Combination Of Granulocyte-macrophage Colony-stimulating Factor Research Articles

IL-2/GM-CSF enhances CXCR3 expression in CAR-T cells via the PI3K/AKT and ERK1/2 pathways.

To investigate the effects of cytokines IL-2 and GM-CSF on CXCR3 expression and chemotaxis of CAR-T cells. High lymphocyte infiltration within the tumor is a basic requirement for good results in tumor immunotherapy; C-X-C motif chemokine receptor 3 (CXCR3) is an important factor for the chemotaxis of lymphocytes to tumor tissues. The tumor microenvironment can exhibit diverse cytokine suppression or promote antitumor immunity. Both interleukin (IL)-2 and granulocyte macrophage colony-stimulating factor (GM-CSF) contribute to the regulation of immunosuppression in the tumor microenvironment. However, the effects of IL-2 and GM-CSF on CXCR3 expression on the T cell surface and its mechanisms are not well understood. Here, we explored the effects of polycytokines on CXCR3 expression in chimeric antigen receptor T cells (CAR-T cells) and on HuH-7 in situ hepatocellular carcinoma. Peripheral blood mononuclear cells (PBMCs) were isolated, followed by purifying using CD3 immunomagnetic beads. Cells were divided into three groups. After 24h of activation using CD3/CD28 antibody, T cells were transfected using lentiviral vector, pGC-SV40-EGFP-GPC3-CAR. Three culture methods were used to amplify the transfected T cells. Method 'A' was to incubate T cells with CD3/CD28 antibody; method 'B' was with CD3/CD28 antibody and IL-2 at a final concentration of 1000 U/ml; method 'C' was with method B in addition of GM-CSF at a final concentration of 1000 U/ml. The phosphorylation of MAPK and PI3K/AKT was determined by western blot. The chemotaxis effect of CAR-T cells on Huh-7 HCCIA in situ was assayed by immunofluorescence and immunohistochemistry. The CD3/CD28/IL-2/GM-CSF combination is the most potent for stimulating activated CAR-T cell proliferation and CXCR3 expression in vitro; CD3/CD28/IL-2 induces CAR-T cell expression of CXCR3 through the activation of the PI3K/APK pathway and GM-CSF induces CXCR3 expression in CAR-T cells through the activation of ERK1/2 rather than the p38 MAPK signaling pathway. CAR-GPC3-T cells with high CXCR3 expression showed increased chemotaxis ability to HuH in situ hepatocellular carcinoma, and considerably inhibited the growth of in situ tumors in nude mouse livers. A multi-factorial amplification protocol can effectively improve CXCR3 expression on the surface of activated CAR-T cells in vitro, as well as enhance the chemotaxis ability of CAR-T cells in vivo, which significantly inhibit the growth of liver cancer.

Immune Modulation by Myeloid-Derived Suppressor Cells in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) frequently leads to end-stage renal disease and other life-threatening illnesses. The dysregulation of glomerular cell types, including mesangial cells, endothelial cells, and podocytes, appears to play a vital role in the development of DKD. Myeloid-derived suppressor cells (MDSCs) exhibit immunoregulatory and anti-inflammatory properties through the depletion of L-arginine that is required by T cells, through generation of oxidative stress, interference with T-cell recruitment and viability, proliferation of regulatory T cells, and through the promotion of pro-tumorigenic functions. Under hyperglycemic conditions, mouse mesangial cells reportedly produce higher levels of fibronectin and pro-inflammatory cytokines. Moreover, the number of MDSCs is noticeably decreased, weakening inhibitory immune activities, and creating an inflammatory environment. In diabetic mice, immunotherapy with MDSCs that were induced by a combination of granulocyte-macrophage colony-stimulating factor, interleukin (IL)-1β, and IL-6, reduced kidney to body weight ratio, fibronectin expression, and fibronectin accumulation in renal glomeruli, thus ameliorating DKD. In conclusion, MDSCs exhibit anti-inflammatory activities that help improve renal fibrosis in diabetic mice. The therapeutic targeting of the proliferative or immunomodulatory pathways of MDSCs may represent an alternative immunotherapeutic strategy for DKD.

