In vivo CAR T-cell generation in nonhuman primates using lentiviral vectors displaying a multidomain fusion ligand.

ABSTRACT: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformative efficacy in treating B-cell malignancies. However, high costs and manufacturing complexities hinder their widespread use. To overcome these hurdles, we have developed the VivoVec platform, a lentiviral vector capable of generating CAR T cells in vivo. Here, we describe the incorporation of T-cell activation and costimulatory signals onto the surface of VivoVec particles (VVPs) in the form of a multidomain fusion protein and show enhanced in vivo transduction and improved CAR T-cell antitumor functionality. Furthermore, in the absence of lymphodepleting chemotherapy, administration of VVPs into nonhuman primates resulted in the robust generation of anti-CD20 CAR T cells and the complete depletion of B cells for >10 weeks. These data validate the VivoVec platform in a translationally relevant model and support its transition into human clinical testing, offering a paradigm shift in the field of CAR T-cell therapies.

The transcription factor STAT5 is critical in dendritic cells for the development of TH2 but not TH1 responses.

Dendritic cells (DCs) are critical in immune responses, linking innate and adaptive immunity. We found here that DC-specific deletion of the transcription factor STAT5 was not critical for development but was required for T helper type 2 (TH2), but not TH1, allergic responses in both the skin and lungs. Loss of STAT5 in DCs led to the inability to respond to thymic stromal lymphopoietin (TSLP). STAT5 was required for TSLP-dependent DC activation, including upregulation of the expression of costimulatory molecules and chemokine production. Furthermore, TH2 responses in mice with DC-specific loss of STAT5 resembled those seen in mice deficient in the receptor for TSLP. Our results show that the TSLP-STAT5 axis in DCs is a critical component for the promotion of type 2 immunity at barrier surfaces.

The Mental Health Consequences of George Floyd's Murder in Minneapolis in Black, Latine, and White Communities.

The high-profile police murder of George Floyd is likely to have an aftermath of negative health consequences, particularly among Black people. Our study evaluates the impact of the murder of Mr. Floyd on mental health in Black, Latine, and white communities in Minneapolis, Minnesota. We constructed a panel dataset merging data from the Minnesota Hospital Association, Minnesota Department of Natural Resources, Minneapolis Police Department, and American Community Survey. First, we specify an overall and racial subgroup autoregressive interrupted time-series design to identify the impact of the murder on rates of mental health hospital discharge at the city-level. We then examine the spatial heterogeneity in the impact of the murder by specifying zip code tabulation area (ZCTA)-level panel models. We find a 0.23 per 1,000 increase in mental health conditions among Black people in the immediate post-murder period, followed by a weekly decline (-.007) in mental health diagnoses. We do not find a substantial rate increase in White or Latine residents. Further, our analyses at the ZCTA-week-level corroborate these findings, while showing that the increase for Black residents was global. These findings speak to the traumatizing effects of police violence and the short- and longer-term public health consequences for Black communities.

Felon history and change in U.S. employment rates.

In recent decades, the share of U.S. adults with felony-level criminal records has risen and the growth in the employment rate has slowed. Sociological theories of labeling and stigmatization, as well as economic theories of statistical discrimination, suggest a possible causal connection between the two phenomena. Surveys of employers have shown increasing reliance on criminal background checks, for example, and audit studies reveal explicit discrimination against people with felony-level criminal records. This paper draws on novel, state-level annual measures of individuals with felony-level records to estimate pooled cross-sectional, panel models predicting changes in aggregate employment rates. Estimates from these models indicate that a 1 percentage point increase in the share of a state's adult population with a felony history is associated with 0.3 percentage point increase in non-employment (being unemployed or not in the labor force) among those aged 18 to 54. Subgroup analysis shows that effects are stronger for women and whites. These results suggest that the stigma of a felony record may play an important part in aggregate employment rates as well as in individual hiring practices.

Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids.

