The receptor for type I IFNs is highly expressed on peripheral blood B cells and monocytes and mediates a distinct profile of differentiation and activation of these cells.

Type I interferons (IFNs) are potent regulators of both innate and adaptive immunity. All type I IFNs bind to the same heterodimeric cell surface receptor composed of IFN-alpha receptor (IFNAR-1) and IFN-alpha/beta receptor (IFNAR-2) polypeptides. This study revealed that type I IFN receptor levels vary considerably on hematopoietic cells, with monocytes and B cells expressing the highest levels. Overnight treatment of peripheral blood mononuclear cells (PBMCs) with IFN-alpha2b or IFN-beta led to increased expression on monocytes and B cells of surface markers commonly associated with activated antigen-presenting cells (APCs), such as CD38, CD86, MHC class I, and MHC class II. Five-day exposure of adherent monocytes to granulocyte-macrophage colony-stimulating factor (GM-CSF) plus IFN-alpha or IFN-beta caused the development of potent allostimulatory cells with morphology similar to that of myeloid dendritic cells (DCs) obtained from culture with GM-CSF and interleukin-4 (IL-4) but with distinct cell surface marker profiles and activity. In contrast to IL-4-derived DCs, IFN-alpha-derived DCs were CD14+, CD1a-, CD123+, CD32+, and CD38+ and expressed high levels of CD86 and MHC class II. Development of these cells was completely blocked by an antibody to IFNAR-1. Furthermore, activity of the type I IFN-derived DC in a mixed lymphocyte reaction (MLR) was consistently more potent than that of IL-4-derived DCs, especially at high responder/stimulator ratios. This MLR activity was abrogated by the addition of anti-IFNAR-1 antibody at the start of the DC culture. In contrast, there was no effect of anti-IFNAR-1 on IL-4-derived DCs, indicating that this is a distinct pathway of DC differentiation. These results suggest a potential role for anti-IFNAR-1 immunotherapy in autoimmune diseases, such as systemic lupus erythematosus (SLE), in which the action of excessive type I IFN on B cells and myeloid DCs may play a role in disease pathology.     

Mycobacterium tuberculosis subverts the differentiation of human monocytes into dendritic cells

Intracellular pathogens have developed strategies for evading elimination by the defenses of the host immune system. Here we describe an escape mechanism utilized by Mycobacterium tuberculosis that involves the interference with the generation of fully competent DC from monocytes. We show that monocytes infected with live M. tuberculosis differentiated into mature, CD83+ and CCR7+ DC (Mt-MoDC), but were characterized by a selective failure in the expression of the family of CD1 molecules. These cells also showed levels of MHC class II and CD80 (B7.1) that were reduced in comparison with LPS-matured DC. In addition, Mt-MoDC produced TNF-alpha and IL-10, but were unable to secrete IL-12. The generation of Mt-MoDC required the infection of monocytes with live M. tuberculosis, since infection with heat-killed bacteria partially abrogated the effects on monocyte differentiation. Interestingly, Mt-MoDC revealed an impaired antigen-presentation function as assessed by the reduced capability to induce proliferation of cord blood T lymphocytes. Further, naive T lymphocytes expanded by Mt-MoDC were unable to secrete cytokines, in particular IL-4 and IFN-gamma, suggesting that they could be ineffective in helping the macrophage-mediated killing of intracellular mycobacteria. Our results suggest that the interference with monocyte differentiation into fully competent DC is an evasion mechanism of M. tuberculosis that could contribute to its intracellular persistence by avoiding immune recognition.     

