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T Cell-Mediated, IFN-γ-Facilitated Rejection of Murine B16 Melanomas
Waltraud Böhm, Stefan Thoma, Frank Leithäuser, Peter Möller, Reinhold Schirmbeck and Jörg Reimann
J Immunol July 15, 1998, 161 (2) 897-908;
Waltraud Böhm
Stefan Thoma
Frank Leithäuser
Peter Möller
Reinhold Schirmbeck
Jörg Reimann
The murine melanoma cell line B16.F10 (H-2b) was used to study specific T cell responses that reject tumors. Stable B16 transfectants were established that express viral Ags, either the hepatitis B surface Ag (HBsAg) or the large tumor Ag (T-Ag) of SV40. B16 cells and their transfected sublines were CD40+CD44+ but expressed no (or low levels of the) costimulator molecules CD154 (CD40L), CD48, CD54, CD80, and CD86. Surface expression of MHC class I (Kb, Db) and class II (I-Ab) molecules by B16 cells was low, but strikingly up-regulated by IFN-γ. CD95 (Fas) and CD95 ligand (CD95L (FasL)) were "spontaneously" expressed by B16 cells growing in vitro in serum-free medium; these markers were strikingly up-regulated by IFN-γ. B16 cells coexpressing CD95 and CD95L were irreversibly programed for apoptosis. In vitro, noninduced B16 transfectants stimulated a specific IFN-γ release response, but no cytolytic response (in a 4-h assay) in MHC class I-restricted CTL; in contrast, IFN-γ-induced B16 targets were efficiently and specifically lysed by CTL. In vivo, B16 transfectants were specifically rejected by DNA-vaccinated syngeneic hosts through a T-dependent immune effector mechanism. The tumors showed evidence of massive apoptosis in vivo during the rejection process. The data suggest that CTL-derived IFN-γ enhances an intrinsic suicide mechanism of these tumor cells in addition to facilitating lytic interactions of effectors with tumor targets.
Transplantable murine melanomas are well-established models for the study of experimental cancer therapies. Melanomas express different tumor-associated Ags, which are potential targets for novel designs of therapeutic cancer vaccines. Melanoma-associated Ags include tyrosinase and tyrosine-related protein (TRP)-1/2 (1, 2, 3) or the MAGE and BAGE class of Ags (4, 5, 6, 7, 8, 9). Many immunotherapeutic protocols have been tested using the murine B16 melanoma cell line (and its sublines) that originated in the C57BL/6 (H-2b) mouse strain. B16 melanoma cells express some of these melanoma-associated Ags, and their growth can be controlled in vivo by different classes of specific or nonspecific immune effector cells.
Some tumor cells express the CD95 receptor for the TNF-like CD95 ligand (CD95L).3 These tumors can potentially be eradicated by CD95L produced locally by tumor-infiltrating lymphomyeloid cells (10). Cells from primary or metastatic lesions of some tumors, and from some in vitro established tumor cell lines, express the CD95L; this was shown for colonic adenocarcinomas (11), hepatocellular carcinomas (12, 13), leukemias (14), and melanomas (15). These CD95L-expressing tumor cells can escape immune surveillance by inducing apoptosis of CD95+ immune effector cells (16, 17). Coexpression of CD95 and CD95L by normal and tumor cells has been found. Cells of some activated lymphocyte subsets coexpress CD95 and CD95L. These cells undergo apoptosis (or "activation-induced cell death"), and this mechanism seems to represent one form of a physiologic down-regulation of an immune response (18, 19, 20). Although CD95 is abundantly expressed by hepatocytes, CD95L+ hepatocellular carcinoma cells down-regulate Fas expression to escape apoptosis (12). Colonic adenocarcinoma cells or leukemia and lymphoma cells coexpress CD95 and CD95L but acquire resistance to CD95-induced apoptosis (11, 14).
Although expression of MHC-I/II and costimulator molecules on the surface of noninduced B16 tumor cells is low or absent, primed CTL can play a role in rejecting this tumor. In vitro, presentation of antigenic peptides in the context of MHC-I glycoproteins by B16.F10 cells to CD8+ CTL lines (CTLL) triggers IFN-γ (IFN-γ) release but not specific lysis of the tumor cells by CTL. IFN-γ enhances MHC-I expression by B16.F10 cells and thereby renders them susceptible to CTL-mediated specific lysis. In addition, we describe the cytokine-amplified apoptosis of murine B16.F10 melanoma cells. By enhancing expression of CD95 and CD95L by a subset of B16 cells, CTL-derived IFN-γ irreversibly commits this tumor cell subset to apoptosis. In vivo, T cells efficiently reject B16.F10 tumor cells. This suggests that IFN-γ-dependent apoptosis of CD95L+/CD95+ tumor cells can play a role in tumor rejection in vivo.
C57BL/6J mice (H-2b) were bred and kept under specific pathogen-free conditions in the animal colony of the University of Ulm. Breeding pairs of these mice were obtained from Bomholtgard (Ry, Denmark). Male and female mice were used at 12 to 16 wk of age.
The Rauscher virus-transformed T lymphoma line, RBL5, is derived from a C57BL/6J (H-2b) mouse. The RBL5/S and RBL5/T transfectants (expressing the hepatitis B surface Ag (HBsAg) or the SV40 large tumor Ag (T-Ag)) have been described (21, 22, 23). The H-2b melanoma cell lines B16.F0 and B16.F1 from the American Type Culture Collection (ATCC, Manassas, VA; CRL-6322, CRL-6323) and B16.F10 (from Dr. P. Antonsson, Lund, Sweden) were used. In most experiments, we used the B16.F10 line.
The bovine papilloma virus-based vector BMGneo (24) (a generous gift from Drs. Y. Karasuyama and F. Melchers, Basel, Switzerland) was used to construct the BMG/HBS expression plasmid (22) and the BMG/T-Ag.1 expression plasmid (21). BMG/HBS and BMG/T-Ag.1 vector DNA, or BMGneo vector DNA (without insert), were transfected into B16 cells using the CaPO4 method. Expression of the viral proteins was tested in B16 transfectants labeled with 400 μCi of [35S]methionine (cat. no. SJ1015; Amersham, Braunschweig, Germany) in methionine-free RPMI 1640 medium. Labeled cells were washed twice in PBS and extracted with lysis buffer (120 mM NaCl, 1% aprotinin (Trasylol, cat. no. 48764; Bayer, Leverkusen, Germany), 50 μM leupeptine, 0.5% Nonidet P-40, 10% glycerol, and 50 mM Tris/HCl, pH 8.0) for 30 min at 4°C. Extracts cleared by centrifugation (30 min, 20,000 × g, 4°C) were subsequently incubated for 2 h at 4°C with protein-A Sepharose (cat. no. 17-0780-01; Pharmacia, Freiburg, Germany). The HBsAg protein was precipitated with a polyclonal rabbit anti-HBs antiserum and protein A-Sepharose, and the T-Ag protein was precipitated with the mAb Pab108 (25) and protein A-Sepharose at 4°C. The precipitates were washed extensively in 0.5 M LiCl, 1% Nonidet P-40, and 0.1 M Tris/HCl (pH 9.0). After two additional washes of the complexes in 1× PBS and one wash in 0.1× PBS, they were recovered from protein-A Sepharose in 400 μl of elution buffer (1.5% SDS, 5% β-ME, and 7 mM Tris/HCl, pH 6.8). Following an incubation for 30 min at 37°C, SDS-denatured eluates were lyophilized and dissolved in 30 μl of aqueous solution of 7% β-ME, 10% glycerol and bromophenol blue. After boiling for 2 min, samples were analyzed by SDS-PAGE.
IFN-γ treatment of B16 cells
B16 cells were cultured for 12 to 48 h in serum-free medium containing 20 to 400 U/ml mouse rIFN-γ (cat. no. 1276905, Boehringer Mannheim, Mannheim, Germany). Cells were harvested and washed, then used in flow cytometry (FCM) analyses or cytotoxic assays.
