Culture In GM-CSF Research Articles

Regulation of DC metabolism by nitric oxide in murine GM-CSF cultures.

The CD11c+ MHCII+ compartment within GM-CSF cultures consists of a MHCIIlow CD11bhigh population (GM-Macs) and a MHCIIhigh CD11bint population (GM-DCs), with different metabolic profiles. GM-Macs upregulate iNOS and produce nitric oxide (NO) upon TLR activation inhibiting mitochondrial respiration (OXPHOS) while promoting glycolytic metabolism in GM-DCs, which naturally do not express iNOS.

Impact of aging on monocyte subset production by bone marrow progenitors

Abstract Monocytes can patrol the circulation or be recruited into tissues to become macrophages or monocyte-derived dendritic cells (moDCs). We previously showed that monocytes arise via two independent pathways in mouse bone marrow: Granulocyte-Monocyte Progenitors (GMPs) produce neutrophils and monocytes, and Monocyte-DC Progenitors (MDPs) yield monocytes and conventional and plasmacytoid DCs (cDCs and pDCs). Notably, both pathways produce monocytes that can differentiate into macrophages, but moDCs arise exclusively via the MDP pathway. We also showed that the microbial stimuli LPS and CpG promote monocyte production by GMPs and MDPs, respectively. We are now investigating how aging impacts the balance of monocyte production by GMPs and MDPs. scRNAseq analysis revealed elevated Cd74 and H2 gene expression by steady-state classical monocytes from old mice compared to young mice, with a larger proportion of monocytes expressing these genes. Consistent with this, we observed proportionally more monocytes expressing CD74 and MHCII in the bone marrow and circulation of old mice by flow cytometry, and a higher yield of CD11c+ MHCII+ moDCs in GM-CSF cultures of MDPs and monocytes from old bone marrow. These findings are indicative of elevated moDC production during aging, perhaps reflecting the more inflammatory microenvironment. In ongoing studies, we are investigating whether imbalanced monocyte production by the GMP and MDP pathways underlies increased moDC production during aging.

VLA-1 Binding to Collagen IV Controls Effector T Cell Suppression by Myeloid-Derived Suppressor Cells in the Splenic Red Pulp.

Myeloid-derived suppressor cells (MDSCs) represent a major population controlling T cell immune responses. However, little is known about their molecular requirements for homing and T cell interaction to mediate suppression. Here, we investigated the functional role of the homing and collagen IV receptor VLA-1 (α1β1-integrin) on in vitro GM-CSF generated murine MDSCs from wild-type (WT) and CD49a/α1-integrin (Itga1 −/−) gene-deficient mice. Here, we found that effector (Teff) but not naive (Tn) CD4+ T cells express VLA-1 and monocytes further up-regulated their expression after culture in GM-CSF when they differentiated into the monocytic subset of resting MDSCs (R-MDSCs). Subsequent activation of R-MDSCs by LPS+IFN-γ (A-MDSCs) showed increased in vitro suppressor potential, which was independent of VLA-1. Surprisingly, VLA-1 deficiency did not influence A-MDSC motility or migration on collagen IV in vitro. However, interaction times of Itga1 −/− A-MDSCs with Teff were shorter than with WT A-MDSCs on collagen IV but not on fibronectin substrate in vitro. After injection, A-MDSCs homed to the splenic red pulp where they co-localized with Teff and showed immediate suppression already after 6 h as shown by inhibition of T cell proliferation and induction of apoptosis. Injection of A-MDSCs from Itga1−/− mice showed equivalent homing into the spleen but a reduced suppressive effect. Interaction studies of A-MDSCs with Teff in the subcapsular red pulp with intravital two-photon microscopy revealed also here that MDSC motility and migration parameters were not altered by VLA-1 deficiency, but the interaction times with Teff were reduced. Together, our data point to a new role of VLA-1 adhesion to collagen IV as a prerequisite for extended contact times with Teff required for suppression.