Regulation of human CD4(+) alphabeta T-cell-receptor-positive (TCR(+)) and gammadelta TCR(+) T-cell responses to Mycobacterium tuberculosis by interleukin-10 and transforming growth factor beta

Mycobacterium tuberculosis is the etiologic agent of human tuberculosis and is estimated to infect one-third of the world's population. Control of M. tuberculosis requires T cells and macrophages. T-cell function is modulated by the cytokine environment, which in mycobacterial infection is a balance of proinflammatory (interleukin-1 [IL-1], IL-6, IL-8, IL-12, and tumor necrosis factor alpha) and inhibitory (IL-10 and transforming growth factor beta [TGF-beta]) cytokines. IL-10 and TGF-beta are produced by M. tuberculosis-infected macrophages. The effect of IL-10 and TGF-beta on M. tuberculosis-reactive human CD4(+) and gammadelta T cells, the two major human T-cell subsets activated by M. tuberculosis, was investigated. Both IL-10 and TGF-beta inhibited proliferation and gamma interferon production by CD4(+) and gammadelta T cells. IL-10 was a more potent inhibitor than TGF-beta for both T-cell subsets. Combinations of IL-10 and TGF-beta did not result in additive or synergistic inhibition. IL-10 inhibited gammadelta and CD4(+) T cells directly and inhibited monocyte antigen-presenting cell (APC) function for CD4(+) T cells and, to a lesser extent, for gammadelta T cells. TGF-beta inhibited both CD4(+) and gammadelta T cells directly and had little effect on APC function for gammadelta and CD4(+) T cells. IL-10 down-regulated major histocompatibility complex (MHC) class I, MHC class II, CD40, B7-1, and B7-2 expression on M. tuberculosis-infected monocytes to a greater extent than TGF-beta. Neither cytokine affected the uptake of M. tuberculosis by monocytes. Thus, IL-10 and TGF-beta both inhibited CD4(+) and gammadelta T cells but differed in the mechanism used to inhibit T-cell responses to M. tuberculosis.     

Dendritic cells derived from BCG-infected precursors induce Th2-like immune response

Human monocytes can differentiate into dendritic cells (DCs) according to the nature of environmental signals. We tested here whether the infection with the live tuberculosis vaccine bacillus Calmette-Guerin (BCG), which is known to be limited in preventing pulmonary tuberculosis, modulates monocyte and DC differentiation. We found that monocytes infected with BCG differentiate into CD1a- DCs (BCG-DCs) in the presence of granulocyte macrophage-colony stimulating factor and interleukin (IL)-4 and acquired a mature phenotype in the absence of maturation stimuli. In addition, BCG-DCs produced proinflammatory cytokines (tumor necrosis factor alpha, IL-1beta, IL-6) and IL-10 but not IL-12. BCG-DCs were able to stimulate allogeneic T lymphocytes to a similar degree as DCs generated in the absence of infection. However, BCG-DCs induced IL-4 production when cocultured with human cord-blood mononuclear cells. The induction of IL-4 production by DCs generated by BCG-infected monocytes could explain the failure of the BCG vaccine to prevent pulmonary tuberculosis.     

IFNalpha treatment generates antigen-presenting cells insensitive to atorvastatin inhibition of MHC-II expression

IFNalpha is an immunostimulatory cytokine involved in the development of autoimmunity. Atorvastatin, a cholesterol-lowering drug, also possess immunomodulatory effects, being able to downregulate IFNgamma-induced MHC-II expression. In this study we evaluated the role of IFNalpha in monocyte differentiation to antigen-presenting cells (APCs) and the effect of atorvastatin during this process. Results showed that IFNalpha-treated monocytes differentiated into dendritic-like cells (IFNalpha-DLCs) characterized by a CD86(high), CD1a(low), MHC-II(high) and CD14(low) expression, consistent with the phenotype of mature DCs and with similar ability to uptake antigens and induce T cell responses to mature IL-4/GM-CSF-DCs. Interestingly enough, the presence of atorvastatin did not inhibit IFNalpha-induced HLA-DR expression, although this drug markedly reduced the ability of IFNalpha-DLCs to induce T cell responses. These results confirm the immunostimulatory role of IFNalpha by promoting the generation of APCs and provide new clinical applications of statins as modulators of autoimmune responses in patients with high pathological or pharmacological IFNalpha levels.     

Vitamin D differentially regulates B7.1 and B7.2 expression on human peripheral blood monocytes.

