text:
Performance of anti-CD19 chimeric antigen receptor T cells in genetically defined classes of chronic lymphocytic leukemia
Immune cell therapies and immune cell engineering
http://orcid.org/0000-0002-8155-9927Veronika Mancikova1,2,
Helena Peschelova1,
Veronika Kozlova1,2,
Aneta Ledererova1,2,
Adriana Ladungova1,
Jan Verner1,2,
Tomas Loja1,
Frantisek Folber2,
Jiri Mayer1,2,
Sarka Pospisilova1,2 and
Michal Smida1,2
1 Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic
2 Department of Internal Medicine-Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic
Correspondence to Dr Veronika Mancikova; veronika.mancikova{at}ceitec.muni.cz; Dr Michal Smida; michal.smida{at}ceitec.muni.cz
Background While achieving prolonged remissions in other B cell-derived malignancies, chimeric antigen receptor (CAR) T cells still underperform when injected into patients with chronic lymphocytic leukemia (CLL). We studied the influence of genetics on CLL response to anti-CD19 CAR T-cell therapy.
Methods First, we studied 32 primary CLL samples composed of 26 immunoglobulin heavy-chain gene variable (IGHV)-unmutated (9 ATM-mutated, 8 TP53-mutated, and 9 without mutations in ATM, TP53, NOTCH1 or SF3B1) and 6 IGHV-mutated samples without mutations in the above-mentioned genes. Then, we mimicked the leukemic microenvironment in the primary cells by '2S stimulation' through interleukin-2 and nuclear factor kappa B. Finally, CRISPR/Cas9-generated ATM -knockout and TP53-knockout clones (four and seven, respectively) from CLL-derived cell lines MEC1 and HG3 were used. All these samples were exposed to CAR T cells. In vivo survival study in NSG mice using HG3 wild-type (WT), ATM -knockout or TP53-knockout cells was also performed.
Results Primary unstimulated CLL cells were specifically eliminated after >24 hours of coculture with CAR T cells. '2S' stimulated cells showed increased survival when exposed to CAR T cells compared with unstimulated ones, confirming the positive effect of this stimulation on CLL cells' in vitro fitness. After 96 hours of coculture, there was no difference in survival among the genetic classes. Finally, CAR T cells were specifically activated in vitro in the presence of target knockout cell lines as shown by the production of interferon-γ when compared with control (CTRL) T cells (p=0.0020), but there was no difference in knockout cells' survival. In vivo, CAR T cells prolonged the survival of mice injected with WT, TP53-knockout and ATM-knockout HG3 tumor cells as compared with CTRL T cells (p=0.0485, 0.0204 and <0.0001, respectively). When compared with ATM-knockout, TP53-knockout disease was associated with an earlier time of onset (p<0.0001), higher tumor burden (p=0.0002) and inefficient T-cell engraftment (p=0.0012).
Conclusions While in vitro no differences in survival of CLL cells of various genetic backgrounds were observed, CAR T cells showed a different effectiveness at eradicating tumor cells in vivo depending on the driver mutation. Early disease onset, high-tumor burden and inefficient T-cell engraftment, associated with TP53-knockout tumors in our experimental setting, ultimately led to inferior performance of CAR T cells.
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http://dx.doi.org/10.1136/jitc-2019-000471
Chronic lymphocytic leukemia (CLL) is the most common adult B-cell leukemia characterized by high genetic and clinical diversity. Genetic mutations are major predictor factors of prognosis, time-to-first-treatment, as well as response to treatment in CLL.1 For instance, patients with TP53 alterations display poor prognosis with shorter time-to-first-treatment and resistance to chemoimmunotherapy (based on fludarabine, cyclophosphamide and anti-CD20 antibody rituximab) routinely used to manage CLL.2 Therefore, these patients are nowadays rather treated with targeted inhibitors (ibrutinib, idelalisib or venetoclax) that show some capacity to induce response in these difficult-to-treat patients.3 Apart from TP53 aberrations, several other genetic defects have been associated with aggressive CLL course, including the unmutated immunoglobulin heavy-chain gene variable ( IGHV) region and mutations in ATM, SF3B1, and NOTCH1. Ultimately, several factors, including IGHV mutational status, genomic changes, patient age, disease stage and the presence of comorbidities, are used nowadays to select the most appropriate treatment option for each patient.4 However, with the exception of allogeneic transplantation, CLL remains incurable.
One possibly curative option could be chimeric antigen receptor (CAR) T-cell immunotherapy. CAR T cells are prepared by genetic modification of patients' T cells. Tumor specificity is imposed on these cells by introducing a synthetic gene coding for a receptor composed of an antigen-binding domain derived from a B-cell receptor fused to T-cell activation domains (such as CD28 or 4-1BB5). This modification reprograms T cells to target selected antigen on the surface of malignant cells. Since its application in CLL is so far limited to clinical trials, only patients with relapsed and/or refractory (r/r) disease have been treated with this therapy. Using CAR T cells targeting CD19 has shown durable complete remissions in these heavily pretreated patients, but only in up to 29% of them.6 7 In general, such favorable response among patients with CLL is much lower when compared with patients with other r/r B-cell malignancies treated with anti-CD19 CAR T cells, where 37%–55% of them reach durable complete remissions.8 9 Some of the possible reasons for this disproportion are inhibitory tumor environment of CLL and larger tumor burdens in patients with CLL at the moment of treatment (reviewed in Lorentzen and Straten10). Additionally, attributes of the final CAR T-cell product, including T-cell fitness, phenotypical differentiation and metabolic program, impact the ultimate therapeutic outcome.11 Apart from these, individual disease-specific characteristics that would distinguish responders from those who will not benefit from CAR T-cell treatment have not been described so far.11 However, the CLL clinical trials have been done only with small numbers of patients and could be underpowered to detect some associations. Thus, the impact of individual genetic aberrations on the response of CLL cells to CAR T-cell therapy has not been reliably evaluated.
Herein, we have comprehensively assessed the effect of various clinically relevant mutations on the response of CLL to CAR T cells in several in vitro and in vivo disease models. In vitro, anti-CD19 CAR T cells were similarly effective at eliminating CLL model cell lines and primary CLL cells of various genetic backgrounds. In vivo, CAR T cells were able to prolong survival of all studied genetic backgrounds but with different curative rate, which closely reflected the disease severity and was lowest in the TP53-knockout group.
Human primary samples, cell lines and culture conditions
Buffy coats from healthy individuals were purchased from the Department of Transfusion and Tissues of University Hospital Brno (UHB). RosetteSep Human T Cell Enrichment Cocktail (StemCell) was used to isolate primary T cells according to manufacturer's protocol. Thirty-two primary CLL samples were selected from the biobank of the Department of Internal Medicine, Hematology and Oncology, UHB. All patients fulfilled the "International Workshop on Chronic Lymphocytic Leukemia"/National Cancer Institute diagnostic criteria for CLL.12 Collected peripheral blood samples were subjected to gradient centrifugation to obtain high-purity B lymphocytes (>98%) using Ficoll-Paque PLUS (GE Healthcare) combined with RosetteSep Kits (StemCell) to deplete non-B cells and subsequently vitally frozen. The selection criteria were based on availability of samples' genetic information in order to compile genetically defined groups. All selected samples had been screened by Sanger and/or next-generation sequencing for somatic hypermutation in IGHV 13 14 and mutations in TP53,15 16 ATM,17 NOTCH1 18 and SF3B1.19 Only cases with highly abundant TP53 and ATM mutations were included. Conversely, wild-type (WT) cases had no mutation detected above the threshold of a respective method used. All primary cells (T and CLL cells) were cultivated in serum-free AIM-V medium (Thermo Fisher Scientific). T cells were stimulated by interleukin (IL)-2 (50 U/mL, Miltenyi Biotech) and Dynabeads Human T-activator CD3/CD28 (ratio 1:3, bead:cell; Thermo Fisher Scientific). For '2S' stimulation of CLL cells, resiquimod (1 µg/mL, Sigma) and IL-2 (500 U/mL) were supplemented to the cells 3 days prior to starting any experiments.
HG3 (a generous gift from Dr Rosenquist, Sweden), MEC1 (DSZM) and Lenti-X 293T (TakaraBio Inc.) cell lines were used herein. HG3 is WT in TP53 gene20, while MEC1 has a truncated TP53 allele resulting from c.949_950insC mutation and the other allele is deleted21. Both cell lines have unmutated IGHV. HG3 was maintained in RPMI-1640 medium (Biosera), MEC1 in Iscove's Modified Dulbecco's Medium (Biosera) and Lenti-X in Dulbecco's Modified Eagle's Medium (Biosera). All media were supplemented with 10% (v/v) heat‑inactivated fetal bovine serum (FBS; Biosera) and 1% (v/v) penicillin/streptomycin (MP Biomedicals).
Lentiviral plasmids, CAR virus production and gene transduction
Sequence encoding anti-CD19 CAR was previously described22 and synthetized in BlueHeron Biotech Company (USA). This construct was subsequently cloned into BamHI and SalI sites of the lentiviral vector pRRL-CMV-GFP-sin18 (originally generated by D Trono, purchased from Addgene), in which the CMV promotor had been exchanged for EF1α promotor by molecular cloning. The sequence of each genetic fragment was confirmed by Sanger sequencing. Additionally, helper plasmids pCMV-VSV-G and pCMV-dR8.91 were from Addgene (originally generated by D Trono).
To generate lentiviral particles, we first treated Lenti-X cells with 25 µM Chloroquine Diphosphate (Sigma) 5 hours prior to transfection. Subsequently, we used polyethylenimine (PEI, 1 mg/mL; Polysciences) in a ratio of 3:1 (PEI:DNA) for transfection, where we combined the plasmids in equimolar ratio and used a total of 3 µg of DNA per 8×105 Lenti-X cells. The medium was replaced 3 hours later. Conditioned lentiviral medium was harvested 48 and 72 hours later, frozen and stored at −80°C. Lenti-X Concentrator (TakaraBio) was used to concentrate the conditioned medium according to the manufacturer's protocol. After centrifugation, the pellets were resuspended in AIM-V medium (1/100th of the original volume) and used immediately to transduce target T cells. A 1.5 mL Eppendorf tube was coated with 0.2 mL of Retronectin (TakaraBio) diluted to 100 µg/mL for 2 hours at room temperature and then incubated with 2% bovine serum albumin (Sigma) for 30 min. T cells (8×105), which were prestimulated with IL-2 and CD3/CD28 24 hours prior to transduction, were resuspended in the retronectin-coated tube in 0.5 mL of concentrated virus supplemented with Polybrene (10 µg/mL, Merck) and centrifuged at 1000 g at 32°C for 2 hours. Immediately afterwards, viral medium was replaced. T cells were then left to expand until further use (the total in vitro cultivation time never exceeded 10 days).
