Low Doses Of GM-CSF Research Articles

Inhibition of PI3K/C/EBPβ axis in tolerogenic bone marrow-derived dendritic cells of NOD mice promotes Th17 differentiation and diabetes development

Dendritic cells (DCs) are key regulators of the adaptive immune response. Tolerogenic dendritic cells play a crucial role in inducing and maintaining immune tolerance in autoimmune diseases such as type 1 diabetes in humans as well as in the NOD mouse model. We previously reported that bone marrow-derived DCs (BM.DCs) from NOD mice, generated with a low dose of GM-CSF (GM/DCs), induce Treg differentiation and are able to protect NOD mice from diabetes. We had also found that the p38 MAPK/C/EBPβ axis is involved in regulating the phenotype, as well as the production of IL-10 and IL-12p70, by tolerogenic GM/DCs. Here, we report that the inhibition of the PI3K signaling switched the cytokine profile of GM/DCs toward Th17-promoting cytokines without affecting their phenotype. PI3K inhibition abrogated the production of IL-10 by GM/DCs, whereas it enhanced their production of IL-23 and TGFβ. Inhibition of PI3K signaling in tolerogenic GM/DCs also induced naive CD4+ T cells differentiation toward Th17 cells. Mechanistically, PI3K inhibition increased the DNA-binding activity of C/EBPβ through a GSK3-dependent pathway, which is important to maintain the semimature phenotype of tolerogenic GM/DCs. Furthermore, analysis of C/EBPβ−/− GM/DCs demonstrated that C/EBPβ is required for IL-23 production. Of physiological relevance, the level of protection from diabetes following transfusion of GM/DCs into young NOD mice was significantly reduced when NOD mice were transfused with GM/DCs pretreated with a PI3K inhibitor. Our data suggest that PI3K/C/EBPβ signaling is important in controlling tolerogenic function of GM/DCs by limiting their Th17-promoting cytokines.

Analysis of the Application of Regulatory Dendritic Cells in Kidney Transplantation

Kidney transplantation (KT_x) is the optimum therapy for end-stage renal diseases. However, long-term usage of immunosuppressive agents results in various toxicities and side effects, which has been a major obstacle for recipients. How to reduce the dosages of immunosuppressive agents has become a problem that desperately needs to be solved. Among potential methods, cell therapy has great potential, and regulatory dendritic cells (DC_reg) have attracted special attention for their tolerogenic ability. Currently, some DC-based clinical trials are ongoing or have been completed at several research centers, including an immune tolerance trial in KT_x named “The One Study”, in which autologous peripheral blood mononuclear cells were isolated and stimulated with low doses of GM-CSF or immunosuppressive agents. DC_reg generated with different induction methods or from different cell sources may function in different ways in different organs.

Upregulation of Heme Oxygenase-1 Endues Immature Dendritic Cells With More Potent and Durable Immunoregulatory Properties and Promotes Engraftment in a Stringent Mouse Cardiac Allotransplant Model.

Heme oxygenase-1 (HO-1) is critical for the ability of immature dendritic cells (imDCs) to suppress T-cell responses. Induction of high HO-1 expression may markedly improve the tolerogenic capacity of imDCs. Here, we generated bone marrow-derived DCs (BMDCs) from BALB/c mice with low doses of GM-CSF and IL-4. The adherent BMDCs were obtained as imDCs. Upregulation of HO-1 in imDCs (HO-1hi-imDCs) was achieved by cobalt protoporphyrin treatment. HO-1hi-imDCs proved to be more maturation-resistant than conventional imDCs, with an enhanced ability to inhibit allogeneic T-cell proliferation stimulated by anti-CD3/CD28 antibodies. When donor-derived DC adoptive transfer was performed in a stringent mouse cardiac allotransplant model, the extent of graft prolongation observed with HO-1hi imDCs was superior to that obtained with conventional imDCs. T-cell activation and proliferation in cardiac allograft recipients was more strongly suppressed in the HO-1hi imDC transfusion group than that in the untreated imDC group. Furthermore, donor HO-1hi imDCs were able to maintain a status of high HO-1 expression and survived longer in the recipient spleens than did untreated imDCs after adoptive transfer. In vitro-generated HO-1hi imDCs had an enhanced tolerogenic capacity to modulate alloimmune responses both in vitro and in vivo, and thus may offer a novel antigen-specific and cost-effective strategy to induce transplant tolerance.