The evolutionary survival of Mycobacterium tuberculosis, the cause of human tuberculosis, depends on its ability to invade the host, replicate, and transmit infection. At its initial peripheral infection site in the distal lung airways, M. tuberculosis infects macrophages, which transport it to deeper tissues. How mycobacteria survive in these broadly microbicidal cells is an important question. Here we show in mice and zebrafish that M. tuberculosis, and its close pathogenic relative Mycobacterium marinum, preferentially recruit and infect permissive macrophages while evading microbicidal ones. This immune evasion is accomplished by using cell-surface-associated phthiocerol dimycoceroserate (PDIM) lipids to mask underlying pathogen-associated molecular patterns (PAMPs). In the absence of PDIM, these PAMPs signal a Toll-like receptor (TLR)-dependent recruitment of macrophages that produce microbicidal reactive nitrogen species. Concordantly, the related phenolic glycolipids (PGLs) promote the recruitment of permissive macrophages through a host chemokine receptor 2 (CCR2)-mediated pathway. Thus, we have identified coordinated roles for PDIM, known to be essential for mycobacterial virulence, and PGL, which (along with CCR2) is known to be associated with human tuberculosis. Our findings also suggest an explanation for the longstanding observation that M. tuberculosis initiates infection in the relatively sterile environment of the lower respiratory tract, rather than in the upper respiratory tract, where resident microflora and inhaled environmental microbes may continually recruit microbicidal macrophages through TLR-dependent signalling.

Measurement accuracy and uncertainty in plant biomechanics.

All scientific measurements are affected to some degree by both systematic and random errors. The quantification of these errors supports correct interpretation of data, thus supporting scientific progress. Absence of information regarding reliability and accuracy can slow scientific progress, and can lead to a reproducibility crisis. Here we consider both measurement theory and plant biomechanics literature. Drawing from measurement theory literature, we review techniques for assessing both the accuracy and uncertainty of a measurement process. In our survey of plant biomechanics literature, we found that direct assessment of measurement accuracy and uncertainty is not yet common. The advantages and disadvantages of efforts to quantify measurement accuracy and uncertainty are discussed. We conclude with recommended best practices for improving the scientific rigor in plant biomechanics through attention to the issues of measurement accuracy and uncertainty.

MiR-155-regulated molecular network orchestrates cell fate in the innate and adaptive immune response to Mycobacterium tuberculosis.

The regulation of host-pathogen interactions during Mycobacterium tuberculosis (Mtb) infection remains unresolved. MicroRNAs (miRNAs) are important regulators of the immune system, and so we used a systems biology approach to construct an miRNA regulatory network activated in macrophages during Mtb infection. Our network comprises 77 putative miRNAs that are associated with temporal gene expression signatures in macrophages early after Mtb infection. In this study, we demonstrate a dual role for one of these regulators, miR-155. On the one hand, miR-155 maintains the survival of Mtb-infected macrophages, thereby providing a niche favoring bacterial replication; on the other hand, miR-155 promotes the survival and function of Mtb-specific T cells, enabling an effective adaptive immune response. MiR-155-induced cell survival is mediated through the SH2 domain-containing inositol 5-phosphatase 1 (SHIP1)/protein kinase B (Akt) pathway. Thus, dual regulation of the same cell survival pathway in innate and adaptive immune cells leads to vastly different outcomes with respect to bacterial containment.

A Quarter Century of Participation in School-Based Extracurricular Activities: Inequalities by Race, Class, Gender and Age?

Extracurricular activity participation is linked to positive development, but it is also a setting for inequality. Using a quarter century of data from Monitoring the Future (N = 593,979; 51% female; 65% non-Hispanic white; 13% non-Hispanic black; 12% Hispanic; 4% non-Hispanic Asian/Pacific Islander; 7% other race), this article documents patterns and trends in school-based extracurricular participation by race, social class, gender, and age, and their links to academic and substance use outcomes. Findings reveal differences by race and confirm a division by social class that has worsened over time. Further, girls are gaining on boys and surpass them in some types of school-based activities. Participation is linked to better academic outcomes and less substance use, affirming the importance of redressing the inequalities revealed.

Cutting edge: allergen-specific CD4 T cells respond indirectly to thymic stromal lymphopoietin to promote allergic responses in the skin.

Thymic stromal lymphopoietin (TSLP) is an epithelial-derived cytokine that has been implicated in the initiation of allergic responses. CD4 T cells and dendritic cells are able to respond to TSLP in vitro; however, there has not been a careful dissection of the spatiotemporal response to TSLP by CD4 T cells in vivo during an allergic response. Previous work has suggested a requirement for TSLP in amplifying Th2 responses during allergen challenge by direct action on CD4 T cells; however, these studies did not determine whether there is an effect of TSLP on CD4 T cells during allergen sensitization. In this study we demonstrate an indirect role for TSLP on CD4 T cells during sensitization and challenge phases of an allergic response. This indirect effect of TSLP on CD4 T cells is due in part to the presence of TSLP exclusively in the allergen-sensitized and -challenged skin, rather than the draining lymph nodes.

Comparison of BG-Sentinel® Trap and Oviposition Cups for Aedes aegypti and Aedes albopictus Surveillance in Jacksonville, Florida, USA.

