anti-Gr1 Antibody Research Articles

IL-25 prevents and cures fulminant hepatitis in mice through a myeloid-derived suppressor cell-dependent mechanism

Fulminant hepatitis (FH) is a disease characterized by massive destruction of hepatocytes with severe impairment of liver function. The pathogenesis of FH is not fully understood, but hyperactivity of T cells and macrophages with excessive production of cytokines are important hallmarks of the condition. In this study, we investigated the role of interleukin (IL)-25 in FH. IL-25 expression was evaluated in patients with FH and in livers of mice with FH induced by D-galactosamine (D-Gal) and lipopolysaccharide (LPS). Mice were treated with IL-25 before D-Gal/LPS-induced FH and before or after concanavalin A (ConA)-induced FH. Mononuclear cells were isolated from livers of mice treated with or without IL-25 and analyzed for GR1(+) CD11b(+) cells. CFSE-labeled T cells were cocultured with GR1(+) CD11b(+) cells and their proliferation was evaluated by flow cytometry. Mice were also treated with a depleting anti-GR1 antibody before IL-25 and D-Gal/LPS administration. IL-25 was constitutively expressed in mouse and human liver and down-regulated during FH. IL-25 prevented D-Gal/LPS-induced FH and this effect was associated with increased infiltration of the liver with cells coexpressing GR1 and CD11b. In vitro studies showed that GR1(+) CD11b(+) cells isolated from mice given IL-25 inhibited T-cell proliferation. Consistently, in vivo depletion of GR1(+) cells abrogated the protective effect of IL-25 in experimental D-Gal/LPS-induced FH. IL-25 was both preventive and therapeutic in ConA-induced FH. IL-25 expression is markedly reduced during human and experimental FH. IL-25 promotes liver accumulation of GR1(+) CD11b(+) cells with immunoregulatory properties.

Reply to Honjo et al.: Functional relevant expression of Toso on granulocytes

Functional relevant expression of Toso on granulocytes. (A) Toso expression. Splenocytes from WT and Toso−/− mice were immunostained with anti-CD19 (B cells) and anti-Gr1 (granulocytes) antibody in combination with DAPI and anti-Toso antibody after pretreatment with Fc receptor block. One representative staining of cells gated on Gr1hi DAPI− cells (Left) or CD19+ DAPI− cells is shown (Right). (B) Quantification of FACS stainings derived from A (n = 4/group). (C–E) WT recipient mice were irradiated and reconstituted with BM from WT mice (CD45.1; group 1), Toso−/− mice (CD45.2; group 2), or a 1:1 mixture of WT and Toso−/− BM (group 3). At 30 d posttransplantation, peripheral blood cells were stimulated with different concentrations of fMLP (Formyl-Methionyl-Leucyl-Phenylalanine) and granulocytes (Gr1+ cells) were analyzed after 30 min for CD45.1 expression (anti-CD45.1), CD45.2 expression (anti-CD45.2), reactive oxygen species production (dihydrorhodamine staining), and degranulation (side scatter). Representative FACS …

The Stimulated Innate Resistance Event in Bordetella pertussis Infection Is Dependent on Reactive Oxygen Species Production

The exacerbated induction of innate immune responses in airways can abrogate diverse lung infections by a phenomenon known as stimulated innate resistance (StIR). We recently demonstrated that the enhancement of innate response activation can efficiently impair Bordetella pertussis colonization in a Toll-like receptor 4 (TLR4)-dependent manner. The aim of this work was to further characterize the effect of lipopolysaccharide (LPS) on StIR and to identify the mechanisms that mediate this process. Our results showed that bacterial infection was completely abrogated in treated mice when the LPS of B. pertussis (1 μg) was added before (48 h or 24 h), after (24 h), or simultaneously with the B. pertussis challenge (10(7) CFU). Moreover, we detected that LPS completely cleared bacterial infection as soon as 2 h posttreatment. This timing suggests that the observed StIR phenomenon should be mediated by fast-acting antimicrobial mechanisms. Although neutrophil recruitment was already evident at this time point, depletion assays using an anti-GR1 antibody showed that B. pertussis clearance was achieved even in the absence of neutrophils. To evaluate the possible role of free radicals in StIR, we performed animal assays using the antioxidant N-acetyl cysteine (NAC), which is known to inactivate oxidant species. NAC administration blocked the B. pertussis clearance induced by LPS. Nitrite concentrations were also increased in the LPS-treated mice; however, the inhibition of nitric oxide synthetases did not suppress the LPS-induced bacterial clearance. Taken together, our results show that reactive oxygen species (ROS) play an essential role in the TLR4-dependent innate clearance of B. pertussis.