Experience with Treatment of Pulmonary Alveolar Proteinosis from a Tertiary Care Centre in North India

Pulmonary alveolar proteinosis (PAP) is a disorder characterised by accumulation of lipids and proteins in the alveoli, with the resultant symptoms ranging from indolent subclinical disease to progressive respiratory failure. We retrospectively studied five patients with PAP managed at our center between January 2007 and April 2010, with whole lung lavage (WLL) and/or subcutaneous granulocyte macrophage-colony stimulating factor (GM-CSF) therapy. Patients undergoing WLL under general anaesthesia were supplemented with three months of GM-CSF therapy. Pre- and post-lavage symptom assessment was performed with a 10-point, symptom-based visual analogue scale. Their mean age was 37.6-7.0 years; there were four males. Diagnosis of PAP [idiopathic (n=3); secondary to Nocardia (n=1)] was established by surgical lung biopsy in four patients who presented with respiratory failure. Three patients with idiopathic PAP (n=3) were treated with a combination of GM-CSF and WLL; one patient with secondary PAP was treated with antibiotics alone. In another patient transbronchial lung biopsy was used to diagnose PAP and GM-CSF alone was administered. All patients were followed up for a median period of two years (range 0.5-3 years). Significant improvement was achieved in all the patients with therapeutic WLL and/or GM-CSF. Whole lung lavage appeared to be an effective and safe therapy in patients with PAP. Efficacy of simultaneous administration of GM-CSF and WLL in the treatment of PAP merits further study.

Granulocyte macrophage-colony stimulating factor and keratinocyte growth factor control of early stages of differentiation of oral epithelium.

Oral epithelial differentiation is known to be directed by underlying fibroblasts, but the responsible factor(s) have not been identified. We aimed here to identify fibroblast‐derived factors responsible for oral epithelial differentiation. Primary normal human oral keratinocytes and fibroblasts were isolated from healthy volunteers after informed consent (n = 5) and 3D‐organotypic (3D‐OT) cultures were constructed. Various growth factors were added at a range of 0.1‐100 ng/ml. 3D‐OTs were harvested after ten days and assessed histologically, by immunohistochemistry and the TUNEL method. Epithelium developed in 3D‐OT without fibroblasts showed an undifferentiated phenotype. Addition of granulocyte macrophage‐colony stimulating factor (GM‐CSF) induced expression of cytokeratin 13 in suprabasal cell layers. Admixture of GM‐CSF and keratinocyte growth factor (KGF) induced, in addition, polarization of epidermal growth factor (EGF) receptor and β1‐integrin to basal cell layer and collagen IV deposition. Terminal differentiation with polarization of TUNEL‐positive cells to superficial layers occurred only in the presence of fibroblasts in collagen gels either in direct contact or at distance from normal oral keratinocytes. Taken together, these results show that major aspects of oral epithelial differentiation are regulated by the synergic combination of GM‐CSF and KGF. However, the terminal stage seems to be controlled by other yet unidentified fibroblast‐derived diffusible factor(s).

Nano-hydroxyapatite-evoked immune response synchronized with controllable immune adjuvant release for strengthening melanoma-specific growth inhibition

Concerns about the potential systematic toxicity limit the extensive application of traditional therapeutic drugs for melanoma therapy, nano-hydroxyapatite (nHA) with good biocompatibility and anti-tumor ability could be an alternative choice. In this study, nHA was employed as an anti-tumor biomaterial due to its tumor-specific toxicity. Meanwhile, granulocyte-macrophage colony-stimulating factor (GM-CSF) served as the immune adjuvant to activate the immune response. The delivery platform was fabricated by co-encapsulation of both nHA and GM-CSF into a biocompatible thermosensitive PLGA-PEG-PLGA hydrogel. The results showed that the bio-activities of nHA and GM-CSF could be well-maintained within the hydrogel. Interestingly, the addition of nHA could attenuate the burst release of GM-CSF due to possible protein absorption capacity of nHA, which is beneficial for GM-CSF sustainable release at the tumor site, achieving boosted and prolonged anti-tumor immunity. The in vitro and in vivo data demonstrated that nHA/GM-CSF hydrogel exhibited greater potency to inhibit tumor growth via enhanced CD8+ T-cell response compared with hydrogel and nHA hydrogel groups, contributed by the synergistic effects of nHA and GM-CSF. Overall, the strategy combining nHA and immune adjuvant shows great promise, which largely broadens the choice of combinational therapies for melanoma. Statement of significanceNano-hydroxyapatite (nHA) has been confirmed to specifically inhibit melanoma tumor growth and induce immune response. However, its antitumor efficiency and immunity-evoking capacity are limited. In this study, granulocyte-macrophage colony-stimulating factor (GM-CSF) was introduced to serve as the immune adjuvant. Both of them were encapsulated into a biocompatible thermosensitive PLGA-PEG-PLGA hydrogel. The addition of nHA could attenuate the burst release of GM-CSF due to the interaction with nHA, which is beneficial for GM-CSF sustainable release at tumor site, achieving boosted and prolonged anti-tumor immunity. Anti-tumor immune response could be activated due to the release of tumor-associated antigen and tumor debris induced by the specifically tumor inhibition effect of nHA and GM-CSF. The combination of nHA and GM-CSF could play synergistic inhibiting effect on tumor growth via boosting and prolonging anti-tumor immunity.

