Citations

The high cost and complexity of CAR-T therapy has spurred efforts to generate CAR-T cells directly within patients via systemic infusion of serum-stable lentiviral vector particles that target T cells in situ for CAR transgene delivery. To ensure effective in vivo delivery, these vectors need to show resistance to neutralization by human serum, demonstrate transduction specificity for T cells, and support subsequent expansion of CAR-T cells. Lentiviral vectors (LV) pseudotyped with VSV G-glycoprotein (G-WT) transduce target cells via the LDL receptor (LDLR), which is ubiquitously expressed at variable levels and is upregulated on activated T cells. G-WT LVs are widely used to generate therapeutic CAR-T cells ex vivo, following purification and ex vivo activation of the T cells. To generate LVs capable of targeting circulating T cells in vivo, we utilized rational engineering of the VSV-G glycoprotein to design a G-CD3. This modified G protein avoids LDLR recognition, incorporates a CD3-specific single-chain antibody for targeted recognition of T cells, and minimizes LV particle inactivation upon exposure to human serum. G-CD3 LVs were shown to target and transduce resting T cells in peripheral blood mononuclear cell (PBMC) cultures with very high efficiency. The G-CD3 LVs powerfully activated T cells, evident from their increased CD25 expression, and the infection was not hindered in the presence of fresh, complement-active human serum (25%). Additionally, G-CD3 LVs efficiently transduced T cells in fresh human whole blood without apparent off-target transduction of non-T cell lineages. In vivo evaluation of G-CD3 LV efficiency was performed in humanized NSG mice by intravenous (IV) or intraperitoneal (IP) delivery of G-CD3 LVs encoding anti-CD19 CAR and GFP transgenes. Both IV and IP delivery of G-CD3 LV led to in vivo CAR T cell generation with peak levels of 17% of total circulating human CD45+ cells on day 21 post vector administration. The in vivo generated CD19-CAR-T cells were fully functional as evidenced by the disappearance of endogenous human CD19+ B cells from peripheral blood. Tissue analysis of spleen, bone marrow, and liver at the time of sacrifice (day 39 post LV injection), showed anti-CD19 CAR-T in all three tissues accompanied by an absence or dramatic reduction in tissue-resident human CD19+ B cells. In contrast, CAR-T cells were not detected at any time point in humanized NSG mice injected with G-WT LV, regardless of whether the virus was administered IV or IP. In summary, these data highlight the potential applicability of fully retargeted, serum stable LVs for in vivo gene delivery or genome editing, offering promising prospects for future human gene therapy applications.

Background Inconsistent nomenclature and anatomical descriptions of regional anesthetic techniques hinder scientific communication and engender confusion; this in turn has implications for research, education and clinical implementation of regional anesthesia. Having produced standardized nomenclature for abdominal wall, paraspinal and chest wall regional anesthetic techniques, we aimed to similarly do so for upper and lower limb peripheral nerve blocks. Methods We performed a three-round Delphi international consensus study to generate standardized names and anatomical descriptions of upper and lower limb regional anesthetic techniques. A long list of names and anatomical description of blocks of upper and lower extremities was produced by the members of the steering committee. Subsequently, two rounds of anonymized voting and commenting were followed by a third virtual round table to secure consensus for items that remained outstanding after the first and second rounds. As with previous methodology, strong consensus was defined as ≥75% agreement and weak consensus as 50%–74% agreement. Results A total of 94, 91 and 65 collaborators participated in the first, second and third rounds, respectively. We achieved strong consensus for 38 names and 33 anatomical descriptions, and weak consensus for five anatomical descriptions. We agreed on a template for naming peripheral nerve blocks based on the name of the nerve and the anatomical location of the blockade and identified several areas for future research. Conclusions We achieved consensus on nomenclature and anatomical descriptions of regional anesthetic techniques for upper and lower limb nerve blocks, and recommend using this framework in clinical and academic practice. This should improve research, teaching and learning of regional anesthesia to eventually improve patient care.