Single-cell knockout clones' production and their verification
MEC1 bears an inherent TP53 impairment. To be able to use it to generate various genetically distinct single-cell clones with either TP53 or ATM knocked out, we first had to reintroduce the TP53 WT allele under an inducible promotor. pCMV-Neo-Bam p53 WT plasmid (#16434, Addgene) was digested by BamHI, and the digestion product (TP53 WT gene) was cloned into a pLenti CMV/TO Hygro empty plasmid (#17484, Addgene) under a doxycyclin-inducible promotor. Lentiviral particles were produced as described previously; MEC1 cells were transduced and selected by 500 μg/mL hygromycin (InvivoGen) for 14 days. Afterwards, the lentiGuide-Puro two-vector system, composed of single guide RNA (sgRNA) containing lentiGuide-Puro vector (#52963, Addgene) and Cas9 expressing LentiCas9-Blast vector (#52962, Addgene), was used to generate CRISPR knockouts of ATM and TP53 genes. The following sgRNAs were cloned into lentiGuide-Puro:
5′-GGATGCTGTTCTCAGACTGA-3′ targeting exon 6 of ATM
5′-TCGACGCTAGGATCTGACTG-3′ targeting exon 2 of TP53
5′-TATCTGAGCAGCGCTCATGG-3′ targeting exon 5 of TP53
Transfection and virus harvesting were performed as described previously. Cas9-transduced cells (HG3 and MEC1 p53 wt) were selected by 10 µg/mL blasticidin (InvivoGen) for 7 days. Immediately afterwards, cells were transduced by lentiviruses containing the sgRNA of interest and selected by 10 µg/mL puromycin (InvivoGen, #ant-pr-1) for 4 days. In order to obtain monoclonal cell populations, the selected cells were plated into 96-well plates as 1 cell per well using the limiting dilution method.
Sanger sequencing and CRISP-ID tool (http://crispid.gbiomed.kuleuven.be/) were used to detect editing events. In the cell clones harboring indels, the loss of the respective protein was verified by western blotting. Only clones with no band visible after prolonged exposure were selected for further study. For ATM protein detection, no prior stimulation was needed. In case of p53, a 24-hour treatment with 2 µM doxorubicin was performed to stabilize the p53 protein. Prior to the treatment with doxorubicin, MEC1 p53 wt cells were also incubated with 2 µg/mL doxycycline for 24 hours in order to induce the WT p53 expression. In order to assess the accumulation of p21, the knockout cells were incubated with 2 µM doxorubicin for 24 hours, lysed and subjected to western blotting. Additionally, the phosphorylation of KAP1 was determined by western blotting in ATM-knockout cells incubated with 1 µM doxorubicin for 4 hours. The antibodies used in the study are listed in online supplementary table 1.
[jitc-2019-000471supp001.pdf]
Cytotoxicity in vitro assay and cytokine production assay
The antileukemic in vitro activity of CAR T cells was assessed in 4-day cultures using 1:5 ratio of target:effector cells. For this assay, the collection of knockout clones, as well as the set of primary CLL cells, were used as targets. In the case of vitally frozen primary CLL cells, these were thawed, and their viability was assessed by flow cytometry. A part of CLL cells was subsequently 2S stimulated and used in cytotoxicity assay only 72 hours later, while the other part was directly mixed with T cells. In detail, 2×104 live B cells were combined with 1×105 T cells (control (CTRL) or CAR) in 96-well U-bottom plates in 200 µL of AIM-V medium without any supplements. The target B-cell survival was monitored at 24 and 96 hours by flow cytometry. The amount of retrieved CD19+ cells from the mixture of B cells with CTRL T cells was set as 100% and compared with the amount of CD19+ cells cultured with CAR T cells. Additionally, supernatants from 4-day coculture of knockout cell clones and CTRL/CAR T cells were collected, and production of interferon (IFN)-γ was measured by ELISA using Human IFN-γ Standard TMB ELISA Development Kit (PeproTech) following manufacturer's protocol.
In vivo studies
Mouse experiments were carried out with equal numbers of male and female NOD-scid IL2Rgnull (NSG) mice (Charles River Laboratories, France) 8–12 weeks old per experimental group.
To assess the penetrance of individual knockout clones, 2.5×106 tumor cells from each individual clone or WT HG3 and MEC1 cells were injected via tail vein in a total volume of 0.1 mL phosphate-buffered saline. For further studies, we preferentially selected clones derived from the HG3 cell line, which had the most similar disease-onset time. Two experimental settings to assess CAR T-cell in vivo performance were used: low-tumor and high-tumor burden settings. These scenarios differed in the number of tumor cells (5×105 cells and 2×106 cells, respectively). Moreover, CTRL or CAR T cells were injected intravenously 5 or 7 days later (low or high scenarios, respectively). Mice were closely monitored for any manifestation of the disease and were sacrificed when symptoms of severe disease (lethargy, excessive weight loss, change in fur quality, apathy and hind leg paralysis) occurred. Spleen was harvested, mechanically homogenized and passed through a 30 µm nylon filter, and erythrocytes were lysed using Tris-buffered ammonium chloride. Samples were then analyzed by staining with fluorescently conjugated monoclonal antibodies. Blood samples from the surviving CAR-treated animals were obtained from the tail vein approximately a week after the mice regained their starting weight. DNA from blood and spleen tissues was extracted using NucleoSpin Tissue DNA extraction kit (Macherey-Nagel) following the manufacturer's instructions.
Detection of integrated CAR transgene by quantitative PCR
Quantitative real-time PCR was performed on 150 ng of DNA with three replicates for each sample using the 2X Taqman Gene Expression Master Mix (Applied Biosystems). Specific primers and probe were designed to amplify the junction region between the light and heavy chain of the single-chain variable fragment of anti-CD19 antibody (clone FMC63) that is contained within the CAR construct:
Forward primer: 5′-ACAGGGTAATACGCTTCCGT-3′
Reverse primer: 5′-CCCTGAGACAGTGCATGTGA-3′
MGB probe: 5′-FAM CCACCTGTGATCTCCAGCTTGGTC-3′
PCR with real-time fluorescence detection was performed on a 384-well Thermo Scientific QuantStudio 12 k (Thermo Fisher Scientific). The number of copies for each vector, expressed as copies/ng DNA, was determined by comparison of the measured cycle threshold for each well to the cycle threshold of a standard curve prepared by dilution of a linearized receptor-encoding plasmid spiked into 150 ng of CTRL mouse genomic DNA (obtained from a healthy NSG animal) per well. The R2 value of the standard curve was 0.9945, and the slope was −3.4134 (corresponding to efficiency of reaction of 96%). Known spike controls ranged from 1×101 to 1×107 copies per well.
Flow-cytometric analysis
All flow-cytometric analyses were performed using FACS Verse flow cytometer (BD Biosciences). Antibodies used in the study are listed in online supplementary table 1. To confirm the CAR expression, double staining was used: biotinylated goat antimouse polyclonal IgG antibody (Thermo Fisher Scientific) was visualized with AlexaFluor647-conjugated streptavidin (Invitrogen). In all flow-cytometric analyses, cell viability was monitored with 7-aminoactinomycin D (Invitrogen). Data were analyzed in FlowJo V.10 software.
All statistical analyses were performed using software incorporated in GraphPad Prism V.5. Specific statistical tests used for different study variables are described in the figure legends. All tests were two-sided. P values of <0.05 were considered statistically significant.
In vitro anti-CD19 CAR T cells are similarly effective at eliminating primary CLL cells of various genetic backgrounds
For the present study, we selected the anti-CD19 CAR incorporating 4-1BB costimulation molecule in the design.5 22 Our optimized protocol led to the production of a high yield of CAR-expressing T cells (>85%, online supplementary figure 1A). The functionality of CAR T cells was validated in various in vitro tests (online supplementary figure 1B). In order to comprehensively assess the prognostic value of CLL genetic background on the response to CAR T-cell therapy, we compiled a set of genetically defined 32 primary CLL samples. Of these, 26 were IGHV unmutated (9 ATM-mutated, 8 TP53-mutated, and 9 without mutations in ATM, TP53, NOTCH1 or SF3B1) and 6 bore hypermutation in the IGHV gene but no mutations in the other studied genes. After thawing, no difference in cell viability was observed among the genetic classes (online supplementary figure 2). B cells were then either directly mixed with T cells or stimulated through IL-2 and nuclear factor kappa B-activating signals23 prior to exposure to T cells in order to mimic a leukemic microenvironment and thus increase the in vitro fitness. After 96 hours of cocultivation, while there still were viable unstimulated CLL cells surviving in culture with CTRL T cells, no viable unstimulated B cells were detected in the mixture with CAR T cells (figure 1A). 2S stimulation proved to have a positive effect on CLL in vitro fitness, as some CLL cells survived even in coculture with CAR T cells (figure 1B). However, after 96 hours of coculture, no significant differences in the survival of the different genetic classes were observed (mean percentage of surviving unmutated IGHV WT cells, 2%; TP53-mutated, 1.5%; ATM-mutated, 3.3%; and IGHV-mutated WT cells, 3.2%; figure 1B). Unsurprisingly, exposure to CAR T cells led to a gradual decrease in the number of target CLL cells throughout the cocultivation period in both studied set-ups (figure 1C).
Cytotoxicity of anti-CD19 CAR T cells against primary CLL cells. (A) T cells from a single donor were transduced and mixed with primary unstimulated CLL cells of various genetic backgrounds in 1:5 target:effector ratio. Target cells are color-coded as follows: IGHV-unmutated samples that had an ATM mutation are in blue; those with a TP53 mutation are in red; and those without mutations in ATM, TP53, NOTCH1 or SF3B1 are in green. IGHV-mutated samples without mutations in the above-mentioned genes are in black.This color coding is maintained throughout the manuscript. Cytotoxicity was measured by flow cytometry at 24 and 96 hours of coculture. Results are expressed as percentage of CD19+ leukemia cell recovery after 4 days of culture relative to culture with CTRL T lymphocytes. (B) Primary CLL cells of various genetic backgrounds stimulated with interleukin-2 and nuclear factor kappa B were mixed with CAR/CTRL T cells in 1:5 target:effector ratio and cytotoxicity is measured as in A. (C) Target elimination was gradual over the cultivation period. CAR, chimeric antigen receptor; CLL, chronic lymphocytic leukemia; CTRL, control; IGHV, immunoglobulin heavy-chain gene variable; n.s., not significant; WT, wild type.