FCM analyses
For FCM studies, cells were suspended in PBS/0.3% (w/v) BSA supplemented with 0.1% (w/v) sodium azide. Nonspecific binding of Abs to FcR was always blocked by preincubating cells with the mAb 2.4G2 (cat. no. 01241D) directed against the FcγIII/II (CD16/CD32) (2 μg mAb/106 cells/100 μl). Cells were incubated with 0.5 μg/106 cells of the relevant mAb for 20 min at 4°C and washed twice. In most experiments, cells were subsequently incubated with a second-step reagent for 20 min at 4°C. Three-color FCM analyses were performed on a FACScan (Becton Dickinson, Mountain View, CA). The forward narrow angle light scatter was used as an additional parameter to facilitate exclusion of dead cells and aggregated cell clumps. The following reagents and mAbs from PharMingen (Hamburg, Germany) were used: FITC-conjugated or biotinylated anti-CD3ε mAb 145-2C11 (cat. no. 01084D, 01082D), FITC-conjugated anti-CD4 (L3T4) mAb H129.19 (cat. no. 09004D), biotinylated anti-CD4 (L3T4) mAb RM4-5 (cat. no. 01062D), anti-CD8α (Ly-2) mAB 53-6.7 (cat. no. 01042D), FITC-conjugated anti-CD54 mAb 3E2 (cat. no. 01224D), FITC-conjugated anti-CD44 (Pgp-1) mAb IM7 (cat. no. 01224D), phycoerythrin (PE)-conjugated anti-CD80 (B7.1) mAb 16-10A1 (cat. no. 0965B), biotinylated anti-CD86 mAb GL-1 (cat. no. 09272D), biotinylated anti-CD95 (Fas) mAb Jo2 (cat. no. 15402D), FITC-conjugated anti-H2-Kb mAb AF6-88.5 (cat. no. 06104D), biotinylated anti-H2-Db mAb 28-14-8 (cat. no. 06232D), FITC-conjugated anti-IAb mAb 25-9-17 (cat. no. 06254D), PE-conjugated anti-CD48 mAb HM48-1 (cat. no. 09175B), biotinlated anti-CD40 mAb 3/23 (cat. no. 09662D), biotinylated anti-CD40L (gp39) mAb MR-1 (cat. no. 09022D), Via-Probe (7-AAD; cat. no. 34321X), Annexin V-PE-conjugated (cat. no. 65875X), Annexin V-FITC-conjugated (cat. no. 65874X), and PE-, cychrome-, and FITC-conjugated streptavidin (cat. no. 13025D, 13024D). The anti-CD95L (FasL) mAb N-20 (cat. no. sc-834) was obtained from Santa Cruz Biotechnology (Heidelberg, Germany).
HBsAg- and T-Ag-encoding expression vectors used for DNA immunization
The construction of the expression plasmid pCMV-1/T has been described (26). The expression plasmid pCI/S (containing the small HBsAg) was constructed by cloning the HBsAg-encoding Xho-I/Bgl-II fragment of HBV, subtype ayw (from the plasmid pTKTHBV2, a generous gift of Dr. M. Meyer, Munich, Germany) into the pCI vector (cat. no. E1731, Promega, Heidelberg, Germany) cut with Xho-I/Bam-HI. COS-7 cells (CRL-1651, ATCC) were transfected with 30 μg plasmid DNA/106 cells using the CaPO4-method. Cells were cultured for 2 d, and expression of the viral proteins by these cells was detected by immunoprecipitation as described above. HBsAg and T-Ag proteins were expressed in transiently transfected COS-7 cells (data not shown). Plasmid DNA used for immunization was purified by anion exchange chromatography using the Qiagen maxiprep kit (Qiagen, Hilden, Germany). Naked plasmid DNA suspended at 1 μg/μl in PBS was injected.
Reverse transcriptase-PCR
Total RNA was isolated from nontransfected or transfected B16 cells (Qiagen minikit, cat. no. 14123) and reverse transcribed to cDNA using a cDNA synthesis kit (Stratagene, Frankfurt, Germany cat. no. 200420). Aliquots from each cDNA preparation were PCR-amplified using the forward/reverse primers and the conditions previously described (27), and the buffer and polymerase from Stratagene (cat. no. 600153-81, 600153-82). The 0.54-kb fragment was resolved on a 2% agarose gel.
DNA vaccination
We injected 50 μl PBS containing 50 μg plasmid DNA into each regenerating tibialis anterior muscle 5 days after the injection of cardiotoxin (Latoxan; Rosans, France) as described (28). All mice received one bilateral intramuscular injection. Mice injected with pCMV-1/T plasmid DNA or pCI/S plasmid DNA developed a potent T-Ag- or HBsAg-specific CTL response (23, 26, 28). Noninjected mice or mice injected with pCI or pCMV-1 plasmid DNA (without insert) were used as controls.
Subcutaneous transplantation of tumor cells
B16.F10, B16.F10/S, and B16.F10/T cells were cultured in serum-free medium (UltraCulture; BioWhittaker, Walkersville, MD, cat. no. 12-725F). The cells were transplanted into immunocompetent, syngeneic C57BL/6J mice by injecting titrated numbers of cells (103-106 cells/mouse) in 50 μl PBS s.c. into the left lateral flank of age- and sex-matched mice. Tumor growth was measured every second or third day. Mice bearing tumors with a diameter >1 cm were sacrified (according to the regulation for animal experimentations of Baden-Württemberg). Data were plotted and statistically evaluated using the GraphPad Prism version 2 software (see Tables I and II).
Spleen cells were obtained from C57BL/6 mice primed 3 weeks before to T-Ag by DNA vaccination. Into B16.F10/T tumor-bearing mice, we injected i.p. 2 × 107-primed splenic (B cell-depleted) T cells. This led to an easily measurable regression of the 3 to 6-mm tumor in most treated mice. Regressing tumors were removed for histopathologic examination 2 to 3 days after T cell transfer.
In vivo suppression of CD4+ or CD8+ T cells in mice
CD4+ or CD8+ T cell subsets were suppressed in mice by repeated injections of the anti-CD4 mAb YTS 191.1 or the anti-CD8 mAb YTS 169.4. Two days before the tumor cell transplantation, mice were i.p. injected with 200 μl PBS containing 500 μg purified Ab. At 5-day intervals (i.e., day 3 and day 8 after tumor cell transplantation) mice were again injected with 250 μg of the respective Abs. FCM analyses of PBMC populations demonstrated that 90 to 98% of T cells expressing the respective phenotype were deleted in treated mice (data not shown).
CTL lines
Spleen or lymph node cells were suspended in α-MEM (Life Technologies, Berlin, Germany) supplemented with 10 mM HEPES buffer, 5 × 10−5 M 2-ME, antibiotics, and 10% v/v FCS (Pan Systems, Aidenbach, Germany). To the culture medium was further added 2% v/v of a selected batch of Con A-stimulated rat spleen cell supernatant. In upright 25-cm2 tissue culture flasks in a humidified atmosphere/5% CO2 at 37°C, 3 × 107 responder cells were cocultured with 1.5 × 106 syngeneic RBL5/S or RBL5/T transfectants (irradiated with 20,000 rad) in 10 ml of medium. CD3+CD8+ T blasts harvested from restimulated cultures were separated on a discontinuous density gradient and restimulated twice weekly (at 5 × 104 cells/ml) with irradiated RBL5/S or RBL5/T transfectants. These CTL lines (CTLL) were grown for 2 to 6 wk in vitro. Cells from all lines were cytolytic and displayed the CD3+CD4−CD8+TCRαβ+ phenotype.
Medium was conditioned by coculturing 106/ml CTLL.T cells (or CTLL.S cells) with 106/ml B16.F10.T cells (or B16.F10.S cells) for 12 to 24 h at 37°C. The medium was cleared after the incubation (15 min, 2000 × g) and stored at 4°C. In the experiments described, the medium was used at 5% v/v. The mAb R4-6A2 neutralizes mouse IFN-γ bioactivity; it was added to cultures to obtain a final concentration of 20 μg/ml. In some experiments, the IFN-γ bioactivity in conditioned medium was neutralized by incubating it with 20 μg/ml of mAb R46A2 for 12 h at 4°C.
In vitro culture and cytotoxic assay
CTL harvested after 5 days of in vitro culture were washed, and serial dilutions of these effector cells were cocultured with 2 × 105 51Cr-labeled targets in 200-μl round-bottom wells. After a 4-h incubation at 37°C, 100 μl of supernatant were collected for gamma counting. The percentage of specific release was calculated as [(experimental release − spontaneous release)/(total release − spontaneous release)] × 100. Total counts were measured by resuspending target cells. Spontaneously released counts were always <15% of the total counts. Data shown are the mean of triplicate cultures. The SEM of triplicate data was always <15% of the mean.
Histology studies
Cells undergoing apoptosis were detected in situ by labeling DNA strand breaks using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL). Briefly, formalin-fixed and paraffin-embedded tissue sections were dewaxed and digested with proteinase K (20 μg/ml). After inactivating endogenous peroxidase with H2O2, the labeling reaction was conducted using 10 U TdT (Promega) and 2 μM digoxigenin-11-dUTP (Boehringer Mannheim) in 50 μl TdT-buffer (0.5 M cacodylix acid, sodium salt, pH 6.8; 1 mM CoCl2, 0.5 mM DTT; 0.05% (w/v) BSA; 0.15 M NaCl). Labeled cells were detected using sheep anti-digoxigenin Fab (5 μg/ml; Boehringer-Mannheim) followed by horseradish peroxidase-conjugated F(ab′)2 fragment of donkey anti-sheep IgG (0.2 mg/ml; Dianova, Hamburg, Germany). Bound horseradish peroxidase was visualized by the substrate 3-amino-9-ethylcarbazole (0.1 mg/ml in 0.17 M sodium acetate, pH 5.2, plus 0.01% H2O2). After counterstaining in Mayer's hematoxylin, representative tissue areas were photographed under a Zeiss Axiophot microscope.