Control of GM-CSF-Dependent Dendritic Cell Differentiation and Maturation by DEF6 and SWAP-70.

Although GM-CSF has been widely used in dendritic cell (DC) research, the mechanisms, factors, and signals regulating steady-state differentiation and maturation of GM-CSF-dependent DCs are insufficiently known. We found that the absence, individually or combined, of the related proteins DEF6 and SWAP-70 strongly enhances differentiation of murine GM-CSF-derived DCs. Contrasting SWAP-70, control through DEF6 does not depend on RHOA activation. DEF6 deficiency leads to expression of the DC-specific transcription factor ZBTB46 and prolonged STAT5 activation in GM-CSF cultures. SWAP-70 and DEF6-mediated restriction of DC differentiation converges mechanistically at the NF-κB pathway. DEF6 acts at early stages of DC differentiation in CD115-cKIT+ myeloid DC progenitors, whereas SWAP-70 acts subsequently. SWAP-70 and DEF6 regulate steady-state DC cytokine expression as well as in vivo accumulation in lymphatic tissue of migratory DCs. Our studies thus elucidate previously unknown roles of two closely related factors with distinct and complementary activities in DC differentiation and steady-state DC function.

GM-CSF Culture Revisited: Preparation of Bulk Populations of Highly Pure Dendritic Cells from Mouse Bone Marrow.

Dendritic cells (DC) loaded with specific peptides are strongly immunogenic for T cells and can be used for cancer immunotherapy. For immunogenic tumors such as melanoma, injection of autologous DC loaded with tumor cell extracts or peptides can induce tumor regression but in only a small proportion of patients. Nevertheless, recent studies on the efficacy of checkpoint blockade for boosting antitumor immunity plus advances in defining tumor neoantigens are stimulating renewed interest in DC immunotherapy. Despite intensive investigation, however, preparation of bulk populations of mature DC has proved difficult, and most preparations contain a significant proportion of potentially tolerogenic immature DC. In this study, we have modified the well-established GM-CSF culture system to prepare substantial quantities of highly pure (>95%) mature DC from mouse bone marrow cells and defined their progenitors. We show that obtaining high yields and purity of DC are heavily dependent on cell density in the cultures and the tempo of addition of growth and maturation stimuli. When loaded with specific peptide, the DC are strongly immunogenic for CD4 and CD8 T cells in vivo and elicit effective antitumor immunity.

Anti-melanoma vaccinal capacity of CD11c-positive and -negative cell populations present in GM-CSF cultures derived from murine bone marrow precursors

We have initially shown that DC/ApoNec vaccine can induce protection against the poorly immunogenic B16F1 melanoma in mice. The population of DC obtained for vaccination after 7days culture with murine GM-CSF is heterogeneous and presents about 60% of CD11c+ DC. Therefore, our purpose was to identify the phenotype of the cells obtained after differentiation and its immunogenicity once injected. DC were separated with anti-CD11c microbeads and the two populations identified in terms of CD11c positivity (DC+ and DC−) were also studied. Approximately 26.6% of the cells in DC+ fraction co-expressed CD11c+ and F4/80 markers and 75.4% were double positive for CD11c and CD11b markers. DC+ fraction also expressed Ly6G. DC− fraction was richer in CD11c-/F4/80+ macrophages (44.7%), some of which co-expressed Ly6G (41.8%), and F4/80-/Ly6-G+ neutrophils (34.6%). Both DC+ and DC− fractions displayed similar capacity to phagocyte and endocyte antigens and even expressed levels of MHC Class II and CD80, CD83 and CD86 costimulatory molecules similar to those in the DC fraction. However, only DC/ApoNec vaccine was capable to induce protection in mice (p<0.01). After 24h co-culture, no detectable level of IL-12 was recorded in DC/ApoNec vaccine, either in supernatant or intracellularly. Therefore, the protection obtained with DC/ApoNec vaccine seemed to be independent of the vaccine's ability to secrete this inflammatory cytokine at the time of injection. In conclusion, we demonstrated that all cell types derived from the culture of mouse bone marrow with GM-CSF are necessary to induce antitumor protection in vivo.