The BG-Sentinel® (BGS) trap and oviposition cups (OCs) have both proven effective in the surveillance of Aedes species. This study aimed to determine which of the 2 traps could best characterize the relative population sizes of Aedes albopictus and Aedes aegypti in an urban section of Jacksonville, FL. Until 1986, Ae. aegypti was considered the dominant container-breeding species in urban northeastern Florida. Since the introduction of Ae. albopictus, Ae. aegypti has become almost completely extirpated. In 2011, a resurgence of Ae. aegypti was detected in the urban areas of Jacksonville; thus this study initially set out to determine the extent of Ae. aegypti reintroduction to the area. We determined that the BGS captured a greater number of adult Ae. aegypti than Ae. albopictus, while OCs did not monitor significantly different numbers of either species, even in areas where the BGS traps suggested a predominance of one species over the other. Both traps were effective at detecting Aedes spp.; however, the BGS proved more diverse by detecting over 20 other species as well. Our results show that in order to accurately determine vectorborne disease threats and the impact of control operations on these 2 species, multiple trapping techniques should be utilized when studying Ae. aegypti and Ae. albopictus population dynamics.

Role for topoisomerase 1 in transcription-associated mutagenesis in yeast.

High levels of transcription in Saccharomyces cerevisiae are associated with increased genetic instability, which has been linked to DNA damage. Here, we describe a pGAL-CAN1 forward mutation assay for studying transcription-associated mutagenesis (TAM) in yeast. In a wild-type background with no alterations in DNA repair capacity, ≈50% of forward mutations that arise in the CAN1 gene under high-transcription conditions are deletions of 2-5 bp. Furthermore, the deletions characteristic of TAM localize to discrete hotspots that coincide with 2-4 copies of a tandem repeat. Although the signature deletions of TAM are not affected by the loss of error-free or error-prone lesion bypass pathways, they are completely eliminated by deletion of the TOP1 gene, which encodes the yeast type IB topoisomerase. Hotspots can be transposed into the context of a frameshift reversion assay, which is sensitive enough to detect Top1-dependent deletions even in the absence of high transcription. We suggest that the accumulation of Top1 cleavage complexes is related to the level of transcription and that their removal leads to the signature deletions. Given the high degree of conservation between DNA metabolic processes, the links established here among transcription, Top1, and mutagenesis are likely to extend beyond the yeast system.

Carjacking and homicide in Minneapolis after the police killing of George Floyd: Evidence from an interrupted time series analysis.

There is abundant research showing the disproportionate impacts of violence on health in disadvantaged neighborhoods, making an understanding of recent violent crime trends essential for promoting health equity. Carjackings have been of particular interest in the media, although little research has been undertaken on this violent crime. We use interrupted time series models to examine the impact of the police killing of George Floyd on the spatiotemporal patterns of carjacking in Minneapolis in relation to neighborhood disadvantage. To provide grounding, we compare our results to the well-studied patterns of homicides. Results indicate that carjackings both increased and dispersed spatially after the murder of George Floyd and subsequent social unrest, more so than homicides. Socially disadvantaged neighborhoods experienced the greatest absolute increase while more advantaged neighborhoods saw a greater relative increase. The challenge ahead is to identify policy responses that will effectively curb such violence without resorting to harsh and inequitable policing and sentencing practices.

Lead optimization of an acylhydrazone scaffold possessing antiviral activity against Lassa virus.

Previously we reported the optimization of antiviral scaffolds containing benzimidazole and related heterocycles possessing activity against a variety of arenaviruses. These series of compounds were discovered through an HTS campaign of a 400,000 small molecule library using lentivirus-based pseudotypes incorporated with the Lassa virus envelope glycoprotein (LASV GP). This screening also uncovered an alternate series of very potent arenavirus inhibitors based upon an acylhydrazone scaffold. Subsequent SAR analysis of this chemical series involved various substitutions throughout the chemical framework along with assessment of the preferred stereochemistry. These studies led to an optimized analog (ST-161) possessing subnanomolar activity against LASV and submicromolar activity against a number of other viruses in the Arenaviridae family.

Discovery and optimization of potent broad-spectrum arenavirus inhibitors derived from benzimidazole and related heterocycles.