Expression of the viral proteins HBsAg and SV40 T-Ag in B16 melanoma cells
B16.F10 cells were transfected with the episomal expression constructs BMG/HBS or BMG/T-Ag.1. Control transfectants were transfected with BMGneo vector DNA without insert. Transfected cells were selected for 5 to 10 wk in vitro. Immunoprecipitation studies revealed stable expression of the nonglycosylated p24 and the glycosylated gp27 form of the HBsAg protein by B16.F10/S transfectants, or of the T-Ag protein (complexed to p53) by the B16.F10/T transfectants (Fig. 1⇓). Cells from these lines, and from sublines derived from these lines, showed stable expression of the viral proteins for longer than 8 mo. The level of expression of HBsAg and T-Ag by B16.F10/S or B16.F10/T transfectants was comparable to that of the RBL5/S and RBL5/T transfectants (Fig. 1⇓) described previously (21, 22).
Expression of the viral proteins in B16.F10 melanoma cells. B16.F10 melanoma cells were transfected with the (HbsAg-encoding) BMG/HBS plasmid DNA, or the (T-Ag-encoding) BMG/T-Ag.1 plasmid DNA, or BMGneo vector DNA (containing no insert). Expression of the viral proteins was detected in cells (selected for 8 wk) by immunoprecipitating the Ag from cell lysates using specific Abs (see Materials and Methods for details). Control immunoprecipitations from lysates of RBL5/S and RBL5/T transfectants are included into the SDS-PAGE analysis.
Surface phenotype of noninduced and IFN-γ-induced B16 cells
B16 melanoma cell lines (derived from H-2b C57BL/6 mice) cultured in serum-supplemented or serum-free medium expressed only very low levels of the MHC-I molecules Kb and Db and the MHC-II molecule I-Ab on the cell surface. Expression of Kb, Db, and I-Ab was strikingly up-regulated in cells stimulated with IFN-γ (Fig. 2⇓). Similar data were obtained with nontransfected B16.F10 cells and the transfected B16.F10/S and B16.F10/T sublines (data not shown).
Surface expression of costimulating molecules by B16.F10 cells. B16.F10 cells cultured in serum-free medium were unstimulated (−IFN-γ) or stimulated for 12 h with 50 U/ml IFN-γ (+IFN-γ). Surface expression of the MHC-I molecules Kb and Db, of the MHC-II molecules I-Ab, and of the costimulator molecules CD40, CD40L, CD44, CD48, and CD54 was measured using the mAb listed in the Materials and Methods.
We analyzed the surface expression of T cell-costimulating molecules in noninduced and IFN-γ-induced (nontransfected or transfected) B16 cells cultured in either serum-supplemented or serum-free medium. Nonpretreated and IFN-γ-treated B16 cells express CD40 molecules of the NGFR superfamily on the cell surface, but no (or very low levels of) CD40L (Fig. 2⇑). High surface expression of CD44 was detectable in noninduced and IFN-γ-induced B16 cells (Fig. 2⇑). Expression of the costimulator molecules CD48 (interacting with murine CD2), CD54/ICAM-1 (interacting with LFA-1), CD80 (B7-1), and CD86 (B7-2) (interacting with CD28/CTLA4) on the surface of nonpretreated and IFN-γ-treated B16 cells was low or absent (Fig. 2⇑, and data not shown). The B16 cells, therefore, expressed the CD40+CD40L−CD44+CD48−CD54−CD80−CD86− phenotype. Similar data were found by analyzing B16.F10 melanoma cells and their transfected sublines cultured in serum-supplemented or serum-free medium.
Inducible surface expression of CD95 (Fas) and CD95L (FasL) in B16 cells
In vitro-cultured B16 cells express CD95 (Fas) and CD95L (FasL) on the cell surface (Fig. 3⇓). The fractions of CD95+ and CD95L+ B16 cells were always higher when cells were cultured in serum-supplemented than in serum-free medium. IFN-γ up-regulated CD95 and CD95L surface expression on B16 cells (Fig. 3⇓). Expression of CD95L by noninduced and IFN-γ-induced B16 cells was confirmed at the RNA level by RT-PCR analyses (Fig. 3⇓). Two-color FCM analyses showed that a fraction of CD95+ B16 cells coexpressed CD95L, while another fraction of B16 cells displayed the CD95+CD95L− surface phenotype (Fig. 3⇓).
Surface expression of CD95 and CD95L by B16.F10 cells. B16.F10 cells growing in serum-free medium were unstimulated (−IFN-γ) or stimulated for 12 h with 50 U/ml IFN-γ (+IFN-γ). Surface expression and coexpression of CD95 (Fas)/CD95L (FasL) was determined using the mAb listed in Materials and Methods. In RT-PCR analyses of unstimulated or stimulated B16.F10 cells, mRNA of CD95L (FasL) was evident.
In IFN-γ-stimulated cultures of B16 cells, the fraction of CD95+CD95L+ B16 cells was increased two- to threefold. This subset was nonviable (stained with the nucleic acid dye 7-AAD) and apoptotic (reacted with the apoptosis marker annexin V and reacted in TUNEL stain). From the cell sorter-purified CD95+CD95L+ subset, no cells could be expanded in vitro and no viable cells were recovered 12 to 24 h after sorting, indicating that this B16 subset is irreversibly programmed for death. Ligation of surface CD95 with Ab on CD95+CD95L+ B16 cells may have triggered apoptosis, but this seems unlikely in view of the observation that cell sorter-purified CD95+CD95L− B16 cells readily grew in vitro (data not shown). Cells of the transfected B16.F10/S and B16.F10/T sublines showed a similar surface expression pattern of CD95 and CD95L (data not shown).
B16 cells stimulate IFN-γ release by CTL and are susceptible to specific CTL-mediated lysis
CTLL specific for MHC-I-binding epitopes of the T-Ag or the HBsAg were obtained from DNA-vaccinated C57BL/6 mice (23, 26). To detect specific IFN-γ release of CD8+ CTLL in response to the class I-restricted presentation of epitopes of the two viral proteins by the transfected melanoma cells, CTL were cocultured for 4 to 24 h with nontransfected B16.F10 cells or transfected B16.F10/T or B16.F10/S cells (or control RBL5, RBL5/T, or RBL5/S cells) (Fig. 4⇓). Transfected RBL5 cells supported a specific IFN-γ release response by CTL detectable after 4 h and 12 h; transfected B16 cells supported a specific IFN-γ release response by CTL detectable after 12 h but not 4 h. The latter response was detectable after coculture with B16.F10/S or B16.F10/T transfectants harvested from serum-supplemented or serum-free cultures (data not shown).
IFN-γ release by CTLL in response to stimulation by B16.F10 tumor cell transfectants. Representative data using two MHC-I-restricted CTLL specific for either HBsAg (CTLL.S) or SV40 T-Ag (CTLL.T) are shown that were generated in C57BL/6 mice by DNA vaccination. CTLL were stimulated for 4, 12, or 24 h with transfected B16.F10 or (positive control) transfected RBL5 cells. Controls included CTLL stimulated with nontransfected B16.F10 or RBL5 cells, transfected RBL5 cells, or B16.F10 cells cultured without CTLL, and supernatant in which IFN-γ bioactivity was neutralized by the mAb R4-6A2 (anti-IFN-γ). IFN-γ in conditioned medium was measured by ELISA.
In short term 4-h cytotoxic assays, CTLL did not specifically lyse B16.F10/S or B16.F10/T targets (Fig. 5⇓, C and D) but efficiently and specifically lysed RBL5/T and RBL5/S control targets (Fig. 5⇓, A and B). IFN-γ-treated B16.F10/S and B16.F10/T transfectants were efficiently and specifically lysed by the respective CTLL in a 4-h assay, indicating that the up-regulated Kb/Db surface expression allowed the specific cytolytic interaction of the targets with the effector cells in a short term assay (Fig. 5⇓, C and D). When the coculture of CTL with (noninduced) B16.F10/S and B16.F10/T targets was extended to 12 h, efficient and specific CTL lysis was observed (Fig. 5⇓, E and F).
Specific CTL-mediated lysis of transfected B16.F10 cells. CTLL specific for HBsAg (A, C, E) or T-Ag (B, D, F) were generated in C57BL/6 mice by DNA vaccination. In a 4-h 51Cr release assay, these CTLL specifically lysed IFN-γ-pretreated, but not nonpretreated, transfected B16.F10 targets (C, D). In a 12-h assay, these CTLL showed specific cytolytic reactivity toward noninduced B16.F10 transfectants (E, F). E:T ratios are 20:1, 10:1, 5:1, and 2.5:1.