Epidermal Hyperplasia and Appendage Abnormalities in Mice Lacking CD109

CD109, a glycosylphosphatidylinositol-anchored glycoprotein, is highly expressed in several types of human cancer tissues, in particular, squamous cell carcinomas. In normal human tissues, human CD109 expression is limited to certain cell types including myoepithelial cells of the mammary, lacrimal, salivary, and bronchial glands and basal cells of the prostate and bronchial epithelium. Although CD109 has been reported to negatively regulate transforming growth factor-β signaling in keratinocytes in vitro, its physiologic role in vivo remains largely unknown. To investigate the function of CD109 in vivo, we generated CD109-deficient (CD109(-/-)) mice. Although CD109(-/-) mice were born normally, transient impairment of hair growth was observed. At histologic analysis, kinked hair shafts, ectatic hair follicles with an accumulation of sebum, and persistent hyperplasia of the epidermis and sebaceous glands were observed in CD109(-/-) mice. Immunohistochemical analysis revealed thickening of the basal and suprabasal layers in the epidermis of CD109(-/-) mice, which is where endogenous CD109 is expressed in wild-type mice. Although CD109 was reported to negatively regulate transforming growth factor-β signaling, no significant difference in levels of Smad2 phosphorylation was observed in the epidermis between wild-type and CD109(-/-) mice. Instead, Stat3 phosphorylation levels were significantly elevated in the epidermis of CD109(-/-) mice compared with wild-type mice. These results suggest that CD109 regulates differentiation of keratinocytes via a signaling pathway involving Stat3.

Regulation of hematopoietic stem cells using protein transduction domain–fused Polycomb

The Polycomb-group complex is a chromatin regulatory factor that is classified into two different complexes: Polycomb repressive complex 1 and 2. Components of Polycomb repressive complex 1 are involved in the self-renewal of hematopoietic stem cells. Bmi1, one of these components, maintains the immaturity of neural and cancer stem cells as well as that of hematopoietic stem cells. We constructed recombinant protein transduction domain (PTD)-Polycomb proteins and transduced them into murine bone marrow (BM) cells. We designed and fused the PTD-protein transduction domain to three proteins (i.e., green fluorescent protein, Bmi1, and Mel18). Murine BM cells were incubated for 48 h and each PTD-Polycomb protein was added. Then, we analyzed the function of hematopoiesis using the colony assay and transplantation. BM cells exposed to PTD-Bmi1 showed an increased number of colonies. In contrast, BM cells exposed to PTD-Mel18 or to both proteins showed a decreased number of colonies. Hematopoietic cells derived from PTD-Bmi1-transduced BM cells were significantly increased in the peripheral blood at 6 weeks after transplantation. Moreover, 80% of mice transplanted with PTD-Bmi1-transduced BM cells died at 8 to 24 weeks after transplantation. However, only a few early deaths were observed in the mice transplanted with BM cells exposed to both PTD-Bmi1 and PTD-Mel18. We expect that hematopoietic stem cells could proliferate after transduction with PTD-Bmi1, but this may generate undesirable effects, e.g., tumorigenesis. Thus, Bmi1 and Mel18 have opposing functions and are present in distinct complexes.

Higher numbers of Gr1hiCD11b+ cells suppress B cell differentiation and autoantibody production in male BWF1 mice (72.7)

Abstract (NZBxNZW) F1 (BWF1) mice spontaneously develop a lupus-like disease characterized by elevated levels of anti-nuclear autoantibodies (ANA) and systemic inflammation and with a similar female bias as observed in humans. Bone marrow (BM) chimera studies have shown intrinsic disease-driving properties of female BM cells and subsequent flow-based analyses identified significantly less Gr1hiCd11b+ myeloid cells in BWF1 female mice as compared to males. As Gr1hiCD11b+ cells have been suggested to be immunosuppressive, we hypothesized that these cells suppress B cell activation and differentiation and protect against lupus-like disease in male BWF1 mice. We analyzed the effect of male and female Gr1hiCD11b + cells on 1) cytokine-driven B cell differentiation in vitro, 2) antibody responses to immunization, and 3) spontaneous autoantibody production. We found that Gr1hiCD11b+ cells from both male and female 4 wk old BWF1 mice significantly suppressed cytokine-driven plasma cell differentiation in vitro in a dose dependent manner. Likewise, treatment of male BWF1 mice with depleting anti-Gr1 antibody resulted in elevated antibody response to Np-CGG immunization. And finally, spontaneous production of ANA were significantly increased in male BWF1 mice during and after anti-Gr1 antibody treatment, with ANA levels comparable to those of unmanipulated BWF1 females. These data suggest a new and important role for immunosuppressive Gr1hiCD11b+ cells during antibody-mediated autoimmunity.