Pembrolizumab (PEM) plus granulocyte macrophage colony stimulating factor (GM-CSF) in advanced biliary cancers (ABC): Final outcomes of a phase 2 trial.

444 Background: Immune checkpoint inhibitors (ICI) have limited activity as monotherapy in unselected patients with ABC; the objective response rate (ORR) of PEM was 5.8% in the multicenter phase 2 KEYNOTE-158 trial. GM-CSF modulates immune cells including monocytes and the innate immune response and has demonstrated safety and prolonged survival (OS) in combination with ipilimumab in melanoma. This phase 2 trial was designed to evaluate the efficacy and safety of PEM in combination with GM-CSF in ABC. Methods: Single-center, phase 2 trial with Simon’s 2-stage design and expansion cohort (EC) (NCT02703714). Key eligibility: ABC with progression/intolerance on ≥ 1 standard therapy, no prior ICI, bilirubin ≤1.5xULN. Treatment: PEM 200 mg IV Q21 days plus 2 cycles of GM-CSF 250 µg SC D1-14 Q21 days in cycles 2 and 3 (Stage 1 and EC) or in cycles 1 and 2 (Stage 2). Primary endpoint was objective response rate (ORR) by RECIST 1.1, H0 5% vs. H1 20%. Key secondary endpoints were safety, progression-free survival (PFS), OS, PD-L1 expression. Exploratory endpoints included relationship between efficacy outcomes and anatomic subsite, risk factors, and tumor genotype. Results: Overall, 42 patients enrolled between 5/2016-12/2019: n = 9 in Stage 1, n = 18 in Stage 2, and n = 15 in EC. Overall: median age 61; female 67%; intrahepatic (ICC) 67%, extrahepatic (ECC) 26%, gallbladder (GBC) 7%; stage IV 90%; hepatitis B/C virus positive (HBV/HCV+) 24%; microsatellite instability (MSI-H)/stable (MSS)/unknown n = 1/35/6. Immune-related (ir)AE occurred in 69%, grade 3/4 treatment-related (tr)AE in 10%, all-cause serious (S)AE in 36%, and trSAE in 7%. ORR was 12% (95% CI: 4, 26), with median PFS of 63 days (95% CI: 55, 125), PFS at 6 months in 27% (95% CI: 14, 43), and median OS 3of 93 days (95% CI: 243, 573). There was no significant difference in ORR, PFS, or OS by anatomic subsite or tumor PD-L1 expression; HBV/HCV+ showed trends toward higher ORR (30% vs 6%, p = 0.08) and longer median PFS (276 vs 63 days, p = 0.06) and OS (1033 vs 323 days, p = 0.052). Conclusions: The combination of PEM plus GM-CSF was safe and well-tolerated with higher ORR than expected for PEM monotherapy in a predominantly MSS ABC population but did not meet target ORR threshold for efficacy. ORR and median PFS and OS were highest in patients with underlying HBV or HCV infection. Immune profiling of on-treatment biopsies and peripheral blood are ongoing to ascertain influence of both PEM and GM-CSF on circulating and tumor immune microenvironment. Clinical trial information: NCT02703714.

Murine Ex Vivo Cultured Alveolar Macrophages Provide a Novel Tool to Study Tissue-Resident Macrophage Behavior and Function.

Tissue-resident macrophages are of vital importance as they preserve tissue homeostasis in all mammalian organs. Nevertheless, appropriate cell culture models are still limited. Here, we propose a novel culture model to study and expand murine primary alveolar macrophages (AMs), the tissue-resident macrophages of the lung, invitro over several months. By providing a combination of granulocyte-macrophage colony-stimulating factor, TGFβ, and the PPARγ activator rosiglitazone, we maintain and expand mouse ex vivo cultured AMs (mexAMs) over several months. MexAMs maintain typical morphologic features and stably express primary AM surface markers throughout invitro culture. They respond to microbial ligands and exhibit an AM-like transcriptional profile, including the expression of AM-specific transcription factors. Furthermore, when transferred into AM-deficient mice, mexAMs efficiently engraft in the lung and fulfill key macrophage functions, leading to a significantly reduced surfactant load in those mice. Altogether, mexAMs provide a novel, simple, and versatile tool to study AM behavior in homeostasis and disease settings.

