Increase In CD11b Research Articles

Abstract 19493: In Type 1 Diabetes Decreased Bone Marrow Neuronal Support Shifts Normal Hematopoiesis Towards the Generation of (C-C Motif) Receptor 2 Monocytes

We previously showed that in both type 1 (T1D) and type 2 diabetes loss of bone marrow innervation leads to circadian dysregulation of hematopoietic stem cell (HSC) release from bone marrow (BM) into the circulation. In this study, we extended these findings to humans with T1D and used T1D rodents to examine the role of key neurotransmitters/neuropeptides in the differentiation and the fate of HSCs. Diabetic (n=5) and control (n=5) subjects underwent a 2 hr blood sampling for 24 hr. Diabetic patients showed an increase in the amplitude of the peak and a phase shift (6 hrs) of the peak compared to controls (p<0.001). In marked contrast, CD34 + cells release in diabetics showed no circadian pattern while controls did. In the long bones of T1D mice, immunodetection of calcitonin gene related peptide was reduced by 65% (p<0.05), neuropeptide Y by 67% (p<0.05), acetylcholine by 51% (p<0.05), and somatostatin by 50% (p<0.05). BM cells from T1D mice showed depletion of Side Population-HSCs but increased numbers of monocyte/macrophage colony forming units. BM supernatant of T1D mice showed increased levels (2.5-fold, p<0.002) of macrophage-colony stimulating factor (M-CSF/CSF-1, a stimulator of monocyte generation) and increased levels IL-1β (2-fold, p<0.05) and IL-27 (3-fold, p<0.01). Chimeric mice (transplanted with gfp-expressing HSC) were made T1D using streptozotocin (n=12) and compared to controls (n=12). T1D retinas, kidneys and brains (enzymatically-dissociated and analyzed by flow cytometry) showed a 10-fold, 6-fold, and 4-fold increase in CD11b + /CD45hi/ (C-C motif) receptor 2 (CCR2 + ) populations compared to controls. Immunohistochemistry of these tissues showed lba + resident microglia that exhibited a ramified morphology suggesting an inflammatory activation by extravasated BM cells. Our data suggests that in diabetic subjects, circadian dysregulation of monocyte and CD34 + cell release exists. Our rodent studies support that the loss of BM neurotransmitter regulation (bone marrow neuropathy) may shift hematopoietic towards the generation of excessive numbers of CCR2 + monocytes and reduced reparative endothelial progenitors which can contribute towards both the vascular and tissue pathology observed in diabetes.

Tle4 Is Critical for Proper B- Cell Differentiation and Maintenance of the Murine Bone Marrow Niche

AbstractAbstract 1202Transducin-like/Enhancer of Split 4 (TLE4), a homolog of the master developmental regulator in Drosophila, Groucho, is a co repressor previously described by our group as the putative tumor suppressor lost in a subset of t(8:21) AML patients also harboring 9q deletion. TLE4 has been found to be of fundamental importance in the repressive function of multiple highly conserved transcriptional complexes, including Hes1, Tcf/Lef, CSL, Runx1-3, and Pax5, all of which have been shown to be intimately connected to normal steady state hematopoiesis and deregulated in malignancy. Owing to its deletion in leukemia and its ability to confer functionality to a variety of signaling networks, our lab sought to investigate its role in hematopoiesis by generating a murine knockout model for Tle4.While normal at birth, within 2–3 weeks of age knockout (KO) animals become runted and eventually moribund by the time of weaning. Gross examination of the spleen and thymus in 3 week old animals reveals significant atrophy of these organs with severe abnormalities in normal tissue architecture. Tle4−/− mice lack clear demarcations between medullary and cortical regions of the thymus, in addition to having a significant increase in apoptosis seen via TUNEL staining. FACS analysis of the thymus shows CD4/CD8 double positive cells are heavily reduced in KOs, with an increase in both single positive mature populations. Additionally, there is a partial, but not complete, block in T cell progenitor development through TCR rearrangement (DN2-DN3 stages). Structural defects are seen in the spleen as well, with a failure in the formation of follicular zones. B cells are significantly reduced with an increase in CD11b+ cells in the spleen, in addition to a shift in the percentages of B1, B2, and marginal zone B cells in some animals. Histological examination of the long bones reveals dramatic abnormalities, both in hematopoietic and bone lineages. Tle4−/− mice exhibit a reduction in calcified bone, a dysplastic and reduced hypertrophic zone, and reabsorption of the trabecular region. These mice also have a gradual clearing of the marrow itself, with 2 week old animals first showing evidence of cytopenia that then progresses to full marrow failure by 3–3.5 weeks of age. The bone marrow exhibits a marked leukocytopenia, with a clear B lymphocytopenia as in the spleen. Furthermore, within the bone marrow, there is an increase in the number of CD4 and decrease in the number of CD8 T cells. ProB staging of developing B cells also trends towards a partial arrest in the transitions from the A through C fractions. When analyzed for progenitor populations, KO mice show severe reductions in the number of common lymphoid progenitors, while myeloid progenitors show no particular shifts in population frequency. Finally, there is a significant decrease in HSCs, defined as lin-Sca1+Ckit++ (LSK).In order to assess whether the hematological phenotype observed in these mice are of a cell intrinsic or extrinsic nature, transplants were performed using embryonic day 13.5 fetal liver and 2 week old bone marrow. Analysis of recipient animals 16 weeks after transplant revealed leukocytopenia with a specific reduction in B cells in the blood, however the T cell and LSK defects seen in donor animals was not observed. Furthermore, when LSK CD34+ Flt3+ or – cells from KO or WT 2 week old animals were plated on OP9 cells over-expressing Deltex1 or GFP, there were no defects in T, B, or CD11b+ cell production.The unique spectrum of phenotypic consequences concomitant to Tle4 deletion argues for a complex yet fundamental role in the maintenance of the hematopoietic niche. As illustrated by the ability to recapitulate some yet not all of the KO hematological aberrations in transplants, it appears that Tle4's involvement in lineage commitment is heterotypic in nature, an unsurprising finding considering its direct involvement in multiple highly conserved signaling pathways. Considering its principal role in those pathways, continued efforts to investigate the role the TLE family of corepressors play in the bone marrow niche may prove vital in reworking our paradigms for both normal and malignant hematopoiesis. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Accumulation of Myeloid-Derived Suppressor Cells in Bone Marrow in Multiple Myeloma Induces Tumor-Specific Immune Suppression and Promotes Tumor Growth