The role of NKT cells in immune evasion of liver metastases arising from intraocular tumors (74.12)

Abstract Uveal melanoma is the most common intraocular tumor in adults within the United States. Half of the patients develop liver metastases, which is almost universally fatal. We have previously demonstrated that liver natural killer (NK) cell-antitumor immunity was suppressed in mice bearing ocular tumors, and natural killer T (NKT) cells were responsible, in part, for liver NK cell suppression. This study examines the role NKT cells in promoting liver metastases arising from intraocular tumors. We showed that when wild type and NKT-deficient C57BL/6 mice were injected with B16LS9 melanoma cells, NKT-deficient mice developed less liver metastasis than wild type mice. The cytotoxicity of liver NK cells was also assessed. Results indicated that NK cells from NKT-deficient mice were more cytotoxic compared to NK cells from wild type mice. However, in-vitro assays suggested that NKT cells were unable to directly suppress NK cell-mediated cytotoxicity. To investigate the potential role of a third-party cell, myeloid derived suppressor cells (MDSCs) were depleted by anti-GR1 antibody. Surprisingly, tumor bearing MDSC-depleted mice developed more liver metastases than isotype-treated mice. Our results imply that liver NKT cells suppress NK cell cytotoxicity through a GR1+ third-party cell population other than MDSCs. We are currently investigating whether liver NKT cells impart their immunosuppressive function through GR1+ neutrophils.

Mitigating Age-Related Immune Dysfunction Heightens the Efficacy of Tumor Immunotherapy in Aged Mice

Although cancer tends to affect the elderly, most preclinical studies are carried out in young subjects. In this study, we developed a melanoma-specific cancer immunotherapy that shows efficacy in aged but not young hosts by mitigating age-specific tumor-associated immune dysfunction. Both young and aged CD4(+)CD25(hi) regulatory T cells (Treg) exhibited equivalent in vitro T-cell suppression and tumor-associated augmentation in numbers. However, denileukin diftitox (DT)-mediated Treg depletion improved tumor-specific immunity and was clinically effective only in young mice. DT-mediated Treg depletion significantly increased myeloid-derived suppressor cell (MDSC) numbers in aged but not young mice, and MDSC depletion improved tumor-specific immunity and reduced tumor growth in aged mice. Combining Treg depletion with anti-Gr-1 antibody was immunologically and clinically more efficacious than anti-Gr-1 antibody alone in aged B16-bearing mice, similar to Treg depletion alone in young mice. In contrast, DT increased MDSCs in young and aged mice following MC-38 tumor challenge, although effects were greater in aged mice. Anti-Gr-1 boosted DT effects in young but not aged mice. Aged antitumor immune effector cells are therefore competent to combat tumor when underlying tumor-associated immune dysfunction is appropriately mitigated, but this dysfunction varies with tumor, thus also varying responses to immunotherapy. By tailoring immunotherapy to account for age-related tumor-associated immune dysfunctions, cancer immunotherapy for aged patients with specific tumors can be remarkably improved.

311 ELUCIDATING MECHANISMS OF SUNITINIB RESISTANCE IN METASTATIC RENAL CELL CARCINOMA BY STUDYING ITS EFFECT ON MYELOID DERIVED SUPPRESSOR CELLS (MDSC) IN MURINE TUMOR MODELS