A Combination of GM-CSF and Released Factors from Gamma-Irradiated Tumor Cells Enhances the Differentiation of Macrophages from Bone Marrow Cells and Their Antigen-Presenting Function and Polarization to Type 1.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes dendritic cell differentiation from precursors, and consequently, enhances the antigen presentation process and adaptive immune responses. With such functions, GM-CSF has been used as immunotherapy in combination with radiotherapy for cancer treatment to augment the survival and activity of immune cells. However, an immune-suppressive tumor microenvironment may cause anergy of T cells. It has also been reported that GM-CSF contributes to the development of myeloid-derived suppressor cells from the precursors. In this study, to analyze the combined effect of GM-CSF and released factors from cancer cells after gamma-ray irradiation on bone marrow cell differentiation and dynamics, we established an in vitro culture system using mouse bone marrow cells, GM-CSF, and conditioned medium from gamma ray irradiated mouse melanoma B16 cells at 24 Gy. We analyzed the gene expression changes of the bone marrow-derived cells on day 6. The results showed that GM-CSF dose-dependently enhanced the differentiation of macrophages from bone marrow cells, their antigen-presenting function and polarization to type I. The results implied the induced macrophages from the bone marrow may potentially contribute to tumor immune responses in a systemic manner when GM-CSF is boosted during photon-beam radiation therapy.

Identification of dendritic cell precursors in the culture of bone marrow with hematopoietic cytokines

Abstract Dendritic cells (DCs) play crucial parts in a variety of immune responses by capturing and presenting antigens to naïve T cells and by producing the subsequent T-cell activation or tolerance. In vitro culture systems of DCs have been widely used. The culture of bone marrow (BM) in the presence of a hematopoietic cytokine, Fms-like tyrosine kinase 3 ligand (FLT3L), produces plasmacytoid DCs (pDCs) and classical DCs (cDCs), whereas the culture of BM with granulocyte-macrophage colony-stimulating factor (GM-CSF) generates DCs and macrophages considered to possess inflammatory phenotypes. In the present study, we examined the culture of BM with both cytokines, i.e., FLT3L and GM-CSF in combination and identified 4 different subsets in CD11c⁺ MHCII+ cells. Meanwhile, in the culture of BM with FLT3L, we discovered a novel population of CD11c⁺ MHCIIhi cells that possess a limited capacity of antigen-presenting activity, suggesting precursors for DCs. This novel population of DC precursors from the BM culture with FLT3L was isolated and evaluated for their potential to differentiate to DCs in the BM culture containing both FLT3L and GM-CSF. Consequently, we demonstrated that the presence of a novel population of DC precursors in the in vitro culture system of DCs.

Combined GM-CSF and G-CSF administration mobilizes CD4+ CD25hi Foxp3hi Treg in leukapheresis products of rhesus monkeys.

Early phase clinical trials are evaluating the feasibility, safety, and therapeutic potential of ex vivo expanded regulatory T cells (Treg) in transplantation. A limitation is the paucity of naturally occurring Treg numbers in peripheral blood. Hence, protracted ex vivo expansion is required to obtain sufficient Treg in order to meet target cell doses. Because cytokine administration has been used successfully to mobilize immune cells to the peripheral blood in experimental and clinical studies, we hypothesized that granulocyte macrophage-colony-stimulating factor (GM-CSF) and granulocyte-CSF (G-CSF) administration would enhance Treg percentages in leukapheresis products of rhesus monkeys. Following combined GM-CSF and G-CSF administration, the incidence of Treg in peripheral blood and leukapheresis products was elevated significantly, where approximately 3.7×106 /kg CD4+ CD25hi Foxp3hi or 6.8×106 /kg CD4+ CD25hi CD127lo Treg can be collected from individual products. Mobilized Treg expressed a comparable repertoire of surface markers, chemokine receptors, and transcription factors to naïve monkey peripheral blood Treg. Furthermore, when expanded ex vivo, mobilized leukapheresis product and peripheral blood Treg exhibited similar ability to suppress autologous CD4+ and CD8+ T cell proliferation. These observations indicate that leukapheresis products from combined GM-CSF- and G-CSF-mobilized individuals are a comparatively rich source of Treg and may circumvent long-term ex vivo expansion required for therapeutic application.