Defective Flt3L driven DC development in the absence of Ikaros (111.35)

Abstract Ikaros null (Ik-) mice have impaired dendritic cell (DC) development. This defect is recapitulated when Ik- bone marrow (BM) is cultured with Flt3L, a DC growth factor. Ik- BM cultures generate significantly fewer DCs than their wild type (WT) counterparts. This phenotype is specific to Flt3L-driven DC developments as Ik- GM-CSF cultures generate normal numbers of DCs. Time course gene expression analyses of Flt3L-cultures show that genes important for DC development such as Irf4, Irf8 and Spib are variably under-expressed in both Ik- progenitors and mature DCs, with Spib being the most affected. These data demonstrate that low expression of DC genes is not solely due to decreased DC differentiation and suggest that Ikaros may regulate their expression, which we are now exploring. The DCs that develop in Ik- Flt3L cultures are also qualitatively different from their WT counterparts. They have an activated phenotype, implicating Ikaros in regulation of DC function as well. Enforced expression of full-length Ikaros (Ik1) in Ik- BM cannot restore Flt3L-driven DC development, suggesting a different Ikaros isoform may be needed for DC rescue. Studies now underway will define Ikaros isoform expression during DC development and determine if enforced expression of discrete Ikaros isoforms or the Flt3 receptor itself can restore Flt3L-driven DC development in Ik- BM. These studies will place Ikaros relative to Flt3 signals during DC development and define its molecular role.

C/EBPβ mediates the differentiation of peritoneal, but not splenic or liver, tissue macrophages (153.28)

Abstract Macrophages are dispersed throughout the body but exhibit tissue-specific phenotypes and functions. The extracellular and transcriptional pathways that regulate the generation of tissue-specific macrophages are largely unknown. In mice, the peritoneal cavity contains two distinct populations of macrophages, small (CD11b+F4/80lowCD93-) and large (CD11b+F4/80hiCD93+) peritoneal macrophages (SPM and LPM, respectively). Here, we show that mice deficient for the transcription factor C/EBPβ have increased numbers of SPM compared to control littermates, but lack LPM. In C/EBPβ-sufficient and deficient mice, however, splenic (MOMA-1+ and F4/80+) and liver (F4/80+) macrophage compartments are equivalent. When adoptively transferred into C/EBPβ-/- mice, wild-type SPM give rise to LPM, revealing a differentiative pathway from SPM to LPM that requires C/EBPβ. A role for C/EBPβ in the differentiation of certain macrophages is also evident in vitro, as bone marrow cells from C/EBPβ-sufficient and -deficient mice produce macrophages equivalently in cultures containing M-CSF, but C/EBPβ-/- bone marrow cells fail to generate normal macrophages in GM-CSF cultures. These findings demonstrate that tissue-specific macrophages arise through distinct transcriptional pathways, and reveal C/EBPβ as a master regulator of differentiation for a restricted subset of tissue macrophages.

SHIP Is Required for Dendritic Cell Maturation

Although several groups have investigated the role of SHIP in macrophage (M) development and function, SHIP's contribution to the generation, maturation, and innate immune activation of dendritic cells (DCs) is poorly understood. We show herein that SHIP negatively regulates the generation of DCs from bone marrow precursors in vitro and in vivo, as illustrated by the enhanced expansion of DCs from SHIP(-/-) GM-CSF cultures, as well as increased numbers of DCs in the spleens of SHIP-deficient mice. Interestingly, however, these SHIP(-/-) DCs display a relatively immature phenotype and secrete substantially lower levels of IL-12 after TLR ligand stimulation than wild type DCs. This, in turn, leads to a dramatically reduced stimulation of Ag-specific T cell proliferation and Th1 cell responses in vitro and in vivo. This immature phenotype of SHIP(-/-) DCs could be reversed with the PI3K inhibitors LY294002 and wortmannin, suggesting that SHIP promotes DC maturation by reducing the levels of the PI3K second messenger phosphatidylinositol-3,4,5-trisphosphate. These results are consistent with SHIP being a negative regulator of GM-CSF-derived DC generation but a positive regulator of GM-CSF-derived DC maturation and function.

