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En este artículo se analizarán cuatro canciones de Parabellum (“Madre muerte”, “Bruja maldita”, “666 engendro” y “Guerra, monopolio, sexo”) a partir de un enfoque basado en tres temas: la relación entre Parabellum y la poesía moderna, su crítica a la sociedad y su visión respecto de la violencia. Los postulados de Hugo Friedrich son un referente teórico importante, ya que hay relaciones entre la poética propuesta por los ultrametaleros de Parabellum y el estilo de la lírica moderna. Además, serán pertinentes algunas citas sobre Baudelaire y Nietzsche, esto con la intención de analizar las particularidades ideológicas de Parabellum. Esta banda representa una época de excesiva violencia, pero también al arte como un eslabón que posibilita afirmar la vida, en esta ocasión, por medio de la figura del ultrametalero. Parabellum, a partir de la belleza decadente, es una banda que refleja un posible escape de la realidad vacua, pero también una liberación.

The success of chimeric antigen receptor (CAR)–mediated immunotherapy in acute lymphoblastic leukemia (ALL) highlights the potential of T-cell therapies with directed cytotoxicity against specific tumor antigens. The efficacy of CAR T-cell therapy depends on the engraftment and persistence of T cells following adoptive transfer. Most protocols for T-cell engineering routinely expand T cells ex vivo for 9 to 14 days. Because the potential for engraftment and persistence is related to the state of T-cell differentiation, we hypothesized that reducing the duration of ex vivo culture would limit differentiation and enhance the efficacy of CAR T-cell therapy. We demonstrated that T cells with a CAR-targeting CD19 (CART19) exhibited less differentiation and enhanced effector function in vitro when harvested from cultures at earlier (day 3 or 5) compared with later (day 9) timepoints. We then compared the therapeutic potential of early versus late harvested CART19 in a murine xenograft model of ALL and showed that the antileukemic activity inversely correlated with ex vivo culture time: day 3 harvested cells showed robust tumor control despite using a 6-fold lower dose of CART19, whereas day 9 cells failed to control leukemia at limited cell doses. We also demonstrated the feasibility of an abbreviated culture in a large-scale current good manufacturing practice–compliant process. Limiting the interval between T-cell isolation and CAR treatment is critical for patients with rapidly progressing disease. Generating CAR T cells in less time also improves potency, which is central to the effectiveness of these therapies. Cancer Immunol Res; 6(9); 1100–9. ©2018 AACR.

Table S1 and Figures S1 -S8

The trucking industry is the backbone of domestic supply chains. In the context of the COVID-19 pandemic, road transportation has been essential to guarantee the supply of basic goods to confined urban areas. However, the connectivity of the trucking network can also act as an efficient virus spreader. This paper applies network science to uncover the characteristics of the trucking network in one major Latin American country −Colombia− and provides evidence on freight networks’ ability to spread contagious diseases spatially. Network metrics, official COVID-19 records at the municipality level, and a zero-inflated negative binomial model are used to test the association between network topology and confirmed COVID-19 cases. Results suggest that: (i) the number of COVID-19 cases in a municipality is linked to its level and type of network centrality; and (ii) being a port-city and a primary economic hub in the trucking network is associated with a higher probability of contracting earlier a pandemic. Based on these results, a risk-based approach is proposed to help policymakers implement containment measures.

Road congestion and air pollution are key challenges for quality of life in urban settings. This research leverages highly disaggregated crowdsourced data from Latin America to study the effect of road congestion on levels of carbon monoxide, nitrogen dioxide, and particulate matter in four of the most congested cities in developing countries: Bogota, Buenos Aires, Mexico City, and Santiago. Based on a panel data econometric approach with over 4.4 billion records from Waze and hourly data from 54 air monitoring stations for 2019, our two-stage least square model shows a cumulative increase of 0.6% in response to a 1% of road congestion on the three air pollutants. Moreover, we find a nonlinear relationship between road congestion and air quality and estimate the threshold above which the effect decays. This study provides evidence that supports public policies designed to make urban mobility more sustainable by implementing measures to reduce road congestion in developing contexts.

The process of a virus spread is inherently spatial. Even though Latin America became the epicenter of the COVID-19 pandemic in May 2020, there is still little evidence of the relationship between urban mobility and virus propagation in the region. This paper combines network analysis of mobility patterns in public transportation with a spatial error correction model for Santiago de Chile. Results indicate that a 10% higher number of daily public transportation trips received by an administrative unit in the city was associated with a 1.3% higher number of confirmed COVID-19 cases per 100,000 inhabitants. Following these findings, we propose an empirical method to identify and classify neighborhoods according to the level and type of risk for COVID-19-like disease propagation, helping policymakers manage mobility during the initial stages of an epidemic outbreak.