OX40L/Jagged1 Cosignaling by GM-CSF–Induced Bone Marrow-Derived Dendritic Cells Is Required for the Expansion of Functional Regulatory T Cells

Abstract Earlier, we had demonstrated that treatment with low dose of GM-CSF can prevent the development of experimental autoimmune thyroiditis (EAT), experimental autoimmune myasthenia gravis, and type 1 diabetes, and could also reverse ongoing EAT and experimental autoimmune myasthenia gravis. The protective effect was mediated through the induction of tolerogenic CD11C+CD8α− dendritic cells (DCs) and consequent expansion of Foxp3+ regulatory T cells (Tregs). Subsequently, we showed that GM-CSF acted specifically on bone marrow precursors and facilitated their differentiation into tolerogenic dendritic cells (DCs; GM-CSF–induced bone marrow-derived DCs [GM-BMDCs]), which directed Treg expansion in a contact-dependent manner. This novel mechanism of Treg expansion was independent of TCR-mediated signaling but required exogenous IL-2 and cosignaling from DC-bound OX40L. In this study, we observed that OX40L-mediated signaling by GM-BMDCs, although necessary, was not sufficient for Treg expansion and required signaling by Jagged1. Concurrent signaling induced by OX40L and Jagged1 via OX40 and Notch3 receptors expressed on Tregs was essential for the Treg expansion with sustained FoxP3 expression. Adoptive transfer of only OX40L+Jagged1+ BMDCs led to Treg expansion, increased production of IL-4 and IL-10, and suppression of EAT in the recipient mice. These results showed a critical role for OX40L- and Jagged1-induced cosignaling in GM-BMDC–induced Treg expansion.

Antagonistic regulation of macrophage phenotype by M-CSF and GM-CSF: Implication in atherosclerosis

ObjectivesWe characterized the transcriptional profiles of GM-CSF- (GM-MØ) and M-CSF-induced macrophages (M-MØ) and investigated in situ a subset of differentially expressed genes in human and mouse atherosclerotic lesions. Methods and resultsUsing microarrays we identified a number of genes and biological processes differentially regulated in M-MØ vs GM-MØ. By varying in culture the M-CSF/GM-CSF ratio (0–10), a spectrum of macrophage phenotypes was explored by RT-QPCR. M-CSF (10ng/ml) stimulated expression of several genes, including selenoprotein-1 (SEPP1), stabilin-1 (STAB1) and CD163 molecule-like-1 (CD163L1) which was inhibited by a low dose of GM-CSF (1ng/ml); M-CSF inhibited the expression of pro-platelet basic protein (PPBP) induced by GM-CSF. Combining Tissue Microarrays/quantitative immunohistochemistry of human aortic lesions with RT-QPCR expression data either from human carotids vs mammary non-atherosclerotic arteries or from the apoE null mice normal and atherosclerotic aortas showed that, STAB1, SEPP1 and CD163L1 (M-CSF-sensitive genes) and PPBP (GM-CSF-sensitive gene) were expressed in both human arterial and apoE null mice atherosclerotic tissues. ConclusionA balance between M-CSF vs GM-CSF defines macrophage functional polarisation and may contribute to the progression of atherosclerosis.