CRISPR/Cas9 generated TP53-knockout and ATM-knockout single-cell clones recapitulate the results from primary CLL cells in vitro
Primary CLL cells strive in vitro and, so far, no successful strategy for their reliable xenotransplantation has been described. Therefore, we used the CRISPR/Cas9 system to generate a collection of cell lines with a specific genetic background from commercially available CLL-derived cell lines (HG3 and MEC1). We aimed to introduce TP53 or ATM mutations that would result in disruption of the targeted gene expression and thus to generate a set of genetically distinct knockout single-cell clones. However, MEC1 cells already express a truncated p53. Since constitutive WT p53 introduction into p53-defficient tumors could induce senescence24 25, we introduced WT TP53 under an inducible promotor. Short-term expression of WT p53 protein could be induced by treating the cells with doxycycline, and it was further stabilized upon DNA damage triggered by doxorubicin (online supplementary figure 3A). These cells were denoted as 'MEC1 p53 WT' and used subsequently to generate individual MEC1 ATM -knockout and TP53-knockout clones. In total, we were able to create 7 TP53 single-cell knockout clones (three and four, respectively) and four ATM single-cell knockout clones (three and one, respectively) from both HG3 and MEC1 cells. As shown in figure 2A, no expression of p53 was detectable upon induction of DNA damage in the TP53-knockout cells, and there was a complete loss of ATM protein in the generated knockouts. Even the downstream effector genes were not activated in the generated cell lines upon adequate stimulus (p21 in case of TP53 knockouts, p21 and pKAP1 in ATM knockouts; online supplementary figure 3B), demonstrating true functional impairment of the targeted genes and their downstream signaling pathways in our generated cell lines. In vitro, no other obvious phenotypical changes were associated with acquiring the desired gene mutations; no major differences in cell proliferation were noted (online supplementary figure 3C); and no differences in expression of serine protease inhibitor PI-9, an inhibitor of granzyme B-mediated T-cell cytotoxicity, were found (online supplementary figure 3D).
Cytotoxicity of anti-CD19 CAR T cells against cell lines with specific genetic background. (A) Indel mutations were induced in TP53 (in red) and ATM (in blue) genes of HG3 and MEC1 cell lines using CRISPR/Cas9. These mutations resulted in loss of the respective protein as demonstrated by western blotting. (B) Production of IFN-γ, as measured by ELISA, was significantly induced when CAR T cells were cultured with ATM-knockout and TP53-knockout cell lines as compared with CTRL T cells (Kruskal-Wallis test, p=0.0020). (C) Cytotoxicity of CAR T cells against generated TP53-knockout and ATM-knockout cell lines was measured in a 96 hours of coculture by flow cytometry. Results are expressed as percentage of CD19+ leukemia cell recovery after 4 days of culture relative to culture with CTRL T lymphocytes. (D) Target elimination over the cultivation period differed between TP53-knockout and ATM-knockout clones. Fifty-seven percent of TP53-knockout cells were able to proliferate in coculture with CAR T cells contrasted with 25% of ATM-knockout cells. CAR, chimeric antigen receptor; CTRL, control; IFN, interferon; n.s., not significant.
When mixed with available knockout cell lines, CAR T cells, but not CTRL T cells, became specifically activated in their presence as evidenced by the production of IFN-γ (p=0.0020, figure 2B). In line with previous observations from primary CLL cells, after 96 hours of in vitro coculture with CAR T cells, a similar proportion of CD19+ target cells were surviving in the two genetically defined groups (mean percentage of surviving tumor cells was 12.9% and 14% in TP53 and ATM knockouts, respectively; figure 2C). However, when we examined the target elimination in time, we observed that TP53-knockout clones more often exhibited the ability to maintain proliferation in coculture with CAR T cells than their ATM-knockout counterparts (4/7 TP53 clones vs 1/4 ATM clone, figure 2D).
CAR T cells prolong survival of all studied genetic classes in vivo, but with different curative rates
Since primary CLL cells engraft inefficiently in immunocompromised mice, we chose to evaluate the in vivo efficacy of anti-CD19 CAR T-cell therapy in NSG mice injected with CRISPR/Cas9 generated knockout cell lines. First, we evaluated the kinetics of the disease of all generated cell lines as well as the original HG3 and MEC1 cells in vivo (figure 3A, HG3 cell line and knockout clones derived from it are depicted with off-color shapes). In this model system, intravenous injection of tumor cell lines led to development of progressive leukemia with significant involvement of the spleen, liver and kidneys. Two HG3-derived TP53-knockout clones and an additional ATM clone did not engraft (clones 2, 3 and 6, respectively) even though these mice were followed for 60 days. In the remaining cases, the kinetics of TP53-knockout-driven disease clearly showed a more severe course than the ATM-knockout one (median days of succumbing to the disease were 19 and 27, respectively; p=0.0262), similar to the original cell lines (median day 20).
TP53-knockout and ATM-knockout clones induce differently severe progressive leukemia in immunodeficient NSG mice. (A) To assess the penetrance of individual knockout clones, 2.5×106 knockout tumor cells or WT HG3 and MEC1 cells were injected intravenously into NSG mice. HG3 and cell lines derived from it are depicted in off-color shapes. Time of disease-related death differed significantly between TP53-knockout and ATM-knockout cell lines (Mann-Whitney test, p=0.0262). (B) Experimental set-up of high-tumor burden setting showing timing and amount of intravenously transferred tumor and CAR/CTRL T cells in NSG mice. (C) Morbidity was assessed by regular weighting of the animals. It is possible to appreciate from this graphics that TP53-knockout disease develops earlier (Mann-Whitney test, p<0.0001) and shows a more aggressive course than ATM-knockout one. (D) No significant difference in the disease morbidity was observed among mice injected with CTRL T cells belonging to either TP53 or ATM group at the time of sacrification (left panel; day of sacrification 17–18 or 23, respectively; Mann-Whitney test; p=0.2786). Animals with TP53-knockout disease showed significantly higher tumor burden, as assessed from the number of CD19+ cells detected in spleens of the deceased animals (Mann-Whitney test, p=0.0002). (E) Kaplan-Meier curves showing survival of mice. P values were calculated with two-tailed log-rank Mantel-Cox tests. (F) The number of copies of anti-CD19 CAR vector in spleen and blood DNA from CAR treated mice. Spleens from TP53-knockout mice were harvested at their sacrification due to progressive disease (days 17 and 18), as well as spleens from two ATM-knockout animals (days 24 and 26, in the graphics depicted in off-color). Spleens from remaining mice with ATM-knockout disease were obtained at the termination more than 6 weeks later. Blood samples were obtained from cured animals approximately 1 week after regaining their original weight (day 29). Significantly more copies of CAR vector were detected in spleens of mice with ATM-knockout disease (Mann-Whitney test, p=0.0012). CAR, chimeric antigen receptor; CTRL, control; n.s., not significant; NSG, NOD-scid IL2Rgnull; WT, wild type.
We selected HG3-derived clones 1 (TP53 knockout) and 4 (ATM knockout) for further in vivo studies. First, we inoculated NSG mice with 2×106 tumor cells (this setting was termed 'high-tumor burden'; figure 3B). Seven days later, CAR or CTRL T cells were injected via tail vein. On day 14, mice injected with the TP53-knockout clone started showing signs of the disease, which was quickly progressing in the following days (figure 3C). In line with the kinetics experiment, ATM-knockout disease onset was delayed (figure 3C, p<0.0001), and its progression was slower. At the moment of succumbing to the disease (median days 16 and 23 in TP53 and ATM CTRL mice, respectively), there was no significant difference in the disease morbidity among the animals injected with CTRL T cells from the two genetic classes (assessed here by weight loss, figure 3D). However, mice injected with the TP53-knockout clone had a significantly higher tumor burden compared with the ATM knockout (p=0.0002, figure 3D). Due to these disease-related differences, the positive treatment effect was solely apparent in mice with ATM-knockout disease (p<0.0001, figure 3E). Quantification of the integrated CAR-expressing vector in spleens of the treated mice additionally uncovered inefficient engraftment of CAR T cells in the animals with TP53-knockout tumors, plausibly caused by shorter time for their proliferation in these mice (p=0.0012, figure 3F). Eighty-five percent of mice bearing ATM-knockout disease treated with CAR T cells regained their original weight, were considered cured and afterwards had high levels of CAR T cells detectable in their circulation (figure 3F).
Finally, in an attempt to alleviate the disease burden caused by the TP53 knockout, we inoculated NSG mice with 5×105 tumor cells (setting termed 'low-tumor burden') and started the treatment 5 days later (figure 4A). In this setting, mice injected with TP53-knockout cells started showing signs of the disease only 1 day later than in the above-mentioned high-tumor burden scenario (day 15, figure 4B). Similarly, severe disease course was observed among mice injected with HG3 WT cells. On the other hand, the low-tumor cell dose was suboptimal to induce leukemia with ATM-knockout cells, since only 4/7 of animals injected with CTRL T cells developed the disease. Nevertheless, in this setting, the treatment partially prolonged survival of animals with TP53-knockout disease (p=0.0204), ATM-knockout disease (p=0.0217), as well as those injected with HG3 WT tumor cells (p=0.0485, figure 4C). One of five (20%) mice injected with HG3 WT cells and treated with CAR T cells was considered cured of disease due to regaining of the original weight and high levels of CAR T cells in blood (assessed by flow cytometry, online supplementary figure 4).
CAR T-cell treatment prolongs the survival of all genetic groups, but with different curative rates. (A) Experimental set-up of low-tumor burden setting showing the timing and amount of intravenously transferred tumor and CAR/CTRL T cells in NSG mice. (B) Morbidity was assessed in the study by regular weighting of the animals. Even in this setting, disease resulting from injecting HG3 WT and TP53-knockout cells develops early and shows a more aggressive course when compared with ATM-knockout disease. (C) Kaplan-Meier curves showing survival of mice. P values were calculated with two-tailed log-rank Mantel-Cox tests. CAR, chimeric antigen receptor; CTRL, control; WT, wild type.