Resumen Introduccion La pandemia de COVID-19 ha ocasionado cerca de 25 millones de casos en el mundo Se ha descrito que los pacientes asintomaticos pueden ser fuentes de transmision Sin embargo, es dificil detectarlos y no es claro su papel en la dinamica de transmision del virus, lo que obstaculiza la implementacion de estrategias para la prevencion Objetivo Describir el comportamiento de la infeccion asintomatica por SARS-CoV-2 en una cohorte de trabajadores del Aeropuerto Internacional El Dorado "Luis Carlos Galan Sarmiento" de Bogota, Colombia Materiales y metodos Se diseno una cohorte prospectiva de trabajadores del Aeropuerto El Dorado El seguimiento se inicio en junio de 2020 con una encuesta a cada trabajador para caracterizar sus condiciones de salud y trabajo Cada 21 dias se tomo una muestra de hisopado nasofaringeo para detectar la presencia del SARS-CoV-2 mediante reaccion en cadena de la polimerasa con transcriptasa inversa (RT-PCR) Se analizo el comportamiento del umbral del ciclo (cycle threshold) de los genes ORFlab y N segun el dia de seguimiento Resultados En los primeros tres seguimientos de la cohorte se encontro una incidencia de la infeccion por SARS-CoV-2 del 16,51 % La proporcion de contactos positivos fue del 14,08 % La mediana del umbral del ciclo fue de 33,53 Conclusion Se determinaron las caracteristicas de la infeccion asintomatica por el SARS-CoV-2 en una cohorte de trabajadores La deteccion de infectados asintomaticos sigue siendo un reto para los sistemas de vigilancia epidemiologica Introduction: The 2019 coronavirus pandemic (COVID-19) has caused around 25 million cases worldwide Asymptomatic patients have been described as potential sources of transmission However, there are difficulties to detect them and to establish their role in the dynamics of virus transmission, which hinders the implementation of prevention strategies Objective: To describe the behavior of asymptomatic SARS-CoV-2 virus infection in a cohort of workers at the El Dorado "Luis Carlos Galan Sarmiento" International Airport in Bogota, Colombia Materials and methods: A prospective cohort of 212 workers from the El Dorado airport was designed The follow-up began in June, 2020 A survey was used to characterize health and work conditions Every 21 day, a nasopharyngeal swab was taken to identify the presence of SARS-CoV-2 using RT-PCR We analyzed the behavior of the cycle threshold (ORFlab and N genes) according to the day of follow-up Results: In the first three follow-ups of the cohort, we found an incidence of SARS-CoV-2 infection of 16 51% The proportion of positive contacts was 14 08% The median threshold for cycle threshold was 33 53 Conclusion: We characterized the asymptomatic SARS-CoV-2 infection in a cohort of workers The identification of asymptomatic infected persons continues to be a challenge for epidemiological surveillance systems

En las últimas décadas se ha intensificado la búsqueda de materia prima proveniente de recursosrenovables para la producción de polímeros, elastómeros, resinas, espumas, etc., que disminuyan el uso industrial de compuestos petroquímicos. En este trabajo se usó el aceite extraído de la semilla de higuerilla (Ricinus communis) por su contenido de ácido ricinoleico (92,3 %), por su fácil producción y por no ser comestible; la síntesis del poliuretano adhesivo se hizo a través de reacciones de transesterificación y polimerización; con él se pegaron láminas de Guaduaangustifolia Kunth y se determinaron las características mecánicas de la estructura; se obtuvo que la resistencia a la compresión paralela a la fibra es de 43±3 (MPa), y la cortante paralela a la fibra es de 5,9±0,9 (MPa). La determinación de la relación molar adecuada y el seguimiento de la reacción durante la síntesis se hicieron con espectroscopía infrarroja con transformada de Fourier y la estructura del producto final se analizó con difracción de rayos X. A través de calorimetría diferencial de barrido se obtuvo la temperatura de transición vítrea del producto de -26.4 °C y se usó la técnica de relajación térmica para medir su calor específico, mientras que su difusividad térmica y el coeficiente de dilatación térmica se estimaron con la técnica fotoacústica.

As transit ridership continues to fall in many cities across the globe, key policy debates continue around whether Uber and other ride-hailing services are contributing to this trend. This research explores the effects of the introduction of ride-hailing to Colombian cities on public transportation ridership using Ubers timeline as case study. We test the hypothesis that ride-hailing may either substitute or compete with public transit, particularly in cities with large transit service gaps in coverage or quality. Our analysis builds on historic transit ridership data from national authorities and uses a staggered difference-in-difference model that accounts for fixed effects, seasonality, socioeconomic controls, and the presence of integrated transport systems. Despite large reductions in transit ridership in most cities, our results suggest that Uber is not statistically associated with the observed drop in ridership. Moreover, consistent with evidence from previous research, public transit reforms implemented between 2007 and 2015 throughout Colombian cities appear to have contributed substantially to the declines in transit ridership observed across the country. Findings in this paper inform policy-targeted insights and contribute to current debates of the links between ride-hailing and public transit in cities in Latin America.

Transport Network Companies (TNCs) have become a popular alternative for mobility due to their ability to provide on-demand flexible mobility services. By offering smartphone-based, ride-hailing services capable of satisfying specific travel needs, these modes have transformed urban mobility worldwide. However, to-date, few studies have examined the impacts in the Latin American context. This analysis is a critical first step in developing policies to promote efficient and sustainable transport systems in the Latin-American region. This research examines the factors affecting the adoption of on-demand ride services in Medellín, Colombia. It also explores whether these are substituting or competing with public transit. First, it provides a descriptive analysis in which we relate the usage of platform-based services with neighborhood characteristics, socioeconomic information of individuals and families, and trip-level details. Next, factors contributing to the election of platform-based services modeled using discrete choice models. The results show that wealthy and highly educated families with low vehicle availability are more likely to use TNCs compared to other groups in Medellín. Evidence also points at gender effects, with being female significantly increasing the probability of using a TNC service. Finally, we observe both transit complementary and substitution patterns of use, depending on the context and by whom the service is requested.

Este documento presenta el primer análisis exhaustivo para la región acerca las características y costos de la congestión urbana en América Latina y el Caribe, con resultados para las áreas metropolitanas de Bogotá (Colombia), Buenos Aires (Argentina), Ciudad de México (México), Lima (Perú), Montevideo (Uruguay), Río de Janeiro (Brasil), San Salvador (El Salvador), Santiago (Chile), Santo Domingo (República Dominicana) y Sao Paulo (Brasil). Utiliza big data y ciencia de datos para elucidar la dinámica de la congestión en cada ciudad y sus costos directos e indirectos para la sociedad. Con el fin de mitigar estos impactos, el documento reúne cinco grupos de medidas de política pública: (i) instrumentos de gestión de tráfico; (ii) políticas que restringen el uso del vehículo particular; (iii) políticas que promueven el uso del transporte público, el transporte activo y el transporte compartido; (iv) planificación integrada de la movilidad y el uso del suelo; y (v) políticas para la gestión de la logística urbana. Concluye con recomendaciones sobre las medidas más adecuadas y la secuenciación de las mismas para reducir la congestión en las grandes ciudades y las megaciudades de la región.