The hormonal active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1, 25(OH)2D3), inhibits (through an unknown mechanism) the ability of monocytes/macrophages to induce T-cell activation. For T cells to be optimally activated, recognition of antigen/major histocompatibility complexes (MHC) by the T-cell receptor (TCR) must be accompanied by a second costimulatory signal. Considerable experimental data now suggest that this costimulatory signal is predominantly generated by B7.1 and/or B7.2 molecules, expressed on antigen-presenting cells (APC), when engaged to their counter-receptor, CD28, present on T cells. To determine whether the inhibitory effect of 1,25(OH)2D3 on monocytes/macrophages might involve modulation of the expression of B7.1 and B7.2 molecules, we analysed (by flow cytometry) the influence of 1,25(OH)2D3 and an analogue, KH 1060, on the expression of these two molecules at the surface of resting human peripheral blood monocytes. In parallel, we tested the effect of these two agents on human monocyte expression of cell-surface markers (CD14 and CD4) and antigen-presenting molecules (MHC class I and MHC class II). Our results showed that both 1,25(OH)2D3 and KH 1060 inhibited the basal expression of B7.2 in a dose- and time-dependent manner, without affecting B7.1. Moreover, these two compounds increased CD14 and reduced MHC class II and CD4 expression. Furthermore, the effect of 1,25(OH)2D3 on B7 molecule expression in combination with lipopolysaccharide (LPS) or cytokines, including interleukin-10 (IL-10), interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), was studied. The 1,25(OH)2D3-induced B7.2 down-regulation was still detectable when monocytes were activated by IL-10, IFN-gamma and TNF-alpha but not with LPS. Moreover, the induction of B7.1 by TNF-alpha was inhibited by addition of 1, 25(OH)2D3. We conclude that the ability of 1,25(OH)2D3 to decrease B7.2 expression on human monocytes might contribute to its inhibitory effect on APC-dependent T-cell activation and to its immunosuppressive properties observed in autoimmune diseases and organ transplantation.     

Diverse functional activity of CD83+ monocyte-derived dendritic cells and the implications for cancer vaccines

Antigen-loaded dendritic cells (DCs) provide key regulatory signals to T cells during a developing antitumor response. In addition to providing costimulation, mature DC provides cytokine and chemokine signals that can define the T1 vs T2 nature of the antitumor T-cell response as well as whether T cells engage in direct interactions with tumor cells. In serum-free culture conditions that hasten the differentiation of monocytes into mature DCs, certain agents, such as CD40L, accelerate phenotypic maturation (e.g., CD83 and costimulatory molecule expression) without influencing the acquisition of Dc1/Dc2 characteristics. In contrast, exposure to serum-free medium and interferon-gamma (IFN-gamma) rapidly influences CD83+ DCs to secrete high levels of IL-12, IL-6, and MIP-1beta, and promotes Dcl differentiation. In contrast, CD83+ DCs matured in serum-free medium in the absence of IFN-gamma, or in the presence of calcium signaling agents, prostaglandin-E2, or IFN-alpha, produce no IL-12, scant IL-6, and prodigious IL-8, MDC, and TARC, and promote Dc2 differentiation. T cells sensitized via IL-12-secreting, peptide-pulsed DCs secrete cytokines when subsequently exposed to relevant peptide-pulsed antigen-presenting cells (APCs) or to HLA-compatible tumor cells endogenously expressing the peptide. In contrast, T cells sensitized via IL-12 nonsecreting DC were limited to antigenic reactivation through APC contact rather than tumor cell contact. Therefore, the development of antitumor responses can be dramatically influenced not only by costimulation, but also by the cytokine and chemokine production of DCs, which must be considered in the development of cancer vaccines.     