In a 4-h assay, B16.F10 transfectants (but not nontransfected control targets) incubated for 12 h either with recombinant IFN-γ, or with medium conditioned by CTLL (stimulated for 12 h with noninduced B16.F10 transfectants) were specifically lysed by CTL (Fig. 6⇓A). Elimination of IFN-γ bioactivity from medium conditioned by CTLL with the neutralizing R4-6A2 Ab suppressed the activity of these supernatants to render B16.F10 transfectants susceptible to CTL lysis in a 4-h assay. CTL-derived IFN-γ generated during coculture of CTLL with noninduced B16.F10 transfectants hence seems to render melanoma cells susceptible to specific cytolytic attack in a 12-h assay. This was confirmed in experiments that showed that the specific cytolysis of B16.F10 transfectants in a 12-h assay was blocked by neutralizing endogenously generated IFN-γ bioactivity in the cultures (Fig. 6⇓B). Similar data were obtained using B16.F10/T or B16.F10/S transfectants (data not shown).
Specific CTL lysis of B16.F10 transfectants is IFN-γ dependent. A, Medium (sup) was conditioned by coculturing 106/ml CTLL.T cells with 106/ml B16.F10.T cells for 12 h at 37°C. The medium was cleared after the incubation (15 min, 2000 × g) and was used at 5% v/v; its IFN-γ content is shown in Figure 4⇑. The mAb R4-6A2 (anti-IFN-γ), which neutralizes mouse IFN-γ, was added to some cultures at a final concentration of 20 μg/ml; its neutralization activity is shown in Figure 4⇑. CTLL.T lysed B16.F10/T targets treated with either sup or rIFN-γ. Sup in which the IFN-γ bioactivity was neutralized did not induce susceptibility to lysis in B16.F10/T targets. B, During a 12-h assay, B16.F10/T targets were lysed only when IFN-γ activity was present in the cultures; neutralization of this activity suppressed the generation of a lytic interaction between CTL and the melanoma targets.
When B16.F10/S and B16.F10/T "effector" cells were cocultured at E:T ratios of 10:1 to 1:1 with radiolabeled CTLL "target" cells in a 12-h assay, no specific lysis of the T cell targets was detectable. We thus found no evidence for specific cytolytic reactivity of the tumor cells against the immune effector cells (data not shown).
Tumorigenicity of the B16 melanoma cell line and its transfected sublines in syngeneic, immune, or nonimmune hosts
Subcutaneous transfer of 103 to 106 B16 cells into C57BL/6 mice induced aggressively growing, lethal tumors in most transplanted animals (Table I⇓). The B16 transfectants expressing either the HBsAg or the T-Ag showed a similar aggressive growth in vivo. Only when low numbers of B16.F10/S transfectants were transplanted did a significantly reduced number of animals develop melanomas (Table I⇓).
Subcutaneous growth of transfected B16 cells in syngeneic hosts
Growth of nontransfected B16.F10 cells and transfected B16.F10/S and B16.F10/T cells was tested in DNA-vaccinated hosts. The data in Figure 7⇓ and Table II⇓ demonstrate that DNA vaccination specifically protected mice against the aggressive growth of a melanoma that expressed the viral Ag against which the host was immunized. MHC-I expression was readily detectable on transfected B16 cells freshly isolated from melanomas growing in immune hosts. These data confirm observations in other mouse tumor models (26, 29, 30, 31, 32). Although MHC-I surface expression is low and MHC-II surface expression is undetectable in FCM analyses of noninduced B16 cells, tumor eradication operated efficiently in immune hosts.
Subcutaneous growth of nontransfected and transfected B16.F10 melanoma cells in immune, syngeneic hosts. C57BL/6 mice were DNA vaccinated against HBsAg or T-Ag. These mice showed efficient, MHC-I-restricted CTL reactivity against the respective viral Ags. These mice were transplanted with 103, 104, 105, or 106 nontransfected B16.F10 or transfected B16.F10/S or B16.F10/T tumor cells. The fraction of tumor-free/tumor-bearing mice was scored for 50 days posttransplantation. Mice with tumors of a diameter >1 cm were sacrificed.
Subcutaneous growth of transfected or nontransfected B16 melanoma cells in DNA-vaccinated, syngeneic hosts
Rejection of transfected B16.F10 melanomas by immune hosts required CD4+ and CD8+ T cells. The CD4+ T cell subset, the CD8+ T cell subset, or both subsets were in vivo deleted in vaccinated mice by Ab treatment before/after tumor cell transfer (Table III⇓). Mice were monitored for 4 mo posttreatment/post-tumor cell transfer for the appearance of tumors. The (almost complete) elimination of T cells from the immune host abolished its capacity to specifically resist tumor growth, while the selective elimination of only CD4+ T cells or only CD8+ T cells did not. Rejection in this system is hence T dependent, and requires both CD4+ and CD8+ T cell subsets. Similar observations were made using B16.F10/T or B16.F10/S transfectants.
T-dependent rejection of transfected B16.F10 melanoma cells in immune hosts
T cells were prepared from mice primed, 3 wk previously, by DNA vaccination to either T-Ag or HBsAg. The cells were adoptively transferred into syngeneic hosts bearing a melanoma that had reached a diameter of 3 to 6 mm (derived from transfected, T-Ag-expressing B16.F10 cells). Transfer of T-Ag-specific but not HBsAg-specific T cell populations led to regression of s.c. growing melanomas. The histopathology of regressing tumors indicated that a large fraction of the tumor cells underwent apoptosis detectable by the TdT-mediated in situ TUNEL technique (Fig. 8⇓). Apoptotic cells were located predominantly at the border to the adjacent stromal tissue, whereas few apoptotic events were detectable in the central areas of tumor nodules. The fraction of apoptotic cells was >10-fold higher in tumor-bearing mice transplanted with T cells specifically immune to the viral Ag that the tumor cells expressed (i.e., the SV40 T-Ag) than in mice transplanted with T cells primed to an irrelevant control viral nucleoprotein. In vivo, rejection of melanomas by primed T cells is thus accompanied by apoptosis of the tumor cells. Injection of 100 U of mouse rIFN-γ into growing B16.F10 melanomas in vivo did not yield a similar picture.
Extensive apoptosis is evident during T-dependent regression of B16 melanomas. Splenic (B cell-depleted) T cells primed to T-Ag (left panel) or to HbS-Ag (right panel) were adoptively transferred into B16.F10/T tumor-bearing mice. Tumors were removed 2 days after T cell transfer and fixed in formalin. Apoptosis was detected using the TUNEL technique. Numerous apoptotic melanoma cells are visible, predominantly at the margin of the tumor (left). In contrast, only a few apoptotic bodies were detected after adoptive transfer of T cells primed to the irrelevant Ag HBsAg (right). Each photomicrograph is representative of a total of four tumors analyzed. Original magnification, ×400.
Stable B16.F10 melanoma transfectants expressing either the HBsAg protein or the T-Ag nucleoprotein were established. In H-2b mice, HBsAg is a "weak" immunogen for class I-restricted CTL, and T-Ag is a "strong" immunogen for this T cell subset (23, 26). Transplantation of genetically modified tumor cells that stably express well defined, CTL-stimulating Ags has been used in many experimental systems to mimick immune responses to tumor-associated Ags. The efficacy of tumor-rejecting CTL responses have been studied with B16 cells transfected with Ag-encoding expression plasmids (32, 33, 34). In our system, the parental B16.F10 line and its transfected sublines showed a similar tumorigenicity in vivo in syngeneic C57BL/6 hosts. Only after transfer of 104 B16.F10/S cells/mouse did we observe a statistically significant, lower incidence of tumor development (Table I⇑); we have published similar data on the P815 mastocytoma system (29).
DNA vaccination efficiently primes HBsAg- or T-Ag-specific, class I-restricted CTL responses to HBsAg and to T-Ag in H-2b mice (23, 26), but transfer of B16.F10/S or B16.F10/T cells into naive C57BL/6 mice did not (data not shown). This observation at least partly explains the aggressive in vivo tumor growth of transfected B16.F10/S or B16.F10/T cells. The low immunogenicity of nontransfected or transfected B16.F10 melanoma cells may be due to their deficient costimulator and cytokine expression profile, as well as to their inefficient MHC-restricted epitope presentation. The B16.F10 line used showed no (or very low) expression of the costimulator molecules CD48 (the ligand for CD2), CD54/ICAM-1 (binding LFA-1), CD80/B7-1, and CD86/B7-2 (binding CD28 or CTLA4). Expression of transfected B7-1/CD80 and/or B7-2/CD86 genes in B16 melanoma cells has been reported to induce anti-tumor immunity and to have an antimetastatic effect (35, 36, 37). It has been reported that B16 cells express IL-2 and the IL-2 receptors (38), but we found no IL-2 bioactivity in medium conditioned by the B16.F10 cells that we used (data not shown). Surface expression of MHC-I and MHC-II molecules by B16.F10 cells was low, but was strikingly up-regulated by IFN-γ treatment. This melanoma cell line is thus inefficient in the immunogenic, MHC-restricted epitope presentation to T cell precursors.