A series of potent arenavirus inhibitors sharing a benzimidazole core were previously reported by our group. SAR studies were expanded beyond the previous analysis, which involved the attached phenyl rings and methylamino linker portion, to include modifications focused on the benzimidazole core. These changes included the introduction of various substituents to the bicyclic benzimidazole ring system along with alternate core heterocycles. Many of the analogs containing alternate nitrogen-based bicyclic ring systems were found to retain antiviral potency compared to the benzimidazole series from which we derived our lead compound, ST-193. In fact, 21 h, built on an imidazopyridine core, possessed a near tenfold increase in potency against Lassa virus pseudotypes compared to ST-193. As found with the benzimidazole series, broad-spectrum arenavirus activity was also observed for a number of the analogs discovered during this study.

Discovery and optimization of potent broad-spectrum arenavirus inhibitors derived from benzimidazole.

A chemically diverse library of about 400,000 small molecules was screened for antiviral activity against lentiviral pseudotypes with the Lassa virus envelope glycoprotein (LASV GP) gene incorporated. High-throughput screening resulted in discovery of a hit compound (ST-37) possessing a benzimidazole core which led to a potent compound series. Herein, we report SAR studies which involved structural modifications to the phenyl rings and methylamino linker portion attached to the benzimidazole core. Many analogs in this study possessed single digit nanomolar potency against LASV pseudotypes. Compounds in this benzimidazole series also exhibited nanomolar antiviral activity against pseudotypes generated from other arenavirus envelopes indicating the potential for development of a broad-spectrum inhibitor. Ultimately, lead compound ST-193 was identified and later found to be efficacious in a lethal LASV guinea pig model showing superior protection compared to ribavirin treatment.

Foxp3(+) regulatory T cells in tuberculosis.

The immune response to Mycobacterium tuberculosis (Mtb) must be tightly regulated to mount a sufficient response to limit bacterial growth and dissemination while avoiding excessive inflammation that could damage host tissues. A wide variety of cell types, cell surface molecules, and cytokines are likely to contribute to this regulation, but recent studies have revealed that a subset of CD4 T cells expressing the transcription factor Foxp3, called regulatory T (reg) cells, play a critical role [1-3]. Although the first reports of T reg cells in tuberculosis (TB) occurred only recently (i.e., 2006) [4, 5], we have already gained many insights into their activity during TB. While it is likely that T reg cells do play some beneficial roles by preventing inflammation-mediated damage to host tissues during TB, this aspect of their function has not been well studied to date. What is clear, however, is that during the initial T cell response to Mtb infection, Mtb induces the expansions of T reg cells that delay the onset of adaptive immunity, suggesting that Mtb has hijacked T reg cell-mediated immune suppression to allow it to replicate unabated in the lung until T cells finally arrive [6]. In this chapter, we will first provide an overview of the delayed T cell response to Mtb and a brief introduction to regulatory T cells. We will then review what is known about T reg cells from observations in human populations, discuss mechanistic insights revealed in the mouse model, and speculate about the relevance of this understanding for future efforts to prevent and treat TB.

Temporal and spatial shifts in gun violence, before and after a historic police killing in Minneapolis.

OBJECTIVE: To determine the impact of the police murder of George Floyd in Minneapolis, MN on firearm violence, and examine the spatial and social heterogeneity of the effect. METHODS: We analyzed a uniquely constructed panel dataset of Minneapolis Zip Code Tabulation Areas from 2016-2020 (n = 5742), consisting of Minnesota Hospital Association, Minneapolis Police Department, Minneapolis Public Schools, Census Bureau, and Minnesota Department of Natural Resources data. Interrupted time-series and random effects panel models were used to model the spatiotemporal effects of police killing event on the rate of firearm assault injuries. RESULTS: Findings reveal a rising and falling temporal pattern post-killing and a spatial pattern in which disadvantaged, historically Black communities near earlier sites of protest against police violence experienced the brunt of the post-killing increase in firearm assault injury. These effects remain after adjusting for changes in police activity and pandemic-related restrictions, indicating that rising violence was not a simple byproduct of changes in police behavior or COVID-19 response. CONCLUSIONS: The results suggest that the increases in firearm violence as a result of police violence are disproportionately borne by underserved communities.

Evaluation of "Caserotek" a low cost and effective artificial blood-feeding device for mosquitoes.