BackgroundAdoptive cell therapy with chimeric antigen receptor T cells (CAR-T) has become a standard treatment for patients with certain aggressive B cell malignancies and holds promise to improve the care of patients suffering from numerous other cancers in the future. However, the high manufacturing cost of CAR-T cell therapies poses a major barrier to their broader clinical application. Among the key cost drivers of CAR-T production are single-use reagents for T cell activation and clinical-grade viral vector. The presence of variable amounts of contaminating monocytes in the starting material poses an additional challenge to CAR-T manufacturing, since they can impede T cell stimulation and transduction, resulting in manufacturing failure.MethodsWe created K562-based artificial antigen-presenting cells (aAPC) with genetically encoded T cell stimulation and costimulation that represent an inexhaustible source for T cell activation. We additionally disrupted endogenous expression of the low-density lipoprotein receptor (LDLR) on these aAPC (aAPC-ΔLDLR) using CRISPR-Cas9 gene editing nucleases to prevent inadvertent lentiviral transduction and avoid the sink effect on viral vector during transduction. Using various T cell sources, we produced CD19-directed CAR-T cells via aAPC-ΔLDLR-based activation and tested their in vitro and in vivo antitumor potency against B cell malignancies.ResultsWe found that lack of LDLR expression on our aAPC-ΔLDLR conferred resistance to lentiviral transduction during CAR-T production. Using aAPC-ΔLDLR, we achieved efficient expansion of CAR-T cells even from unpurified starting material like peripheral blood mononuclear cells or unmanipulated leukapheresis product, containing substantial proportions of monocytes. CD19-directed CAR-T cells that we produced via aAPC-ΔLDLR-based expansion demonstrated potent antitumor responses in preclinical models of acute lymphoblastic leukemia and B-cell lymphoma.ConclusionsOur aAPC-ΔLDLR represent an attractive approach for manufacturing of lentivirally transduced T cells that may be simpler and more cost efficient than currently available methods.

In Public Libraries, the memory of the women-in particular diverse women-is a vital element to the collective construction of the localidentity. There for, women voices are a very important historical element in the time of the change, especially in this moment when the country is building a peace process. That is why, orality is the symbol of the liberty, equality and autonomy; thus, the library is a meeting place and a rights scenario, because the people to the fourth location of Bogotá (San Cristóbal), need to build speeches from the women and diverse women -victims or not of the violence-, and to use the knowledge in a social perspective of transformation. This is the point from The Public Library La Victoria and other social actors, (for instance District Women's Secretariat and Collective Archipiélago), are together in a work plan, trying to change the reality of the women and the diverse women in the territory; thorough reading, writing, culture, exchange of knowledge, debates and other participation mechanisms from the library as such. This experience show the work that library had done since 2015, in order to get that women and thediverse women empower themselves, using their voice to build local memory.

Understanding the temporal and spatial dynamics of traffic accidents are a key determinant in their mitigation. This article leverages big data and a Poisson model with fixed effects to understand the causality of traffic congestion on road accidents in ten cities in Latin America: Bogota, Buenos Aires, Lima, Mexico City, Montevideo, Rio de Janeiro, San Salvador, Santiago, Santo Domingo, and Sao Paulo. Analyzing over 10 billion observations in 2019, results show a positive non-linear causality of congestion on the number of accidents. Overall, the results suggest that a 10% reduction in traffic delay would reduce accidents by 3.4%, equivalent to over 72 thousand traffic accidents. Sao Paulo and Mexico City would be particularly benefited, with reductions of 5.4% and 4.7%, respectively. The results of this paper aim to support policymakers in emerging economies in implementing measures to reduce congestion and, with it, the related direct and indirect costs borne by societies.

The chimeric antigen receptor (CAR) technology started out as a tool to understand lymphocyte biology but rapidly developed into a T cell therapeutic agent for the treatment of cancers. Here, we describe the technological advances in the field of CARs and highlight critical components of its success. Additionally, we describe how various laboratories have worked toward developing new, safer, and more potent CARs for cancer.

The success of chimeric antigen receptor (CAR)–mediated immunotherapy in acute lymphoblastic leukemia (ALL) highlights the potential of T-cell therapies with directed cytotoxicity against specific tumor antigens. The efficacy of CAR T-cell therapy depends on the engraftment and persistence of T cells following adoptive transfer. Most protocols for T-cell engineering routinely expand T cells ex vivo for 9 to 14 days. Because the potential for engraftment and persistence is related to the state of T-cell differentiation, we hypothesized that reducing the duration of ex vivo culture would limit differentiation and enhance the efficacy of CAR T-cell therapy. We demonstrated that T cells with a CAR-targeting CD19 (CART19) exhibited less differentiation and enhanced effector function in vitro when harvested from cultures at earlier (day 3 or 5) compared with later (day 9) timepoints. We then compared the therapeutic potential of early versus late harvested CART19 in a murine xenograft model of ALL and showed that the antileukemic activity inversely correlated with ex vivo culture time: day 3 harvested cells showed robust tumor control despite using a 6-fold lower dose of CART19, whereas day 9 cells failed to control leukemia at limited cell doses. We also demonstrated the feasibility of an abbreviated culture in a large-scale current good manufacturing practice–compliant process. Limiting the interval between T-cell isolation and CAR treatment is critical for patients with rapidly progressing disease. Generating CAR T cells in less time also improves potency, which is central to the effectiveness of these therapies. Cancer Immunol Res; 6(9); 1100–9. ©2018 AACR.