Endocarditis-associated oral streptococci promote rapid differentiation of monocytes into mature dendritic cells

Endocarditis is frequently attributable to oral streptococci, but mechanisms of pathogenesis are not well understood, although monocytes appear to be important. High titers of interleukin-12 (IL-12) are produced by peripheral blood mononuclear cells (PBMC) after engaging Streptococcus mutans, but monocytes in developing endocardial vegetations tend to disappear rather than become macrophages. These data prompted the hypothesis that streptococcus-infected monocytes differentiate into short-lived IL-12-producing dendritic cells (DCs) rather than macrophages. PBMC from healthy subjects were stimulated with six isolates of oral streptococci, three nonstreptococcal oral bacteria, or IL-4 plus granulocyte-macrophage colony-stimulating factor, and the appearance of cells with markers typical of mature DCs (CD83(+), CD86(+), CD11c(+), and CD14(-)) was monitored. Supernatant fluids from the PBMC cultures were harvested and IL-12 p70 levels were determined. S. mutans-stimulated monocytes were analyzed for their ability to elicit allogeneic mixed-lymphocyte reactions. All streptococci examined, except one strain of Streptococcus oralis (35037), rapidly induced up-regulation of CD83 and CD86 and a loss of CD14 in the CD11c(+) monocyte population within 20 h. Induction of IL-12 was CD14 dependent and correlated with streptococcal isolates that promoted the DC phenotype. Major histocompatibility complex (MHC) class II expression was up-regulated by S. mutans, and these cells were short-lived and elicited potent allogeneic mixed-lymphocyte reactions typical of DCs. In summary, monocytes stimulated with endocarditis-associated oral streptococci rapidly exhibited the DC phenotype and functions. These data suggest that the initiation of bacterial endocarditis by oral streptococci may involve monocyte-to-DC differentiation, and this may help explain the low levels of macrophages in the site.     

Marked suppression of T cells by a benzothiophene derivative in patients with human T-lymphotropic virus type I-associated myelopathy/tropical spastic paraparesis

In a search for new anti-autoimmune agents that selectively suppress activation of autoreactive T cells, one such agent, 5-methyl-3-(1-methylethoxy)benzo[b]thiophene-2-carboxamide (CI-959-A), was found to be effective. This compound, which is known to suppress tumor necrosis factor alpha (TNF-alpha)-induced CD54 expression, inhibited the primary proliferative response of the T cell to antigen (Ag)-presenting cells (APCs) including allogenic dendritic cells (DCs), autologous Epstein-Barr virus-infected B cells, and human T lymphotropic virus type I (HTLV-I)-infected T cells. Autoreactive T cells from patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) spontaneously proliferate in vitro, and their activation is reported to be associated with CD54 expression. The spontaneous proliferation of T cells from patients with HAM/TSP was entirely blocked by CI-959-A. However, in this study, the T-cell proliferation in 15 patients with HAM/TSP was found to depend more extensively on major histocompatibility complex (MHC) class II and CD86 than on CD54 Ags. Since most important APCs for the development of HAM/TSP are DCs and HTLV-I-infected T cells, the effect of CI-959-A on DC generation and on the expression of surface molecules on activated T cells is examined. CI-959-A suppressed recombinant granulocyte-macrophage colony stimulating factor (GM-CSF)- and recombinant interleukin-4-dependent differentiation of DCs from monocytes and inhibited the expression of CD54 and, more extensively, MHC class II and CD86 Ags. CI-959-A showed little toxicity toward lymphoma or HTLV-I-infected T-cell lines or toward monocytes and cultured DCs. These results suggest that CI-959-A might be a potent anti-HAM/TSP agent.     