Differentiation plasticity of human monocytes in culture

<p>Introduction. Human monocytes are heterogeneous and plastic cell population with the ability to undergo phenotypic and functional changes as a response to a stimulus from a local microenvironment. Our aim was to determine the potential of human monocytes to differentiate into different cell populations depending on two different cytokines (IL-4 and IL-6) added to cultures as well as to compare their phenotypical and functional characteristics. Methods. Peripheral blood mononuclear cells (PBMNC) were isolated from buffy coats of healthy donors. Monocytes, which were separated from PBMNC by plastic adherence, had been cultivated in Dendritic cell (DC), serum free medium for 5 days, either with granulocyte/macrophage colony-stimulating factor (GM-CSF) alone or with GM-CSF, with addition of interleukin 4 (IL-4) or interleukin 6 (IL-6), respectively. After cultivation, phenotypic characteristics of these cells were analyzed by flow cytometry, whereas the levels of produced cytokines in culture supernatants were quantified by ELISA. The potential of differentiated cells to modulate the proliferation of allogeneic T cells was examined by co-cultivation of these cells with PBMNC. Results. GM-CSF differentiated monocytes into M0/M1 macrophages (MØ). The combination of GM-CSF and IL-4 favoured differentiation of immature DC, whereas GM-CSF and IL-6 transformed monocytes into monocytic myeloid derived suppressor cells (M-MDSC). All cell populations expressed typical monocyte/macrophage markers such as CD14, CD11b, CD16 and CD33, HLA-DR, CD209 and CD86, a co-stimulatory marker. DC and M-MDSC expressed CD1a and CD11c, in contrast to M0/M1 MØ. The expression of HLA-DR, CD1a, CD209 and CD86 was highest on DC. The expression of CD33 and CD16 was highest on M-MDSC, followed by lowest expression of HLA-DR. The potential of promoting T-cell proliferation was highest in cultures of PBMNC with DC, whereas M-MDSC had the opposite, suppressive, effect. These differences correlated with highest production of immunosuppressive cytokines such as IL-10, IL-27 and TGF-b by M-MDSC. Conclusion. This study confirmed the differentiation plasticity of human monocytes, which are influenced by cytokines added in cultures. Phenotypic characteristics of these cells correlated with the production of cytokines involved in modulation of T-cell proliferation.</p>

Efficacy of early combination of local radiotherapy and GM-CSF for advanced non-small cell lung cancer treated with icotinib.

Lung cancer is a disease that severely endangers human health. Non-small cell lung cancer (NSCLC) accounts for approximately 4/5 of lung cancers. To investigate the efficacy of early combination of local radiotherapy and granulocyte macrophage colony-stimulating factor (GM-CSF) for advanced NSCLC treated with icotinib. Forty-two patients with stage IV NSCLC complicated with EGFR gene mutation were selected and randomly divided into two groups, with 21 patients in each group. Patients in control group were treated with icotinib, and patients in experimental group were treated with icotinib combined with local radiotherapy and subcutaneous injection of GM-CSF. One-year progression free survival between two groups was compared. Three months after treatment, the efficacy in experimental group was significantly better than that in control group, and objective response rate was 95.24% in experimental group, which was higher than the 71.43% in control group. Patients in experimental group had no differences in white blood cell and neutrophil, but had significantly lower carcino-embryonic antigen and neuron-specific enolase levels and higher CD3+, CD4+, and CD4+/CD8+ than those in control group and before treatment. There were no differences in the proportion of patients with adverse reactions between two groups. One-year progression free survival was significantly better in experimental group than in control group. Early combination of local radiotherapy and GM-CSF has a significant efficacy for advanced NSCLC accounts for approximately 4/5 of lung cancers treated with icotinib, and it can improve patients' autoimmunity and lengthen progression free survival.

Determinants of combination GM-CSF immunotherapy and oncolytic virotherapy success identified through in silico treatment personalization.