In the current era of precision medicine, medical decisions are being tailored to the individual patient based on their predicted response. One example of such personalization from hemato-oncological practice is the preferential treatment of patients with CLL who test positive for aberrations affecting the TP53 gene, with targeted inhibitors3 due to their predicted chemorefractoriness.26 However, no such personalization is possible so far in the case of CAR T-cell therapy, even though roughly only one out of three patients with CLL will respond to this treatment.6 7 Herein, we set out to comprehensively assess for the first time the impact of two major genetic aberrations on the performance of CAR T cells in several CLL disease models. Not only do we study CAR T-cell performance on a genetically defined set of primary samples, but we also use CRISPR/Cas9 system to prepare several TP53-gene and ATM-gene knockout clones from two CLL-derived cell lines. While we do not observe any significant differences in survival of target CLL cells in vitro, in our in vivo disease model, TP53 knockout is associated with earlier time of disease onset, higher tumor burden and inefficient T-cell engraftment, which jointly negatively influence the treatment outcome.
CLL is a disease of many genetic drivers, some of which exhibit oncogenic properties, while others function as tumor suppressors.27 The study of disease characteristics and response to treatment using primary CLL cells is hampered by their decreased in vitro viability and their inefficiency to engraft in immunodeficient mice (reviewed in Bertilaccio et al 28). Nevertheless, in the current study, we worked with a set of genetically characterized primary CLL cells to assess their response to CAR T-cell treatment. All unstimulated CLL cells were efficiently and completely eradicated by CAR T cells at the end point of our experiment, while some of those in which we mimicked microenvironment interactions23 survived the cultivation period. However, the total CLL survival was low (<5%). Therefore, to exclude that the results were affected by the reduced fitness of CLL cells in prolonged in vitro cultures, we decided to prepare genetically homogeneous CLL-derived cell lines bearing clinically relevant mutations. Herein, we focused on TP53 and ATM genes for several reasons. First, they are among the most frequently mutated genes in CLL.29 Second, aberrations in these two genes have been linked with poor prognosis.2 30 Finally, these genes are tumor suppressors (ie, loss or reduction in their function is found in cancer), and thus we could use CRISPR/Cas9 technology to disrupt the gene rather than to introduce any specific mutations, which is technically less challenging.31 Of note, while the survival of the generated knockout cell lines in response to CAR T cells was higher (mean ~15%), we still could not detect any difference in survival according to the genetic background, corroborating the results obtained with primary CLL cells.
The commercial HG3 cell line, which is one of those we used to prepare knockout cells, has been described to be composed of subclones and thus to be heterogeneous.32 Therefore, creation of isogenic cell lines originating from a single cell could lead to opportunistic selection of a phenotype associated with one of the subclones rather than with the generated genetic feature. Indeed, when we transplanted our knockout cell line collection into NSG mice, three clones derived from HG3 cells did not engraft, which could possibly point to them being derived from a specific common subclone. Nevertheless, the remaining clones (both TP53 and ATM knockouts) derived from HG3 cells behaved remarkably similarly to those derived from MEC1 cells in all remaining assays. Therefore, we believe that in these clones, the phenotypes we observe are indeed caused by the introduced mutations and not by a subclonal origin of the cells.
Already the in vitro data hinted the aggressive potential of TP53-knockout cell lines, since they more often maintained the ability to proliferate in coculture with CAR T cells. Still, in vitro modeling does not allow for faithful recapitulation of the disease pathogenesis and the complex in vivo relationships between leukemic cells and the microenvironment,28 which are crucial in CLL. In our in vivo model, we could recapitulate some of the clinical characteristics of TP53-mutated CLL. For instance, NSG mice injected with TP53-knockout clones showed the shortest time to disease manifestation, which is in accordance with TP53-mutated patients displaying shorter time to first treatment.26 Of note, TP53-knockout clones' superior proliferation rate was only apparent in vivo, once again underpinning the positive effect of microenvironment on CLL tumorigenesis. In our model, this aberration was therefore also associated with a higher tumor burden, which has already been connected with failure of the CAR T-cell therapy.33
It is important to acknowledge here that some patients with CLL with TP53-mutated disease have already been successfully treated with CAR T-cell therapy.34 While in our in vivo experimental setting we worked with a well-defined homogeneous model, in the clinical practice, there is a certain degree of heterogeneity among patients with CLL with TP53-mutated disease: some patients with this poor prognosis marker do not suffer from such a quickly progressing disease and can therefore be candidates for CAR T-cell therapy. Similarly, there are patients who do not harbor TP53 aberrations and suffer from an aggressive progressive disease.35 We hypothesize that those could also benefit to a lesser extent from CAR T cells.
Unexpectedly, disease onset in mice injected with ATM-knockout cell lines showed major retardation. ATM plays an important role in double-stranded DNA repair, as well as p53 activation with subsequent cell cycle arrest and/or apoptosis. Thus, its inactivation can cause p53 dysfunction. The ATM gene is localized on the long arm of chromosome 11 in a region, which is very frequently deleted in CLL. Those patients with CLL, whose residual ATM allele is mutated, show poorer clinical outcome.36 In all of our knockout clones, both alleles of ATM gene were affected by indel mutations, which resulted in complete loss of ATM at protein level. Thus, adverse phenotype was anticipated. However, a similarly mild phenotype as we describe here for ATM knockout has been observed in glioma tumorigenesis, where knockout or inhibition of ATM function promoted animal survival as compared with WT cells.37 Similarly, in vivo tumor formation was impaired by ATM knockout in a breast cancer cell line as compared with the WT CTRL.38
Finally, the quality of patient's T cells used to prepare the final CAR T-cell product can impact the treatment efficacy in CLL.11 In our in vivo models, we did observe inefficient engraftment of CAR T cells in mice with TP53-knockout-driven disease. However, the CAR T cells for each of the different stages of the study (in vitro studies, high-tumor and low-tumor in vivo burden) were produced from a single healthy T-cell donor (different for every stage) with a similarly high efficiency. Thus, it is highly improbable that any inherent T-cell defects could be affecting our results. The effect we describe is possibly caused by the shorter time T cells had for proliferation in mice with TP53-knockout-driven tumors. CAR T-cell cytotoxicity has been shown to be blocked by production of high levels of the serine protease PI-9/serpin-6 that irreversibly inactivates granzyme B.39 However, we did not observe any differences in expression of this enzyme among the used cell lines, which could explain the differences we observe in vivo. Further studies could uncover if additional mechanisms (eg, immunosuppressive microenvironment) are also at play in these mice.
This is the first study comprehensively assessing the impact of various clinically relevant genetic aberrations on performance of CAR T cells in CLL. In vitro, anti-CD19 CAR T cells are similarly effective at eliminating CLL model cell lines and primary CLL cells of various genetic backgrounds. In vivo, early disease onset, high-tumor burden and inefficient T-cell engraftment, which were associated with TP53-knockout-driven tumors in our experimental setting, ultimately lead to poorer response to CAR T-cell treatment in this CLL subgroup.
The authors thank Karla Plevova, PhD, for selecting primary lymphocytic leukemia cells for the study, and also all medical doctors and nurses involved in collecting patients' samples (Professor Doubek, Dr Brychtova) and all the patients.
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VM and MS are joint senior authors.
VM, HP and VK contributed equally.
Contributors HP and VK prepared the CRISPR/Cas9 knockout clones; HP performed their functional validation. VM prepared CAR T cells, and VM, HP, VK, ALe and ALa conducted all other experiments. JV supervised in vivo studies. TL supervised flow-cytometry measurements. VM, HP, VK and MS discussed and analyzed the data. All authors contributed to the drafting of the manuscript.
Funding This project has received funding from the European Union's (EU) Horizon 2020 research and innovation program under the Marie Skłodowska-Curie actions and is cofinanced by the South Moravian Region under grant agreement number 665860. This report reflects only the author's view and the EU is not responsible for any use that may be made of the information it contains.
Ethics approval The study has been approved by ethics committee of Masaryk University (number of ethics committee case EKV-2017-008). Chronic lymphocytic leukemia samples were selected from the biobank of the Department of Internal Medicine, Hematology and Oncology, where all blood samples are taken after written informed patient's consent, in accordance with the Declaration of Helsinki, under protocols approved by the hospital ethics committee. All animal experiments were performed with the approval of the institute's ethics committee according to the Law on the Protection of Animals Against Cruelty. The research involving animal models has additionally obtained approval by the Ministry of Education, Youth and Sports (MSMT-25883/2017-2).
Data availability statement Data sharing not applicable as no datasets generated and/or analysed for this study. All data relevant to the study are included in the article or uploaded as supplementary information. No datasets have been generated within this study, and all data relevant to the study have been included in the article figures or supplementary figures.
© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. http://creativecommons.org/licenses/by-nc/4.0/This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.