An improved method to generate equine dendritic cells from peripheral blood mononuclear cells: Divergent maturation programs by IL-4 and LPS

Equine dendritic cells (eqDC) can be generated from peripheral blood monocytes by propagation in GM-CSF and IL-4. Despite similarities with the generation of human DC, we found significant improvements for eqDC generation and functional influences on eqDC maturation. The fractionation of peripheral blood mononuclear cells (PBMC) by two subsequent gradients at densities of 1.090 and 1.077 as well as an adherence step in AIM V ® medium on dishes coated with extracellular matrix components (Primaria ®) improved the purity and yield of DC. After 3 days, eqDC cultures with GM-CSF alone developed into three subsets of (i) MHC II neg cells, (ii) MHC II low immature, endocytic cells and (iii) MHC II high spontaneously mature, non-endocytic DC. The immature DC fraction of the GM-CSF cultures matured, as detected by MHC II up-regulation, upon LPS exposure overnight. DC cultures in GM-CSF plus IL-4 resulted in higher cell yields, a loss of the immature MHC II low population but increased mature MHC II high DC, suggesting maturation. However, the MHC II high DC fraction was still endocytically active and did not lose their endocytic function after LPS treatment. They marginally up-regulated MHC II expression but this did not result in an enhanced stimulation of an allogeneic mixed lymphocyte reaction. However, LPS treatment clearly induced mRNA for IL-12p35 and p40, which was not observed by addition of IL-4 alone. Together our data indicate that IL-4 and LPS induce two different maturation programs. IL-4 induces a semi-maturation where the cells are still endocytic, which can be further matured to secrete cytokines in a second step by LPS.

Phenotypic and functional differentiation of KG-1 into dendritic-like cells

The cell line KG-1 has been used as an in vitro model for human dendritic cell (DC) differentiation. We have investigated the response of KG-1 cells to stimulation with a number of factors known to induce differentiation and/or maturation of DCs in vitro. KG-1 cells showed no differentiation in response to LPS, CpG oligodeoxynucleotide or CD40 ligation. Culture in the presence of TNF- α induced some differentiation, but only treatment with PMA and ionomycin (with or without prior culture in GM-CSF and IL-4) induced morphological and phenotypic changes consistent with DC-like maturation, and even these maximally differentiated KG-1 cells showed lower levels of surface marker expression, macromolecular endocytosis, and ability to stimulate in allogeneic MLR compared with in vitro monocyte-derived DCs. Our data show that KG-1 cells differentiate in vitro into cells with DC-like functional characteristics under the influence of strong inducers of cellular activation, but lack the potency of mature DCs in key aspects of professional antigen presenting cells.

Superior Acute Myeloid Leukemia-Specific T Cell Responses Using Dendritic Cells Pulsed with Apoptotic Bodies, vs.Tumor Lysates or mRNA.