Although inefficient in priming a T cell response, B16 melanoma cells are nevertheless targets for various immune effector cells. In vivo growth of B16 melanomas can be eradicated by natural cell-mediated cytotoxicity (39), activated macrophages (40, 41), or CD8+ CTL (1, 37, 40, 42). Although CD8+ CTL seem to be involved in the rejection of B16 melanomas, CD4+ T cells, and possibly other immune effector mechanisms, seem to contribute to resistance to this tumor because CD8 T cell-depleted, immune mice showed resistance to this tumor. Rejection of B16 melanomas has been reported to be facilitated by the cytokines IL-1β (43, 44), IL-2 (45, 46, 47, 48), IL-4 (49), IL-10 (50, 51), IL-12 (52, 53), granulocyte-macrophage (GM)-CSF (36, 54, 55, 56, 57), monocyte (M)-CSF (52), TNF-α (43), and IFN-γ (40, 41, 58), but inhibited by TGF-β (45, 59). IFN-γ treatment in vivo and in vitro renders B16 melanoma cells susceptible to lysis by activated macrophages (1, 41, 60) and by CTL (this paper). Our data indicate that the interaction of Ag-presenting B16 cells with MHC-I-restricted CTL involved multiple events. 1) Specific MHC-I-restricted recognition of the viral epitopes presented by noninduced B16 cells triggers IFN-γ release by CTL but not their cytolytic program. 2) IFN-γ stimulation of B16 cells up-regulates MHC-I-restricted epitope presentation and allows the specific, cytolytic interaction between effector and target cell. 3) IFN-γ stimulation enhances the CD95/CD95L suicide program of a subset of B16 melanoma cells.
CTL that specifically recognize epitopes of the viral proteins HBsAg or T-Ag, in the context of MHC-I molecules presented by noninduced B16 cells, release IFN-γ. In contrast, the cytolytic program of these CTL (detectable in a 4-h 51Cr release assay) was not triggered by this specific interaction. This was observed using five independently derived CTLL (data not shown). This partial T cell activation may result from low MHC-I expression and/or low antigenicity of the recognized peptide epitope. The phenomenon resembles the selective signaling model observed in altered peptide ligand-induced partial T cell activation (61). IFN-γ produced by CTL in the immediate vicinity of B16 cells stimulates up-regulation of MHC-I- and MHC-II-restricted epitope presentation. This apparently facilitates the specific and efficient cytolysis of melanoma cells by primed CTL. In addition, IFN-γ may also enhance other tumor-rejecting effector mechanism (e.g., macrophages, dendritic cells).
The CTL-mediated lysis of melanoma cells may not be the only or the decisive mechanism of tumor eradication. A subset of nontransfected and transfected B16.F10 cells showed surface expression of CD95 and CD95L. Almost 90% of the B16.F10 cells expressed readily detectable levels of CD95 on the surface after a 12-h stimulation by 30 IU/ml murine IFN-γ. Furthermore, a well-defined 30 to 40% subset of the IFN-γ-stimulated B16.F10 cells coexpressed CD95L and CD95 on the cell surface; this was demonstrated by RT-PCR and by FCM analyses. IFN-γ-stimulated B16 cells that coexpressed CD95 and CD95L were apoptotic. They were stained for the apoptosis markers annexin, by the nucleic acid dye 7-AAD, and by the TUNEL technique; and they could not be maintained in a viable state in vitro. In unstimulated B16.F10 cells growing under serum-free conditions, a fraction of 10 to 20% are continuously eliminated by suicide. The apoptotic fraction of these melanoma cells is higher when cells are cultured in serum (FCS)-supplemented medium. We did not detect a significant fraction of cells dying of fratricide because apoptotic CD95+CD95L− melanoma cells were always below the detection threshold. It is surprising that a tumor cell line adapted to long term culture for many years continuously and "spontaneously" generates an apoptotic subset of progeny. This may represent a "loss by differentiation" event. Analyses using maturation markers of melanocytes may clarify this point. The important point for the design of tumor vaccines is that CTL-derived cytokines can greatly amplify this differentiation program of melanomas to an extent that might reach therapeutic value.
In vivo, specific rejection of transfected B16.F10 melanoma cells was T-dependent; and adoptively transferred, primed T cells induced massive apoptosis and regression in a s.c. growing melanoma. It is not clear to what extent this in vivo apoptosis results from direct CTL attack or from CTL-facilitated suicide. It seems of interest to test whether focusing CTL with a desired profile of cytokines to growing tumor cells is as important for tumor eradication as a direct cytolytic attack of tumor cells by these immune effector cells.
We gratefully acknowledge the excellent technical assistance of T. Krieg and A. Titz. Dr. P. Antonsson (Lund, Sweden) and Dr. H.-U. Weltzien (Freiburg, Germany) kindly provided cell lines. We appreciate the helpful discussions with Dr. H. P. Pircher (Freiburg, Germany).
↵1 This study was supported by a grant from the Wilhelm-Sander-Stiftung to J.R.
↵2 Address correspondence and reprint requests to Dr. Jörg Reimann, Department of Medical Microbiology and Immunology, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany. E-mail address: joerg.reimann{at}medizin.uni-ulm.de
↵3 Abbreviations used in this paper: L, ligand (e.g., CD95L, CD40L, FasL); MHC-I/II, MHC class I/II; T-Ag, large tumor Ag of SV40; HBsAg, hepatitis B surface Ag; FCM, flow cytometry; CTLL, CTL line; PE, phycoerythrin; TdT, terminal deoxynucleotidyl transferase; TUNEL, TdT-mediated dUTP-biotin nick end labeling; 7-AAD, 7-amino-actinomycin D.
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The number of cases of mother and baby deaths and injuries being investigated at NHS hospitals in Shropshire has risen dramatically. Shropshire Defend Our NHS has provided information to the Ockenden inquiry, which led to an increase in its scope. Information and analysis via Pete Gillard and Gill George.
People's Assembly NHS march, central London 3rd February 2018. Photo by Steve Eason.
At least 104 families have now come forward for their cases to be considered as part of an independent inquiry into maternity care at the Shrewsbury and Telford NHS Trust (SaTH) in Shropshire. The Ockenden Review was announced in April 2017 in response to 23 cases, but the number of cases in which "women, infants and new-born babies had died or suffered harm" to be included in the inquiry has kept climbing. The Care Quality Commission (CQC) has now issued an enforcement notice against SaTH over their appalling record in maternity services. The Health Service Journal reports that the numbers are still expected to increase even further.
The causes of the crisis are complex. We're looking here at 'systemic failure' – at a maternity service that has failed to support local women and babies and failed to support the staff who care for them in multiple ways. Responsibility for that rests with the Senior Management Team at SaTH, and with the Head of Midwifery, who seems to have cheerfully gone along with every cost-cutting exercise.
A significant part of the problem is 'culture', in a broad sense. The culture for many years in this service has been one of denial. Babies have died avoidably, and parents have been fobbed off with empty reassurances that 'it was just one of those things'. If you won't acknowledge that a problem exists in the first place, you can't begin to tackle it. There is no culture of learning. The phrase that comes back, frighteningly often, from bereaved parents is that there was a 'cover-up'. There is no established culture of listening to women either. Some of the most heart-breaking cases are when women have known that something is wrong with their baby – and they have been ignored.
That strand of misogyny extends to the treatment of midwives, in a service where most obstetric consultants are men. One obstetric consultant boasted to an expectant mother that he didn't talk to midwives. Another told councillors and the public in a recent meeting that giving birth in a midwife-led unit is no safer than giving birth in a supermarket. Safe maternity care depends on joined-up working: on a relationship of trust and communication between obstetricians and midwives. You can't create that when the attitude from some (male) consultants is one of open contempt towards their (female) midwife colleagues.
Cost-cutting is – unsurprisingly – a big part of the problem. SaTH runs an Obstetric Unit at Telford. Obstetric Units cost a lot of money. The research says that for women with low risk pregnancies, it is actually safer to give birth in a midwife-led unit than it is to use the medicalised environment of an Obstetric Unit. But because it's so expensive to run the Obstetric Unit, SaTH has tried to reduce the 'unit cost' of Obstetric Unit births by forcing women to give birth in that medicalised environment – even when they don't want to and it places them at higher risk. Part of the cost-cutting has been to deny choice to women in rural areas by closing the rural midwife-led units in Bridgnorth, Ludlow and Oswestry. Why? It saves £1.5 million a year. Choice and control – fundamental in child-birth and strongly linked to safety – are secondary.
The cost-cutting extends to staffing. The number of midwives at the main Obstetric Unit in Telford was steadily reduced between 2015 and 2017, as the Trust's financial crisis escalated. When new midwives were finally recruited last year, the Trust focused on employing newly qualified staff because they are cheaper. The number of beds on the Delivery Ward and the Postnatal Ward has been increasingly inadequate, as more and more women are funnelled through the Obstetric Unit conveyor belt. Women report being treated as 'a number on a bed'. Midwives also report the terrible stress of trying to provide decent care when you haven't got the resources to do it.