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August Riseponds: Separating Art and Artist
Riseponds is a monthly series where a group of Risen members are picked, not based on viewpoints, but solely through a "first come first serve" basis to speak on a topic that they're passionate or apathetic about. The randomness serves so readers can gain different viewpoints from different individuals with contrasting experiences.
This month, we've been thinking about separating art from its artist: can people still consume art/media without guilt when it was created by an artist who has done horrible things or is problematic?
Alexis: I think that art is a representation of the artist, first and foremost. Any statement that art makes is a statement that the artist believes in, so you cannot separate the artist from the art. However, nobody is perfect. All artists and therefore all art will have flaws and problems. Trying to consume media of any kind that doesn't upset somebody in some way is impossible: the line between 'upsetting' and 'genuinely problematic' is different for everyone, so pay attention to what people say, but take it with a grain of salt. Be the judge of what media you consume and enjoy what you want, but don't forget to acknowledge that it isn't perfect. It's okay to acknowledge something's flaws while still enjoying it.
It's important to note though, that using the separation of art and artist as an excuse to shamelessly support abusers, homophobes, and generally bad people doesn't mean that consuming that media is okay. It means that you support to the people who made it, no matter what you say to the contrary.
Adele: When bringing up this subject, I dread hearing the "separate the art from the artist" spiel. From my experience, those who uses this argument are either 1) not artists themselves and/or 2) are accepting of said problematic behaviour/actions. I wouldn't exactly call myself an artist, as I don't create art consistently, but I understand that art is an expression of yourself. Some people are more blunt than others in their medium, but I still believe that even making something "pointless" calls into who you are. You can, however, acknowledge the flaws of said person while consuming their art. In the age of "Your Fave Is Problematic" and call out culture, it can be hard to enjoy anything, but acknowledging that what the artist did or said was wrong is always a better option than ignoring or attempting to minimize/ridicule the issue. The thing about being "problematic" is that it is inherently human to be so. Nobody's perfect.
I also feel like it's important to add that deeming someone as "problematic" doesn't mean that you necessarily need to cut them off. I know personally that there are a few artists who have been deemed "problematic" that I don't support personally but I will still consume their art in some way. Sometimes that means listening to their albums or admiring their film work or photography. But it also isn't necessarily bad for you to literally cut them out. There are a few musical artists that I've loved in the past, where I stopped consuming their art and following them as a person because I couldn't forgive their actions. Personally, if an artist I like says or does something that is homophobic (and/or: lesbophobic, biphobic), transphobic, antiblack, racist, ableist, etc., and makes no effort to genuinely apologize for it, then I make no effort to support them in any way.
Vivian: Personally, I think separating the art from the artist is not necessarily a negative thing as long as the art is not an expression of, and has no ties to, the artist's ideals. For example, if this artist is an actor, I think you should be allowed to admire their acting skills even without agreeing with their worldviews or even liking them as a person. However, it is also important to not idolize an artist and defend them regardless of their wrongdoings. We should recognize their respective talents without putting them on a pedestal. People, even those we look up to, are not always in the right or without determining faults. That being said, I find it hard to admire an artist after learning of their "problematic" ways/behaviours. For example, Tim Burton's whitewashed cast has caused me to lose respect for him as a director because a director's decisions do tie in with the way they present their art and their opinions. But behaviour may not always be involved with an artist's respective craft. In other words, we need to be careful when judging an artist's behaviour and determine for ourselves the line between actions that can be outweighed and those that cannot. Mostly, we need to remember that artists are human and make mistakes too, but it's important to separate mistakes from detrimental actions.
Tyler: In my experience, separating the art from the artist has always been something I've done. I wrote an article recently about whether or not black women can truly like rap music because of the fact that so much of it is based on the tearing down of their identities. I spoke about the fact that in the black community, we are raised to like certain musicians and that they can do no wrong because we place their songs on such a high pedestal. We ignore any problematic things artists have done in order to keep them in the culture. I don't think in today's society people are able to consume media from problematic artists without backlash. Oftentimes I do feel guilty for liking some of the music I do. For example, I wholeheartedly agree with the idea that musician R. Kelly is a bad role model and very problematic in his ways. However I cannot deny that I know the words to his songs and sing them when they come on because these are the songs I grew up listening to. I think that if you know right from wrong and can recognize that an artist is problematic then separating the art from the artist is fine.
adele lukasa alexis artist riseponds tyler vivian