Abstract 3954Multiple myeloma (MM) is an incurable malignancy of clonal plasma cells in bone marrow (BM). Accumulation of MM cells in BM is associated with a corresponding dysfunction of local immune responses. The mechanisms involved in the immune suppression remains poorly understood. Myeloid-derived suppressor cells (MDSC) are heterogeneous group of relatively immature myeloid cells characterized by potent immune suppressive activity. However, their role in regulation of immune responses in BM in MM remains unclear.To determine MDSC accumulation in MM patients, BM and blood samples were obtained from newly diagnosed patients and healthy donors. We observed a significant increase in CD11b+ CD33+ CD14− HLA-DRlo MDSCs, but not in CD11b+ CD33+ CD14+ HLA-DRhi monocytes, in the blood and BM of patients as compared to cells with the same phenotype in healthy donors. MDSC isolated from patient BM suppressed T cell responses as measured by IFN-g Elispot assays of mixed leukocyte reactions. Immature myeloid cells with the same phenotype from BM of healthy donor did not inhibit T cells.The possible role of MDSC in MM progression was further evaluated in mouse model of MM. Three different MM cell lines (BCM, DP42 and ATLN) obtained from Dr Van Ness (University of Minnesota) were used. MM tumors were established by i.v. injection of syngeneic mice. We observed an expansion of CD11b+ GR1+ MDSCs (up to 70%) in the BM of tumor-bearing (TB) mice within one week after injection of tumor cells. MDSC purified from BM of TB mice at that time point potently suppressed antigen-specific T cell proliferation and IFN-g production. As tumor progressed, CD138+ MM cells expanded (up to 90%) in the BM replacing normal BM cells including MDSC. However, at later time points (2–3 weeks), MDSC expansion was seen in spleen and lymph nodes of TB mice.To evaluate the possible role of MDSC in MM progression, we used S100A9 knockout (KO) mice. S100A9 plays an important role in MDSC accumulation in cancer and S100A9 KO TB mice had reduced accumulation of these cells. When wild-type (WT) and S100A9 KO mice were inoculated with DP42 cells no differences in tumor growth and survival were observed. S100A9 KO mice had a decrease in MDSC accumulation in the BM of TB mice compared to WT mice. Additionally, MDSC from S100A9 KO mice failed to suppress antigen-specific T cells ex vivo, whereas MDSC from WT TB mice did. To evaluate the development of antigen-specific immune responses in MM, we generated a MM cell line over-expressing the chicken ovalbumin protein (DP42-OVA). S100A9 KO mice injected with the immunogenic DP42-OVA cell line showed significantly delayed tumor growth and improved survival as compared with WT mice. Further analysis of T cell responses in vivo showed increased accumulation of antigen-specific T cells within the BM of S100A9 KO mice, but not WT mice, as early as one week post-tumor inoculation. Antigen-specific T cells in spleens were not detected at that time point. These cells were seen in spleens on S100A9 KO mice only 2–3 weeks after tumor injection.Thus, our study demonstrated that MM is associated with rapid accumulation of MDSC with potent immune suppressive activity against tumor-specific T cells. Our data directly suggest that targeting MDSC in MM may improve antitumor immune response and clinical outcome of the disease. Disclosures:No relevant conflicts of interest to declare.

Angiogenesis inhibition: quo vadis?

Among the novel treatments for gliomas evaluated in 2012, inhibitors of angiogenesis have undoubtedly raised the most interest. Although mature data are awaited from 2 randomized phase III trials exploring agents with different modes of action (bevacizumab, an antibody to vascular endothelial growth factor [VEGF], and cilengitide, an integrin inhibitor), multiple other targeted agents exploring similar or novel targets are also being explored, including agents inhibiting placental growth factor, fibroblast growth factor, and stromal-derived factor-1. According to a press release by Roche, the registration trial for bevacizumab in the treatment of newly diagnosed glioblastoma, AVAGlio, has reached the primary end point of improving progression-free survival, whereas no mature results for the overall survival end point have been made available. These observations, although preliminary, underscore the urgent need to understand what is happening during treatment with anti-angiogenic agents, in what way such treatments modify tumor biology, and whether there is a risk of promoting an even more malignant tumor phenotype after they escape anti-angiogenic treatment. Altogether, there is little doubt that anti-angiogenic agents can be only cytostatic but are unlikely to represent curative approaches, and innovative sequencing strategies involving radiotherapy and classical cytotoxic chemotherapy remain to be further investigated. In the present issue of Neuro-Oncology, Piao et al from Dr. John de Groot`s laboratory at The University of Texas MD Anderson Cancer Center explored the evolution of resistance to anti-VEGF therapy and compared the ligand-sequestering antibody, bevacizumab, with a small molecule inhibitor of tyrosine kinase receptors, sunitinib.1 In the U87MG orthotopic human glioma model, bevacizumab doubled median survival, whereas sunitinib had no effect on survival. However, when sunitinib was combined with bevacizumab, survival was increased over the increase achieved with bevacizumab alone. Both agents reduced vascularity in the tumors, but bevacizumab seemed to be more effective in inhibiting vascularity in the periphery of the tumors, compared with sunitinib. There was earlier revascularization in sunitinib-treated tumors than in bevacizumab-treated tumors that was associated with earlier tumor progression. There was, in general, good correlation between vascularization and tumor cell proliferation. Long-term surviving animals showed enhanced tumor invasiveness. Using carbonic anhydrase 9 as a marker of hypoxia, they found that hypoxia was more prominent in control and sunitinib-treated mice than in bevacizumab-treated mice. An increase in CD11b+/F4/80+ cells was observed earlier in control and sunitinib-treated animals and was, overall, associated with tumor progression and treatment failure. This is a timely study indicating the potential differences between targeting angiogenic ligands versus angiogenic receptors in glioma models. The potential limitations include the study of only one cell line, which is often not considered to represent the full spectrum of glioma biology. However, this study, as similar previous studies in other glioma models, illustrates that a better understanding of interactions between glioma cells and the tumor microenvironment, specifically in the brain, is required to fully exploit the potential value of anti-angiogenic agents in the treatment of glioblastomas. Specifically, it is important to understand tumor-host cell interactions better: where do infiltrating cells come from, what do they do, and how do they overall contribute to the natural course of disease and to therapy resistance and response?

