermis, and dermal DC. LC have classically been thought vital to initiate T cell responses to cutaneous Ags. However, recent data have highlighted the importance of dermal DC in cutaneous immunity, and the requirement for LC has become unclear. To define the relative roles of LC and dermal DC, we and others generated mouse models in which LC were specifically depleted in vivo. Unexpectedly, these studies yielded conflicting data as to the role of LC in cutaneous contact hypersensitivity (CHS). Extending our initial finding, we demonstrate that topical Ag is inefficiently transported to draining lymph nodes in the absence of LC, resulting in suboptimal priming of T cells and reduced CHS. However, dermal DC may also prime cutaneous T cell responses, suggesting redundancy between the two different skin DC subsets in this model.     

Efficient human cytomegalovirus reactivation is maturation dependent in the Langerhans dendritic cell lineage and can be studied using a CD14+ experimental latency model

Studies from a number of laboratories have shown that the myeloid lineage is prominent in human cytomegalovirus (HCMV) latency, reactivation, dissemination, and pathogenesis. Existing as a latent infection in CD34(+) progenitors and circulating CD14(+) monocytes, reactivation is observed upon differentiation to mature macrophage or dendritic cell (DC) phenotypes. Langerhans' cells (LCs) are a subset of periphery resident DCs that represent a DC population likely to encounter HCMV early during primary infection. Furthermore, we have previously shown that CD34(+) derived LCs are a site of HCMV reactivation ex vivo. Accordingly, we have utilized healthy-donor CD34(+) cells to study latency and reactivation of HCMV in LCs. However, the increasing difficulty acquiring healthy-donor CD34(+) cells--particularly from seropositive donors due to the screening regimens used--led us to investigate the use of CD14(+) monocytes to generate LCs. We show here that CD14(+) monocytes cultured with transforming growth factor β generate Langerin-positive DCs (MoLCs). Consistent with observations using CD34(+) derived LCs, only mature MoLCs were permissive for HCMV infection. The lytic infection of mature MoLCs is productive and results in a marked inhibition in the capacity of these cells to promote T cell proliferation. Pertinently, differentiation of experimentally latent monocytes to the MoLC phenotype promotes reactivation in a maturation and interleukin-6 (IL-6)-dependent manner. Intriguingly, however, IL-6-mediated effects were restricted to mature LCs, in contrast to observations with classical CD14(+) derived DCs. Consequently, elucidation of the molecular basis behind the differential response of the two DC subsets should further our understanding of the fundamental mechanisms important for reactivation.     

Langerin is a natural barrier to HIV-1 transmission by Langerhans cells

Human immunodeficiency virus-1 (HIV-1) is primarily transmitted sexually. Dendritic cells (DCs) in the subepithelium transmit HIV-1 to T cells through the C-type lectin DC-specific intercellular adhesion molecule (ICAM)-3-grabbing nonintegrin (DC-SIGN). However, the epithelial Langerhans cells (LCs) are the first DC subset to encounter HIV-1. It has generally been assumed that LCs mediate the transmission of HIV-1 to T cells through the C-type lectin Langerin, similarly to transmission by DC-SIGN on dendritic cells (DCs). Here we show that in stark contrast to DC-SIGN, Langerin prevents HIV-1 transmission by LCs. HIV-1 captured by Langerin was internalized into Birbeck granules and degraded. Langerin inhibited LC infection and this mechanism kept LCs refractory to HIV-1 transmission; inhibition of Langerin allowed LC infection and subsequent HIV-1 transmission. Notably, LCs also inhibited T-cell infection by viral clearance through Langerin. Thus Langerin is a natural barrier to HIV-1 infection, and strategies to combat infection must enhance, preserve or, at the very least, not interfere with Langerin expression and function.     