The STATus of PD-L1 (B7-H1) on tolerogenic APCs

Expression by DCs of co-inhibitory molecules such as programmed death ligand-1 (PD-L1/B7-H1/CD274), a member of the B7 superfamily, is crucial for the downregulation of T-cell responses and the maintenance of immune homeostasis. Exposure of immature DCs to danger-associated molecular patterns (DAMPS) or pathogen-associated molecular patterns (PAMPs) generally results in their maturation and acquisition of immunostimulatory function. However, exposure of DCs to TLR ligands early during their differentiation can inhibit further differentiation and confer tolerogenic properties on these APCs. A report in this issue of The European Journal of Immunology reveals that early inhibition of human DC differentiation from blood monocytes by TLR agonists is associated with a tolerogenic phenotype and Treg generation. The tolerogenic function of these APCs is dependent on MAPK-induced IL-6 and IL-10 production, which drives STAT-3-mediated PD-L1 expression. These observations link IL-10 and IL-6 to PD-L1 expression, providing a new dimension to the anti-inflammatory properties of these cytokines. These findings also have implications for understanding the inherent function of DCs in non-lymphoid tissues such as the liver and lung, where they are exposed to PAMPs that are found constitutively in the local microenvironment.     

Functional repertoire of dendritic cells generated in granulocyte macrophage-colony stimulating factor and interferon-alpha

Monocyte-derived dendritic cells (DCs) generated in granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4-DCs) are used to enhance antitumor immunity in cancer patients, although recent evidence suggests that their functional repertoire may be incomplete; in particular, IL-4-DCs appear unable to induce type 2 cytokine-producing T helper (Th) cells. To assess whether type 1 interferon (IFN) could replace IL-4 and generate DCs with a more complete repertoire, we characterized in detail DCs generated from human monocytes cultured with GM-CSF and IFN-alpha (IFN-DCs). We found that IFN-alpha induces DC differentiation more efficiently than IL-4, yielding similar numbers of DCs in a shorter time and that this differentiation persists upon removal of cytokines. Although IFN-DCs had a more mature immunophenotype than IL-4-DCs, showing higher expression of CD80, CD86, and CD83, they still preserved comparable endocytic and phagocytic capacities and responsiveness to maturation stimuli. IFN-DCs had strong antigen-presenting capacity, inducing intense proliferation of T cells to alloantigens or influenza virus. Moreover, IFN-DCs produced lower levels of IL-12p70 and higher levels of IFN-alpha, IL-4, and IL-10 than IL-4-DCs. As a consequence of this different pattern of cytokine secretion, IFN-DCs induced T cells to produce type 1 (IFN-gamma) and type 2 (IL-4 and IL-10) cytokines, and as expected, IL-4-DCs induced only Th1 differentiation. As immune responses with extreme Th1 bias are considered inadequate for the induction of optimal, systemic antitumor immunity, the ability of IFN-DCs to promote more balanced cytokine responses may suggest the advisability to consider these cells in the development of future, DC-based immunotherapy trials.     

Reciprocal regulation of plasmacytoid dendritic cells and monocytes during viral infection.

Reciprocal regulation of opposing functions characterizes biological systems. We now show that adenovirus-infected plasmacytoid dendritic cells (PDC) inhibit monocyte to myeloid dendritic cell (MDC) differentiation and function, and that adenovirus-infected monocytes inhibit PDC type I interferon secretion. Adenovirus-infected PDC secreted IFN-alpha, beta and omega in an 86:2:1 ratio. PDC type I interferons inhibited MDC differentiation and function (reduced IL-12 secretion, IFN-gamma induction, MLR and CD40 expression, and increased CD1a(+)CD14(+) cells). Type I interferon receptor blocking antibody reversed all PDC effects, and recombinant IFN-alpha, beta or omega replicated all effects, except reduced CD40. Adenovirus-infected monocytes suppressed PDC type I interferon secretion, which was reversed with anti-IL-10 neutralizing antibodies. Exogenous IL-10 suppressed PDC type I interferon secretion without reducing PDC viability. Therefore, monocyte IL-10 regulates PDC type I interferon secretion, and PDC type I interferons inhibit MDC differentiation and function. Such reciprocal regulation of potentially opposing influences may help modulate anti-pathogen immunity.     