αB-Crystallin regulates expansion of CD11b⁺Gr-1⁺ immature myeloid cells during tumor progression.

The molecular chaperone αB-crystallin has emerged as a target for cancer therapy due to its expression in human tumors and its role in regulating tumor angiogenesis. αB-crystallin also reduces neuroinflammation, but its role in other inflammatory conditions has not been investigated. Here, we examined whether αB-crystallin regulates inflammation associated with tumors and ischemia. We found that CD45(+) leukocyte infiltration is 3-fold increased in tumors and ischemic myocardium in αB-crystallin-deficient mice. Notably, αB-crystallin is prominently expressed in CD11b(+) Gr-1(+) immature myeloid cells (IMCs), known as regulators of angiogenesis and immune responses, while lymphocytes and mature granulocytes show low αB-crystallin expression. αB-Crystallin deficiency results in a 3-fold higher accumulation of CD11b(+) Gr-1(+) IMCs in tumors and a significant rise in CD11b(+) Gr-1(+) IMCs in spleen and bone marrow. Similarly, we noted a 2-fold increase in CD11b(+) Gr-1(+) IMCs in chronically inflamed livers in αB-crystallin-deficient mice. The effect of αB-crystallin on IMC accumulation is limited to pathological conditions, as CD11b(+) Gr-1(+) IMCs are not elevated in naive mice. Through ex vivo differentiation of CD11b(+) Gr-1(+) cells, we provide evidence that αB-crystallin regulates systemic expansion of IMCs through a cell-intrinsic mechanism. Our study suggests a key role of αB-crystallin in limiting expansion of CD11b(+) Gr-1(+) IMCs in diverse pathological conditions.

Glioblastoma resistance to anti-VEGF therapy is associated with myeloid cell infiltration, stem cell accumulation, and a mesenchymal phenotype.

Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis. Inhibiting the VEGF-VEGF receptor (R) signal transduction pathway in glioblastoma has recently been shown to delay progression, but the relative benefit and mechanisms of response and failure of anti-VEGF therapy and VEGFR inhibitors are not well understood. The purpose of our study was to evaluate the relative effectiveness of VEGF sequestration and/or VEGFR inhibition on orthotopic tumor growth and the mechanism(s) of treatment resistance. We evaluated, not only, the effects of anti-VEGF therapy (bevacizumab), anti-VEGFR therapy (sunitinib), and the combination on the survival of mice bearing orthotopic gliomas, but also the differential effects of the treatments on tumor vascularity, cellular proliferation, mesenchymal and stem cell markers, and myeloid cell infiltration using flow cytometry and immunohistochemistry. Bevacizumab significantly prolonged survival compared with the control or sunitinib alone. Both antiangiogenic agents initially reduced infiltration of macrophages and tumor vascularity. However, multitargeted VEGFR inhibition, but not VEGF sequestration, rapidly created a vascular gradient and more rapidly induced tumor hypoxia. Re-infiltration of macrophages was associated with the induction of hypoxia. Combination treatment with bevacizumab and sunitinib improved animal survival compared with bevacizumab therapy alone. However, at the time of tumor progression, a significant increase in CD11b(+)/Gr1(+) granulocyte infiltration was observed, and tumors developed aggressive mesenchymal features and increased stem cell marker expression. Collectively, our results demonstrate a more prolonged decrease in tumor vascularity with bevacizumab than with sunitinib, associated with a delay in the development of hypoxia and sustained reduction of infiltrated myeloid cells.

Palmitate induces phenotype changes in monocytes via de novo ceramide synthesis

Ageing is associated with physiological changes such as altered adipose tissue distribution and redox status, elevated metabolic disorder risk and increased free fatty acids. Saturated fatty acids can induce insulin resistance in endothelial cells and monocytes, in part explaining the increase cardiovascular disease risk with elevated fatty acids. The aim of this study is to investigate the effects of the two major physiological fatty acids palmitate and oleate (saturated and monounsaturated fatty acids respectively) on monocyte phenotype. THP-1 monocyte cell surface marker expression, mitochondrial reactive oxygen species, cell viability and caspase-3 were evaluated following 24h treatment with palmitate, oleate or bovine serum albumin. A concentration dependent increase in CD11b (p<0.01), CD36 (p<0.001) and mitochondrial reactive oxygen species (p<0.05) following palmitate but not oleate treatment was determined by flow cytometry. Decreased metabolic viability (p<0.01) was observed with palmitate (300μM), whilst no significant change in caspase-3 was observed. The superoxide dismutase mimetic MnTBAP (200μM) ameliorated the reduced metabolic viability and increased mitochondrial ROS due to palmitate, whilst increased CD11b and CD36 were unaffected. De novo ceramide synthesis inhibitor fumonisin B1 (50μM) prevented the palmitate dependent increase in CD11b (p<0.05) and CD36 (p<0.001). Ceramide converting inhibitors 1S,2R-D-erythro-2-N-myristoylamino)-1-phenyl-1-propanol (d-erythro MAPP 20 μM) and 1-phenyl-2-palmitoylamino-3-morpholino-1- propanol (PPMP 0.05 μM) were used to determine if ceramides or downstream sphingolipids are required for palmitate induced phenotype changes in monocytes. In the presence of MAPP and PPMP, palmitate induced increase in CD11b are sustained suggesting sphingolipids are key players in the observed phenotypic changes. The present study demonstrates in monocytes that palmitate but not oleate increases cell surface marker expression, accompanied by increased mitochondrial ROS and decreased metabolic viability. MnTBap could ameliorate changes in mitochondrial ROS and metabolic viability, but surface antigen expression decreased only by fumonisin B1 suggesting changes require de novo ceramide synthesi.