Apoptotic cells induce immunosuppression through dendritic cells: critical roles of IFN-gamma and nitric oxide

Apoptotic cells induce immunosuppression through unknown mechanisms. To identify the underlying molecular mediators, we examined how apoptotic cells induce immunoregulation by dendritic cells (DC). We found that administration of DC exposed to apoptotic cells (DC(ap)) strongly inhibited the expansion of lymphocytes in draining lymph nodes in vivo and the subsequent Ag-specific activation of these lymphocytes ex vivo. Unexpectedly, DC(ap) supported T cell activation to a similar extent as normal DC in vitro, leading to proliferation and IL-2 production, except that DC(ap) did not support T cell production of IFN-gamma. Surprisingly, when DC(ap) were cocultured with normal DC, they completely lost their ability to support T cell activation, an effect reversed by anti-IFN-gamma or inhibitors of inducible NO synthase (iNOS). As expected, exposure to apoptotic cells rendered DC(ap) capable of producing much more NO in response to exogenous IFN-gamma than normal DC. Furthermore, DC(ap) from iNOS(-/-) or IFN-gammaR1(-/-) mice were not inhibitory in vitro or in vivo. Therefore, the IFN-gamma-induced production of NO by apoptotic cell-sensitized DC plays a key role in apoptotic cell-mediated immunosuppression.     

Variation in the C:P ratio of suspended and settling seston and its significance for P uptake calculations

SUMMARY. 1. In 1981–84 limnocorral (LC) experiments were performed in Lake Lucerne. Switzerland, to manipulate the planktonic community by varying P fertilization and by removing large zooplankton (with a 95 μm screen).
2. The C:P ratios in both suspended and entrapped seston exceeded the 'ideal' C:P ratio of 106 proposed by Redfield, Ketchum & Richards (1963) when P was limiting algal growth.
3. P fertilization could decrease the sestonic C:P ratio to 106 only when P did not limit algal growth; P additions far exceeding the P loading of eutrophic lakes were necessary to obtain this situation.
4. Changes in epilimnetic C:P ratios were usually related to short- term changes in primary production, caused by variable in situ light conditions and turbulence, and subsequent variation in POC concentrations.
5. Entrapped seston in the 95 μm-filtered LCs showed C:P ratios slightly higher than those of suspended seston, indicating fast P release and slower C mineralization in the epilimnetic nutrient cycle.
6. Removing large crustacean zooplankton enhanced epilimnetic P mineralization, and C:P ratios of entrapped seston in the 95 μm-filtered LCs were increased.
7 Detritus formed a relatively high proportion of the seston and amounted to more than two-thirds of the measured POC concentration.
8. Calculations of algal P uptake using information on sestonic C:P ratios and ¹⁴C uptake rates are questionable, as long as detritus cannot be separated from algae and net carbon uptake cannot be accurately measured.     

ASSESSMENT OF ARGOS LOCATION ACCURACY FROM SATELLITE TAGS DEPLOYED ON CAPTIVE GRAY SEALS

The Argos satellite system is commonly used to track and relay behavioral data from marine mammals, but their underwater habit results in a high proportion of locations of non-guaranteed accuracy (location classes (LC) O, A, and B). The accuracy of these locations is poorly documented in marine mammals. We assessed the accuracy of all LCs on four juvenile gray seals fitted with Argos satellite relay data loggers and held in captivity in an outdoor tank for a total of 61 seal-days. Four hundred and twenty-six locations were obtained from seals in captivity, and their latitude and longitude error was assessed before and after filtering, following MConnell et al. (1992). There was significantly more error in longitude than latitude in all LCs except I. C A. The ratio of the standard deviations of longitude : latitude ranged from 1.77 (LC 3) to 2.58 (LC 1). Filtering had very little effect on errors in LCs 3-1, but in the remaining LCs filtering resulted in error reductions ranging from 8% to 63%. In LCs O, A, and B, error reduction was greater in the 95th percentile errors, especially in longitude. The averages of the latitude and longitude 68th percentile errors and those predicted by Argos (in brackets) were 226 (150), 372 (350), and 757 (1000) m for LCs 3, 2, and 1 respectively. Both latitude and longitude errors of LCs > O were normally distributed. Both filtered and unfiltered LC A locations were of a similar accuracy to LC 1 locations, and considerably better than LC O locations.     