Dendritic cells (DCs), which hold promise for cancer immunotherapy have two distinct functions. Immature DCs internalize antigens; while mature DCs trigger native T cell activation. To date, no consensus has been reached concerning the optimal source of tumor antigen nor the mode of antigen uptake for efficient cross-priming of tumor specific response. Reports have demonstrated that human DCs can acquire relevant antigens and stimulate MHC class I-restricted cytotoxic T lymphocytes (CTLs) by phagocytosing apoptotic tumor cells. Apoptosis was established to be the critical trigger of cross-priming in this process. Apoptotic bodies form when cells undergo programmed cell death. In this study, three acute myelogenous leukemia in (AML) antigen preparations were compared in an autologous setting.Apoptotic bodies,Tumor lysateTumor mRNAMethods: DCs were generated by CD14 magnetic selection of peripheral blood progenitor cells (PBPCs) and six days of culture in GM-CSF and IL-4. Tumor lysate was made by repeated freezing and thawing of patient AML cells (leukodepletion product >95% blasts); mRNA was extracted using a kit (Qiagen); apoptotic bodies were generated with UVB irradiation of a fraction of the same AML product. The rate of apoptosis was detected using Annexin V FITC flow cytometry. The DCs were loaded with mRNA by electroporation. The DCs were loaded with lysate and apoptotic bodies by incubation. Autologous T cells stimulated with DCs loaded with the three differing antigen preparations were compared for interferon(INF)-g production in ELISPOT and proliferation assays.Results: The DCs pulsed with apoptotic bodies; lysate and mRNA expressed similar levels of the costimulatory molecules CD80, CD86, and theDC maturation marker CD83. The internalization rate of apoptotic bodies was about 20% detected by DAPI (label the apoptotic bodies) and DC83 double staining. The lysate pulsed DC and mRNA pulsed DCs were above 90% detected by flow of trace FITC- albumin and tracer GFP mRNA from previous experiments. Yet IFN-g secretion of T lymphocytes was statistically significant higher (P<0.05), using apoptotic body-loaded DC as compared to total lysate or mRNA. The T cell proliferation stimulated by apoptotic body-loaded DC was also better than that of lysate or mRNA loaded DCs (p<0.05).Conclusions: These preliminary data demonstrate that apoptotic body-loaded DCs induced better Th1 T cell responses than DCs loaded with AML lysate or mRNA. One potential advantage conferred by the use of apoptotic body loaded DCs is that presentation of intracellular proteins in tumor lysates and mRNA that are irrelevant to the induction of anti-tumor responses are eliminated, which may lead to better antigen presentation and consequently an improved Th1 response.

Cell-Autonomous Upregulation of Dendritic Cell Immunocompetence Is Antigen-Dependent.

Introduction: Dendritic cells (DCs) are the master regulators of the adaptive immune response. In previous experiments, the double-loading of DCs with matched sets of MHC class I and class II repertoires (i.e. mRNA and cell lysate derived from the same source) appeared to enhance T-cell responses by maximizing the availability of T-cell help. T-cell help, provided by accessory CD4+ lymphocytes, elevates DC immunocompetence in response to antigen-specific interactions between the T-cell receptor and the peptide antigen/MHC class II complex. While T-cell help is specific and aids in the priming of only cognate CD8+ effectors, the mechanism governing this specificity is not understood. Here, we have explored the hypothesis that the specificity of T-cell help may be initiated by the DC itself upon detection of an antigen-dependent signal: the loading of the DC with matched sets of class I and class II determinants.Methods: To examine this hypothesis, we doubly-loaded DCs with either matched sets of antigenic determinants derived from acute myelogenous leukemia (AML) mRNA and lysate preparations, or, mismatched sets of antigenic preparations derived from AML products, the human TF-1a erythroblastic cell line, or the murine FBMD-1 stromal cell line. Following loading and maturation, we analyzed DCs for markers of immunocompetence such as IL-12 secretion and CD83 surface expression. Using specific siRNA oligonucleotides, we also examined a possible role for the intracellular CD63 tetraspanin in this process. DCs were generated by CD14 magnetic selection of apheresis products and six days of culture in GM-CSF and IL-4. Analysis of immature DCs at this time point by flow cytometry typically showed a CD3+ content equivalent to or less than that of the isotype control. DCs were then loaded with either matched or mismatched sets of determinants (as described above) and matured.Results: Matched-loaded DCs exhibited a 4-fold increase in IL-12 secretion over unloaded DCs while mismatch-loaded DCs showed only a 0.7-fold increase (similar to controls) (p = 0.0086). Moreover, matched-loaded DCs demonstrated a 21% increase in CD83 surface expression over unloaded/singly-loaded controls while mismatch-loaded DCs showed only a 6% increase (statistically identical to controls) (p = 0.009). In a single-antigen model system, electroporation of CD63 siRNA could reduce IL-12 secretion from matched-loaded DCs by 60% in comparison to matched-loaded DCs electroporated with non-targeting siRNAs. Secretion of non-Th-1 cytokines (i.e. IL-10) was unaffected.Conclusions: It appears that DC immunocompetence may be upregulated in a cell-autonomous, antigen-dependent fashion. Such upregulation is induced by the loading of DCs with matched sets of MHC class I and class II antigenic determinants and does not occur if DCs are loaded with mismatched determinants. CD63 appears to be involved in this process. CD63 is a member of the tetraspanin family of integral membrane proteins, molecules that facilitate the interaction of membrane and intracellular signaling complexes. In DCs, CD63 is localized exclusively to lysosomal exosomes, sites at which MHC class II, MHC class I, and phagocytosed antigens also co-localize. The data are suggestive of a cross-licensing model by which T-cell help might first be solicited by DCs loaded with matched antigenic determinants. Permission granted by a receptive DC, the helper T-cell might then license DC priming of CD8+ responses. Confirmatory studies are in progress.