Differential expression of alternative H2-M isoforms in B cells, dendritic cells and macrophages by proinflammatory cytokines

Major histocompatibility (MHC) class II heterodimers bind peptides generated by degradation of endocytosed antigens and display them on the surface of antigen presenting cells (APCs) for recognition by CD4+ T cells. Efficient loading of MHC class II molecules with peptides is catalyzed by the MHC class II-like molecule H2-M. The coordinate regulation of MHC class II and H2-M expression is a prerequisite for efficient MHC class II/peptide assembly in APCs determining both the generation of the T cell repertoire in the thymus and cellular immune responses in the periphery. Here we show that expression of H2-M and MHC class II genes is coordinately and cell type-specific regulated in splenic B cells, splenic dendritic cells (DCs) and peritoneal macrophages (Mphi) in response to proinflammatory and immunoregulatory cytokines, including GM-CSF, IFN-gamma, TGF-beta2, IL-4, IL-10 and viral IL-10. In addition, ratio-RT-PCR expression analysis of the duplicated H2-Mbeta-chain loci demonstrates for the first time that Mbl and Mb2 genes are differentially expressed in individual APC types. Mb2 is preferentially expressed in IL-4, GM-CSF, IL-10, vIL-10 and IFN-gamma stimulated splenic B cells, whereas splenic DCs express both Mb genes at almost equal levels. In contrast, peritoneal Mphi express predominantly Mb2 but stimulation with IFN-gamma induces a switch towards Mb1 expression. These data suggest a common mechanism that regulates coordinate expression of H2-M and MHC class II genes in professional APCs. Differential expression of Mb1 and Mb2, and by consequence alternative H2-M isoforms (Malphabeta1 or Malphabeta2), may influence the nature of the peptide repertoire presented by different APC types.     

NKT cells direct monocytes into a DC differentiation pathway

Monocytes can differentiate into macrophages or dendritic cells (DCs). The processes that promote their differentiation along one pathway rather than the other remain unknown. NKT cells are regulatory T cells that respond functionally to self and foreign antigens presented by CD1d molecules. Hence, in addition to contributing to antimicrobial responses, they may carry out autoreactively activated functions when there is no infectious challenge. However, the immunological consequences of NKT cell autoreactivity remain poorly understood. We show here that human NKT cells direct monocytes to differentiate into immature DCs. The ability to induce monocyte differentiation was CD1d-dependent and appeared specific to NKT cells. Addition of exogenous antigens or costimulation from IL-2 was not required but could enhance the effect. DC differentiation was a result of NKT cell secretion of GM-CSF and IL-13, cytokines that were produced by the NKT cells upon autoreactive activation by monocytes. NKT cells within PBMC samples produced GM-CSF and IL-13 upon exposure to autologous monocytes directly ex vivo, providing evidence that such NKT cell-autoreactive responses can occur in vivo. These results show that when NKT cells are activated by autologous monocytes, they are capable of providing factors that specifically direct monocyte differentiation into immature DCs. Thus, autoreactively activated NKT cells may contribute to the maintenance of the immature DC population, and microbial infection or inflammatory conditions that activate NKT cells further could stimulate them to promote an increased rate of DC differentiation.     

Interferon-alpha disables dendritic cell precursors: dendritic cells derived from interferon-alpha-treated monocytes are defective in maturation and T-cell stimulation

Dendritic cells (DC) can be derived from monocytes in vitro by culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). It is unknown whether this regimen reflects DC differentiation from blood precursors under physiological conditions. Induction of DC development from monocytes by interferon-alpha (IFN-alpha) may occur in vivo during infection or inflammation and thus may represent a more physiological approach to DC differentiation in vitro. Here, we show that incubation of GM-CSF-cultured monocytes with IFN-alpha does not induce DC differentiation: cells maintain their original phenotype and cytokine secretion pattern. Even after stimulation with pro-inflammatory or T-cell-derived activation signals, IFN-alpha-treated monocytes do not develop DC characteristics. Addition of IL-4 during stimulation of IFN-alpha-treated monocytes results in the rapid development of DC-like cells expressing co-stimulatory molecules, CD83 and chemokine receptor CCR7, indicating that some degree of developmental plasticity is preserved. However, DC pre-activated with IFN-alpha are less effective in inducing allogeneic or antigen-specific autologous T-cell proliferation, produce less IL-12 and express lower levels of CCR7 compared to DC generated by culture with GM-CSF and IL-4. Incubating GM-CSF-cultured monocytes simultaneously with IFN-alpha and IL-4 does not affect phenotypic maturation of DC, but reduces IL-12 production upon pro-inflammatory activation. We conclude that: (1) IFN-alpha fails to induce DC differentiation and thus cannot replace IL-4 in generating DC from monocytes in vitro; and (2) the presence of IFN-alpha prior to or during differentiation of DC from monocyte precursors alters their response to maturation stimuli and may affect their capacity to stimulate T helper type 1 immune responses in vivo.     