Vaccination with a plasmid DNA encoding HER-2/neu together with low doses of GM-CSF and IL-2 in patients with metastatic breast carcinoma: a pilot clinical trial

BackgroundAdjuvant trastuzumab (Herceptin) treatment of breast cancer patients significantly improves their clinical outcome. Vaccination is an attractive alternative approach to provide HER-2/neu (Her2)-specific antibodies and may in addition concomitantly stimulate Her2-reactive T-cells. Here we report the first administration of a Her2-plasmid DNA (pDNA) vaccine in humans.Patients and MethodsThe vaccine, encoding a full-length signaling-deficient version of the oncogene Her2, was administered together with low doses of GM-CSF and IL-2 to patients with metastatic Her2-expressing breast carcinoma who were also treated with trastuzumab. Six of eight enrolled patients completed all three vaccine cycles. In the remaining two patients treatment was discontinued after one vaccine cycle due to rapid tumor progression or disease-related complications. The primary objective was the evaluation of safety and tolerability of the vaccine regimen. As a secondary objective, treatment-induced Her2-specific immunity was monitored by measuring antibody production as well as T-cell proliferation and cytokine production in response to Her2-derived antigens.ResultsNo clinical manifestations of acute toxicity, autoimmunity or cardiotoxicity were observed after administration of Her2-pDNA in combination with GM-CSF, IL-2 and trastuzumab. No specific T-cell proliferation following in vitro stimulation of freshly isolated PBMC with recombinant human Her2 protein was induced by the vaccination. Immediately after all three cycles of vaccination no or even decreased CD4+ T-cell responses towards Her2-derived peptide epitopes were observed, but a significant increase of MHC class II restricted T-cell responses to Her2 was detected at long term follow-up. Since concurrent trastuzumab therapy was permitted, λ-subclass specific ELISAs were performed to specifically measure endogenous antibody production without interference by trastuzumab. Her2-pDNA vaccination induced and boosted Her2-specific antibodies that could be detected for several years after the last vaccine administration in a subgroup of patients.ConclusionThis pilot clinical trial demonstrates that Her2-pDNA vaccination in conjunction with GM-CSF and IL-2 administration is safe, well tolerated and can induce long-lasting cellular and humoral immune responses against Her2 in patients with advanced breast cancer.Trial registrationThe trial registration number at the Swedish Medical Products Agency for this trial is Dnr151:785/2001.

Immature and Maturation-Resistant Human Dendritic Cells Generated from Bone Marrow Require Two Stimulations to Induce T Cell Anergy In Vitro

Immature dendritic cells (DC) represent potential clinical tools for tolerogenic cellular immunotherapy in both transplantation and autoimmunity. A major drawback in vivo is their potential to mature during infections or inflammation, which would convert their tolerogenicity into immunogenicity. The generation of immature DC from human bone marrow (BM) by low doses of GM-CSF (lowGM) in the absence of IL-4 under GMP conditions create DC resistant to maturation, detected by surface marker expression and primary stimulation by allogeneic T cells. This resistence could not be observed for BM-derived DC generated with high doses of GM-CSF plus IL-4 (highGM/4), although both DC types induced primary allogeneic T cell anergy in vitro. The estabishment of the anergic state requires two subsequent stimulations by immature DC. Anergy induction was more profound with lowGM-DC due to their maturation resistance. Together, we show the generation of immature, maturation-resistant lowGM-DC for potential clinical use in transplant rejection and propose a two-step-model of T cell anergy induction by immature DC.

Low doses of GM-CSF (molgramostim) and G-CSF (filgrastim) after cyclophosphamide (4 g/m2) enhance the peripheral blood progenitor cell harvest: results of two randomized studies including 120 patients