espanolCon el fin de evaluar el efecto de la educacion y de la experiencia sobre los ingresos laborales en Colombia, se estimo un modelo minceriano cuadratico por Minimos Cuadrados en Dos Etapas, con variable instrumental para controlar el problema de seleccion, basado en la informacion de la Gran Encuesta Integrada de Hogares (GEIH) entre los anos 2009 y 2015. Para el caso, se realiza una revision bibliografica y de hechos estilizados, con el fin de proporcionar un acercamiento a la teoria referente a la hipotesis planteada. Asi mismo, se realiza un conjunto de ejercicios econometricos, en aras de verificar empiricamente la relacion directa entre educacion e ingreso laboral, en particular, el retorno a la educacion en el sector urbano, obtenido a partir de la semielasticidad, que los premios educativos reflejan sobre el logaritmo de los salarios. Se encuentra que, culminar los estudios secundarios y universitarios aumenta los salarios recibidos; que ser hombre, tiene tambien un efecto positivo sobre los ingresos; pero que, el premio que reciben por culminar los estudios, es mayor para las mujeres que para los hombres. EnglishIn order to evaluate the effect of education and experience on labor income in Colombia, Two-Stage Least Squares, estimated a quadratic mincerian model, with an instrumental variable to control the selection bias, with base on information from the Great Integrated Household Survey (GIHS) between 2009 and 2015. A bibliographic review and stylized facts were carried out, to provide an approach to the theory regarding the hypothesis raised. In addition, econometric exercises were executed, to verify empirically the direct relation between education and labor income, in particular, the return of education in the urban sector, obtained from the semi elasticity that the educational premiums reflect on the logarithm of wages. It is found that culminating secondary and university studies increases the salaries received, that being a man also has a positive effect on income, but that the premium they receive by completing the studies is higher for women than for men.

Tolerance to self-antigens prevents the elimination of cancer by the immune system1,2. We used synthetic chimeric antigen receptors (CARs) to overcome immunological tolerance and mediate tumor rejection in patients with chronic lymphocytic leukemia (CLL). Remission was induced in a subset of subjects, but most did not respond. Comprehensive assessment of patient-derived CAR T cells to identify mechanisms of therapeutic success and failure has not been explored. We performed genomic, phenotypic and functional evaluations to identify determinants of response. Transcriptomic profiling revealed that CAR T cells from complete-responding patients with CLL were enriched in memory-related genes, including IL-6/STAT3 signatures, whereas T cells from nonresponders upregulated programs involved in effector differentiation, glycolysis, exhaustion and apoptosis. Sustained remission was associated with an elevated frequency of CD27+CD45RO–CD8+ T cells before CAR T cell generation, and these lymphocytes possessed memory-like characteristics. Highly functional CAR T cells from patients produced STAT3-related cytokines, and serum IL-6 correlated with CAR T cell expansion. IL-6/STAT3 blockade diminished CAR T cell proliferation. Furthermore, a mechanistically relevant population of CD27+PD-1–CD8+ CAR T cells expressing high levels of the IL-6 receptor predicts therapeutic response and is responsible for tumor control. These findings uncover new features of CAR T cell biology and underscore the potential of using pretreatment biomarkers of response to advance immunotherapies. An IL-6/STAT3 signature and memory CD8 T cell subset in preinfusion chimeric antigen receptor–expressing T cells associate with response in patients with high-risk chronic lymphocytic leukemia.

The recent clinical success of CD19-directed chimeric antigen receptor (CAR) T cell therapy in chronic and acute leukemia has led to increased interest in broadening this technology to other hematological malignancies and solid tumors. Now, advances are being made using CAR T cell technology to target myeloma antigens such as B cell maturation antigen (BCMA), CD138, and kappa-light chain as well as CD19 on putative myeloma stem cells. To date, only a limited number of multiple myeloma patients have received CAR T cell therapy but preliminary results have been encouraging. In this review, we summarize the recently reported results of clinical trials conducted utilizing CAR T cell therapy in multiple myeloma (MM).

We examined the formation, participation, and functional specialization of virus-reactive Foxp3+ regulatory T cells (Tregs) in a mouse model of influenza virus infection. “Natural” Tregs generated intrathymically, based on interactions with a self-peptide, proliferated in response to a homologous viral Ag in the lungs and, to a lesser extent, in the lung-draining mediastinal lymph nodes (medLNs) of virus-infected mice. In contrast, conventional CD4+ T cells with identical TCR specificity underwent little or no conversion to become “adaptive” Tregs. The virus-reactive Tregs in the medLNs and the lungs of infected mice upregulated a variety of molecules associated with Treg activation, as well as acquired expression of molecules (T-bet, Blimp-1, and IL-10) that confer functional specialization to Tregs. Notably, however, the phenotypes of the T-bet+ Tregs obtained from these sites were distinct, because Tregs isolated from the lungs expressed significantly higher levels of T-bet, Blimp-1, and IL-10 than did Tregs from the medLNs. Adoptive transfer of Ag-reactive Tregs led to decreased proliferation of antiviral CD4+ and CD8+ effector T cells in the lungs of infected hosts, whereas depletion of Tregs had a reciprocal effect. These studies demonstrate that thymically generated Tregs can become activated by a pathogen-derived peptide and acquire discrete T-bet+ Treg phenotypes while participating in and modulating an antiviral immune response.

En cuanto al efecto de la geografia en el comportamiento socialmente violento, este estudio introduce la idea de que la fragmentacion geografica esta asociada con mas terrorismo, dada su cercana relacion con el role del gobierno y las condiciones socioeconomicas de las personas. Nosotros consideramos un panel de 128 paises entre 1971 y 2005 usando metodologias para datos de conteo. Esto nos permite proveer evidencia robusta de un efecto consistente, incluso cuando diferentes controles son incluidos. Nuestra estimacion de referencia indica que un pais 1% mas geograficamente fragmentado es asociado con un incremento de 1.38 en el promedio de numero de ataques terroristas.