Hypercalcemia induces a proinflammatory phenotype in rat leukocytes and endothelial cells

Endothelial plasma membrane lipid microdomains, so called lipid rafts/caveolae, are rich in neutral glycosphingolipid, globotriaosylceramide, Gb3Cer, or CD77. Several plasma membrane Ca(2+) channels and pumps are located in lipid rafts/caveolae. Increased Ca(2+) influx could cause the development of an endothelial proinflammatory phenotype. Therefore, the aim of this study was to estimate the effects of hypercalcemia in rats by determination of CD77 expression on CD34+ endothelial cells in the heart, kidney, and vena cava. In addition, potential proinflammatory calcium effect was estimated by CD11b and CD15s expression on leukocytes. To achieve hypercalcemia, Sprague-Dawley male rats were given CaCl2 solution with a concentration of 1.5 % elemental calcium during 14 days. CD77 expression on CD34+ endothelial cells in cell suspensions of the heart, kidney, and vena cava, as well as leukocyte expression of CD11b and CD15s in hypercalcemic and control rats were determined by flow cytometry. Ionized calcium concentration in plasma was 1.37 ± 0.01 mM in hypercalcemic vs. 1.19 ± 0.03 mM in control rats. Hypercalcemic group showed statistically significantly decreased proportion of endothelial CD34+ CD77- cells in the kidney and vena cava in parallel with increase of CD11b and CD15s leukocyte proinflammatory markers. In conclusion, it is tempting to speculate that plasma membranes of glycosphingolipid CD77- endothelial cells are poorer in caveolae lipid microdomains and therefore weaker in controlling of Ca(2+) influx. The percentage of CD11b+ CD15s+ leukocytes could be a measure of proinflammatory effects of mild hypercalcemia.

Lactoferrin Regulates an Axis Involving CD11b and CD49d Integrins and the Chemokines MIP-1α and MCP-1 in GM-CSF-Treated Human Primary Eosinophils

Eosinophils are multifunctional immune cells that contribute to innate and adaptive immune/repair responses. Lactoferrin (LF) is an iron-binding protein indicated to alter cell adhesion and immune function by receptor-mediated interactions or by participating in redox mechanisms. The eosinophil adhesion molecules, αMβ2 and α4β1, are differentially expressed following exposure to the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) and various redox agents. We hypothesized that LF can alter the function and production of proteins involved in adhesion/migration. Utilizing eosinophil peroxidase activity or fluorescent labeling adhesion assays, LF reduced GM-CSF-induced eosinophil adhesion in the presence of fibronectin or vascular adhesion molecule-1 compared with GM-CSF treatment alone. Flow cytometric analysis of eosinophil αM (CD11b) and α4 (CD49d) integrins revealed that cotreatments (24 h) with LF plus GM-CSF induced a significant increase in CD11b compared with control and GM-CSF treatments but a significant decrease in CD49d compared with control and GM-CSF treatments. These changes in CD11b and CD49d levels were significantly correlated with the increased production of chemokines (macrophage inflammatory Protein-1α, monocyte chemotactic protein-1) and an identified increase in S100A9 production. Thus, LF release at sites of inflammation may alter eosinophil recruitment/activation and possibly the progression of diseases such as cancer and asthma where significant eosinophil influx has been described.

Palmitate promotes monocyte atherogenicity via de novo ceramide synthesis

Elevated plasma free fatty acids (FAs) are associated with increased risk of cardiovascular disease. This study investigates the effects of the saturated FA palmitate and unsaturated FA oleate on monocyte phenotype and function. Incubation of human U937 and THP-1 monocytes with palmitate for 24h increased cell surface expression of integrin CD11b and scavenger receptor CD36 in a concentration-dependent manner with some decrease in mitochondrial reducing capacity at high concentration (300μM). Monocytes incubated with palmitate, but not oleate, showed increased uptake of oxidized LDL and increased adhesion to rat aortic endothelium, particularly at bifurcations. The palmitate-induced increase in CD11b and CD36 expression was associated with increased cellular C16 ceramide and sphingomyelin, loss of reduced glutathione, and increased reactive oxygen species (ROS). Increased monocyte surface CD11b and CD36 was inhibited by fumonisin B1, an inhibitor of de novo ceramide synthesis, but not by the superoxide dismutase mimetic MnTBap. In contrast, MnTBap prevented the mitochondrial ROS increase and metabolic inhibition due to 300μM palmitate. This study demonstrates that in viable monocytes, palmitate but not oleate increases expression of surface CD11b and CD36. Palmitate increases monocyte adhesion to the aortic wall and promotes uptake of oxidized LDL and this involves de novo ceramide synthesis.

Effect of antiangiogenic therapy on tumor-associated macrophages in recurrent glioblastoma.