The use of a combination of G-CSF and GM-CSF versus G-CSF alone, after cyclophosphamide (4 g/m2) was compared in two randomized phase III studies, including 120 patients. In study A, 60 patients received 5 x 2 microg/kg/day of G-CSF and GM-CSF compared to 5 mug/kg/day of G-CSF. In study B, 60 patients received 2.5 x 2 microg/kg/day G-CSF and GM-CSF compared to G-CSF alone (5 microg/kg/day). With the aim to collect at least 5 x 10(6)/kg CD34 cells in a maximum of three large volume leukapherises (LK), 123 LK were performed in study A, showing a significantly higher number of patients reaching 10 x 10(6)/kg CD34 cells (21/29 in G+GM-CSF arm vs 11/27 in G-CSF arm, P=0.00006). In study B, 109 LK were performed, with similar results (10/27 vs 15/26, P=0.003). In both the study, the total harvest of CD34 cells/kg was twofold higher in G-CSF plus GM-CSF group (18.3 x 10(6) in study A and 15.85 x 10(6) in study B) than in G-CSF group (9 x 10(6) in study A and 8.1 x 10(6) in study B), a significant difference only seen in multiple myeloma, with no significant difference in terms of mobilized myeloma cells between G-CSF and GM-CSF groups.

Experimental study on induction of tolerance to experimental autoimmune myasthenia gravis by immature dendritic cells.

To investigate the effect of immature dendritic cells (iDCs) on experimental autoimmune myasthenia gravis (MG), iDCs were generated in low dose of GM-CSF, and then they were pulsed with acetylcholine receptor (AchR) and transferred to allogeneic rats. After 3 weeks, all rats were immunized with AchR and complete Freund's adjuvant (CFA) and observed for the corresponding indices of MG for 7 weeks. Our results showed that compared with mature DCs (mDCs) generated at high dose of GM-CSF plus additional stimulation by lipopolysaccharide, iDCs expressed significantly lower levels of MHC-II, CD80 and CD86, and their ability to uptake FITC-Dextran was stronger but the ability of stimulating proliferation of allogeneic T cells were weaker. Like controls, after immunization, all rats transferred with iDCs, mDCs and AchR-pulsed mDCs showed typical symptoms in 4 to 7 weeks. The amplitude of electromyogram wave dropped obviously, the level of serum AchRab increased and neuromuscular junction showed typical damage of MG. In contrast, no conspicuous changes were noted in rats transferred with AchR-pulsed iDCs. The results suggest that iDCs could be generated by inducing bone marrow precursors in low dose of GM-CSF, AchR-pulsed iDCs could induce tolerance of EAMG. The dysfunction of DCs may play an important role in the initiation and maintenance of normal immune response in MG.

Prolongation of Heart Allograft Survival by Immature Dendritic Cells Generated from Recipient Type Bone Marrow Progenitors

Recent studies suggest that particular dendritic cells (DC) subpopulations may be tolerogenic. To test the capacity of different DC subpopulations to modulate allograft rejection, we generated two distinct populations of rat bone marrow-derived DCs (BMDC) with low doses of GM-CSF and IL-4. The non-adherent population (nBMDC), which are the 'classical' DCs was able to stimulate naive allogeneic T cells and could be induced to completely mature using various stimuli. In contrast, the adherent population (aBMDC), which displayed an immature phenotype, was unable to stimulate T cells and was more resistant to maturation. We found that syngeneic aBMDCs, injected one day before transplantation, induced significant prolongation of heart allograft survival and decreased anti-donor humoral and cellular responses. Similarly, syngeneic aBMDCs inhibited T-cell responses to KLH in the spleen but not in lymph node in a KLH immunization model without graft. This effect was not antigen specific and could be reversed using an inhibitor of inducible nitric oxide synthase. This compartmentalized inhibition could be in part explained by the fact that the majority of syngeneic adherent cells administered intravenously were found in the spleen with some of them reaching the T-cell areas. These data suggest that syngeneic aBMDCs can modulate immune responses.

Long-term follow-up of idiotype vaccination in human myeloma as a maintenance therapy after high-dose chemotherapy.