The objective of this research is piloting and evaluating the introduction of teaching innovations for better learning in a general microeconomics course for undergraduate students. The assessment is based on the Self Determination Theory and student-centered methodologies (SDT)(Deci and Ryan, 1985; Deci et al., 1991a), which remark the individual motivation as crucial for learning. The piloting of the instrument was implemented with a sample of 323 students grouped into 14 classes, giving evidence that the most relevant course problem is that students do not feel that what they are learning is really useful in their specific professions, despite the fact that they agree with the importance to know about such topics. This piloting allowed us to validate the used instrument which implements two psychometric scales: Knowledge transfer and self-determination scales. The impact evaluation was implemented for a sample of 204 students, distributed in treated and control groups. The assignment of students to treatment is random. Results provide evidence that the intervention significantly improves the self-determined motivation as well as the academic performance of students, although in a modest magnitude.

Autologous T cells modified to recognize novel antigen targets are a novel form of therapy for cancer. We review the various potential forms of observed and hypothetical toxicities associated with genetically modified T cells. Despite the focus on toxicities in this review, re-directed T cells represent a powerful and highly effective form of anti-cancer therapy; we remain optimistic that the common toxicities will become routinely manageable and that some theoretical toxicity will be exceedingly rare, if ever observed.

NF-κB is pivotal for transactivation of cell-cycle regulatory, cytokine, and adhesion molecule genes and is dysregulated in many cancers, neurodegenerative disorders, and inflammatory diseases. Proteins with pyrin and/or caspase recruitment domains have roles in apoptosis, innate immunity, and inflammation. Many pyrin domain (PYD) proteins modulate NF-κB activity as well as participate in assembling both the perinuclear “apoptotic speck” and the pro-IL1β/IL-18-converting inflammasome complex. “Pyrin-only” proteins (POP) are attractive as negative regulators of PYD-mediated functions and one such protein, POP1, has been reported. We report the identification and initial characterization of a second POP. POP2 is a 294 nt single exon gene located on human chromosome 3 encoding a 97-aa protein with sequence and predicted structural similarity to other PYDs. Highly similar to PYDs in CATERPILLER (CLR, NLR, NALP) family proteins, POP2 is less like the prototypic pyrin and ASC PYDs. POP2 is expressed principally in peripheral blood leukocytes and displays both cytoplasmic and nuclear expression patterns in transfected cells. TNF-α-stimulated and p65 (RelA)-induced NF-κB-dependent gene transcription is inhibited by POP2 in vitro by a mechanism involving changes in NF-κB nuclear import or distribution. While colocalizing with ASC in perinuclear specks, POP2 also inhibits the formation of specks by the CLR protein CIAS1/NALP3. Together, these observations demonstrate that POP2 is a negative regulator of NF-κB activity that may influence the assembly of PYD-dependent complexes.

137 Background: The adoptive transfer of autologous T cells genetically modified to express a CD19-specific, 4-1BB/CD3ζ-signaling CAR (CTL019) has shown remarkable activity and induces long-term remissions in a subset of patients with relapsed/refractory chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). In ALL, CTL019 induces a complete response (CR) in over 90% of patients while in CLL 25% of patients obtain a CR. It is not fully understood why only certain patients respond to therapy. Methods: We employed next generation sequencing of RNA (RNAseq) to identify predictive indicators of response to CTL019. We performed RNAseq on leukapheresis and manufactured product T cells prior to re-infusion from 35 CLL and 7 pediatric ALL patients with heavily pre-treated and high-risk disease. To characterize potency, we performed RNAseq on the infusion product after stimulation with the CAR. Results: We find that durable remission in CLL is associated with gene expression signatures of early memory and T-effector cells, while T cells from non-responding patients are enriched in signatures of T-regulatory cells, terminal differentiation, and exhaustion. In following the results from CLL, we find that pediatric ALL manufactured T cells are significantly enriched for an early memory, naïve T cell state and all achieved a CR. In parallel in vitro experiments, stimulation of the infusion product further demonstrated that CTL019 cells from CRs have an increased capacity for activation upon stimulation. We tested if we could extend these observations to identify a phenotype of T cells that is predictive of response prior to CTL019 manufacturing and find that the signatures predictive of response at the pre-infusion stage are also observed at the earlier leukapheresis time point. Conclusions: These findings suggest that intrinsic T cell fitness dictates response to CAR T cells. These gene expression signatures, along with additional immunological biomarkers, may be used to identify which patients are most likely to respond to adoptive transfer strategies and suggest manufacturing modifications that might potentiate the generation of maximally efficacious infusion products. Clinical trial information: NCT01029366, NCT01747486, NCT01626495.

We have previously shown that AIDS-associated lymphomas and lymphoma cell lines contain mitochondrial genome concatemers not present in normal T-lymphocytes. Since cellular homeostasis and energy production rely heavily on mitochondrial DNA (mtDNA) stability, mutations in the mtDNA have long been linked to the development of various types of cancers. In most of the cases, however, neoplastically transformed cells harbor non-mutated mtDNA. Herein, we propose an alternative mechanism that shows how the formation of mitochondrial genome concatemers may promote oncogenic transformation of normal lymphoid progenitor cells when no mtDNA mutations or chromosomal aberrations are present. We detected high reactive oxygen species (ROS) levels in the lymphoma samples tested despite no identification of putative mutations in the coding mtDNA. We propose that the formation of atypical mtDNA configurations (i.e. dimers and concatemers) interferes with normal mitochondrial function. Unstable mitochondria lead to abnormal assembly and dysfunction of the oxidative phosphorylation (OXPHOS) complexes, eventually leading to oxidative stress from elevated production of intracellular ROS. ROS have been reported to activate transcription factors associated with cellular proliferation and apoptosis inhibition. Therefore, we hypothesize that formation of mitochondrial genome concatemers can augment endogenous ROS levels capable of promoting oncogenic transformation of normal lymphoid progenitor cells.