Murine respiratory tract dendritic cells: isolation, phenotyping and functional studies

Respiratory tract dendritic cells (RTDC) form a contiguous subepithelial network within the nasorespiratory tract bridging innate and acquired immunity and have been implicated in nasal mucosal tolerance induction. Discrepancies exist between isolation techniques with respect to phenotype and function of RTDC. Therefore, the aim of this study was to modify previous methods to provide a consistent isolation method whilst maintaining good cell viability and enriched cell numbers so as to facilitate further phenotype and functional studies of murine RTDC. RTDCs isolated by enzyme digestion, Percoll density gradient centrifugation and overnight GM-CSF culture followed by MACS separation retain an archetypical immature dendritic cell phenotype, characterised by MHCII low CD40 neg CD86 neg CD80 neg CD11c low cell surface expression. Splenic-derived DC (SDC) isolated conformed to a day 1 in vitro phenotype; MHCII low CD40 neg CD86 low CD80 neg CD11c low but can further mature phenotypically in vitro. Both RTDC and SDC processed and presented antigen efficiently to T cells in vitro. Using such modified isolation procedures for RTDCs, we have developed a consistent method of RTDC enrichment, which maintains the immature phenotype and functional antigen presenting capability.

Interleukin-3 in Cooperation with Transforming Growth Factor β Induces Granulocyte Macrophage Colony Stimulating Factor Independent Differentiation of Human CD34+ Hematopoietic Progenitor Cells into Dendritic Cells with Features of Langerhans Cells

Recently, we have reported that M-CSF in cooperation with TGF-beta1 can induce Langerhans cell (LC) development from hematopoietic progenitor cells (HPCs) without GM-CSF. In the present study, we examined whether TGF-beta1 changes the differentiation of HPCs induced by IL-3 towards LC development. We cultured HPCs in a serum-free medium in the presence of IL-3 and a combination cytokines including Flt3L, SCF, and TNF-alpha with or without TGF-beta1. DCs induced by the IL-3 culture (IL-3 DCs) did not significantly differ from those induced by the GM-CSF culture (GM-CSF DCs). Namely, both expressed CDla, F-cadherin, and Langerin in the presence of TGF-beta1 and stimulated allogeneic T cells at a similar magnitude. In contrast to GM-CSF DCs, IL-3 DCs lacked the expression of Birbeck granules (BGs) in spite of their expression of Langerin. When we compared the expression of Langerin between these two DCs, however, it became clear that both Langerin protein and mRNA were significantly lower in IL-3 DCs than in GM-CSF DCs. These studies again demonstrated the ability of TGF-beta1 to polarize the differentiation of HPCs induced by IL-3 towards LC development, although IL-3 DCs were unable to form BGs partly because of their poor ability to induce Langerin.