Entomological research studies on mosquito vector biology, vector competence, insecticide resistance, dispersal, and survival (using mark-release-recapture techniques) often rely on laboratory-reared mosquito colonies to produce large numbers of consistently reared, aged, and sized mosquitoes. We developed a low-cost blood feeding apparatus that supports temperatures consistent with warm blooded animals, using commonly available materials found in low resource environments. We compare our system ("Caserotek") to Hemotek and glass/membrane feeding methods. Two experiments were conducted with Aedes aegypti (Linnaeus 1762) and one with Anopheles darlingi (Root 1926) (Diptera: Culicidae); 3 replicates were conducted for each experiment. Aedes aegypti female mosquitoes were provided chicken blood once per week for 30 min (Experiment #1) for 14 days or 1 hour (Experiment #2) for 21 days. Anopheles darlingi were fed once for 1 hour (Experiment #3). Blood-feeding rates, survival rates, and egg production were calculated across replicates. Caserotek had a significantly higher 30-min engorgement rate (91.1%) than Hemotek (47.7%), and the glass feeder (29.3%) whereas for 1-hour feeding, Hemotek had a significantly lower engorgement rate than either of the other two devices (78% versus 91%). Thirty-day survival was similar among the feeding devices, ranging from 86% to 99%. Mean egg production was highest for the Caserotek feeder (32 eggs per female) compared to the glass feeder and Hemotek device (21-22 eggs per female). Our new artificial feeding system had significantly higher blood feeding rates than for more expensive artificial systems and was equivalent to other fitness parameters. Caserotek only requires the ability to boil water to maintain blood temperatures using a Styrofoam liner. It can be easily scaled up to large production facilities and used under austere conditions.

Preclinical proof of concept for VivoVec, a lentiviral-based platform for in vivo CAR T-cell engineering.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformational outcomes in the treatment of B-cell malignancies, but their widespread use is hindered by technical and logistical challenges associated with ex vivo cell manufacturing. To overcome these challenges, we developed VivoVec, a lentiviral vector-based platform for in vivo engineering of T cells. UB-VV100, a VivoVec clinical candidate for the treatment of B-cell malignancies, displays an anti-CD3 single-chain variable fragment (scFv) on the surface and delivers a genetic payload that encodes a second-generation CD19-targeted CAR along with a rapamycin-activated cytokine receptor (RACR) system designed to overcome the need for lymphodepleting chemotherapy in supporting successful CAR T-cell expansion and persistence. In the presence of exogenous rapamycin, non-transduced immune cells are suppressed, while the RACR system in transduced cells converts rapamycin binding to an interleukin (IL)-2/IL-15 signal to promote proliferation. METHODS: UB-VV100 was administered to peripheral blood mononuclear cells (PBMCs) from healthy donors and from patients with B-cell malignancy without additional stimulation. Cultures were assessed for CAR T-cell transduction and function. Biodistribution was evaluated in CD34-humanized mice and in canines. In vivo efficacy was evaluated against normal B cells in CD34-humanized mice and against systemic tumor xenografts in PBMC-humanized mice. RESULTS: In vitro, administration of UB-VV100 resulted in dose-dependent and anti-CD3 scFv-dependent T-cell activation and CAR T-cell transduction. The resulting CAR T cells exhibited selective expansion in rapamycin and antigen-dependent activity against malignant B-cell targets. In humanized mouse and canine studies, UB-VV100 demonstrated a favorable biodistribution profile, with transduction events limited to the immune compartment after intranodal or intraperitoneal administration. Administration of UB-VV100 to humanized mice engrafted with B-cell tumors resulted in CAR T-cell transduction, expansion, and elimination of systemic malignancy. CONCLUSIONS: These findings demonstrate that UB-VV100 generates functional CAR T cells in vivo, which could expand patient access to CAR T technology in both hematological and solid tumors without the need for ex vivo cell manufacturing.

Dibutyl phthalate-induced thymic stromal lymphopoietin is required for Th2 contact hypersensitivity responses.

Thymic stromal lymphopoietin (TSLP) is an IL-7-related cytokine, produced by epithelial cells, that has been linked to atopic dermatitis and asthma; however, it remains unclear how TSLP shapes the adaptive immune response that causes these allergic disorders. In this study, we demonstrate a role for TSLP in a Th2 model of contact hypersensitivity in mice. TSLP is required for the development of Th2-type contact hypersensitivity induced by the hapten FITC in combination with the sensitizing agent dibutyl phthalate. TSLPR-deficient mice exhibited a dramatically reduced response, including markedly reduced local infiltration by eosinophils, Th2 cytokine production, and serum IgE levels, following FITC sensitization and challenge. The reduced response by TSLPR-deficient mice is likely due to decreased frequency and reduced T cell stimulatory function of skin-derived Ag-bearing FITC(+)CD11c(+) dendritic cells in draining lymph nodes following FITC sensitization. These data suggest that skin-derived dendritic cells are direct or indirect targets of TSLP in the development of type 2 immune responses in the skin, where TSLP drives their maturation, accumulation in skin draining lymph nodes, and ability to induce proliferation of naive allergen-specific T cells.