The adoptive transfer of autologous T cells genetically modified to express a CD19-specific, 4-1BB/CD3z-signaling CAR (CTL019) has shown remarkable activity and induce long-term remissions in a subset of patients with relapsed/refractory chronic lymphocytic leukemia (CLL). To date, little is known about predictive indicators of efficacy. This study was designed to evaluate biomarkers of clinical response to CTL019 in CLL. We studied forty-one patients with advanced, heavily pre-treated and high-risk CLL who received at least one dose of CTL019 cells. We show that in vivo expansion and persistence are key quality attributes of CTL019 cells in CLL patients who have complete responses to therapy; in 2 patients responses are sustained beyond five years and accompanied by the persistence of functional CTL019 cells. Furthermore, durable remissions were associated with transcriptomic signatures of early memory T cells, while T cells from non-responding patients were enriched in genes belonging to known pathways of terminal differentiation and exhaustion. Polychromatic flow cytometry also demonstrated a significantly higher level of T cell exhaustion markers on the infused CAR T cells and reduced CD27 expression in non-responding patients. Accordingly, the combined assessment of PD1 and CD27 expression on CD8+ CTL019 cells in the infusion product accurately predicted response to treatment. Restimulation of the infusion product through the CAR further demonstrated that CTL019 cells from complete responders secreted significantly higher levels of several cytokines, including CCL20, IL-21, IL-22, IL-17, and IL-6, suggesting that the STAT3 signaling pathway may play a role in potentiating the enhanced potency of CTL019 cells. To identify a phenotype of T cells that is predictive of response prior to CTL019 manufacturing, we initially retrospectively evaluated the proportions of naïve, stem cell memory, central memory, effector memory and effector cells at the time of leukapheresis and observed either marginally significant or no significant correlations with clinical outcome. A systematic, unbiased analysis of the same biomarker panel revealed that the frequency of CD27+CD45RO- cells in the CD8+ T cell population correlated significantly with complete and durable responses to this therapy. Analysis of the infusion products using the same flow cytometric panel showed that most (>95%) of T cells expressed CD45RO at the end of the manufacturing run; CD27 expression frequencies, however, were maintained at the same level as in the leukapheresis. Together, these findings suggest that intrinsic T cell fitness dictates both response and resistance to highly active engineered CAR T cells. Thus, enrichment of T cells with optimal differentiation potential and proliferative capacity by timing of collection or culture modification might potentiate the generation of maximally efficacious infusion products. These data and additional immunological biomarkers may be used to identify which patients are most likely to respond to adoptive transfer strategies, leading to an enhanced personalized approach to cellular therapy. Disclosures Fraietta: Novartis: Patents & Royalties: Novartis, Research Funding. Lacey:Novartis: Research Funding. Wilcox:Novartis: Research Funding. Bedoya:Novartis: Research Funding. Chen:Novartis: Research Funding. Orlando:Novartis: Employment. Brogdon:Novartis: Employment. Hwang:Novartis: Research Funding. Frey:Novartis: Research Funding; Amgen: Consultancy. Pequignot:Novartis: Research Funding. Ambrose:Novartis: Research Funding. Levine:Novartis: Patents & Royalties, Research Funding; GE Healthcare Bio-Sciences: Consultancy. Bitter:Novartis: Employment. Porter:Genentech: Employment; Novartis: Patents & Royalties, Research Funding. Xu:Novartis: Research Funding. June:Immune Design: Consultancy, Equity Ownership; University of Pennsylvania: Patents & Royalties; Celldex: Consultancy, Equity Ownership; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Tmunity: Equity Ownership, Other: Founder, stockholder ; Pfizer: Honoraria; Johnson & Johnson: Research Funding. Melenhorst:Novartis: Patents & Royalties: Novartis, Research Funding.

Introduction For chimeric antigen receptor T cell-based (CAR-T) and engineered T cell receptor (TCR) immunotherapies, T cell expansion methods and phenotype/s of transplanted T cells may heavily influence clinical outcomes. Much current focus is on the potential of defined CD4+/CD8+ T cell populations vs bulk, and on the potential superiority of CAR-T cells from naïve (TN) or central memory (TCM) versus effector memory (TEM) cells. Many commercial T cell activation and expansion methods utilize rigid magnetic beads bound to antibodies against CD3 and CD28 as substrates. These methods are often associated with high costs and licensing restrictions for clinical and commercial applications. Additionally, de-beading processes can be highly complex and inefficient, adding additional time, costs and risks. It has been shown that substrate rigidity influences T cell expansion and phenotype. We hypothesized that a novel phase-change substrate could modulate expanded T cell phenotype/s and address de-beading challenges. Methods An alginate-based phase-change hydrogel was synthesized and coated onto magnetic beads to form hydrogel-coated particles of approximately 10 µm diameter. This hydrogel, in the presence of chelating agents, rapidly dissolves, enabling removal magnetic bead removal. The coated particles were conjugated with streptavidin (SA) and bound to biotinylated antibodies against CD3 (OKT3) and CD28 (28.2) to form CD3/CD28 hydrogel particles (CD3/CD28-HP). Human CD3+ T cells from peripheral blood were seeded (Day 0) at 1x10E6 cells/mL in 24 well plates (n=3) in complete RPMI medium supplemented with IL-2. To each well, 25 µL of CD3/CD28-HP were added per 0.5x10E6 cells in a single stimulation. Media addition or change of culture vessel occurred each 2-3 days. Following expansion, chelating agent was added and magnetic beads removed. Flow cytometry was used to assess cell viability and expression of phenotypic markers including CD3, CD4, CD8, CD45RA and CCR7. ELISA was used to measure secretion of IL-2, IL-4, and IFNγ. Residual magnetic beads were counted via hemocytometer. Results CD3/CD28-HP promoted significant T cell expansion of 0.3, 1.4, 2.4, 4.8 and 6.6 population doublings (PD) by Days 2, 5, 6, 9, and 13 respectively (p Phenotypic markers were assessed on Days 6 and 13. Expansion using CD3/CD28-HP led to significantly more CD8+ cells and significantly fewer CD4+ cells versus the starting population on both days (p Following de-beading of expanded cells, cell recovery was 96% for the CD3/CD28-HP-expanded cells and 93% for cells expanded using commercial magnetic bead-based expansion product. Additionally, in de-beaded cells, fewer residual magnetic particles were present in the CD3/CD28-HP-expanded population than in cells expanded via the commercial magnetic bead-based expansion product. Conclusions These data demonstrate the utility of a novel phase-change hydrogel system to efficiently induce T cell proliferation, promote expansion of functional T cells expressing markers associated with CD8+, TN and TCM phenotypes, and to separate expanded cells efficiently from magnetic beads. In future studies, we will determine if T cells expanded using this method show increased stemness and persistence in in vivo models, and further explore the possibilities of this novel system for rapid expansion and recovery of specific T cell subtypes. Disclosures Jesuraj: Quad Technologies: Employment, Other: stock options. Cole:Quad Technologies: Employment, Other: Stock Options. Wells:Quad Technologies: Employment, Other: Stock Options. Qin:Quad Technologies: Employment, Other: Stock options. Kevlahan:Quad Technologies: Employment, Equity Ownership. Maus:Novartis: Patents & Royalties: related to CTL019, Research Funding. Ball:Quad Technologies: Employment, Other: Stock Options.