redpajama_set_name: RedPajamaCommonCrawl


Finding your way in a strange New York City neighborhood can be frightening. Finding your way in a strange neighborhood while not knowing English can be tougher still. Finding your way in a strange neighborhood while not knowing English as an immigrant unfamiliar with New York City ways can be even more scary.
So, imagine finding your way in a strange New York City neighborhood as an immigrant speaking no English while blind! Terrifying.
Fortunately, Catholic Guild for the Blind offers special, free services for immigrants and refugees who are blind or visually impaired. These services are open to all immigrants, documented and undocumented, refugees, including individuals who are not eligible for state or federally funded programs due to their immigration status.
Often, when a visually impaired person arrives in the United States, they face challenges acclimating to their new environment due to difficulty communicating, navigating an unfamiliar environment and traveling to destinations independently. In addition, they may not know that services are available to them and may benefit from guidance and links to social services.
These classes include specialized instructional materials for individuals with vision loss.
These classes teach white can skills for identification, traveling in unfamiliar environments, movement in home surroundings, street crossings and using public transportation.
This provides links to community resources including Catholic Charities Immigration and Refugee services, food pantry services, soup kitchens and resources specifically for the disabled.


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maintainers with opinions on the matter.


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Where Mr Green is trying to stand out from the competition is by taking the 'game responsibly' angle seriously. They offer players to cap a limit on their bets, which most other websites do as well. The difference here is that they provide the offer upfront.
Mr Green has been attracting more and more players by offering generous bonuses, cash prizes, and free spins. They even have a loyalty points system that is accumulated by simply logging on. Based on the number of points, players are eligible to enter various cash draws.
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Ariadne is formally crowned Queen, but with Pasiphae still at large and the city struggling in the aftermath of battle, Atlantis remains vulnerable indeed. So when an enigmatic stranger arrives offering salvation, he proves hard to resist. But his proposal is far from straightforward – will the cost be too much for Ariadne to bear?
As plans progress for the royal wedding, Ariadne must undertake a journey to Telemon's homeland of Aegina. With the kingdom still beset by Colcheans, Jason is not the only one who fears for the Queen's safety. Determined to do what is best for her people, Ariadne continues regardless. But as the voyage progresses, it becomes clear that the royal retinue faces enemies at every turn. Has the young Queen's luck finally run out?
As the dust settles, the gang find themselves dispersed around the ancient necropolis. And they soon realize they are not alone…the space is crawling with a magical army of the undead. The race is on to find Jason before he falls prey to the flesh eating monsters. As Jason battles for his life, he finds an unlikely ally in the mysterious Medea –but can he really trust her?
As Ariadne lies on her deathbed, Jason's guilt is almost too much to bear. He resolves to do all he can to save her and embarks on a dangerous quest into the unknown. After a daring encounter with the ancient Grey Sisters, Jason's mission brings him face to face with Medea and Pasiphae once more. Can he thwart their powerful magic to save his love, or do they have other plans?


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text:
Who is Verified on Twitter, and Why Does it Matter?
by Shanley Kane on August 18th, 2016
Veve Jaffa's article on Twitter verification earlier this week got me thinking. With new, expanded Verification policies (misleadingly) painted as an anti-online harassment strategy — inasmuch as Verified users have had access to improved filters and safety features — it's an opportune moment to think about the politics of WHO gets "status" on social media, and what it tells us about how social platforms function.
So: who is Verified on Twitter? I put out a quick inquiry online, and was excited when @_anti_climacus began a preliminary inquiry into the gender distribution of Verified users. The test used the public streaming API to collect a large sample of Verified accounts, and the Genderize REST API — which lets researchers approximate gender data by comparing first names against a large database — to come up with some initial stats. In the end, the test sampled 21,000 Verified accounts, and the results are concerning: to start, many more men are verified than women, with women making up only about a third of individual Verified users, and just 20% of all Verified accounts (including brand/corporate accounts). Of course, this also indicates that a huge number of verified accounts are not people at all, but brands, institutions, mainstream media and corporations:
Of course, it should be noted that this data is from early and rough investigation; it should be taken as preliminary inquiry only, indicating the need for more rigorous research — particularly, research across larger number of accounts, research that incorporates race, sexuality as well as a more expansive view of gender, and research with a more rigorous process of design and peer review. However, this early data is completely consistent with a platform that has constantly prioritized white men and corporate users over marginalized people.
Who gets verification status is important, because status is just one of many ways that large social platforms shape our attention and influence the public based on their own value system. Specifically, Verification is tied to increased visibility on the platform in myriad ways: One, the users who are generally recommended upon signing up/logging in to Twitter are Verified accounts. Further, on many large/mainstream media sites, Verified users are more likely to be cited, quoted and/or interviewed in news articles, trend pieces, listicles, etc. It's likely that Verified users are able to grow their audience more quickly due to public perception of the "Verified" badge; for privileged users, this audience is often tied to an increase in influence, economic opportunity, access to positions of power, etc. In the media, entertainment, fashion, tech and other industries, status symbols — even those as seemingly arbitrary as a blue check — do contribute to an individual's perceived social value… and, in turn, to their professional support system, socioeconomic status, career trajectory, and other material effects.
Considering Verification has been in place for seven full years, we can't underestimate the cumulative effects over time of a system that has consistently verified some users over all other users. This is particularly poignant when we see how Twitter's growth has been driven by marginalized, unaffiliated women (as Shaadi Devereaux highlights in "Why These Tweets Are Called My Back"); Black Twitter (as Lesli-Ann Lewis notes, "Twitter has become a cultural force largely because of the clout, reach and wit of its Black users"); and various movements and disruptions led by marginalized activists. Yet, these are the users consistently cut out of consideration, protection, respect, and status by Twitter the company; yet another way tech business models are built on the labor of marginalized people.
Some might argue that the current demographics of Verified users are irrelevant, reflecting past Verification processes that are currently being replaced by an expanded application system. However, these arguments ignore the fact that Verification remains at Twitter's discretion… even though now more people can apply, getting the check still requires meeting Twitter's criteria. Thus, the new system doesn't necessarily represent any departure from the prior value system; in fact, the newly opened verification process suggests that, contrary to any change in underlying logics, this system just makes applying those same values more efficient and scalable for Twitter's staff. If the value system that Twitter employs to determine Verified status has remained unchanged, we can expect privileged men and brands to continue to be the primary recipients and beneficiaries of status.
Certainly, ongoing reports from victims of severe harassment having their applications rejected indicates that victims of abuse aren't at the center of these changes. So, if not increasing safety, what then are the goals of Twitter's newly expanded verification? As we can see in preliminary data, a large portion of Verified users are brands; an expanded program would suggest that brand management, monetization through advertising, and the continued reification of corporations over people is part-and-parcel. This is consistent with Twitter's investment in tooling, which has offered advanced features to, for example, social media teams working on corporate accounts, while individuals targeted by abuse on the platform are left with no recourse. The predominance of Verified brands on Twitter further brings its reputation as a progressive space into question; while eager to capitalize on its purported role in social movements, an ongoing focus on advertisers, brands and corporate users while neglecting marginalized ones shows that this trend is more the result of activist ingenuity and subversion more than intentional design. With Twitter's growth practically stalled, one would think they'd turn their focus to individual users rather than brands, lest the "free speech wing of the free speech party" become a deserted hell-hole of brands trolling each other for inclusion in a plagiarized Buzzfeed listicle… Then again, no one asked me. (They never do…)
More than anything, preliminary Verified demographics shows the importance of looking holistically at diversity in tech. While diversity in tech efforts tend to focus narrowly on the full-time employees of tech companies, that leaves out huge swathes of the picture, especially dynamics in the user base. In the case of Twitter, we must look at user demographics, we must look at the demographics of abusers vs targets, who is influential on the platform and who is rewarded on the platform (not always the same thing), and who is Verified. Especially as the verification program expands and Twitter ups its investment in Verification as a core strategy, it's particularly worth monitoring if Verification status is distributed in a fair and equitable way that recognizes and values the diversity of its users.
PS: If anyone is interested in launching a more rigorous investigation of Verified demographics on Twitter, please get in touch!


Today Tonight is giving you the chance to win one of 10 family passes (for a family of four) to the 2017 Royal Adelaide Show.
We'll feature some of the best videos on Today Tonight.
Entries close Sunday, August 27, at 11.59pm.