You have accessJournal of UrologyKidney Cancer: Basic Research II1 Apr 2012311 ELUCIDATING MECHANISMS OF SUNITINIB RESISTANCE IN METASTATIC RENAL CELL CARCINOMA BY STUDYING ITS EFFECT ON MYELOID DERIVED SUPPRESSOR CELLS (MDSC) IN MURINE TUMOR MODELS Kevin Chandler, Charles Tannenbaum, and James Finke Kevin ChandlerKevin Chandler Cleveland, OH More articles by this author , Charles TannenbaumCharles Tannenbaum Cleveland, OH More articles by this author , and James FinkeJames Finke Cleveland, OH More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.370AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES We find that the tyrosine kinase receptor (TKR) inhibitor sunitinib, a front line treatment for metastatic renal cell carcinoma (RCC), reduces MDSC numbers in patient blood and tumor. Given the ability of MDSCs to synthesize both immunosuppressive- and TKR-independent angiogenic molecules, MDSC recruitment may be a means by which tumors become resistant to current anti-angiogenic treatments directed at tyrosine kinase signaling alone. Our goal is to elucidate the role of MDSC persistence in sunitinib resistant murine tumor models, and to assess whether specific tumor- or bone marrow (BM)-derived cytokines protect MDSCs from sunitinib therapy by modulating survival and/or immunosuppressive or angiogenic gene expression. METHODS Here we used the sunitinib sensitive RENCA (renal) and resistant 4T1 (mammary) murine tumor lines. Mice bearing 10mm tumors were treated or not with sunitinib (40mg/kg) for 6 days. Thereafter single cell suspensions of BM and tumor were stained with anti-Gr1 and anti-CD11b antibodies to assess the number and percentages of neutrophilic (n-MDSC, Gr1+hiCD11b+) and monocytic (m-MDSC, Gr1+lowCD11b+) MDSC subsets by FACS. RNA from FACS-sorted n-MDSCs and m-MDSCs from BM was subjected to real time PCR to assess the effect of sunitinib on gene expression. RESULTS n-MDSCs and m-MDSCs decreased 20 fold and 10 fold, respectively, in sunitinib-treated RENCA tumors as compared to untreated controls, whereas neither MDSC subpopulation was detectably diminished in sunitinib-treated 4T1 tumors, or in the BM of mice carrying either sunitinib-treated RENCA or 4T1 tumors. Interestingly, of the multiple angiogenic and immunosuppressive genes that were assessed in BM MDSCs, the only significant modulation observed was a 4 fold stimulation of BV8 expression by the m-MDSC subset in the sunitinib-treated 4T1-bearing animals. CONCLUSIONS The persistence of MDSCs within tumor and BM may represent a mechanism of cancer resistance to sunitinib. Further studies will elucidate the means by which MDSCs persist despite sunitinib treatment, and will determine if overcoming MDSC accumulation affects outcomes. It may be that increased expression of GM-CSF, G-CSF, or IL-1 beta within 4T1 tumors will correlate with MDSC survival and persistence. In addition it may be the case that sunitinib mediates its antitumor effects by inhibiting MDSC gene expression in susceptible tumors. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e126 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kevin Chandler Cleveland, OH More articles by this author Charles Tannenbaum Cleveland, OH More articles by this author James Finke Cleveland, OH More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Myeloid-Derived Suppressor Cell Inhibition of the IFN Response in Tumor-Bearing Mice

Our group and others have determined that immune effector cells from patients with advanced cancers exhibit reduced activation of IFN signaling pathways. We hypothesized that increases in immune regulatory cells termed myeloid-derived suppressor cells (MDSC) could interfere with the host immune response to tumors by inhibiting immune cell responsiveness to IFNs. The C26 murine adenocarcinoma model was employed to study immune function in advanced malignancy. C26-bearing mice had significantly elevated levels of GR1(+)CD11b(+) MDSC as compared with control mice, and splenocytes from tumor-bearing mice exhibited reduced phosphorylation of STAT1 (P-STAT1) on Tyr(701) in response to IFN-α or IFN-γ. This inhibition was seen in splenic CD4(+) and CD8(+) T cells as well as natural killer cells. In vitro coculture experiments revealed that MDSC inhibited the IFN responsiveness of splenocytes from normal mice. Treatment of C26-bearing mice with gemcitabine or an anti-GR1 antibody led to depletion of MDSC and restored splenocyte IFN responsiveness. Spleens from C26-bearing animals displayed elevated levels of iNOS protein and nitric oxide. In vitro treatment of splenocytes with a nitric oxide donor led to a decreased STAT1 IFN response. The elevation in nitric oxide in C26-bearing mice was associated with increased levels of nitration on STAT1. Finally, splenocytes from iNOS knockout mice bearing C26 tumors exhibited a significantly elevated IFN response as compared with control C26 tumor-bearing mice. These data suggest that nitric oxide produced by MDSC can lead to reduced IFN responsiveness in immune cells.