Adoptive cell therapy employing T cells equipped with a chimeric antigen receptor (CAR) containing a single chain antibody fragment fused to T cell signaling domains 4-1BB and CD3zeta (CTL019) has shown great potency against various hematopoietic malignancies, e.g. B cell acute lymphoblastic leukemia (ALL). However, it has not shown the same response rate in other malignancies such as chronic lymphocytic leukemia (CLL). We recently demonstrated that the in vivo expansion and persistence of CAR T cells is an important predictor of response to CTL019 in CLL (PMID: 26333935) and ALL (Thudium et al., ASH 2016; Fraietta et al., ASH 2016). Furthermore, it is well known that prolonged culture of T cells negatively impacts the in vivo expansion of the adoptively transferred cells. We therefore hypothesized that minimizing the ex vivo manipulation of T cells would improve the efficacy of CAR T cells. We tested this hypothesis by generating CART19 cells using our standard 9-day manufacturing process plus two abbreviated versions. Cells from normal donors (n=9) and from patients with adult ALL (n=6) were stimulated on day 0 followed by transduction with the CAR19-encoding lentiviral vector on day 1. Cells were harvested on days 3, 5, and 9. Cryopreserved aliquots were evaluated for T cell differentiation using polychromatic flow cytometry, cytokine secretion profile using Luminex, cytolytic ability against a leukemia cell line (NALM6), proliferative ability upon restimulation with CD19-expressing target cells, and in vivo control of our well-established xenogeneic ALL model employing NALM6 as the target. Our data show that all cultures contain a substantial proportion (40%-80%) of na•ve-like CD45RO-CCR7+ T cells that progressively differentiate leading to the accumulation of predominantly (60%-90%) central memory T cells by the end of expansion. Comparative assessment of the CART19 cells at all three time points demonstrated that the cells from the shorter cultures displayed a superior in vitrocytolytic activity, and proliferative response compared to the standard process. In addition,the cells from our standard and shortened cultures all secreted comparable levels of type I cytokines (i.e. IFN-g, IL-2, and TNF-α). Importantly, we investigated the therapeutic potential of cells harvested at day 3 versus later time points. We treated NALM6 xenograftmice with a low dose (0.5 x106 CAR+ T cell I.V.) or standard dose (3 x106 CAR+ T cell I.V.).We demonstrate that day 3 CART19 cells show superior anti-leukemic activity compared to day 5 or day 9 cells. Additionally, we show that mice treated at a low dose with day 3 cells exhibit the greatest anti-leukemic efficacy compared with day 9 cells where the latter fail to control leukemia (Figure 1). Our preclinical findings provide evidence that extended ex vivo manipulation of T cells negatively affects their in vivo potency.In summary, we show that limiting T cell culture ex vivo to the minimum required for lentiviral transduction provides the most efficacious T cells for adoptive T cell immunotherapy. Figure 1 Figure 1. Disclosures Ghassemi: Novartis: Research Funding. Scholler:Novartis: Patents & Royalties; University of Pennsylvania: Patents & Royalties: FAP-CAR US Patent 9,365,641 for targeting tumor microenvironment. Nunez-Cruz:Novartis: Research Funding. Barrett:Novartis: Research Funding. Bedoya:Novartis: Patents & Royalties. Fraietta:Novartis: Patents & Royalties: Novartis, Research Funding. Lacey:Novartis: Research Funding. Levine:GE Healthcare Bio-Sciences: Consultancy; Novartis: Patents & Royalties, Research Funding. Grupp:Novartis: Research Funding. June:Johnson & Johnson: Research Funding; Tmunity: Equity Ownership, Other: Founder, stockholder ; University of Pennsylvania: Patents & Royalties; Pfizer: Honoraria; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Immune Design: Consultancy, Equity Ownership; Celldex: Consultancy, Equity Ownership. Milone:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding.