One less local player will be on the Lobos bench this coming basketball season.
At a regularly scheduled mid-summer, program update press conference on Wednesday, UNM men's basketball coach Paul Weir announced former Valley High School star point guard Anthony Chavez, one of three in-state walk-on players on the Lobos roster to start the offseason, has decided to move on.
The Journal reached out to Chavez for comment, but he did not respond.
It is unclear if Chavez might pursue playing at another school.
SCHEDULE: While it remains close, the Lobos' full non-conference schedule is still not done, Weir said.
OTHER PLAYER UPDATES: Junior college point guard Keith McGee is "in Albuquerque," but not yet eligible to join the Lobos, Weir said.
The coach said McGee is still wrapping up summer classes and tentatively put an Aug. 9 timetable on when he might be able to begin workouts.
As for Carlton Bragg, the former Kansas Jayhawk and Arizona Sun Devil who transferred to UNM in January, he is still not being allowed to talk to the media, though he is fully cleared by the NCAA to practice and participate in all team activities.
Weir said the team is still waiting on the NCAA's decision on a waiver that would allow Bragg to play at the beginning of the season (he's currently eligible to start playing in December after the fall semester ends) and added that Bragg, the 6-foot-10 power forward who is not listed on the team's web site, has not yet met the coach's requirements to play.
"He's still working through the expectations that I have for him to one day get onto the court," Weir said.
Bragg was a full participant in Wednesday's practice, at one point finishing a fast break with a ferocious dunk after taking a 50-foot bounce pass through traffic from a sprinting Vance Jackson, another Lobo transfer who sat out last season after being a starter at Connecticut as a freshman.
SPORTS CUTS: Two hours prior to Wednesday's press conference, Weir posted on his Twitter account a prepared statement addressing recent sports cuts at UNM.
During the press conference, Tuesday's comments by UNM football coach Bob Davie were brought up by a reporter and Weir was asked if, as Davie claimed, he was worried his sport might have been one of the ones on the chopping block.
The Mountain West requires for membership universities field football, volleyball, and men's and women's basketball.
SOMEWHERE ELSE: Weir wants to play a regular season or exhibition game each year somewhere outside of Albuquerque, and not including the annual game with rival New Mexico State.
Nothing is set in stone, he said, but said he's talked to enough fans in Santa Fe, Gallup, Farmington, etc., that people around the state are as passionate about the program as those in Albuquerque are.
"I stole the idea from (New Mexico State University Athletic Director) Mario Moccia, who came here to play games," said Weir, referring to NMSU playing an annual regular season "home" game in Rio Rancho at the Santa Ana Star Center.
The goal, Weir said, is statewide outreach.
"(We want) to continue to embrace our community and thank different areas of the state of New Mexico and bring Lobo basketball to them instead of always asking them to come to us," Weir said.
HOWL YES? Weir said he's open to renewing the Lobo Howl, the preseason, public practice/exhibition event that was discontinued last year after dwindling attendance. The Howl usually has events like a dunk contest, a 3-point contest and involves the UNM women's team.
RECRUITING SEASON: July isn't just a hot recruiting period for college coaches looking for players, apparently.
Weir took to Twitter on Wednesday posting a link to an open, full-time marketing position on the UNM jobs web site that will focus on basketball.
Earlier in the day, he mentioned the team has sold 600 new season tickets so far (he said it's hard to have a good grasp just yet on renewal numbers). He then said there was a marketing position open in athletics that could help the program.
Lobos sophomore Makuach Maluach, left, defends senior guard Anthony Mathis at a workout session Wednesday in Dreamstyle Arena — The Pit. Media watched the workouts after a press conference with coach Paul Weir.


Abstract: In order for e-services to work, a certain level of dependability is necessary in the consumers and providers involved. I draw on an unusual source - microcredit schemes - in order to find ways of promoting this dependability. Microcredit schemes lend money to people who normal banks won't lend to, because they're too poor. However, the default rate on loans from microcredit schemes can be lower than the default rate on loans from normal banks. I put forward hypotheses as to why this might be. These hypotheses suggest some ways to promote dependability in the context of e-services. Finally, I discuss whether e- services can be used by microcredit organizations.


Noble Reveal is a condominium project, developed by Noble Development, located at 36 Thanon Ekkamai, Khwaeng Phra Khanong Nuea, Khet Watthana, Krung Thep Maha Nakhon 10110. Noble Development is also the developer behind Noble Ambience Sarasin, Real BY Noble and Noble Reform. Construction of Noble Reveal was completed in 2011. Condominium comprises of a single building, having 27 floors and includes 273 units.


text:
United Nations Court Tells Japan to Stop Killing Whales
Published March 31, 2014 by Jennifer O'Connor. Last Updated July 29, 2015.
Whales in the Southern Ocean can swim in peace for now, after the International Court of Justice—the United Nations' judicial body—suspended Japan's annual massacre.
Despite worldwide condemnation, Japan has continued to slaughter whales in defiance of the International Whaling Commission moratorium on commercial whaling, which went into effect in 1986. Even though whale flesh is widely available in Japan, the thinly disguised commercial whaling operation has been defended as "scientific research." The court found that those claims were unfounded. People have been mesmerized watching the Sea Shepherd Conservation Society's actions to interrupt the hunt on the television show Whale Wars.
"In light of the fact the [research program] has been going on since 2005, and has involved the killing of about 3,600 minke whales, the scientific output to date appears limited," said U.N. Presiding Judge Peter Tomka.
But the fight isn't over yet. While Japan must suspend the carnage in the Southern Ocean, the court's ruling does not stop the country's second, smaller killing program in the northern Pacific. And both Norway and Iceland brazenly continue to kill minke whales.


Change Agent, Are You Searching For Justice, Wondering What It's All For?
As a change agent, I have committed my life to teaching and sharing the skills and strategies to navigate difficult situations and create truly inclusive, socially just organizations while maintaining the passion and energy critical to create meaningful, sustainable change.
But today, I am struggling to use these tools in my own life.
This roller coaster of extreme emotions from Alabama last week to the tax scam this week… from feeling so useful this week as I coached so many brilliant, talented people to waking up today wondering what it is all for….
And yet I know we will move through these challenging times as we have before. As I celebrate another birthday next week in my 6th decade, I remember all the challenging times in our country, our world and in my own life that seemed so desperately bleak and overwhelming.
And with hard work, persistent dedication, and deep love and faith in the world we envision, we made progress. Slowly, surely, over time, I believe there have been many shifts and changes that shore up the foundation to build the justice and liberation I know in my soul is possible.
Together, we CAN do this hard thing.
As Carrie Newcomer sings, "You can do this hard thing." Since 1st hearing her song a year ago after the disastrous national election, her words have been a comforting, motivating mantra for me.
And today I am repeating them over & over again to counter-balance all the thoughts of grief and rage and hopelessness that seep into my consciousness.
I will feel these feelings so they float by like clouds and don't stay stuck in my body and psyche, depleting my energy and passion once again… but today it feels harder than yesterday to find the inner resources to get up and keep going.
But I will – and we will – together.
Doing our part to bring in more light each day to those around us.
Fostering greater enlightenment with our families, our colleagues, our neighbors, our communities.
Living in our strength, utilizing our unique power to help move toward a world of true equity, justice, and liberation.
Mentoring and supporting others to step up into their power, live in their strength, and spread their light.
Of course, I'm talking about justice for all! Not just for the very wealthy and those with political power, but truly for each and every human being across race, class, disability status, sexuality, gender identity, religion and spirituality, ethnicity and culture, and every other "difference" that is used to privilege a very few at the expense and dehumanization of the vast majority of people on this earth. Bring the light into your heart, move from a grounding of love and faith and get up each day and recommit your full being to do these hard things.
I would love to have you join us so you can not only take better care of yourself (after all, you can't give from an empty cup) – but so that you can take what you'll learn and share it with others. Click here to start 2018 off strong by signing up for my newest course.


text:
using System;
#if (MF_FRAMEWORK_VERSION_V4_2 || MF_FRAMEWORK_VERSION_V4_3)

#else
using System.Collections.Generic;
using System.Linq;
using System.Text;
#endif

namespace LifxLib.Messages
{
    public class LifxSetDimAbsoluteCommand : LifxCommand
    {
        private UInt16 mDimLevel;
        private UInt32 mDurationMilliseconds;
        private const UInt16 PACKET_TYPE = 0x68;

        public LifxSetDimAbsoluteCommand(UInt16 dimLevel, UInt32 durationMilliseconds)
            : base(PACKET_TYPE, null)
        {
            mDimLevel = dimLevel;
            mDurationMilliseconds = durationMilliseconds;
        }

        #region ILifxMessage Members

        public override byte[] GetRawMessage()
        {
            byte[] bytes = new byte[6];

            Array.Copy(BitConverter.GetBytes(mDimLevel), bytes, 2);
            Array.Copy(BitConverter.GetBytes(mDurationMilliseconds), 0, bytes, 2, 4);

            return bytes;
        }

        #endregion
    }
}

redpajama_set_name: RedPajamaGithub


TIP! Make sure you're able to say what your home business is about in a short sentence. Cultivating the skill of being succinct when describing your business will impress possible new customers.
From articles to paid programs, news and magazines, there is a lot out there to learn about starting your own home business enterprise. Be certain you choose wisely. The following article will help you to understand the reality of what it really takes to run a home business enterprise.
Running a home business enterprise takes hard work and dedication. What will your niche be? Your niche can be anything, though it helps if you are familiar with the subject first. Research different businesses; don't risk everything without knowing what you're doing. Be certain to network whenever possible with other work from home business entrepreneurs.
TIP! Establish a difference between work and personal time by creating a set schedule of work hours. You could ignore business calls after a certain hour, for instance.
TIP! Make sure that your business mail goes to a PO Box address. Don't put your real address on the Internet under any circumstances.
One possible moneymaker is offering lessons for an area in which you are skilled. A lot of people prefer to take lessons privately instead of through a school with rigid schedules. Things like music, art, academic subjects and hobbies can be taught from behind your computer easily and effectively.
An office is necessary when you are based at home for work purposes. The space need not be large, but it should be attractive. Set up a room that inspires you to work hard and makes you feel comfortable and calm. Size is the last thing you should worry about.
TIP! Make use of affiliate marketing. You can trade links with other business websites, since this is a great way to build up your web traffic.
Get business cards to add to your credibility. There are some Internet sites where you can get business cards for free. Include all of your important information, such as your business name, your name, contact number, email and website address. Your cards should also provide online contact info, like your site URL and your email. The more ways the customers can contact you, the better the business.
TIP! Communicate appropriately with your home business customer base; share information without overwhelming them. Newsletters that are rich in content are a great way to feature discounts and specials.
Be brief when writing the objective of your business. The main idea is that you want to establish your company's goals and purpose for existing. You should be able to tell people what you are all about and why you stand out.
TIP! It is not hard to promote a business once you know what you are doing. One great option may be to set up a website to promote your home business.
Your home business website needs to have a catchy theme which will make it easy to remember. Avoid domain names that are long or hard to spell. Simple and catchy is always best when creating domain names.
TIP! Your exposure to the rest of the web has much to do with your search engine rank. Search engine optimization can be an excellent method for owners of home businesses to market their sites online and increase their audience size.
As you can see, launching a successful online business is possible if you are willing to work hard. Succeeding in a home-based business will take a lot of hard work on your part, and will depend on following the practices of good business sense.