It's a toxic mix. It creates an environment where women are less likely to be heard, where midwives don't get the training and support and back-up they need – and where signs that something is going wrong and a baby is in distress are much more likely to be missed. The NHS is very good at finding scapegoats when people are harmed by inadequate care. The danger here is that individual midwives will become convenient targets. This would be utterly wrong. Responsibility for the tragedies in SaTH's maternity service lies with SaTH's Chief Executive Simon Wright – and he is the man who must be held accountable.
Much of what is emerging now is only in the public domain because of the heroism of bereaved parents who have fought for the truth and for justice for their babies. The parents of Kate Stanton-Davies who died in 2009 had to fight for eight years to get an apology. The independent report into Kate's death said the trust "abdicated its responsibility". On 19th September, the Chief Executive of SaTH was being questioned by the Health Overview and Scrutiny Committee over maternity deaths. He and his Director of Nursing chose not to say that SaTH had just been issued with an enforcement order over unsafe maternity care by the CQC. It was only because a health activist from Shopshire Defend Our NHS raised it from the floor at the end of the meeting that the committee was made aware. The health bosses had conveniently made a quick exit. Still, after all these years, the culture of dishonesty remains.
The news from Shropshire comes in the same week as the launch of a report by Maternity Action on migrant women's experiences of maternity care in the NHS (which is available in full here: What Price Safe Motherhood?). As Home Secretary, Theresa May introduced charges for NHS secondary care under the raft of 'hostile environment' policies intended to marginalise migrants in every corner of society. Those who were made ineligible for free NHS treatment are charged the full cost of their care, as well as an additional 50% punitive surcharge. The charges reduce the likelihood that migrant women receive essential care, due to financial inaccessibility and fears of being reported to the Home Office. The hostile environment policies also contribute to stress and anxiety, which in turn has a negative effect on pregnancy outcomes.
The Maternity Action report details the experiences of 16 women with a range of immigration statuses who were deemed ineligible for free NHS care, some mistakenly. Women have avoided care due to fears of being detained or deported, been charged thousands of pounds they are in no position to pay, and received constant coercive phone calls from debt repayment companies. The report concluded that "NHS charging regulations are unworkable, unjust and harmful to women, especially those who are destitute, living in poverty or unable to earn a living." Back in July, the same group wrote an open letter to the Health Secretary, signed by hundreds of people (including health practitioners and prominent trade unionists), demanding an end to charges for maternity care. They were ignored.
These events are testament to the racist and misogynistic weaponisation of the NHS under Theresa May's hostile environment, and to the harm which is callously allowed to continue in the context of austerity and marketisation. The Chief Executive of SaTH must resign. There must be justice for the Shropshire families whose lives have been irreversibly changed – and there must be a safe service for women and babies going forward. Every aspect of the hostile environment and all charges for healthcare must be abolished.


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A few passing clouds. Low near 65F. Winds light and variable..
A few passing clouds. Low near 65F. Winds light and variable.
Photo provided
BY CALVIN SILVERS Herald correspondent
OSKALOOSA — This week's featured softball player is Oskaloosa freshman, Faith Elizabeth DeRonde. DeRonde is the daughter of Jeremy and Sarah DeRonde.
DeRonde is number 10 on this year's softball team and finds herself primarily on the pitching mound. DeRonde can also be seen making tremendous plays in the outfield as she plays center field.
DeRonde was born on June 19, 2003, in Oskaloosa and has lived in Oskaloosa her whole life.
DeRonde began playing t-ball around the age of five and played for a few traveling teams when she became of age. At the age of nine, she started playing for the Oskaloosa Sweet Heat, then at the age of thirteen, she began playing for traveling teams in Des Moines. DeRonde was a pitcher and played first base for her respected traveling teams.
The ability for DeRonde to experience the sport of softball outside of Oskaloosa had a huge impact on her.
"My favorite thing about playing for traveling teams in Des Moines was the ability to meet new people," DeRonde said. "I met a lot of new friends and I still keep in touch with them."
To DeRonde, nothing beats the moments she's experienced on the Oskaloosa varsity softball team, and her favorite moment came from playing Newton this year.
"My favorite memory was high school softball came in the 12th inning against Newton when I hit the ball over to win the game," DeRonde said. "It was really amazing, especially being able to help your team like that as a freshman."
As the season is coming to the very end, DeRonde is still pushing to accomplish her goals she set out for herself and her team at the beginning of the season.
"The overall goal is for the team to make state," DeRonde said. "To get where we want to be I made it a goal to help contribute to the team whether I'm on or off the field."
While DeRonde made a lot of softball friends from Des Moines, she couldn't forget about her life long friends she's made in Oskaloosa. When asked whom she'd want by her side in a zombie apocalypse, the choice was easy.
"I'd definitely want Ashley Kindley by my side," DeRonde said. "She's like the mom of the team. She always knows what to do."
DeRonde also wanted to show her appreciation and love for her past teammate, Anna Jones.
"If I could bring back one past teammate to join me for another year, It'd be Anna," DeRonde said. "She was always outgoing and crazy, and I loved that about her. She had the best personality."
DeRonde is always seen having a good time and enjoying a laugh with her teammates. When asked whom she believes the funniest person on the team is, she was quick to say one name in particular.
"Olivia Gordon is easily the funniest person on this year's softball team," DeRonde said. "Everything she says is just so funny. Half the time her jokes are so stupid you can't help but laugh at them."
As for DeRonde's pregame rituals, she likes to listen to the genre rap. While she doesn't have a favorite song, she listens to rap because it gets her pumped up for the games. Her favorite snack to eat before games are dried noodles. She'll always bring different kinds to eat in the dugout during games.
Besides playing in front of a wild Oskaloosa crowd, DeRonde looks forward to the 20-minute travel to play the Pella Dutch.
"I love playing at Pella," DeRonde said. "It's always fun to beat the Dutch in Pella, it makes the win that much sweeter."
Besides softball, DeRonde can be found on the volleyball court or running track. DeRonde says she really looks forward to playing volleyball in the fall.
"I love playing volleyball with my Des Moines team and friends," DeRonde said. "The ability to play the sport competitively and at a high level makes the game fun. I love the challenge it brings."
When DeRonde is not playing sports, she likes to swim with her friends, play Spike Ball, and go shopping at Lululemon.
As DeRonde is wrapping up her first year of high school, she said her favorite memory came from being a new face in high school.
"Meeting all the upperclassman was a fun experience," DeRonde said. "They're all super nice and amazing people."
DeRonde's favorite subject in school is math because it comes easy to her. As for her favorite teacher, DeRonde chose one teacher she can always count on being there for her.
"Mrs. Dolash is by far my favorite teacher," DeRonde said. "She will listen and help you with anything. Even if it's not about schoolwork."
To end the interview, DeRonde shared a quote she has always stuck to. The quote goes, "stay humble." DeRonde believes that staying humble allows things to come easier to you than when you boast about your accomplishments.
Faith Elizabeth Deronde
Oskaloosa Sweet Heat
Dolash


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Nick's Website
NICHOLAS PAYTON PRESENTS 'SKETCHES OF SPAIN' WITH THE SINFONIEORCHESTER BASEL ON SEPTEMBER 24 ON BMF Records™
NEW ORLEANS, September 10, 2013 — On September 24th, for his 40th birthday week, Grammy-winning trumpeter Nicholas Payton offers the world a present worthy of his immense skills: a live concert performance of the iconic Miles Davis-Gil Evans collaboration, Sketches of Spain via his BMF Records imprint. Framing Payton's virtuosic playing is a 19-piece ensemble comprised of 15 brass and woodwinds and a harpist drawn from the Sinfonieorchester Basel with conductor Dennis Russell Davies, and Payton's long-time collaborators Vicente Archer on bass, Marcus Gilmore on drums, and Daniel Sadownick on percussion.
Sketches of Spain is Payton's second release on BMF, which launched in February 2013 with #BAM Live at Bohemian Caverns, on which Archer and drummer Lenny White joined Payton playing trumpet and Fender Rhodes piano simultaneously. Here Payton focuses exclusively on the trumpet, which he places to his lips for around 90% of the 40-minute suite. In the process, he reaffirms his stature as a peerless master of his primary instrument. He rises to and transcends not only the physical challenge of functioning throughout the marathon at consistent levels of execution and creativity, but also the conceptual challenge of addressing and refracting into his own argot Davis' indelible declamations from one of the second-largest selling recordings of all time.
"At this point, Miles is my favorite musician, period," Payton says. "I understand his music so intimately that for me it's not even about a style, but conjuring a spirit that at any moment I can call upon to walk through me. I've dealt with the idea of being influenced by this person but I feel I'm in a space where my voice has developed so much that I don't feel haunted by Miles' ghost. I can be free to create and use whatever part of his spirit when it's necessary, and other times magnify certain things that are not necessarily directly related to Miles.