Langerhans cells derived from genetically modified human CD34+ hemopoietic progenitors are more potent than peptide-pulsed Langerhans cells for inducing antigen-specific CD8+ cytolytic T lymphocyte responses

Sustained Ag expression by human dendritic cells (DCs) is an attractive means of optimizing Ag presentation for stimulating durable cellular immunity. To establish proof of principle, we used Langerhans cell (LC) progeny of retrovirally transduced CD34(+) hemopoietic progenitor cells to stimulate responses against the HLA-A*0201-restricted influenza matrix peptide (fluMP). Retroviral transduction of CD34(+) hemopoietic progenitor cells, during pre-expansion by thrombopoietin, c-kit ligand, and FLT-3 ligand, on recombinant fibronectin, but in the absence of FCS, resulted in gene expression by 20-30% of the LCs. Expression persisted at least 28 days, with little decline (<30%) over that time. Retroviral transduction did not alter the phenotype or potent immunogenicity of normal mature DCs. FluMP-transduced LCs stimulated a 130-fold expansion of T cells reactive with HLA-A*0201-fluMP tetramers, even at LC:T cell ratios of 1:100-150 and lower, whereas fluMP-pulsed LCs stimulated only a 30-fold expansion. FluMP-transduced LCs also stimulated higher IFN-gamma secretion (100-123 spot-forming cells/10(5) CD8(+) T cells) than did fluMP-pulsed LCs (10-91 spot-forming cells/10(5) CD8(+) T cells). CD8(+) T cells stimulated by transduced LCs did not react preferentially with retrovirally transduced targets, indicating that the responses targeted only the immunizing influenza and not the retroviral vector Ags, even though these could have provided nonspecific helper epitopes presented by the transduced LCs. These data demonstrate that gene-transduced LCs maintain the activated phenotype as well potent immunogenicity typical of mature DCs. LCs genetically modified to express fluMP are also more potent stimulators of Ag-specific CD8(+) T cell responses than are peptide-pulsed LCs.     

Antigen-independent immune responses after dendritic cell vaccination

The ability of cultured, antigen-loaded dendritic cells (DCs) to induce antigen-specific T cell immunity in vivo has previously been demonstrated and confirmed. Immune monitoring naturally focuses on immunity against vaccine antigens and may thus ignore other effects of DC vaccination. Here we therefore focused on antigen-independent responses induced by DC vaccination of renal cell carcinoma patients. In addition to the anticipated response against the vaccine antigen KLH, vaccination with CD83⁺ monocyte-derived DCs resulted in a strong increase in the ex vivo proliferative and cytokine responses of PBMCs stimulated with LPS or BCG. In addition, LPS strongly enhanced the KLH-induced proliferative and cytokine response of PBMCs. Moreover, proliferative and cytokine responses of PBMCs stimulated with the homeostatic cytokines IL-7 and IL-15 were also clearly enhanced after DC vaccination. In contrast to LPS induced proliferation, which is well known to depend on monocytes, IL-7 induced proliferation was substantially enhanced after monocyte depletion indicating that monocytes limit IL-7 induced lymphocyte expansion. Our data indicate that DC vaccination leads to an increase in the ex vivo responsiveness of patient PBMCs consistent with a DC vaccination induced enhancement of T cell memory. Our findings also suggest that incorporation of bacterial components and homeostatic cytokines into immunotherapy protocols may be useful in order to enhance the efficacy of DC vaccination and that monocytes may limit DC vaccination induced immunity. Supported by a grant to Martin Thurnher from the kompetenzzentrum medizin tirol (kmt), a center of excellence.     

Peroxisome proliferator-activated receptor gamma inhibits the migration of dendritic cells: consequences for the immune response

The migration of dendritic cells (DCs) from the epithelia to the lymphoid organs represents a tightly regulated multistep event involved in the induction of the immune response. In this process fatty acid derivatives positively and negatively regulate DC emigration. In the present study we investigated whether activation of peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors activated by naturally occurring derivatives of arachidonic acid, could control DC migration from the peripheral sites of Ag capture to the draining lymph nodes (DLNs). First, we show that murine epidermal Langerhans cells (LCs) express PPAR gamma, but not PPAR alpha, mRNA, and protein. Using an experimental murine model of LC migration induced by TNF-alpha, we show that the highly potent PPAR gamma agonist rosiglitazone specifically impairs the departure of LCs from the epidermis. In a model of contact allergen-induced LC migration, PPAR gamma activation not only impedes LC emigration, and their subsequent accumulation as DCs in the DLNs, but also dramatically prevents the contact hypersensitivity responses after challenge. Finally, after intratracheal sensitization with an FITC-conjugated Ag, PPAR gamma activation inhibits the migration of DCs from the airway mucosa to the thoracic LNs and also profoundly reduces the priming of Ag-specific T lymphocytes in the DLNs. Our results suggest a novel regulatory pathway via PPAR gamma for DC migration from epithelia that could contribute to the initiation of immune responses.     