Airway epithelial IL-15 transforms monocytes into dendritic cells

IL-15 has recently been shown to induce the differentiation of functional dendritic cells (DCs) from human peripheral blood monocytes. Since DCs lay in close proximity to epithelial cells in the airway mucosa, we investigated whether airway epithelial cells release IL-15 in response to inflammatory stimuli and thereby induce differentiation and maturation of DCs. Alveolar (A549) and bronchial (BEAS-2B) epithelial cells produced IL-15 spontaneously and in a time- and dose-dependent manner after stimulation with IL-1beta, IFN-gamma, or TNF-alpha. Airway epithelial cell supernatants induced an increase of IL-15Ralpha gene expression in ex vivo monocytes, and stimulated DCs enhanced their IL-15Ralpha gene expression up to 300-fold. Airway epithelial cell-conditioned media induced the differentiation of ex vivo monocytes into partially mature DCs (HLA-DR+, DC-SIGN+, CD14+, CD80-, CD83+, CD86+, CCR3+, CCR6(+), CCR7-). Based on their phenotypic (CD123+, BDCA2+, BDCA4+, BDCA1(-), CD1a-) and functional properties (limited maturation upon stimulation with LPS and limited capacity to induce T cell proliferation), these DCs resembled plasmacytoid DCs. The effects of airway epithelial cell supernatants were largely blocked by a neutralizing monoclonal antibody to IL-15. Thus, our results demonstrate that airway epithelial cell-conditioned media have the capacity to differentiate monocytes into functional DCs, a process substantially mediated by epithelial-derived IL-15.     

Role of the cytokine environment and cytokine receptor expression on the generation of functionally distinct dendritic cells from human monocytes

Myeloid dendritic cells (DC) and macrophages evolve from a common precursor. However, factors controlling monocyte differentiation toward DC or macrophages are poorly defined. We report that the surface density of the GM-CSF receptor (GM-CSFR) alpha subunit in human peripheral blood monocytes varies among donors. Although no correlation was found between the extent of GM-CSFR and monocyte differentiation into DC driven by GM-CSF and IL-4, GM-CSFR expression strongly influenced the generation of CD1a(+) dendritic-like cells in the absence of IL-4. CD1a(+) cells generated in the presence of GM-CSF express CD40, CD80, MHC class I and II, DC-SIGN, MR, CCR5, and partially retain CD14 expression. Interestingly, they spontaneously induce the expansion of CD4(+) and CD8(+) allogeneic T lymphocytes producing IFN-gamma, and migrate toward CCL4 and CCL19. Upon stimulation with TLR ligands, they acquire the phenotypic features of mature DC. In contrast, the allostimulatory capacity is not further increased upon LPS activation. However, by blocking LPS-induced IL-10, a higher T cell proliferative response and IL-12 production were observed. Interestingly, IL-23 secretion was not affected by endogenous IL-10. These results highlight the importance of GM-CSFR expression in monocytes for cytokine-induced DC generation and point to GM-CSF as a direct player in the generation of functionally distinct DC.     