Adoptive T cell immunotherapy involves the isolation, ex vivo expansion and reinfusion of patient T cells. The efficacy of adoptive immunotherapy is dependent on the ability of T cells to engraft, expand and persist upon adoptive transfer. In this therapy, T cells are cultured ex vivo using natural or artificial antigen presenting cells that deliver signal 1 (TCR/CD3) and signal 2 (e.g. CD28 co-stimulation) along with exogenously added cytokines. IL-2 is the most commonly used cytokine for ex vivo T cell culture; however, there is renewed interest in IL-7 and IL-15 due to their ability to enhance the survival and proliferation of stem cell memory (Tscm) and central memory (Tcm) T cells. We show that primary human T cells freshly isolated from peripheral blood are heterogeneous with substantial numbers of Tscm and Tcm cells in addition to effector differentiated T cells. During ex vivo culture, these cells progressively differentiate into a population of T cells with a predominantly CD45RO+, CD27-, CCR7- effector differentiated phenotype. Exogenous IL-7 and IL-15 delay this transition in T cell phenotype and preserve a greater proportion of Tscm and Tcm cells in the final ex vivo culture product. We hypothesize that limited ex vivo culture of T cells in the presence of IL-7 and IL-15 rather than IL-2 will enhance engraftment and persistence of T cells in vivo contributing to enhanced efficacy in adoptive transfer. We show that T cells can be harvested and viably frozen from ex vivo cultures as early as day 3 following activation. Early activated T cells expressing a chimeric antigen receptor targeting CD19 (CART-19) show potent yet specific cytotoxicity and cytokine production in vitro. We investigated the therapeutic potential of cells harvested at day 3 versus later time points using a Nalm-6 leukemic cell xenograft mouse model. We demonstrate that day 3 CART-19 cells show potent anti-leukemic activity compared to day 5 or day 9 cells. Comparing CART19 cells cultured in either IL-2 or IL-7/15, we show that mice treated at a 10-fold lower dose with day 3 cells cultured in IL-7/15 exhibit the greatest anti-leukemic efficacy compared with day 9 cells where the latter fail to control leukemia. In summary, we show that limiting T cell culture ex vivo to the minimum required for lentiviral transduction in the presence of IL-7 and IL-15 provides the most efficacious T cells for adoptive T cell immunotherapy.

Adoptive T cell immunotherapy involves the isolation, ex vivo expansion and reinfusion of T cells, and is dependent on T cell engraftment and persistence for efficacy. IL-2 is the most commonly used cytokine for ex vivo T cell culture; however, there is renewed interest in IL-7 and IL-15 due to their ability to enhance the survival and proliferation of stem cell memory (Tscm) T cells. Peripheral blood (PB) contains substantial numbers of Tscm, central memory (Tcm) and effector subsets. During ex vivo culture, PB T cells differentiate into cells with a predominantly CD45RO+, CD27−, CCR7− effector phenotype. Exogenous IL-7/15 delays this transition in phenotype and preserves a greater proportion of Tscm and Tcm cells. We hypothesize that limited ex vivo T cell culture in the presence of IL-7/15 rather than IL-2 will enhance engraftment and persistence of T cells in vivo. We show that T cells can be harvested from ex vivo cultures as early as day 3 (d3) following activation. T cells expressing a chimeric antigen receptor targeting CD19 (CART-19) show potent yet specific cytotoxicity in vitro. We investigated the therapeutic potential of cells harvested at d3 versus later time points using a Nalm-6 leukemic cell xenograft mouse model. We demonstrate that d3 CART-19 cells show potent anti-leukemic activity compared to day 5 or day 9 cells. Comparing CART19 cells cultured in IL-2 or IL-7/15 for 3 or 9 days, we show that mice treated with d3 cells cultured in IL-7/15 exhibit the greatest anti-leukemic efficacy at a 10-fold lower dose compared with day 9 cells. In summary, we show that limiting T cell culture ex vivo to the minimum required for lentiviral transduction, in the presence of IL-7/15, provides the most efficacious T cells for adoptive immunotherapy.

Since most oncogenic viruses persist as extrachromosomal covalently closed circular DNA (cccDNA) in tumor cells, we developed an assay to visualize and identify cccDNA in primary lymphomas. We identified concatemers of the mitochondrial genome in all samples analyzed, but not in normal lymphocytes. One AIDS-associated lymphoma (EL) was further studied in detail as its mitochondrial genome consisted of tandem head-to-tail duplications. Insertion of C-residues was noted near the origin of replication of EL mtDNA. EL cells responded weakly to Fas-apoptotic stimulus, displayed reduced mitochondrial activity and mass, and produced higher levels of reactive oxygen intermediates. Screening of several AIDS-associated lymphomas and established lymphoid cell lines also revealed the presence of mitochondrial genome concatemers consisting of interlinked monomer molecules. Taken together, our results suggest that formation of mtDNA concatemers is associated with oncogenic transformation in lymphoid cells.

We have examined the formation, participation and functional specialization of virus-reactive Foxp3+ regulatory T cells (Tregs) in a mouse model of influenza virus infection. “Natural” Tregs generated intra-thymically based on interactions with a self-peptide proliferated in response to a homologous viral antigen in the lungs, and to a lesser extent in the lung-draining mediastinal LN (medLN), of virus-infected mice. By contrast, conventional CD4+ T cells with identical TCR specificity underwent little or no conversion to become “adaptive” Tregs. The virus-reactive Tregs in the medLN and the lungs of infected mice upregulated a variety of molecules associated with Treg activation, and also acquired expression of molecules (T-bet, Blimp-1 and IL-10) that confer functional specialization to Tregs. Notably, however, the phenotypes of the T-bet+ Tregs obtained from these sites were distinct, since Tregs isolated from the lungs expressed significantly higher levels of T-bet, Blimp-1 and IL-10 than did Tregs from the medLN. Adoptive transfer of antigen-reactive Tregs led to decreased proliferation of anti-viral CD4+ and CD8+ effector T cells in the lungs of infected hosts, while depletion of Tregs had a reciprocal effect. These studies demonstrate that thymically-generated Tregs can become activated by a pathogen-derived peptide and acquire discrete T-bet+ Treg phenotypes while participating in and modulating an antiviral immune response.