Activation of dendritic cells by human papillomavirus-like particles through TLR4 and NF-kappaB-mediated signalling, moderated by TGF-beta

Human papillomavirus-like particles (HPV-VLP) are a candidate vaccine for prevention of HPV infection, and also are a candidate for an immunogenic delivery system for incorporated antigen. VLP activate in vitro generated dendritic cells (DC) but not Langerhans cells (LC); however, the mechanism of this activation is unknown. We have shown that uptake and activation of DC by VLP involves proteoglycan receptors and can be inhibited by heparin. Heparin has been shown to activate DC by signalling through Toll-like receptor 4 (TLR4) and nuclear factor (NF)-kappaB. The pathway of DC activation by VLP was further investigated in the present study. Exposure to VLP induced costimulatory molecule expression, RelB translocation and IL-10 production by DC but not by LC. The lack of LC activation was reversible when TGF-beta was removed from the LC medium. VLP-induced induction of costimulatory molecule expression, RelB activation and cytokine secretion by DC was blocked by inhibition of NF-kappaB activation, heparin or TLR4 mAb. The data provide evidence that HPV-VLP signal DC through a pathway involving proteoglycan receptors, TLR4 and NF-kappaB, and shed light on the mechanism by which VLP stimulate immunity in the absence of adjuvants in vivo. LC may resist activation in normal epithelium abundant in TGF-beta, but not in situations in which TGF-beta concentrations are reduced.     

Dendritic cells are host cells for mycobacteria in vivo that trigger innate and acquired immunity.

In the present study, we investigated in vivo the infection and APC functions of dendritic cells (DC) and macrophages (Mphi) after administration of live mycobacteria to mice. Experiments were conducted with Mycobacterium bovis bacillus Calmette-Guerin (BCG) or a rBCG expressing a reporter Ag. Following infection of mice, DC and Mphi were purified and the presence of immunogenic peptide/MHC class II complexes was detected ex vivo on sorted cells, as was the secretion of IL-12 p40. We show in this study that DC is a host cell for mycobacteria, and we provide an in vivo detailed picture of the role of Mphi and DC in the mobilization of immunity during the early stages of a bacterial infection. Strikingly, BCG bacilli survive but remain stable in number in the DC leukocyte subset during the first 2 wk of infection. As Ag presentation by DC is rapidly lost, this suggests that DC may represent a hidden reservoir for mycobacteria.     

TGF-beta 1 prevents the noncognate maturation of human dendritic Langerhans cells

TGF-beta 1 is critical for differentiation of epithelial-associated dendritic Langerhans cells (LC). In accordance with the characteristics of in vivo LC, we show that LC obtained from human monocytes in vitro in the presence of TGF-beta 1 1) express almost exclusively intracellular class II Ags, low CD80, and no CD83 and CD86 Ags and 2) down-regulate TNF-RI (p55) and do not produce IL-10 after stimulation, in contrast to dermal dendritic cells and monocyte-derived dendritic cells. Surprisingly, while LC exhibit E-cadherin down-regulation upon exposure to TNF-alpha and IL-1, TGF-beta 1 prevents the final LC maturation in response to TNF-alpha, IL-1, and LPS with respect to Class II CD80, CD86, and CD83 Ag expression, loss of FITC-dextran uptake, production of IL-12, and Ag presentation. In sharp contrast, CD40 ligand cognate signal induces full maturation of LC and is not inhibited by TGF-beta 1. The presence of emigrated immature LCs in human reactive skin-draining lymph nodes provides in vivo evidence that LC migration and final maturation may be differentially regulated. Therefore, due to the effects of TGF-beta 1, inflammatory stimuli may not be sufficient to induce full maturation of LC, thus avoiding potentially harmful immune responses. We conclude that TGF-beta 1 appears to be responsible for both the acquisition of LC phenotype, cytokine production pattern, and prevention of noncognate maturation.