Dendritic cells generated in the presence of interferon-alpha stimulate allogeneic CD4+ T-cell proliferation: modulation by autocrine IL-10, enhanced T-cell apoptosis and T regulatory type 1 cells

Dendritic cells (DCs) generated in the presence of IFN-alpha (IFN-DCs) exhibit high expression of major histocompatibility and co-stimulatory molecules and a potent ability to stimulate CD8(+) T-cell responses. Here, we found that IFN-DCs were more potent stimulators of bulk and purified CD8(+) T-cell proliferation, as compared with IL-4-DCs. In contrast, IFN-DCs were less efficient than IL-4-DCs in stimulating allogeneic CD4(+) T-cell proliferation, due to a weak induction of naive CD4(+)CD45RO(-) T-cell proliferation by these DCs. However, both DC populations induced similar levels of proliferation of memory CD4(+)CD45RO(+) T cells. IFN-DCs and IL-4-DCs exhibited a similar phenotype and production of IL-10 following maturation induced by CD40 ligation. In contrast, IFN-DCs produced higher levels of IL-10 during the first days of differentiation. In addition, neutralization of IL-10 during the differentiation of DCs increased the expression of DC-LAMP and MHC class II by IFN-DCs, and the ability of IFN-DCs to stimulate allogeneic CD4(+) T-cell proliferation at similar levels, than IL-4-DCs. Independently of IL-10 production, IFN-DCs were found to induce higher levels of CD4(+)T-cell apoptosis, this effect being more sticking on naive T cells. Finally, we demonstrated that IFN-DCs induced a differentiation bias of naive CD4(+) T cells towards Th1 and Tr1 cells, compared to IL-4-DCs. Taken together, these results indicate that, despite the induction of Tr1 cells and enhanced apoptosis of naive CD4(+) T cells, IFN-DCs are potent stimulators of CD8(+) and memory CD4(+) T cells, and induce a strong polarization of naive CD4(+) T cells towards Th1 cells, further supporting their use in immune-based therapy.     

Generation and characterization of ovine dendritic cells derived from peripheral blood monocytes

Dendritic cells (DCs) are professional antigen‐presenting cells with a highly immunostimulatory function and the capacity to activate naïve T cells. In recent years the rapid progress in mouse and human DC research can be mainly attributed to the generation of DCs from precursor cells in vitro, although a lack of reagents has hampered DC research in many large animal models. Here we describe the generation and characterization of ovine monocyte‐derived DCs in vitro. In addition to the characteristic morphology and non‐adherence of DCs, peripheral blood mononuclear cell monocytes cultured with ovine granulocyte–macrophage colony‐stimulating factor (GM–CSF) and interleukin‐4 (IL‐4) expressed CD11c and major histocompatibility complex (MHC) class II, but did not express CD14. High levels of endocytosis and an ability to stimulate antigen‐specific proliferation of CD4 T lymphocytes were also demonstrated.     

Differentiation of monocytes into CD1a- dendritic cells correlates with disease progression in HIV-infected patients

Monocyte differentiation into dendritic cells (DCs) depends on microenvironmental conditions. In this study, the capacity of human monocytes to differentiate into mature DCs and their ability to induce an antiviral immune response was investigated in HIV-infected patients. In healthy subjects, monocytes differentiate into CD1a+ DCs in the presence of granulocyte macrophage colony-stimulating factor and interleukin (IL)-4 and matured in the presence of lipopolysaccharide. Here, we found that in 30% and 45% of HIV-infected white and African subjects, respectively, monocytes gave rise to a homogeneous CD1a* DC population. In the patients who gave rise only to the CD1a* DCs, this population spontaneously produced IL-10 but not IL-12, and induced a T helper 2-like immune response when cultured with human T cells isolated from cord blood mononuclear cells. In patients with monocytes differentiated into CD1a* DCs, a high percentage of HIV-specific CD4 T cells producing IL-4 were seen in the peripheral blood. Furthermore, differentiation of monocytes into DCs with CD1a* phenotype correlated with low CD4 T-cell counts and high viral loads in HIV-infected subjects. These results suggest that the differentiation of monocytes into CD1a* DCs may be a phenotypic marker associated with progression of the disease.