Oncolytic virotherapies, including the modified herpes simplex virus talimogene laherparepvec (T-VEC), have shown great promise as potent instigators of anti-tumour immune effects. The OPTiM trial, in particular, demonstrated the superior anti-cancer effects of T-VEC as compared to systemic immunotherapy treatment using exogenous administration of granulocyte-macrophage colony-stimulating factor (GM-CSF). Theoretically, a combined approach leveraging exogenous cytokine immunotherapy and oncolytic virotherapy would elicit an even greater immune response and improve patient outcomes. However, regimen scheduling of combination immunostimulation and T-VEC therapy has yet to be established. Here, we calibrate a computational biology model of sensitive and resistant tumour cells and immune interactions for implementation into an in silico clinical trial to test and individualize combination immuno- and virotherapy. By personalizing and optimizing combination oncolytic virotherapy and immunostimulatory therapy, we show improved simulated patient outcomes for individuals with late-stage melanoma. More crucially, through evaluation of individualized regimens, we identified determinants of combination GM-CSF and T-VEC therapy that can be translated into clinically-actionable dosing strategies without further personalization. Our results serve as a proof-of-concept for interdisciplinary approaches to determining combination therapy, and suggest promising avenues of investigation towards tailored combination immunotherapy/oncolytic virotherapy.

Intravenous delivery of granulocyte-macrophage colony stimulating factor impairs survival in lipopolysaccharide-induced sepsis.

BackgroundCell-based therapies with bone marrow-derived progenitor cells (BMDPC) lead to an improved clinical outcome in animal sepsis models. In the present study we evaluated the ability of granulocyte macrophage-colony stimulating factor (GM-CSF) to mobilize BMDPC in a lipopolysaccharide (LPS)-induced sepsis model and thereby its potential as a novel treatment strategy.MethodsMale Wistar rats received LPS (25μg/kg/h for 4 days) intravenously and were subsequently treated with GM-CSF 12.5μg/kg (0h,24h,48h,72h). As control groups, rats were infused with sodium chloride or GM-CSF only. Clinical and laboratory parameters, proinflammatory plasma cytokines as well as BMDPC counts were analyzed. Cytokine release by isolated peripheral blood mononuclear cells from rat spleen upon incubation with LPS, GM-CSF and a combination of both were investigated in vitro.ResultsIn vivo, rats receiving both LPS and GM-CSF, showed a reduced weight loss and increased mobilization of BMDPC. At the same time, this regime resulted in an increased release of proinflammatory cytokines (IL-6, IL-8) and a significantly increased mortality. In vitro, the combination of LPS and GM-CSF showed a significantly increased IL-6 release upon incubation compared to incubation with LPS or GM-CSF alone.ConclusionsGM-CSF did not have a beneficial effect on the clinical course in our LPS-induced sepsis model. It synergistically promoted inflammation with LPS and probably thereby impaired survival.

MCP-1, KC-like and IL-8 as critical mediators of pathogenesis caused by Babesia canis.

Canine babesiosis caused by the intraerythrocytic protozoan parasite Babesia canis is a tick-borne disease characterized by a host response that involves both cellular and humoral immunity. This study focuses on the secretion of cytokines Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Keratinocyte Chemotactic-like (KC-like), Interleukins (IL)-2, IL-7, IL-8, IL-10, IL-15, IL-18 and Monocyte Chemotactic Protein-1 (MCP-1) in babesiosis caused by Babesia canis upon treatment with Imizol®. We assessed time dependent changes in cytokine levels and tested whether these changes correlate with pathogenesis of the disease. Sixteen healthy dogs and 31 dogs infected with Babesia canis, of which 18 showed complications, were treated with Imizol®. One dog died during the study (3.2%). Longitudinal study was perfomed by monitoring dogs at the first day of presentation (day 1) and 6 days later (day 7). Our results show that higher MCP-1 levels on day 1 are positively associated with the occurrence of complications, (complicated vs. uncomplicated; p = 0.00016). A similar pattern was observed for KC-like on day 1 (p = 0.0326) and day 7 (p = 0.044). Moreover, babesiosis caused by B. canis produced a steady increase in IL-8 levels with a moderate to strong negative correlation with erythrocyte counts and hematocrit in uncomplicated diseased dogs only (Spearman's rank correlation coefficient rs = -0.582 and rs = -0.598 respectively). Like for MCP-1, KC-like levels also differed in complicated and uncomplicated diseased dogs on day 1 (p = 0.03236) and day 7 (p = 0.044). Furthermore, KC-like levels were strongly correlated with IL-8 levels (rs = 0.663–0.7) and non-segmented neutrophil counts (rs = 0.572–0.732) in both diseased groups. Analysis of ROC suggests the use of serum levels of MCP-1 and IL-7 as predictors of the occurrence of complications with an AUC of 0.906 and 0.896 respectively and linear combinations of MCP-1, KC-Like, IL-7 and GM-CSF with values up to AUC = 0.983. Cytokine cluster analysis presented in this study can contribute to a better understanding of the pathogenesis of babesiosis and serve as a prognostic tool for the early detection of cases with highest likelihood of developing complications. Overall, our studies show that infection by B. canis elicits a cytokine pattern that is distinct from that observed with B. rossi, and that some of the inflammatory mediators can be useful to predict complications. Our results also suggest targets for the development of novel therapeutic strategies in babesiosis caused by B. canis.