Immunotherapy of NOD mice with bone marrow-derived dendritic cells.

We evaluated two bone marrow-derived dendritic cell (DC) populations from NOD mice, the murine model for type 1 human diabetes. DCs derived from GM-CSF [granulocyte/macrophage colony-stimulating factor] + interleukin (IL)-4 cultures expressed high levels of major histocompatibility complex (MHC) class II, CD40, CD80, and CD86 molecules and were efficient stimulators of naive allogeneic T-cells. In contrast, DCs derived from GM-CSF cultures had low levels of MHC class II costimulation/activation molecules, were able to take up mannosylated bovine serum albumin more efficiently than GM + IL-4 DCs, and were poor T-cell stimulators. The two DC populations migrated to the spleen and pancreas after intravenous injection. To determine the ability of the two DC populations to modulate diabetes development, DCs were pulsed with a mixture of three islet antigen-derived peptides or with medium before injection into prediabetic NOD mice. Despite phenotypic and functional differences in vitro, both populations prevented in vivo diabetes development. Pulsing of the DCs with peptide in vitro did not significantly improve the ability of DCs to prevent disease, which suggests that DCs may process and present antigen to T-cells in vivo. In addition, we detected GAD65 peptide-specific IgG1 antibody responses in DC-treated mice. Overall, these results suggest that a Th2 response was generated in DC-treated mice. This response was optimal when using GM + IL-4 DCs, which suggests that the balance between regulatory Th2 and effector Th1 cells may have been altered in these mice.

Type I IFNs Enhance the Terminal Differentiation of Dendritic Cells

This study identifies type I IFNs as activating cytokines in a serum-free system in which human dendritic cells (DC) were generated from CD34+ progenitor cells. After 14 days of culture in GM-CSF, TNF-alpha, and IL-4, CD34+ progenitors gave rise to a population of large, immature DC expressing CD1a and CD11b but lacking CD14, CD80, CD83, CD86, and CMRF44. During the next 2 wk, this population spontaneously matured into nonadherent, CD1a(low/-), CD11b(low/-), CD14-, CD80+, CD83+, CD86+, CMRF44+ DC with high allostimulatory activity in the MLR. To examine which factors influenced this maturation, 25 different cytokines or factors were added to the immature DC culture. Only type I IFNs (alpha or beta) accelerated this maturation in a dose-dependent manner, so that after only 3 days the majority of large cells acquired the morphology, phenotype, and function characteristics of mature DC. Furthermore, supernatants from cultures containing spontaneously maturing DC revealed low levels of endogenous IFN production. Because of the similarity of the activation of DC in our culture system with the phenotypic and functional changes observed during Langerhans cells activation and migration in vivo, we investigated the effect of IFN-alpha on human Langerhans cell migration. IFN-alpha also activated the migration of human split skin-derived DC, demonstrating that this effect was not limited to DC derived in vitro from hemopoietic progenitor cells. DC activation by type I IFNs represents a novel mechanism of immunomodulation by these cytokines, which could be important during antiviral responses and autoimmune reactions.

Expression of the co-stimulatory molecule B7 on melanoma cells.

The induction of T-cell responses against tumor cells is believed to depend on both recognition of antigen and receipt of co-stimulatory signals from interaction of ligands such as B7 with its receptors CD28 or CTLA-4 on T cells. In the present study the expression of B7 on cultured human melanoma cells was studied at the mRNA level by reverse PCR analysis and surface expression by flow cytometric analysis with monoclonal antibodies (MAbs). PCR analysis revealed mRNA for B7 in 3 of 6 (50%) cultured primary melanoma and 8 of 19 (42%) cultures of metastatic melanoma. Analysis of B7 expression by flow cytometry using the BB1 MAb revealed low levels of expression in 3 of 10 melanoma that had mRNA for B7. In 2 of the latter (but not 4 other PCR+ lines) expression could be increased by culture in GM-CSF, IL-2, IFN-gamma and IFN-alpha 2. Our results indicate that although mRNA for B7 is present in 40-50% of melanoma cell lines, expression at the protein level is at low or undetectable levels in the majority of the cell lines. Expression of B7 protein was also not detected in studies on tissue sections from 11 primary and 9 metastatic melanomas.