The aim of this work was to evaluate the long-term immunological and clinical impact of idiotype (Id) vaccination in multiple myeloma (MM) patients in first remission after high-dose chemotherapy. A total of 15 patients received a series of subcutaneous (s.c.) injections of autologous Id, conjugated to keyhole limpet hemocyanin (KLH) and in association with low doses of GM-CSF. The median duration of follow-up was 110 months from diagnosis. The vaccine induced immune responses that lasted almost 2 years after the end of treatment. Antibody responses included anti-KLH IgM and IgG (90% of patients), anti-KLH IgE (30%), anti-GM-CSF IgG (20%), anti-Id IgG (20%), and anti-Id IgE (30%). Id-specific delayed type hypersensitivity skin tests were positive in 85% of tested patients. Following vaccination, a progressive recovery of T-cell receptor (TCR) diversity was observed and the loss of oligoclonality was significantly correlated with the remission duration. Although Id/KLH conjugates did not eliminate the residual tumor burden, the median progression-free survival, and overall survival were 40 and 82 months, respectively. A retrospective case-matched analysis showed similar results in patients treated with IFN-alpha alone or in association with steroids. This vaccine formulation can overcome Id-specific immune tolerance by inducing clinical responses that are worthy of further investigation.

Incidence of GM-CSF antibodies in cancer patients receiving GM-CSF for immunostimulation.

We have assessed the immunogenicity profile of GM-CSF in patients with either colorectal carcinoma (CRC) at different stages of disease or with multiple myeloma who were given recombinant human GM-CSF (Escherichia coli-derived) combination therapy. Metastatic CRC patients received a colon carcinoma-reactive antibody and high doses of GM-CSF (425--500 microg/day for 10 days), while other CRC patients and those with myeloma received low doses of GM-CSF (75--80 microg/day for 4 days) as an adjuvant along with appropriate tumor antigens. We found that 55% of the patients (11/20) given high doses of GM-CSF developed GM-CSF-reactive antibodies in comparison with an incidence of only 16% (4/25) in patients given low doses of GM-CSF. None of the patients developed neutralizing antibodies and so the biological effects of GM-CSF were not compromised. A majority of patients (80%) (36/45) also developed antibodies to E. coli proteins that were present as trace contaminants in the GM-CSF product. Treatment with recombinant GM-CSF products, therefore, may induce antibodies against this cytokine depending on the regimen and the amounts used. In this study, multiple immunizations with low doses of GM-CSF was associated with a low incidence of GM-CSF antibodies, which did not neutralize the effect of the cytokine. This therapeutic strategy was effective in inducing adjuvant-type effects and needs to be explored in further clinical trials with this cytokine.

IL-3, GM-CSF and CSF-1 modulate c-fms mRNA more rapidly in human early monocytic progenitors than in mature or transformed monocytic cells.

We have previously shown that a low concentration of CSF-1 (1 U/ml) can trigger human immature monocytic progenitor proliferation in the presence of low concentrations of IL3 (1.7 U/ml). No c-fms down-regulation was observed during this early cell activation. In contrast, 20 U/ml of CSF-1, active on late monocytic cell growth, down-regulated c-fms mRNA expression in immature progenitors and monocytes derived from bone marrow CD34+ cells in culture. We have now extended this study to include the effects of various concentrations of GM-CSF, IL3 and G-CSF on c-fms expression. We observed that high doses of GM-CSF or IL3 down-modulated c-fms mRNA, whereas low doses of GM-CSF or IL3, which were active on early monocytic growth, had no such effect. Similar results were observed at the protein level. In contrast, whatever the concentration, G-CSF had no effect on c-fms mRNA or protein levels. We further observed that the more immature the c-fms expressing progenitors, the faster the down-modulation of this receptor. This was observed within less than 1 hour for immature bone marrow cells, 6 hours for peripheral blood monocytes and even longer for transformed monocytic cells. These results suggest that oncogene expression can be regulated much more rapidly in immature progenitors than was previously observed in mature cells or transformed cell lines.

Treatment of cyclic neutropenia with very low doses of GM-CSF

Treatment of cyclic neutropenia with very low doses of GM-CSF