"We're now 54 years from that piece, and music has evolved. That gives me a frame of reference that allows me to do things Miles didn't think about doing, because it was a new work then. So I didn't necessarily feel bound by the blueprint. Whatever Sketches of Spain meant to Miles then, I want to be able to take that just as far as my imagination will allow."
As an example, consider Payton's sublime interpretation of the melody on the suite-opening "Concierto De Aranjuez," composed by Joaquín Rodrigo, or his brisk improvisation on "The Pan Piper," movement three, which Evans fleshed out from a Peruvian Indian folk theme. Then there's his use of multiphonics on "Saeta," the fourth movement, on which Davis, depicting a lone woman's emotional singing of the Passion of Christ, produced one of the singular performances of his career. On the quasi-flamenco final section, "Solea," Payton produces a masterpiece 12-minute solo, revealing old-master control of dynamics and compression-release strategies as he sustains momentum, "It's like a snowball collecting to the point where you reach the other side of the peak of the mountain… It's like an avalanche."
Spurring Payton's imagination are the orchestra's well-wrought tempi and tone colors and the state-of-the-art rhythmic impetus provided by Gilmore, Sadownick and Archer. "As opposed to [earlier Davis-Evans classics] Miles Ahead and Porgy and Bess, swinging rhythms are not employed in this piece," Payton says. "So the orchestral musicians who are not versed in playing from a Black American rhythmic perspective can just play—for lack of a better word—music."
"Paul Chambers and Jimmy Cobb [the bassist and drummer on the original Sketches of Spain] are part of the fabric of our musical DNA—and what we like to listen to, period. But although we're deferential to the arrangement, I didn't want us to be too deferential to the style. What's going to set this apart from Miles is for my band to play like they play. The music has changed and evolved. The best way for us to pay respect is to pretend we're getting this piece for the first time, and interpret in the way and with the language we've created within ourselves, the same as Miles' band did within their bag."
To address each piece according to its own strengths and inherent characteristics from a hereand- now perspective is an approach not dissimilar to Payton's procedures on his classic GRAMMY® award-winning 1997 collaboration with legendary trumpeter, and one of Payton's musical grandfathers, Doc Cheatham, entitled Doc Cheatham & Nicholas Payton, or on Dear Louis and Gumbo Nouveau, on which Payton channeled and updated the spirit of traditional and vernacular New Orleans music with such palpable flair, individuality and freshness that, by his mid-twenties, connoisseurs considered him a major figure on the trumpet timeline.
"The idea of taking the best of the past and bringing it into today's context has been a thread throughout everything I've done," Payton says. "As a kid playing in brass bands, or in my teens when I played Louis Armstrong and "King" Oliver material with Wynton Marsalis at Lincoln Center, I always saw myself as doing something more modern. I always fought heavily against being categorized."
This is one reason why Payton finds it so liberating to document his musical production on BMF, on which he will release his recently composed Black American Symphony —also performed by the Sinfonieorchester Basel.
"Instead of looking for an opportunity that's suited for me, I can create the opportunity the way I want from start to finish," he says. "I don't have to develop a sales pitch when I want to do a project. I can do whatever I want, and do it the way I want. I can see where my money is being spent, how it's coming back, exactly how many I'm selling. To me, it is the business manifestation of everything that I talk about in terms of Black American Music. Now I have a physical home for the ideas that I espouse conceptually. It seems right that I would have a company at this time to further the other things that I talk about. This is the physical manifestation of taking control of my music, putting it out and branding myself, as opposed to other people branding me."
Artist: Nicholas Payton
Nicholas Payton with the Sinfonieorchester Basel
Album: Sketches of Spain
Catalog Number: BMF-002
Label: BMF Records
Cover Art: "Jezebel" by Allen Stringfellow (1923-2004)
Nick Online
Nicholas Payton – Afro-Caribbean Mixtape
BUY NOW: iTunes | BandCamp
Nicholas Payton – Textures
BUY NOW: Amazon | CDBaby | iTunes
Nicholas Payton – Letters
BUY NOW: CDBABY | iTunes
Nicholas Payton – Numbers
Nicholas Payton – Sketches of Spain
Nicholas Payton – #BAM Live at Bohemian Caverns
On Why Black Artists Are Dying Too Soon—The Real Epidemic
My Soul Brother, Roy Hargrove…
On Humanity…
On Louis Armstrong…
The Nicholas Payton Quintet: Live at the Village Vanguard 1997
Artwork for Textures — by Anastasia Pelias
Selections from "Textures"
Black Keys — article by Jennifer Odell
CALLING ALL ARTISTS/LOVERS: First International #BAM Masterclass in New York City
An Adversarial Katrinaversary and the Delusional Post-Diluvial New Orleans — a Manmade Disaster
What Folks Have To Say About "Letters" . . .
Press release for my upcoming album, "Letters," written by Benny Green…
The Savior of Archaic Pop Meets The Human Jukebox
#tbt: Nicholas Payton & Karriem Riggins — BoomBap in 1997
Nicholas Payton's "Numbers" Mixtape (hosted by The Music Snobs)
Part I: The Rise of Capitalism and The Fall of Culture
On The New Yorker "Satirizing" Sonny
The #BAM Babies in Anticipation of "Numbers"
Video: Bill Stewart with The Nicholas Payton Trio — Live at Blues Alley
Press Release for "Numbers," by Nicholas Payton ft. Butcher Brown
Masters of Funky New Orleans Drumming — Vol. 1
Should I Spotify?
"Snob of the Year," Nicholas Payton Meets The Music Snobs
#BAM @ BAM
On Quincy Jones, Ray Brown, Brownie, Fat Girl, Fab, Art and Helen . . .
Black American Music and the Jazz Tradition
Another Shot in the Arm for Jazz
Album Review: Can Beyoncé's Eponymous Apolcalyptic Album Bring Melody Back To The Mainstream?
On Art, Community, and the Culture of Commerce
Why Jazz Still Isn't Cool: The 2nd #BAMiversary in Review
On Truth and Beauty in the Age of Bullshit
Too Loose in Toulouse
The Complete Nicholas Payton Interview with Richard Scheinin
Why Hiphop Isn't Cool Anymore
Western Thought and the African Dilemma
Has Hiphop and Sampling Killed Music?
Thanks for the love, Guadalajara!
Nicholas Payton Speaks On Yeezus . . .
Live in Anguilla: The Mulgrew Miller Trio with Kenny Garrett & Nicholas Payton
Grew Stepped Out of a Dream: A Few Words on Mulgrew Miller
Bruce Raeburn Interviews Nicholas Payton — May 5, 2013
The Day Those Mothas Ruined Mother's Day for New Orleans
My Two Favorite Quotes Told To Me at Jazz Fest 2013 . . .
Payton's Treatise on 4/4 Swing
NP Bootleg Series: The Nicholas Payton XXX Live in Munich
Sam: #BAM The Official Poster Child of BMF Records
#BAM For Dummies . . .
On Dr. Donaldson Byrd . . .
General Thoughts (1)
Meditations (4)


Students and staff across the Sachem Central School district participated in a collection of heartwarming events and activities last week in recognition of P.S. I Love You Day, an initiative that looks to eliminate bullying and promote mental health awareness.
At elementary schools around the district, students were joined by peer education students from Sachem High School East and North who led inspiring assemblies on kindness and being proud of who you are. As part of the assemblies, peer educators facilitated activities showcasing the power of a compliment and how one simple act of kindness can change someone's day.
During an activity at Cayuga Elementary School in Jaimie DiPalma and Kristin Ruhs' second-grade class, students created kindnessgrams in which students wrote a list of compliments to one of their peers and shared it aloud to the class. With bright smiles, students read and received compliments until the entire class had shared.
At Sachem High School East, peer education students encouraged faculty, students and staff to visit the Peer Education Station where they could design and decorate a puzzle piece that highlighted the qualities that make them unique. The assembled puzzle will remain displayed in the hallway, representing the unique characteristics found in the individuals throughout the school building.
At the conclusion of the week, students and staff dressed in purple to foster kindness, create a welcoming environment and signify that no one is ever alone.


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Cesar Chavez Park
A bike and pedestrian path runs around the water's edge to the bay side from the back of the peninsula this 90 acre park shares with the Marina and Pier, revealing an incredible view of the bay, the city, Alcatraz, the GG Bridge, and Mt. Tamalpais. With steady winds coming off the bay, a large field, and low hills, this is a kite flying Mecca. There is also a kite shop in a truck that parks here most afternoons that sells kites and parts. The park also has picnic areas with BBQ pits, an off-leash dog area, a wetland and wildlife sanctuary.
http://www.yelp.com/biz/cesar-chavez-park-berkeley
Berkeley Rose Garden, on Euclid Avenue
A half-hour's walk north of the UC Berkeley campus. Especially nice for watching the sunset, with a view of the bay.
http://www.ci.berkeley.ca.us/contentdisplay.aspx?id=12048
Indian Rock Park, Indian Rock Ave.