Differential and synergistic effects of human granulocyte-macrophage colony-stimulating factor and human granulocyte colony-stimulating factor on hematopoiesis in human long-term marrow cultures

The ability of granulocyte-macrophage colony-stimulating factor (GM- CSF) and G-CSF to influence hematopoiesis in long-term cultures (LTC) of human marrow was studied by cocultivating light density normal human marrow cells with human marrow fibroblast feeders engineered by retroviral infection to constitutively produce one of these growth factors. Feeders producing stable levels of 4 ng/mL GM-CSF or 20 ng/mL G-CSF doubled the output of mature nonadherent cells. The numbers of both colony forming unit-GM (CFU-GM) and erythroid burst forming unit (BFU-E) in the G-CSF LTC were also increased (twofold and fourfold, respectively, after 5 weeks in culture), but this effect was not seen with the GM-CSF feeders. At the time of the weekly half medium change 3H-thymidine suicide assays showed primitive adherent layer progenitors in LTC to be quiescent in both the control and GM-CSF cultures. In contrast, in the G-CSF cultures, a high proportion of primitive progenitors were in S-phase. A single addition of either recombinant GM- CSF or G-CSF to LTC in doses as high as 80 ng/mL and 150 ng/mL, respectively, failed to induce primitive progenitor cycling. However, three sequential daily additions of 150 ng/mL G-CSF did stimulate primitive progenitors to enter S-phase and a single addition of 5 or 12.5 ng/mL of G-CSF together with 10 ng/mL GM-CSF was able to elicit the same effect. Thus, selective elevation of G-CSF in human LTC stimulates proliferation of primitive clonogenic progenitors, which may then proceed through to the terminal stages of granulopoiesis. In contrast, the effects of GM-CSF in this system appear limited to terminally differentiating granulopoietic cells. However, when both GM- CSF and G-CSF are provided together, otherwise biologically inactive doses show strong stimulatory activity. These findings suggest that the production of both of these growth factors by normal stromal cells may contribute to the support and proliferation of hematopoietic cells, not only in LTC, but also in the microenvironment of the marrow in vivo.

Role of granulocyte/macrophage colony-stimulating factor in the regulation of murine alveolar macrophage proliferation and differentiation.

Granulocyte/macrophage (GM)-CSF is one of the hemopoietic growth factors that stimulates neutrophilic granulocyte and macrophage production by bone marrow progenitor cells. In this study, the effect of GM-CSF on the growth and differentiation of murine pulmonary alveolar macrophages (PAM) was investigated. In the presence of GM-CSF, normal murine PAM were induced to proliferate and develop into macrophage colonies with a dose-response curve similar to that of bone marrow GM colony-forming cells. PAM also responded to CSF-1, a lineage-restricted growth factor, but required much higher doses of CSF-1 and a longer incubation time for optimal colony formation. The proliferative response of PAM to CSF-1, however, was greatly enhanced by the concurrent addition of low doses of GM-CSF. In contrast, low doses of CSF-1 failed to potentiate the proliferative response of PAM to GM-CSF. Macrophages derived from GM-CSF cultures were rounder and less stretched and possessed less FcR-mediated phagocytic activity than cells produced in CSF-1 cultures. A study with hydrocortisone-induced monocytopenia showed that nearly one half of lung macrophages may be sustained by local proliferation of PAM without the continuous migration of blood monocytes. This study suggests that GM-CSF may play a major role in the production of PAM by two modes of action, 1) direct stimulation of cell proliferation and 2) enhancement of their responsiveness to CSF-1, thereby producing more mature and functionally competent macrophages.