Abstract 1694: Systemic granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment increases T cell receptor diversity in localized and metastatic prostate cancer patients

Abstract Granulocyte-macrophage colony-stimulating factor (GM-CSF) is frequently utilized as an adjuvant in cancer immunotherapies, and has known effects as a growth factor. However the extent to which GM-CSF modulates the adaptive immune response, including possible effects on the antigenic repertoire, remains unclear. We used next-generation sequencing of T cell receptor (TCR) beta chain sequences amplified from total RNA from peripheral blood mononuclear cells using consensus primers to assess changes in the circulating antigenic repertoire of prostate cancer patients treated with GM-CSF in multiple clinical trials. Administration of systemic GM-CSF monotherapy to patients with localized prostate cancer prior to planned radical prostatectomy (NCT00305669) results in a significant decline in clonality from the pre-treatment timepoint to the 2-week timepoint on treatment, indicative of increased early repertoire diversity (p=0.039 by Wilcoxon signed rank test). In a separate clinical trial (NCT00064129), the combination of systemic GM-CSF (250 μg/m2/day on days 1-14 of each cycle) with ipilimumab in metastatic castrate-resistant prostate cancer (mCRPC) patients also resulted in a significant decline in clonality from pre-treatment samples to the 2-week timepoint on treatment (p=0.002). In contrast, mCRPC patients who received ipilimumab alone in a separate study (NCT00323882) did not experience a similar decline in clonality after 3 weeks on treatment (p=0.625). In addition, comparison of the dynamics of specific clonotypes between the paired timepoints in the two mCRPC studies demonstrates that while there is no significant difference in the ratios of post-treatment to pre-treatment clonality between studies, patients treated with the combination of iplilimumab and GM-CSF show more repertoire change, with lower Morisita’s distance for all clones found at either timepoint (p=0.023), smaller intraclass correlation coefficient (p=0.028), and a smaller proportion of clonotypes that remain unchanged (defined by +/- 2-fold change for clones found at both timepoints) (p=0.002). These results indicate increased repertoire turnover when GM-CSF is combined with checkpoint inhibition. Hence data from both localized and metastatic prostate cancer, and from monotherapy and combination therapy regimens, supports a role for GM-CSF in inducing early diversification of the TCR repertoire. Citation Format: David Y. Oh, Li Zhang, Jason Cham, Alan Paciorek, Mark Klinger, Malek Faham, Susan F. Slovin, Lawrence Fong. Systemic granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment increases T cell receptor diversity in localized and metastatic prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1694. doi:10.1158/1538-7445.AM2017-1694

Dinutuximab in the Treatment of High-Risk Neuroblastoma in Children

Neuroblastoma is the most common extracranial tumor derived from neural crest cells in childhood, and treatment of high-risk neuroblastoma is a difficulty in oncology field. The discovery of new treatment strategies to treat pediatric patients with high-risk neuroblastoma is important. Dinutuximab (ch14.18; Unituxin), a chimeric human-mouse monoclonal antibody, is approved by Food and Drug Administration in 2015 to be used specifically in the treatment of high-risk neuroblastoma. It binds the disialoganglioside (GD2) antigen on the surface of neuroblastoma cells and induces lysis of GD2-expressed neuroblastoma cells via antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. To enhance its activity, it is used with a combination of granulocyte-macrophage colony-stimulating factor, interleukin 2, and 13-cis-retinoic acid. In this review, we discuss the use of dinutuximab in the treatment of high-risk neuroblastoma.