2010 Background: Antiangiogenic therapy is associated with increased radiographic responses in glioblastoma (GBM), but tumors invariably recur. Tumor associated macrophages (TAMs) have been proposed as a mechanism of resistance to anti-angiogenic therapy in preclinical models. To examine the role of TAMs in recurrent GBM, we analyzed autopsy specimens from patients with or without history of antiangiogenic therapy. Methods: We compared autopsy brain specimens from 17 recurrent GBM patients who received anti-angiogenic treatment and chemoradiation (AAT+) to 7 patients who received chemotherapy and/or radiotherapy without anti-angiogenic therapy, or no treatment (AAT-). TAMs were morphologically and phenotypically identified with flow cytometry and immunohistochemistry (IHC) with CD68, CD11b, CD14, and CD163 markers. All specimens were obtained from the Department of Pathology at Massachusetts General Hospital and clinical information gained through review of the patients’ records. Results: Using flow cytometry, we observed an increase in CD11b+CD14+ cells in the AAT+ patients compared to AAT- patients. Using IHC analysis, we observed a significant increase in CD68+ macrophages in the tumor bulk (p&lt;0.01) and infiltrative areas (p&lt;0.05) in AAT+ versus AAT- patients. We also observed a significant increase in CD11b+ myeloid cells in the tumor bulk (p&lt;0.01) and a significant increase in CD163+ cells in the infiltrative areas (p&lt;0.05) in the AAT+ group. Finally, we noted a trend toward an increase in CD163+ cells in the tumor bulk (p=0.087) in the AAT+ versus the AAT- patients. Conclusions: Patients with recurrent GBM after antiangiogenic therapy showed a significant increase in CD68+ TAMs and in CD11b+ cells in the tumor bulk. Additionally, antiangiogenic treatment induced an increase in CD68+ and CD163+ TAMs in the infiltrative region. These data indicate that TAMs may participate in escape from antiangiogenic therapy and may represent a future therapeutic target in recurrent GBM.

Contribution of an aging environment towards a proinflammatory tumor-like milieu (54.4)

Abstract Chronic inflammation associated with aging is termed inflamm-aging. It manifests as a rise in many proinflammatory mediators and contributes to a decline in immune function. Unregulated inflammasome activation initiates an inappropriate inflammatory response. It is particularly imperative to understand inflammasome dysregulation in the elderly (&amp;gt;65 yrs), since it is a likely basis of immune dysfunction. Analyses of freshly isolated memory CD4+ and CD8+ splenic T cells demonstrated that even without further activation, inflammasome genes were already significantly up-regulated in old mice compared to young, including factors critical to myeloid-derived suppressor cell (MDSC) expansion including S100a8, S100a9, IL-1β, IL-13 and IL-6. A corresponding 2-3 fold increase in CD11b+Gr1+cells was observed in the spleens of old naïve mice compared to young. Importantly, cells with the CD11b+Gr1+ phenotype from old mice, unlike young, were functionally MDSCs and suppressed T cell proliferation. Therefore, the immunosuppressive environment of the tumor-bearing host also appears to be operative in aging, even in the absence of tumor. We hypothesize that constituitive activation of inflammasome complex results in a chronic inflammatory state, altering immune responses in the elderly similarly to the tumor-bearing state. Preemptive reversal of inflammasome-induced MDSCs may enhance T cell immunosurveillance in the elderly, a population with a heightened susceptibility to malignancy.

Potentiation of Apoptosis in MDS/AML by Combination of Azacitidine and the EGFR-Tyrosine Kinase Inhibitor (TKI) erlotinib

Abstract 2790 Background:TKIs were initially developed as targeted therapies that would solely interfere with a “specific” aberrant signaling pathway in malignant cells. However, we and others showed that the EGFR TKI erlotinib (Erlo) has in vivo and in vitro efficacy in MDS and AML (Boehrer et al., Blood, 2008). We also previously observed, in a preliminary study, the potentiation of apoptosis upon combination of Erlo with azacitidine (Aza), but not with decitabine, in HL-60 cells and in cells from a few (five) MDS/AML patients (ASH 2010, 974). We decided to expand this pre-clinical study to define the potential interest of combining different TKI (Dasatinib (Dasa), Sorafenib (Sora) or Erlo) with Aza, now a reference first line treatment in higher risk MDS (Lancet Oncol, 2009). Methods:Erlo (10μM), Dasa (500nM), or Sora (5μM) were combined to Aza (1μM) and apoptosis over-time (24, 48 and 72h) quantified by FACS analysis following DioC3(6)/PI staining in different MDS/AML-derived cell lines (MOLM-13, SKM-1, MV4-11, Kasumi-1). Quantification of apoptosis at 48h or 72h was recapitulated ex vivo in CD34+ cells from patients with MDS (n=12), AML (n=14) or AML post MDS (n=5). For each single drug, as well as the respective combinationsErlo, Aza and Erlo+Aza, the capacity to induce cell cycle arrest (PI staining), cytotoxicity (MTT assay) and decrease of proliferation (Click-it EdU Assay) was assessed concomitantly. Differentiation was assessed by staining with CD11b on day 3 (FACS), evaluation of ROS production at 24/48h by FACS staining with CM-DFCDA (oxidative stress indicator) and HE (Hydroethidine). Functional relevance of apoptosis-related signaling pathways was determined by co-incubation of Aza (and drugs combinations with biochemical inhibitors against MAPK: JNK (SP600125, 10μM), p-38MAPK (SB203580, 10μM) and MEK (U0126, 5μM). Immunoblot analyses of caspase-3, PARP, Mcl-1 and Bcl-xl were performed at 24h and 48h. Results:Whereas co-incubation of Dasa or Sora with Aza did not increase the degree of apoptosis observed with Aza alone, combination of Erlo with Aza had synergistic effects already observed at 24h, an effect that increased over-time (SKM-1 72h (mean PI+ cells, n=3), Erlo: 15%, Aza: 40%, Erlo+Aza: 81% - MOLM-13 72h, Erlo: 21%, Aza: 25%, Erlo+Aza: 64%). The % of cells with intact metabolic activity at 72h (determined by the MTT assay using 2.5μM Erlo and 0.5μM Aza) decreased from 83% (Erlo) and 79% (Aza) to 23% (Erlo+Aza) in SKM-1 (similar results for MOLM-13 cells). To determine if Erlo also impacts on apoptosis in CD34+ cells from patients with MDS or AML, we screened 31 samples and observed a synergistic effect in 5/31 samples (2/12 MDS, 2/14 LAM, 1/5 AML post MDS) and an additive effect in 8/31 samples. Induction of apoptosis was not preceded by differentiation (no increase in CD11b) or production of ROS. On the other hand, analysis of cell cycle distribution at 24h showed that apoptosis was accompanied by an increase in G0/G1 arrest and a decrease in the % of cells in S phase upon incubation with the combination of Erlo+Aza (G0/G1 (mean, n=2) in SKM-1, DMSO: 51%, Erlo: 52%, Aza: 61%, Erlo+Aza: 78%, G0/G1 in MOLM-13, DMSO: 40%, Erlo: 51%, Aza: 38%, Erlo+Aza: 65%) confirmed by the study of newly synthesized DNA before induction of apoptosis (15h) with an alternative BrdU test (Click-EdU assay). Indeed, in SKM-1 cells, proliferation was decreased by 20% upon Erlo or Aza and by 80% with the association. Moreover, cell cycle arrest was associated with an increase in the % of apoptotic cells with a sub-diploid DNA content (about 10% for Erlo or Aza and 32% for Erlo+Aza in SKM-1. Immunoblot analyses confirmed an increase in cleaved PARP and caspase-3 upon incubation with Aza +Erlo as compared to the single agents, as well as a decrease of the anti-apoptotic protein Mcl-1 and Bcl-xl. Incubation of Aza with SP600125 induced the same extent of apoptosis observed with Erlo+Aza, whereas no effect was observed with the p-38MAPK inhibitor SB203580 or the MEK inhibitor U0126. In addition, Erlo decreased phosphorylation of JNK on Thr183/Tyr185, suggesting a potential implication of the JNK pathway in the mechanism of apoptosis. Conclusions:In this study, Erlo was the only TKI tested able to increase sensitivity towards Aza in MDS/AML cell lines and in patient-derived cells. These results suggest a potential clinical interest of combining Aza to Erlo in MDS/AML. Disclosures:Fenaux:Celgene: Honoraria, Research Funding.