Abstract 1788: COX-2 blockade suppresses gliomagenesis by inhibiting CCL2-mediated accumulation of myeloid-derived suppressor cells

Abstract Recent reports in epidemiology suggest yet unexplained roles of immunosurveillance in gliomagenesis and survival of glioma patients. For example, the use of nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to be associated with reduced occurrence of malignant glioma in humans. NSAIDs mediate their biological effects partially by suppression of cyclooxygenase (COX)-2 and its product prostaglandin (PG) E2, both of which are known to induce immunoregulatory cells, such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells, in tumor-bearing hosts. In the current study, using a novel Sleeping Beauty transposon-mediated de novo mouse glioma model, we evaluated our hypothesis that COX-2 blockade by NSAIDs would suppress gliomagenesis by altering chemokine expression and immune cell attraction in glioma microenvironment, thereby promoting anti-glioma immunosurveillance. We chose acetylsalicylic acid (ASA; a non-selective COX-2 inhibitor) and celecoxib (a selective COX-2 inhibitor) as candidates of therapeutic agents. In wild type (WT) mice with developing gliomas, ASA-based treatment delayed glioma development only if the treatment was initiated simultaneously with glioma induction, but not after the tumor was established. In contrast, celecoxib-based treatment inhibited glioma development even if the treatment was initiated later. These data indicate that COX-2 plays an important role in gliomagenesis. Consistently, C57BL/6-background mice deficient for COX-2 gene (Cox2-deficient mice) demonstrated a significant delay in tumor growth when compared with the WT mice. Of importance, both the NSAID-treated and Cox2-deficient mice exhibited a decrease in systemic PGE2 levels when compared with the controls, which was concomitant with decreases in CCL2 and CD11b+Ly6CloLy6G+ MDSCs but increases in CXCL10 and CD8+CD107a+ CTLs in the glioma microenvironment. In addition, MDSC depletion by anti-Gr1 antibody treatment prolonged the survival of the WT mice developing gliomas, suggesting that COX2-induced MDSCs play an important role in gliomagenesis. We finally addressed the impacts of CCL2 and CXCL10 on the attraction of MDSCs and CTLs as well as gliomagenesis. With developing gliomas, Ccl2-deficient mice exhibited a decrease in MDSC accumulation in the glioma microenvironment, whereas Cxcl10-deficient mice exhibited a decrease in CTL accumulation. Taken together, these findings indicate important roles of the COX-2 pathway in gliomagenesis through CCL2-mediated MDSC accumulation and a reduction of CXCL10-mediated CTL infiltration into the glioma microenvironment, and suggest a possibility to develop NSAID-based prophylactic strategies for gliomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1788. doi:10.1158/1538-7445.AM2011-1788

Lymphocytes Are Dispensable In Neutrophil Homeostasis.

AbstractAbstract 2613 Introduction:In contrast to red cell or platelet homeostasis, the feedback mechanisms involved in the control of peripheral neutrophil numbers are incompletely understood. Granulocyte-Colony Stimulating Factor (G-CSF) is generally accepted to be the most important determinant of neutrophil production and release from the bone marrow. Recently, a feedback loop including a specialized subset of T-lymphocytes (Tn cells) has been established to explain the correlation between peripheral neutrophil clearance and increased G-CSF production. Methods:Wild type C57bl/6 mice or NODSCIDcγ−/− received anti-Gr1 or anti-1A8 antibodies to deplete neutrophils. Hematopoietic tissues and peripheral blood were harvested at different times and analyzed on cellular, protein and RNA level. Results:Both anti-Gr1 and 1A8 antibodies reduced neutrophils effectively and persistently in vivo. The reaction on neutrophil depletion in the marrow uniformely consisted of an absolute increase in lin-/Sca1+/c-kit+ (LSK) cells and a shift of myeloid progenitors towards granulocyte-macrophage precursors (GMP) and common myeloid progenitors (CMP) at the expense of megakaryocyte-erythrocyte precursors (MEP). Of note, exogenous G-CSF resulted in identical changes. We therefore hypothesized that neutrophil depletion increases G-CSF through a feedback regulatory loop. Neutrophil depletion with anti-Gr1 antibody in wt mice increased G-CSF levels up to approximately 8-fold. While previous observations suggest that G-CSF may be passively regulated through receptor binding and internalization by mature neutrophils, we also found a 10-fold increase of G-CSF mRNA in the marrow. These findings are consistent with active feedback. Interestingly, the effects of 1A8 antibody on G-CSF were less pronounced. Instead, mice depleted of neutrophils with 1A8 antibody displayed predominant increases of M-CSF, suggesting redundant feedback pathways. Our results in wildtype mice treated with 1A8 antibody confirm previous data by Stark et al. (Immunity 2005; 22: 285–294), including increases of IL-23 and IL-17 after neutrophil depletion. However, when neutrophils were depleted in NODSCIDcγ−/− mice, who lack lymphocytes and NK-cells, both IL-23 and IL-17 remained unchanged, but G-CSF levels still increased markedly. Conclusions:Effective neutrophil depletion by antibodies directed against different neutrophil antigens uniformly results in major shifts in the hematopoietic bone marrow showing an increase in the number of LSK hematopoietic stem cells accompanied by a significant increase in myeloid progenitors at the expense of thrombopoietic, red cell, and lymphoid precursors. Our results demonstrate regulatory feedback loops of neutrophil granulopoiesis culminating in increased production of myelopoiesis stimulating cytokines such as G-CSF, GM-CSF, and M-CSF. The underlying chain of events includes IL-23 and IL-17 in wild type mice as previously described. However, additional redundant pathways exist in lymphocytopenic NODSCIDcγ−/− mice. Disclosures:No relevant conflicts of interest to declare.