Evaluation of the potential therapeutic benefits of macrophage reprogramming in multiple myeloma

AbstractTumor-associated macrophages (TAM) are important components of the multiple myeloma (MM) microenvironment that support malignant plasma cell survival and resistance to therapy. It has been proposed that macrophages (MØ) retain the capacity to change in response to stimuli that can restore their antitumor functions. Here, we investigated several approaches to reprogram MØ as a novel therapeutic strategy in MM. First, we found tumor-limiting and tumor-supporting capabilities for monocyte-derived M1-like MØ and M2-like MØ, respectively, when mixed with MM cells, both in vitro and in vivo. Multicolor confocal microscopy revealed that MM-associated MØ displayed a predominant M2-like phenotype in the bone marrow of MM patient samples, and a high expression of the pro-M2 cytokine macrophage migration inhibitory factor (MIF). To reprogram the protumoral M2-like MØ present in MM toward antitumoral M1-like MØ, we tested the pro-M1 cytokine granulocyte–macrophage colony-stimulating factor (GM-CSF) plus blockade of the M2 cytokines macrophage colony-stimulating factor or MIF. The combination of GM-CSF plus the MIF inhibitor 4-iodo-6-phenyl-pyrimidine achieved the best reprogramming responses toward an M1 profile, at both gene and protein expression levels, as well as remarkable tumoricidal effects. Furthermore, this combined treatment elicited MØ-dependent therapeutic responses in MM xenograft mouse models, which were linked to upregulation of M1 and reciprocal downregulation of M2 MØ markers. Our results reveal the therapeutic potential of reprogramming MØ in the context of MM.

The combination of calcium ionophore A23187 and GM-CSF can safely salvage aged human unfertilized oocytes after ICSI.

Artificial oocyte activation using calcium ionophores and enhancement of embryonic developmental potential by the granulocyte-macrophage colony-stimulating factor (GM-CSF) have already been reported. In this study, we evaluated the synergistic effect of these two methods on aged human unfertilized oocytes after intracytoplasmic sperm injection (ICSI). Then, we cultured the resulting embryos to the blastocyst stage and screened them for chromosomal abnormalities, to assess the safety of this protocol. Aged human oocytes deemed unfertilized after ICSI were activated, either by briefly applying the calcium ionophore A23187 alone (group A) or by briefly applying the ionophore and then supplementing the culture medium with recombinant human GM-CSF (rhGM-CSF) (group B). Next, the development was monitored in a time-lapse incubator system, and ploidy was analyzed by array comparative genomic hybridization (aCGH), after whole embryo biopsy and whole genome amplification. Differences between oocytes and resulting embryos in both groups were evaluated statistically. Oocytes unfertilized after ICSI can be activated with the calcium ionophore A23187 to show two pronuclei and two polar bodies. Addition of rhGM-CSF in the culture medium of A23187-activated oocytes enhances their cleaving and blastulation potential and results in more euploid blastocysts compared to the culture medium alone. This study shows that activating post-ICSI aged human unfertilized oocytes with a combination of a calcium ionophore and a cytokine can produce good-morphology euploid blastocysts.

Systematic review of the use of granulocyte-macrophage colony-stimulating factor in patients with advanced melanoma.

Several immunomodulatory checkpoint inhibitors have been approved for the treatment of patients with advanced melanoma, including ipilimumab, nivolumab and pembrolizumab. Talimogene laherparepvec is the first oncolytic virus to gain regulatory approval in the USA; it is also approved in Europe. Talimogene laherparepvec expresses granulocyte–macrophage colony-stimulating factor (GM-CSF), and with other GM-CSF-expressing oncolytic viruses in development, understanding the clinical relevance of this cytokine in treating advanced melanoma is important. Results of trials of GM-CSF in melanoma have been mixed, and while GM-CSF has the potential to promote anti-tumor responses, some preclinical data suggest that GM-CSF may sometimes promote tumor growth. GM-CSF has not been approved as a melanoma treatment. We undertook a systematic literature review of studies of GM-CSF in patients with advanced melanoma (stage IIIB–IV). Of the 503 articles identified, 26 studies met the eligibility criteria. Most studies investigated the use of GM-CSF in combination with another treatment, such as peptide vaccines or chemotherapy, or as an adjuvant to surgery. Some clinical benefit was reported in patients who received GM-CSF as an adjuvant to surgery, or in combination with other treatments. In general, outcomes for patients receiving peptide vaccines were not improved with the addition of GM-CSF. GM-CSF may be a valuable therapeutic adjuvant; however, further studies are needed, particularly head-to-head comparisons, to confirm the optimal dosing regimen and clinical effectiveness in patients with advanced melanoma.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-016-1860-3) contains supplementary material, which is available to authorized users.