Src Kinase Inhibition by Dasatinib Enhances Targeting of Human AML Stem/Progenitor Cells by Chemotherapeutic Agents

Abstract 4270Tyrosine kinases play a critical role in regulating cellular proliferation, differentiation and survival. We have previously shown that Src family tyrosine kinases (SFKs) including Lyn, Hck and Fgr are abnormally activated in Acute Myeloid Leukemia (AML) cells compared to normal CD34+ hematopoietic progenitors (Dos Santos et al., Blood 2008, 111:2269). Dasatinib is a potent small molecule inhibitor of SFKs and the ABL kinase that is approved for clinical use in Chronic Myeloid Leukemia. However the ability of Dasatinib to target AML is poorly documented. Here we evaluated the potential activity of Dasatanib on AML stem/progenitor cell maintenance and drug sensitivity. We assessed SFK phosphorylation in stem/progenitor populations from AML patients by flow cytometry (n=42). We observed significant increase of SFK phosphorylation in AML compared to normal Lin− CD34+CD38− cells (3.5±1.8 versus 1.7±0.48 fold increase compared to isotype control, p=0.008), Lin− CD34+ CD38+ (3.6±1.6 versus 1.6±0.37, p=0.001) and Lin− CD34− cells (2.9±1.4 versus 1.1±0.4, p=0.001). Treatment with Dasatinib significantly reduced AML stem cell growth in LTCIC assays (56±23% inhibition with 200nM Dasatinib, p=0.003, n=5), and reduced growth of committed progenitors in CFC assays (73.6±13% inhibition with 500nM Dasatinib, and 41.7±10.8% inhibition with 10nM Dasatinib, n=9, p<0,0001), but did not reduce normal LTC-IC (n=4) or CFU-GM (23±9.1% inhibition with 500nM Dasatinib, and 1.3±11.3% with 10nM Dasatinib, n=6, p=0,007). Inhibition of growth was associated with increased apoptosis (41.5% ±10.7 with 200nM Dasatinib versus 26%±8.2 for the control, p=0,004, n=5), reduced proliferation (as evaluated with CFSE labeling, proliferation index 3.02±0.3 for the control and 2.27±0.6 with 200 nM Dasatinib, p=0.01 (n=5), and enhanced differentiation of AML stem/progenitor cells with significant increase in CD11b+ and CD15+ cells and reduction in CD34+, CD33+ and CD71+ cells, (Dasatinib 200 nM n=7). Exposure of AML cells (n=4) to Dasatinib (200nM) resulted in reduced phosphorylation of STAT3 (Y705), STAT5 (Y694) and Akt (Thr308) but did not affect MAPK (Thr202/Y204) phosphorylation. Interestingly, Dasatinib treatment strongly enhanced expression of p53 protein and its downstream targets, including p21 and Bax, in AML specimen (n=7), suggesting that Dasatinib treatment resulted in activation of p53 signaling in AML stem/progenitor cells. As compared to treatment with individual agents, the combination of Dasatinib with either daunorubicin (DNR) or Ara-C resulted in significantly increased apoptosis (14.9%±11 with Dasatinib, 15.8%±8.6 with 0.05μ M DNR and 25% with DNR+ Dasatinib=7, p=0.005), decreased proliferation (proliferation index 3.02±0.3 for control, 2.27±0.6 with 200 nM Dasatinib, 2.32±0.8 with 0.05μ M DNR and 1.29±0.4 for the combination n=5, p=0.01) and reduced CFC growth (n=6, 56.2±6.4% inhibition with 200nM Dasatinib, 35%±5.6 with 0.01μ M DNR, p=0.001, 82%±9.5 with DNR + Dasatinib, p=0.001, 51.8%±8.5 with 0.01μ M Ara-C and 83.2%±4.7 with Ara-C + Dasatinib, p=0.0002). The combination of Dasatinib with chemotherapeutic agents significantly enhanced expression of p53 and downstream targets compared with these agents alone. In conclusion, SFK activity is increased in primary human AML stem and progenitor cells. SFK blockade with Dasatinib reduces maintenance of AML stem and progenitor cells through inhibition of proliferation, induction of apoptosis and enhancement of differentiation. Dasatinib treatment enhances sensitivity of AML stem/progenitor cells to chemotherapeutic agents, potentially through inhibition of Akt and STAT signaling and activation of p53. These results support further preclinical and clinical evaluation of SFK blockade with Dasatinib to enhance AML stem and progenitor cells targeting by chemotherapeutic agents. Disclosures:No relevant conflicts of interest to declare.