Abstract 5311: Myeloid-derived suppressor cells and immune cell responsiveness in tumor-bearing mice

Abstract Conditions that inhibit IFN-induced signal transduction and gene transcription can dampen the host immune system and its ability to recognize and eliminate tumors leading to impaired immunoediting. We hypothesized that in the setting of cancer, myeloid-derived suppressor cells (MDSC) could impair the responsiveness of immune effector cells to interferons. We used the murine adenocarcinoma cell line C26 which induces MDSC generation when implanted in C2DF1 mice. C26-bearing mice had significantly elevated levels of GR1+CD11b+ MDSC (p<0.0001) and plasma IL-6 as compared to controls (p<0.0001). The ability of IFNa or IFNy to stimulate phosphorylation of the STAT1 transcription factor (P-STAT1, a direct measure of IFN action) was measured by intracellular flow cytometry. Induction of P-STAT1 following a 15 minute stimulation of splenocytes with IFNa (102-104 U/mL) was significantly reduced in C26-bearing mice (Mean P-STAT1 = 18.8; Range = 4.0 - 33.4) as compared to control mice (Mean P-STAT1 = 37.9; Range = 27.0 - 50.0; p<0.0001). A similar decrease in P-STAT1 in splenocytes of C26-bearing mice was observed following IFNy stimulation (1 ng/mL; 15 min; p=0.002). This decrease in IFN responsiveness was also observed in cells derived from inguinal lymph nodes following IFN-stimulation. The level of IFNa induced P-STAT1 was inversely correlated with the plasma IL-6 levels (Pearson correlation = −0.635; p=0.019). However, direct exogenous treatment of mice with IL-6 did not decrease splenic IFN response. In vitro co-culture experiments revealed that MDSC inhibited IFN-responsiveness of splenocytes from normal mice. Reduction of MDSC with either gemcitabine or anti-GR1 antibody treatment restored IFN responsiveness in splenocytes from C26-bearing mice as compared to tumor-bearing mice treated with vehicle or isotype control antibody. Spleens from C26-bearing animals displayed elevated levels of iNOS protein and nitric oxide (NO). The elevation in NO in C26-bearing mice was associated with increased levels of nitration on STAT1. In vitro treatment of splenocytes with a nitric oxide donor led to a decreased STAT1 IFN response. Taken together, these data suggest a novel link between MDSC and a decreased molecular response of the immune effectors responsible for promoting functional anti-tumor responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5311.

Abstract 1359: COX2 blockade suppresses glioma-genesis by promoting anti-glioma immunosurveillance

Abstract Recent reports in epidemiology suggest yet unexplained roles of immunosurveillance in glioma-genesis and survival of glioma patients. For example, the use of nonsteroidal anti-inflammatory drugs (NSAID) has been shown to be associated with reduced occurrence of malignant glioma in humans. NSAIDs mediate their biological effects partially by suppression of cyclooxygenase (COX)-2 and its product prostaglandin (PG) E2, both of which are known to promote induction of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg). Using a novel Sleeping Beauty (SB) transposon-mediated de novo mouse glioma model, we evaluated our hypothesis that COX-2 blockade by NSAIDs would suppress glioma-genesis by promoting anti-glioma immunosurveillance. Because acetylsalicylic acid (ASA), in contrast to a selective COX-2 inhibitor celecoxib, did not demonstrate direct growth inhibition on cultured glioma cells at pharmacological concentrations, we utilized ASA to primarily address the impact of COX-2 blockade on immune cells. Neonatal C57BL/6 mice received SB-mediated glioma induction along with ASA (10 mg/kg) or control saline starting either at the time of glioma induction or after the establishment of solid glioma. Interestingly, ASA treatment starting at the time of SB-glioma induction rather than after the establishment of solid tumors reduced the rate and growth of glioma in the wild type (WT) mice. To elucidate the specific contributions of the COX-2 pathway, C57BL/6-background mice deficient for COX-2 gene (Cox2-deficient) received the SB-mediated glioma induction to be evaluated for tumor growth and changes in tumor microenvironment. The Cox2-deficient mice also demonstrated a significant delay in tumor growth when compared with the WT mice. Both the ASA-treated and Cox2-deficient mice demonstrated lower serum levels of PGE2, as well as marked reduction of Gr1+CD11b+ MDSCs and CD4+FoxP3+ Tregs in the glioma microenvironment when compared with the control mice. In addition, MDSC depletion by anti-Gr1 antibody treatment prolonged the survival of the WT mice developing gliomas, suggesting that these cells are at least partially responsible for glioma-genesis. We finally addressed the significance of endogeneous type-1 interferons (IFN) in the effects of COX-2 blockade because the type-1 IFNs are known to be central coordinators in anti-tumor immunosurveillance. To this end, C57BL/6-background mice deficient for type-1 IFN receptors (Ifnar1-deficient) received the SB-mediated glioma induction. In contrast to the WT mice, the Ifnar1-deficient mice did not delay tumor growth in response to ASA treatment. Taken together, these data suggest that COX-2 blockade by NSAIDs may reduce MDSCs and Tregs in the CNS microenvironment partially through the type-1 IFN pathways, thereby improving the immunosurveillance and prevention against gliomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1359.