Indian Rock Park is a 1.18-acre public park located in the northeast part of the city, about one block north of the Arlington/Marin Circle, and straddles Indian Rock Avenue. The central feature of the park is a large rock outcropping on the west side of Indian Rock Ave. You can climb up the rocks on the carved out stairs and gaze at the great vista from the top. Highly recommended for sunsets.
While not visually spectacular, this is a landmark of the 1960's Free Speech Movement. The park has since served mostly as an unofficial residence for Berkeley's homeless. A publicly funded restoration spruced it up a bit, and occasional festivals do still happen here, but on the surface it's still just a mangy patch of trampled grass.
Tilden Park
Offers several inexpensive activities for families with children. Many kilometers of hiking trails of various difficulties are available for free hiking, and include vistas of San Francisco and the Golden Gate Bridge. Home to a 5-inch scale, narrow-gauge working steam train. $8 gets five rides through the woods atop the Berkeley hills, including a tunnel and trestle bridge.
http://www.ebparks.org/Page158.aspx
UC Berkeley Botanical Garden, on Centennial Drive (in the hills above the UC campus).
Open daily 9AM-5PM; closed first Tuesdays of each month and on certain holidays. Part outdoor museum, park, and biological research facility for the University of California, the 34-acre garden hosts over 12,000 different species of plant life. Admission is $5 for adults, $3 for seniors and $1 for children.
http://botanicalgarden.berkeley.edu/
Alternative Music Foundation, 924 Gilman St (8th & Gilman)
North Berkeley is home to one of the world's most famous punk rock clubs, known simply as Gilman, after the street it located on. Shows are Friday and Saturday evenings, and sometimes Sunday afternoons.
http://www.924gilman.org/
Berkeley Repertory Theater (Berkeley Rep)
2025 Addison Street (near Shattuck; 2 blocks north of Berkeley BART station), Hours vary, normally Tu-Su 8PM-11PM, Sa-Su 2PM-4PM (matinee). With its consistently high-quality productions of contemporary and historical plays, the Berkeley Rep is one of the primary centers of theater on the West Coast. The theater is also very pleasant, in a modern way. $35-$55 (varies by night; half-price students and seniors, $20 under 30).
http://www.berkeleyrep.org/
Cal Bears Football-Memorial Stadium
The stadium plays host in the fall season to UC Berkeley football games. But if you want to watch a game for free, head above the canyon to Tightwad Hill, a hill where students and the public gather to watch games. It's also an ideal location for spectacular views of the Bay Area when there's not a game going on. Anyone is free to come as long as they pick up their own trash and don't wear the colors of Stanford University (red).
http://www.calbears.com/facilities/memorial-stadium.html
Long Haul Infoshop
3124 Shattuck Ave (near Ashby BART) is an anarchist library and community space. there is a wide range of books and magazines collected over the years for folks to read, as well as free coffee. on the weekends you may also find political and counter cultural events.
http://thelonghaul.org/
Takara Sake Co. tasting room and museum
708 Addison St. (4th St.). Noon-6 PM daily. Sample flights of Sho Chiku Bai sake and plum wines in a gorgeous tasting room within the brewery. Afterwards, check out the displays of traditional sake-making gear in the sake museum.
http://www.takarasake.com/
Strawberry Cannon
Strawberry Canyon is the most frequented tramp in Berkeley, perhaps because one may stroll along the upper creek bed and lose sight of all that reminds him of a town—forget, for a little while, streets, and houses, and gardens...and books. Running between the walls of the hill, over a tumbled bed of boulders, and through regular tunnels of oak and laurel and willow, and tangled disorder of creeper and fern, Strawberry Creek has an untamed beauty and waywardness that pleases as no garden or park-land can.
http://berkeleyheritage.com/berkeley_landmarks/strawbcanyon.html
Laurence Hall of Science
The Lawrence Hall of Science, UC Berkeley's public science center, has been providing parents, kids, and educators with opportunities to engage with science since 1968. Located at One Centennial Drive, about half a mile from Grizzly Peak Boulevard in the hills above the UC Berkeley campus. They are open 10:00 a.m. to 5:00 p.m. every day and most holidays.$12.00 adults (ages 19–61). $9.00 students/seniors/disabled (ages 7–18; 62-plus). $6.00 children (ages three to six; children under three are admitted free). Free for members, UC Berkeley students and staff.
http://www.lawrencehallofscience.org/
Berkeley Fire Trails
Awesome trail that will give you a great workout and an AMAZING view once you get to the top. Trust us...the hard work will be worth it once you lay your eyes on the beautiful bay area skyline! The 7 mile trail has some steady gradual inclines with a few steep inclines. Wear a hat especially if you plan to hike this in the heat and make sure you bring plenty of water! It takes about 2.5 hrs round trip, with quite a few pit stops to enjoy the view!
http://www.yelp.com/biz/berkeley-fire-trails-berkeley
Little Farm in Tilden
One of the District's three oldest parks, Tilden has been called the jewel of the system, and its recreational activities have become a happy tradition for generations of East Bay youngsters. From a carousel ride and a picnic to a swim at Lake Anza and a stroll through the Botanic Garden, Tilden has variety to delight everyone. Yet there are plenty of quiet places in Tilden's 2,079 acres to shelter the wildlife and preserve natural beauty. Tilden was named for Charles Lee Tilden, first president of the Park District Board of Directors. The park is reached via Canon Drive, Shasta Road, or South Park Drive, all off Grizzly Peak Boulevard in Berkeley.
http://www.ebparks.org/parks/tilden
The Adventure Playground at the Berkeley Marina is a wonderfully unique outdoor facility where staff encourage children to play and build creatively. Come climb on the many unusual kid designed and built forts, boats, and towers. Ride the zip line or hammer, saw, and paint. By providing these low risk activities Adventure Playground creates opportunities for children to learn cooperation, meet physical challenges and gain self confidence.
http://www.ci.berkeley.ca.us/adventureplayground/
Berkeley Pier
Berkeley Marina and Pier is a big area covered by the Bay Area Bay Trail. Though the trail takes you around the areas of the park, it is up to you to search and discover what each part has hidden. The Marina is a small place where you can watch boats enter and leave. The Pier is a great place to go to fish or watch boats or surfers battle the waves.
http://www.abag.ca.gov/bayarea/baytrail/vtour/map5/access/bmarinapier/bm...
Berkeley will fill your itinerary with activities to engage your senses, revive your spirit, and exercise your imagination. This is a city where you can tour a brewery or botanical garden, savor the sunset or a foodie feast, explore a 1,000-acre regional park or a fun science museum. Time to tour!
http://visitberkeley.com/what-to-do/sightseeing-tours
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Q: Probability distribution of a linear function of a continuous random variable If $X$ is a continuous random variable, under what condition does the following relation hold true?
$$\mathbb{P}(X < k)  = \mathbb{P}(aX+b < ak +b)$$
Is the above relation dependent on the PDF of $X$?

A: When $a$ is a positive number.

A: Elaborating on @Michael Burr's comment, if $a>0$ then for any $\omega$,
$$X(\omega) < k \iff aX(\omega) + b < ak + b,$$
so
$$\{X < k\} = \{aX + b < ak + b\} $$
and hence
$$\mathbb P\{X < k\} = \mathbb P\{aX + b < ak + b\}.$$

redpajama_set_name: RedPajamaStackExchange


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Статьи > Know > Embassy of Argentina in Ukraine has honored the memory of Taras Shevchenko and Julio Cortazar
Embassy of Argentina in Ukraine has honored the memory of Taras Shevchenko and Julio Cortazar
Автор: OUTLOOK 18.12.2014 | diplomacy, ambassador, República Argentina
December 17, Embassy of Argentina in Ukraine held a diplomatic reception, dedicated to the memory of two outstanding literary figures: Ukrainian poet Taras Shevchenko and Argentinian writer Julio Cortazar.
This year, two countries celebrated the anniversaries of the birth of these outstanding persons. On this occasion, the Ambassador of Argentina to Ukraine Lila Roldan Vazquez de Moine organized that evening.
During the event Taras Shevchenko's poetry and parts from works of Julio Cortázar in Spanish were declamated. Melodies of Argentine tango from the Quartet Kiev Tango Project" sounded. In addition, there was an opportunity for guests to watch the documentary film " Cortazar " directed by Tristan Brauer.
In her speech, the Ambassador said:
«If there's a way to start to describe the characteristics that distinguish Cortázar as a writer, perhaps his own statement would serve this purpose best. In one interview he said that since his childhood it was both his happiness and misfortune "to not accept things the way that are given to me".»
Finally, Lila Roldan Vazquez de Moine gave some memorable quotes of Argentinian writer:
… what do you want? Love longs for the road, for the wind, it doesn't know how to die in solitude…
… as if you could pick in love, as if it were not a lightning bolt that splits your bones…
… Hope belongs to life, it's life itself, protecting itself…
… How long will we keep believing that happiness is nothing but one of the games of illusion?
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