Interleukin-33 prolongs allograft survival during chronic cardiac rejection

Interleukin-33 (IL-33) stimulates the generation of cells and cytokines characteristic of a Th2 immune response. We examined the effects of IL-33 on allografted heart tissue in a chronic cardiac rejection model, including analysis of the peripheral myeloid and lymphoid compartments. B6.C-H2bm12/KhEg hearts were transplanted into MHC class II-mismatched C57Bl/6J mice; IL-33 was administered daily. Cells from allografts and spleens were isolated for flow cytometry and cultured for cytokine production; some tissues were used for immunohistochemistry. Animals treated with IL-33 showed significantly longer allograft survival, which was associated with a distinct cytokine profile produced by graft-infiltrating cells. Proinflammatory IL-17A production was decreased with IL-33 treatment, while increased levels of IL-5, IL-10, and IL-13 were observed. After IL-33 therapy, flow cytometry showed a direct induction of CD4(+) Foxp3(+) Treg, whereas the number of B220(+) CD19(+) B cells, and circulating, as well as allograft deposited, alloantibodies was reduced. Following IL-33 treatment, a significant decrease in graft-infiltrating CD11b(high) Gr1(high) granulocytes coincided with a significant increase in CD11b(high) Gr1(intermediate) myeloid-derived suppressor cells (MDSC). In conclusion, IL-33 treatment in the setting of chronic rejection promotes the development of a Th2-type immune response that favors MDSC and Treg expansion, reduces antibody-mediated rejection (AMR), and ultimately, prolongs allograft survival.

Study of Camelpox Virus Pathogenesis in Athymic Nude Mice

Camelpox virus (CMLV) is the closest known orthopoxvirus genetically related to variola virus. So far, CMLV was restricted to camelids but, recently, three human cases of camelpox have been described in India, highlighting the need to pursue research on its pathogenesis, which has been hampered by the lack of small animal models. Here, we confirm that NMRI immunocompetent mice are resistant to intranasal (i.n.) CMLV infection. However, we demonstrate that CMLV induced a severe disease following i.n. challenge of athymic nude mice, which was accompanied with a failure in gaining weight, leading to euthanasia of the animals. On the other hand, intracutaneous (i.c.) infection resulted in disease development without impacting the body weight evolution. CMLV replication in tissues and body fluids was confirmed in the two models. We further analyzed innate immune and B cell responses induced in the spleen and draining lymph nodes after exposure to CMLV. In both models, strong increases in CD11b+F4/80+ macrophages were seen in the spleen, while neutrophils, NK and B cell responses varied between the routes of infection. In the lymph nodes, the magnitude of CD11c+CD8α+ lymphoid and CD11c+CD11b+ myeloid dendritic cell responses increased in i.n. challenged animals. Analysis of cytokine profiles revealed significant increases of interleukin (IL)-6 and IL-18 in the sera of infected animals, while those of other cytokines were similar to uninfected controls. The efficacy of two antivirals (cidofovir or HPMPC, and its 2, 6-diaminopurine analog) was evaluated in both models. HPMPC was the most effective molecule affording 100% protection from morbidity. It appeared that both treatments did not affect immune cell responses or cytokine expression. In conclusion, we demonstrated that immunodeficient mice are permissive for CMLV propagation. These results provide a basis for studying the pathogenesis of CMLV, as well as for evaluating potential antiviral therapies in an immunodeficiency context.

Tumor- and organ-dependent infiltration by myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) increase during tumor growth and following cytoreductive therapy resulting in immune dysfunction and tumor escape from host control. We report organ- and tumor-specific expansion of MDSCs, differences in their molecular and membrane phenotypes and T-cell suppressive activity. A significant increase in MDSCs was observed within the spleen, peripheral blood (PB), bone marrow (BM), lungs, and livers of mice bearing orthotopic 4T1, but not CI66 mammary tumors. The PB of 4T1 TB mice had the highest frequency of MDSCs (78.6±2.1%). Similarly, the non-parenchymal cells (NPCs) in the tumor tissue, livers and lungs of 4T1 tumor-bearing (TB) mice had an increased MDSCs frequency. Studies into Gr-1 and Ly-6C staining of MDSCs revealed significant increases in CD11b+Gr-1dullLy-6Chigh and CD11b+Gr-1brightLy-6Clow subsets. The frequency of MDSCs inversely correlated with the CD3+ T-cell frequency in the spleen, and blood of 4T1 TB mice and was associated with a significant decrease in splenic and NPCs IFN-γ and IL-12 transcript levels, as well as significantly increased levels of granulocyte–macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), interleukin-10 (IL-10), interleukin-13 (IL-13), arginase-1 (ARG-1), nitric oxide synthase (NOS-2), vascular endothelial growth factor-A (VEGF-A) transcripts. In summary, MDSCs are significantly increased not only in lymphoid organs, but also in parenchymal organs including lungs and livers of TB mice, where they may facilitate metastasis to these organ sites.