text:
A long way to go for human rights in Asia
Thailand aims to lead Asean in improving protection, but our own record also falls short
Today is International Human Rights Day and also the 60th anniversary of the Universal Declaration of Human Rights (UNDHR). When Thailand signed the UNDHR in 1948, it became the boldest country in Asia by acceding to the universal norms that respect human rights. It was the first Asian country to do so. That was a remarkable undertaking, achieved at a time when Thailand, like other countries, had just emerged from the destruction of World War II. Thailand wanted to join the international community as soon as possible. After officially supporting the Japanese against the Allied forces, Thailand wanted to clean up its recent past by quickly embracing the United Nations and other related international activities. International organisations, including their universal values and shared norms, served as a shield for Thailand against the threat of communism.
Today, a glance at the country's human rights record might lead one to draw the conclusion that progress in this area is still very disappointing. Authorities concerned are still not willing to work for justice. Abuse and disappearances still continue. The well-known case of human rights lawyer, Somchai Neeraphaijit, still unaccounted for, not to mention other recent disappearances involving minorities in the remote provinces of northern and southern Thailand, are examples of this lack of progress.
It is sad that Thailand, which started out as a well-respected member of the international community, and one that supposedly placed a high value on human rights, has ended up this way. The political crisis of the past six months has added to the general perception around the world that Thailand has no rule of law. Time and again our society could easily have slipped into anarchy. Ordinary people are seen on TV being beaten up because of the colour of their T-shirt. It is thus imperative that the new government accord top priority to human rights protection and improve the country's tarnished international image.
It has been over a decade since the National Commission on Human Rights was set up to protect and promote human rights in Thailand. But the NCHR's performance has been dismal. The commission could have done a better job if had been a stronger and less dogmatic leadership team; one that was more focused on civil and political rights.
One of the problems has been the emphasis on collective rights, or community rights. Obviously these collective rights are pivotal, but to provide sufficient protection in this area is almost impossible under the current circumstances. New legislation and human rights education is urgently needed if such an approach is to continue, as it involves the rural masses. An increase in the violation of individual human rights, especially in southern Thailand, shows that protection of individual rights has failed. The state security apparatus continues to ignore the basic rights guaranteed by the Thai Constitution and international laws.
Thailand's human rights ideal should have served as a model for Asean countries to emulate. But the country's dismal record, as well as its poor political leadership over the years, has literally eaten into the country's reputation and creditability. This also helps to explain why Thailand's repeated call for a more liberal and comprehensive approach to human rights within Asean has failed miserably. Only Indonesia and the Philippines see eye to eye with the Thais.
As the current Asean chair, Thailand has been working hard to produce a good terms of reference for the establishment of an Asean human rights body by the end of next year. It is going to be tough because of the grouping's principle of consensus and the fact that the majority of Asean members still prefer a docile mechanism that pays just enough lip service to the protection of human rights and not much more. They say that human rights protection in Asean should be an evolutionary process. This is the reason it has taken the grouping more than a decade to even establish the idea of setting up a human rights mechanism.
How long will it take before there is real protection of human rights in Asean?
If Thailand really wants to uphold human rights values and standards, our government officials must change their mindset now. It is silly to continue to cite how many international bills of rights we have ratified, because these do not help improve the situation on the ground at all. The efficient enforcement of constitutional rights has been lacking.
For instance, Thailand has courageously ratified the UN Convention Against Torture, but torture practices, especially during detention periods, continue unabated. Therefore, it is futile to talk about human rights protection while these practices go on.
In the field of human rights, action speaks louder than words, and in the case of Thailand, we are hearing a loudspeaker.
This entry was posted on Wednesday, December 10th, 2008 at 2:21 pm and is filed under Uncategorized.	You can follow any responses to this entry through the RSS 2.0 feed. You can leave a response, or trackback from your own site.


CASology: Week 266: Weekly Winners!
PJ's sweet card is perfectly balanced with its subtle colors, matching border, and plenty of white space.
1. Jen's Choice: Conil's card cracked me up and sums up perfectly my feelings about missing cake!
2. Hannelie's choice : A pop of color and pattern on gorgeous white space! Congratulations Valentyna!
3. Rachel's Choice: Asa's card is stunning with bold black and white with a touch of color!
5. Jacquie's Choice: Invisible Pink's no line watercoloring is bright, fun & fabulous!
6. Anna's choice: A beautiful take on the cue. The monocromatic color palette makes it perfectly CAS.
7. Janet's choice: Tenia's card pops with colour, dimension and fun.
8. Judy's Choice: I love how Sundaystamper's cupcakes are off the edge paired with a bold sentiment!
9. Melissa's Choice: Maryam's design has lots of white space & her main image is framed with interest!
10. Kymona's Choice: I love the simplicity of Jane's card. The colors are pretty. Congratulations!!
Be sure to stop back in an hour for challenge #267!
Thank you Kymona for picking my card, absolutely delighted! Congratulations to PJ, stunning CAS card, and all the other picks.
Thank you so much for this awesome surprise of being the "winner"! I am completely blown away. I sure do appreciate this.. You have made this gal super happy♥ Congrats to all the amazing honorable mention winners all your work is fabulous.
Thank you so much Joyce for showcasing my card ! You made my day ! Congratulations to the winner and all the other honorable mention !


• Stapler fastens up to 20 sheets, creates a yawning fissure for tacking flexibility and features an antimicrobial finish.
• The performance-driven mechanism has an inner rail for long-term stapling integrity.
• Positive locking latch ensures the confidence and control of a secure stapling position.
• All-metal, die-cast base offers durability and stability. Design also features a contoured cap and padded rubber bottom for comfortable, convenient handheld stapling.
• High-quality, durable desktop stapler belongs in every office worker's set of desktop tools.
• Contemporary, rounded metal stapler fastens up to 20 sheets and The upper portion retracts like a viper for tacking flexibility so you can easily tack up flyers and posters.
• Enhanced for stability, the 747 Business Stapler includes an inner rail that prevents staples from jamming and a positive locking latch to increase precision positioning of each staple.
• All-metal, die-cast base offers durability and stability.
• Design also features a contoured cap and padded rubber bottom for comfortable, convenient handheld stapling.
• Use chisel point staples for efficient and dependable performance in stapling machines that use standard staples.
• Staples fit all standard staplers. Crown measures 1/2" wide.
• The leg length is 1/4". Each staple fastens up to 30 sheets and is made of galvanized iron wire.
• Staples come in strips of 210.
• Stapler offers ergonomic stand-up design that takes up less desk space than conventional desktop staplers.
• Rotating anvil allows stapling and pinning.
• Design also features comfort hand grips and all-metal working parts.
• Stapler fastens up to 20 sheets of 20 lb. paper using standard staples.
• Staple capacity is 210 staples.


Multiple-commissioner Josh from Saxapahaw, NC is back with another goodie, James Cameron's take on the Aliens franchise. One of the all time great sci-fi action movies, it supplies nearly non stop action with a sturdy heroine to build around, lots of colorful characters to die, implacable, terrifying antagonists, and impressive effect and design work. Plus, we eulogize the late, great, Bill "Tons of Peace" Paxton. Thanks again, Josh, this was a lot of fun!
In the final episode of season 3, I cover the use of the Espheni weapon, the status of the war, the attempted relocation to Brazil, Weaver's Achy Breaky Heart and the return of Anne & the baby.
Jim talks about the latest episode of Falling Skies. Are they really fooling anyone with this mole thing? How well did Crazy Lee's plotline work? What's the worst thing about tv battles? Camera and mic upgrades. Do they help?
Jim talks about the 2-hour premiere of season 3 of TNT's apocalyptic alien drama Falling Skies. This is my first piece of video content so the visual and audio quality is terrible, as is the presentation. It will get better, I promise. Hopefully you're here for the insightful television commentary anyway, not the razzle dazzle.