NOD-scid IL2rgamma(null) mouse model of human skin transplantation and allograft rejection.

Transplantation of human skin on immunodeficient mice that support engraftment with functional human immune systems would be an invaluable tool for investigating mechanisms involved in wound healing and transplantation. Nonobese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) readily engraft with human immune systems, but human skin graft integrity is poor. In contrast, human skin graft integrity is excellent on CB17-scid bg (SCID.bg) mice, but they engraft poorly with human immune systems. Human skin grafts transplanted onto immunodeficient NSG, SCID.bg, and other immunodeficient strains were evaluated for graft integrity, preservation of graft endothelium, and their ability to be rejected after engraftment of allogeneic peripheral blood mononuclear cells. Human skin transplanted onto NSG mice develops an inflammatory infiltrate, consisting predominately of host Gr1(+) cells, that is detrimental to the survival of human endothelium in the graft. Treatment of graft recipients with anti-Gr1 antibody reduces this cellular infiltrate, preserves graft endothelium, and promotes wound healing, tissue development, and graft remodeling. Excellent graft integrity of the transplanted skin includes multilayered stratified human epidermis, well-developed human vasculature, human fibroblasts, and passenger leukocytes. Injection of unfractionated, CD4 or CD8 allogeneic human peripheral blood mononuclear cell induces a rapid destruction of the transplanted skin graft. NSG mice treated with anti-Gr1 antibody provide a model optimized for both human skin graft integrity and engraftment of a functional human immune system. This model provides the opportunity to investigate mechanisms orchestrating inflammation, wound healing, revascularization, tissue remodeling, and allograft rejection and can provide guidance for improving outcomes after clinical transplantation.

Implication of macrophages in blood brain barrier disruption during central nervous system infection

Infection of the central nervous system (CNS) with the neurotropic JHM strain of mouse hepatitis virus (JHMV) induces extensive cell infiltration and provides a useful model to study both virus-induced encephalitis and demyelination. Cells recruited into the CNS include components of the innate response, namely neutrophils, NK cells and macrophages, followed by CD4, CD8 T cells and B cells. CD8 T cells are the primary effectors which control virus replication. The exact contribution of macrophages in JHMV pathogenesis remains unclear. Pathogenesis studies in CCR5 deficient mice (a chemokine receptor involved in macrophage and T cell recruitment) suggested a role of macrophages in demyelination. In addition, depletion of both neutrophils and monocytes by the antiGr1 antibody (RB6-8C5) significantly reduced blood brain barrier (BBB) permeability and correlated with an absence of matrix metalloproteinase-9 (MMP9) activity. These data suggested a potential role of neutrophils and macrophages in BBB disruption by MMP9 release.

Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy

The tumor microenvironment consists of stromal cells and extracellular factors that evolve in parallel with carcinoma cells. To gain insights into the activities of stromal cell populations, we developed and applied multicolor imaging techniques to analyze the behavior of these cells within different tumor microenvironments in the same live mouse. We found that regulatory T-lymphocytes (Tregs) migrated in proximity to blood vessels. Dendritic-like cells, myeloid cells and carcinoma-associated fibroblasts all exhibited higher motility in the microenvironment at the tumor periphery than within the tumor mass. Since oxygen levels differ between tumor microenvironments, we tested if acute hypoxia could account for the differences in cell migration. Direct visualization revealed that Tregs ceased migration under acute systemic hypoxia, whereas myeloid cells continued migrating. In the same mouse and microenvironment, we experimentally subdivided the myeloid cell population and revealed that uptake of fluorescent dextran defined a low-motility subpopulation expressing markers of tumor-promoting, alternatively activated macrophages. In contrast, fluorescent anti-Gr1 antibodies marked myeloid cells patrolling inside tumor vessels and in the stroma. Our techniques allow real-time combinatorial analysis of cell populations based on spatial location, gene expression, behavior and cell surface molecules within intact tumors. The techniques are not limited to investigations in cancer, but could give new insights into cell behavior more broadly in development and disease.