Exposure to fractionated dose of 60 Gy affects molecular response of HL-60 cells to irradiation

In this work we evaluated changes in molecular response of human promyelocyte leukemia cells HL-60 and HL-60-IR cells (HL-60 irradiated by 10 cycles of radiation with total dose of 60 Gy, given over a period of 3 months) to irradiation by the dose of 2 and 8 Gy. Analysis of CD11b and apoptosis by flow-cytometry revealed that on 3rd day after irradiation by 8 Gy the HL-60-IR are more resistant to radiation-induced apoptosis and more differentiated (increase in CD11b in non-apoptotic cells) than regular HL-60. We found that both types of cells have high basal level of phosphorylated extracellular signal-regulated kinases Erk1/2 . Irradiation induces decrease in Erk1/2 phosphorylation after 4 and 8 h in both cell types. However, in HL-60-IR cells Erk1/2 phosphorylation is restored faster than in HL-60. Also it was found that in contrary to HL-60 cells, the HL-60-IR cells react to 2 Gy irradiation by p53 independent increase in p21(WAF1/Cip1), and not by activation of checkpoint kinase Chk-2. Therefore we concluded that relatively high dose of radiation (6 Gy) does not lead after 10 repetitive irradiations to eradication of HL-60 cells, but instead increases their radioresistance, increases the ability to differentiate, alters MAPK response, increases amount of p21(WAF1/Cip1), and decreases induction of apoptosis by ionizing radiation. p21(WAF1/Cip1) might prevent apoptosis induction and trigger permanent cell-cycle arrest, which can also contribute to regression of this leukaemia after therapy.

Tim-3/Galectin-9 Pathway: Regulation of Th1 Immunity through Promotion of CD11b+Ly-6G+ Myeloid Cells

IFN-gamma plays a central role in antitumor immunity. T cell Ig and mucin domain (Tim-3) is expressed on IFN-gamma-producing Th1 cells; on interaction with its ligand, galectin-9, Th1 immunity is terminated. In this study, we show that transgenic overexpression of Tim-3 on T cells results in an increase in CD11b(+)Ly-6G(+) cells and inhibition of immune responses. Molecular characterization of CD11b(+)Ly-6G(+) cells reveals a phenotype consistent with granulocytic myeloid-derived suppressor cells. Accordingly, we find that modulation of the Tim-3/galectin-9 (Gal-9) pathway impacts on tumor growth. Similarly, overexpression of Tim-3 ligand, Gal-9, results in an increase in CD11b(+)Ly-6G(+) cells and inhibition of immune responses. Loss of Tim-3 restores normal levels of CD11b(+)Ly-6G(+) cells and normal immune responses in Gal-9 transgenic mice. Our data uncover a novel mechanism by which the Tim-3/Gal-9 pathway regulates immune responses and identifies this pathway as a therapeutic target in diseases where myeloid-derived suppressor cells are disadvantageous.

Abstract B52: Characterizing the MDSCs phenotypes, expansion, and trafficking pattern and associated tumor-induced immune dysfunction

Abstract Among the prerequisites to be effective, immunotherapy must work in the presence of the host immunosuppression. Myeloid-derived suppressor cells (MDSCs) are one of the cellular mediators that have a role in limiting the induction of adaptive immune response and host responses to immunotherapy. MDSCs are an immature, heterogeneous cellular population, including hematopoietic precursors for granulocytes, macrophages, dendritic cells, and endothelial cells. They are infrequent in healthy hosts, but increase during tumor progression as a result of dysregulated myeloid cell expansion and differentiation. Tumor growth is frequently associated with an increased frequency of MDSCs that results in a loss in T-cell number and function through increased ROS and peroxynitrites production and arginase 1 (ARG1) and nitric oxide synthase 2 (NOS-2) upregulation. A better understanding of MDSCs expansion and the regulation of biodistribution and arrest would improve interventional strategies and cancer immunotherapy outcomes. Using five mammary tumor models, we have examined tumor-associated expansion of MDSCs and observed that this occurs in an organ-specific manner. Murine MDSCs can be subdivided into phenotypically and functionally distinct sub-populations according to Gr-1 expression levels; the CD11b+Gr-1 dim MDSCs with monocytic nuclei and strong T-cell suppressive activity, and CD11b+Gr-1bright having ring-shaped nuclei (polymorphonuclear cells) and a lower T-cell suppression profile. These cells can also be subset based on the expression of Ly-6C which is highly expressed on immature, bone marrow-derived cells and combined this Gr-1 expression Our studies revealed a significant increase in the Ly-6Cbright expression on the CD11b+Gr-1dim MDSCs in all organs studied in tumor-bearing mice as compared to non-tumor-bearing mice. In association with the increase in CD11b+Gr-1dimLy-6Cbright cells in the tumor non-parenchymal cells of 4T1 tumor bearing mice, increased transcripts levels of cytokines associated with MDSCs expansion (granulocyte-colony stimulating factor [G-CSF]) and MDSCs-associated immunosuppression (ARG-1, NOS-2, and cyclooxygenase 2 [COX-2]) while lower levels of these cytokines were observed in tumor NPCs from Cl66 tumor-bearing mice and non-tumor-bearing mice. In association with the increase in MDSCs and loss of T-cells in the 4T1 tumor-bearing mice there was a significant decrease in cytokine transcript levels that can enhance immune response (interferon gamma [IFN-γ] and interleukin 12 [IL-12]) from tumor non-parenchymal cells (NPCs) in 4T1 tumor bearing mice as compared to Cl66 tumor bearing mice and non-tumor bearing mice. Studies to assess where the MDSCs were expanding revealed that in 4T1 tumor bearing mice, frequency of the proliferating MDSCs is increased only in the spleen and liver compared to the non-tumor-bearing mice. Our studies also examined the affect of tumor burden on the trafficking of MDSCs. We observed that, after adoptive transfer of MDSCs isolated from 4T1 tumor-bearing mice, they arrest initially within the lungs in non-tumor-bearing mice. While in 4T1 tumor-bearing mice they rapidly transverse the lungs with arrest within the tumor tissue. We and others reported that the increase in MDSCs frequencies is associated with a decrease in CD3+ cells function and numbers. We observed a decrease in frequency of all T-cell subsets in the thymus of tumor bearing mice tested compared to the non-tumor-bearing mice suggesting defects in T-cell maturation associated with tumor burden. We investigated the effect of tumor burden on the phenotype of the CD3+ cells using the Annexin V staining and found that 4T1 tumor bearing mice have increased frequency of Annexin V positive CD3+ T-cells that are infiltrating tumor tissue compared to those in other organs. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B52.