Role of H2-calponin in Regulating Macrophage Motility and Phagocytosis

The actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. Calponin is an actin-associated regulatory protein. Here we report the finding of significant levels of the h2 isoform of calponin in peripheral blood cells of myeloid lineage. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed. Germ line transmission of the Cnn2-flox-neo allele was obtained in chimeras from two independent clones of targeted embryonic stem cells. The insertion of the neo(R) cassette into intron 2 of the Cnn2 gene resulted in a significant knockdown of h2-calponin expression. Removing the frt-flanked neo(R) cassette by FLP1 recombinase rescued the knockdown effect. Cre recombinase-induced deletion of the loxP-flanked exon 2 eliminated the expression of h2-calponin protein. H2-calponin-free mice showed reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free macrophages demonstrated a higher rate of proliferation and faster migration than that of h2-calponin-positive cells, consistent with a faster diapedesis of peripheral monocytes and neutrophils. H2-calponin-free macrophages showed reduced spreading in adhesion culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also significantly increased macrophage phagocytotic activity, suggesting a novel mechanism to regulate phagocyte functions.

Role of Bv8 in neutrophil-dependent angiogenesis in a transgenic model of cancer progression

The secreted Bv8 protein has been recently characterized as a regulator of myeloid cell mobilization and a neutrophil-derived mediator of tumor angiogenesis in several xenografts, but its role in tumor progression in an endogenous setting was unknown. The rat insulin promoter (RIP)-T-antigen (Tag) is a well characterized transgenic mouse model of multistage pancreatic beta-cell tumorigenesis. Also, the role of neutrophils in RIP-Tag angiogenic switching, as assessed by systemic ablation using anti-Gr1 antibodies at different stages of tumor progression, has been recently described. Here, we show that early treatment of RIP-Tag mice with anti-Bv8 antibodies resulted in a significant reduction in the number of angiogenic islets relative to control antibody-treated mice, implicating Bv8 in the angiogenic switch during neoplasia. Histological analysis showed a significant reduction in vascular surface areas in hyperplastic and angiogenic lesions in pancreatic islets from anti-Bv8-treated mice. Anti-Bv8 treatment also inhibited the mobilization and homing of CD11b+Gr1+ cells to the peripheral blood and the emerging neoplastic lesions. However, anti-Bv8 treatment had no effect on tumor vascularization or burden when initiated at later stages of tumor progression. The stage-dependent efficacy of anti-Bv8 treatment appears remarkably similar to that reported after neutrophil ablation, suggesting that Bv8 is an important mediator of neutrophil-dependent angiogenesis in this transgenic model. In summary, our studies verify a role for Bv8 in the mobilization and recruitment of myeloid cells and in the induction of tumor angiogenesis in the early stages of neoplastic progression.

Dendritic cells overexpressing CD95 (Fas) ligand elicit vigorous allospecific T-cell responses in vivo

Dendritic cells (DCs) genetically engineered to overexpress CD95 (Fas) ligand (CD95L-DC) were proposed as tools to induce peripheral tolerance to alloantigens. Herein, we observed that CD95L-DC obtained after retroviral gene transfer in bone marrow (BM) precursors derived from CD95-deficient (lpr/lpr) mice elicit much stronger allospecific type 1 helper T-cell and cytotoxic T-cell activities than control DCs upon injection in vivo, although they induce lower T-cell responses in vitro. Indeed, a single injection of CD95L-DC prepared from C57BL/6 mice was sufficient to prime bm13 recipients for acute rejection of C57BL/6 skin allografts that were otherwise tolerated in the context of this single weak major histocompatibility complex (MHC) class I incompatibility. Massive neutrophil infiltrates depending on interleukin (IL)-1 signaling were observed at sites of CD95L-DC injection. Experiments in IL-1 receptor-deficient mice or in animals injected with depleting anti-Gr1 monoclonal antibody (mAb) established that neutrophil recruitment is required for the development of vigorous T-cell responses after injection of CD95L-DC in vivo.