Background Acute pericarditis (AP) is considered a cardiovascular complication in patients with COVID‐19. We aimed to ass‐ess the incidence, associated complications, and clinical impact of AP on hospitalized patients with COVID‐19. Methods and Results In this retrospective cohort study, International Classification of Diseases, Tenthth Revision, Clinical Modification ( ICD‐10 ) codes were used to identify patients with COVID‐19 with or without AP in the National Inpatient Sample 2020 database. We compared outcomes between AP and non‐AP groups before and after propensity‐score matching for patient and hospital demographics and relevant comorbidities. A total of 211 619 patients with a primary diagnosis of COVID‐19 were identified, including 983 (0.46%) patients who had a secondary diagnosis of AP. Before matching, patients with COVID‐19 with AP were younger (59.93±19.24 years old versus 64.29±16.82 years old) and more likely to have anemia (40.5% versus 19.9%), cancer (6.7% versus 3.6%), and chronic kidney disease (29.3% versus 19.6%) (all P <0.05). After matching, patients with COVID‐19 with AP (n=980), when compared with the matched non‐AP group (n=2936), had higher rates of mortality (21.3% versus 11.1%, P <0.001), cardiac arrest (5.0% versus 2.6%, P <0.001), cardiogenic shock (4.2% versus 0.5%, P <0.001), ventricular arrhythmia (4.7% versus 1.9%, P <0.001), acute kidney injury (38.3% versus 28.9%, P <0.001), acute congestive heart failure (14.3% versus 4.8%, P <0.001), and longer length of stay (7.00±10.00 days versus 5.00±7.00 days, P <0.001) and higher total charges ($75066.5±$130831.3 versus $44824.0±$63660.5, P <0.001). Conclusions In hospitalized patients with COVID‐19, AP is a rare but severe in‐hospital complication and is associated with worse in‐hospital outcomes.

Abstract Multiple myeloma (MM) is a malignancy that is often driven by MYC and that is sustained by IRF4, which are upregulated by super-enhancers. IKZF1 and IKZF3 bind to super-enhancers and can be degraded using immunomodulatory imide drugs (IMiD). Successful IMiD responses downregulate MYC and IRF4; however, this fails in IMiD-resistant cells. MYC and IRF4 downregulation can also be achieved in IMiD-resistant tumors using inhibitors of BET and EP300 transcriptional coactivator proteins; however, in vivo these drugs have a narrow therapeutic window. By combining IMiDs with EP300 inhibition, we demonstrate greater downregulation of MYC and IRF4, synergistic killing of myeloma in vitro and in vivo, and an increased therapeutic window. Interestingly, this potent combination failed where MYC and IRF4 expression was maintained by high levels of the AP-1 factor BATF. Our results identify an effective drug combination and a previously unrecognized mechanism of IMiD resistance. Significance: These results highlight the dependence of MM on IKZF1-bound super-enhancers, which can be effectively targeted by a potent therapeutic combination pairing IMiD-mediated degradation of IKZF1 and IKZF3 with EP300 inhibition. They also identify AP-1 factors as an unrecognized mechanism of IMiD resistance in MM. See related article by Neri, Barwick, et al., p. 56. See related commentary by Yun and Cleveland, p. 5. This article is featured in Selected Articles from This Issue, p. 4

Across multiancestry groups, we analyzed Human Leukocyte Antigen (HLA) associations in over 176,000 individuals with Parkinson’s disease (PD) and Alzheimer’s disease (AD) versus controls. We demonstrate that the two diseases share the same protective association at the HLA locus. HLA-specific fine-mapping showed that hierarchical protective effects of HLA-DRB1 *04 subtypes best accounted for the association, strongest with HLA-DRB1 *04:04 and HLA-DRB1 *04:07, and intermediary with HLA-DRB1 *04:01 and HLA-DRB1 *04:03. The same signal was associated with decreased neurofibrillary tangles in postmortem brains and was associated with reduced tau levels in cerebrospinal fluid and to a lower extent with increased Aβ42. Protective HLA-DRB1 *04 subtypes strongly bound the aggregation-prone tau PHF6 sequence, however only when acetylated at a lysine (K311), a common posttranslational modification central to tau aggregation. An HLA-DRB1 *04-mediated adaptive immune response decreases PD and AD risks, potentially by acting against tau, offering the possibility of therapeutic avenues.

We introduce tumor connectomics, a novel MRI-based complex graph theory framework that describes the intricate network of relationships within the tumor and surrounding tissue, and combine this with multiparametric radiomics (mpRad) in a machine-learning approach to distinguish radiation necrosis (RN) from true progression (TP). Pathologically confirmed cases of RN vs. TP in brain metastases treated with SRS were included from a single institution. The region of interest was manually segmented as the single largest diameter of the T1 post-contrast (T1C) lesion plus the corresponding area of T2 FLAIR hyperintensity. There were 40 mpRad features and 6 connectomics features extracted, as well as 5 clinical and treatment factors. We developed an Integrated Radiomics Informatics System (IRIS) based on an Isomap support vector machine (IsoSVM) model to distinguish TP from RN using leave-one-out cross-validation. Class imbalance was resolved with differential misclassification weighting during model training using the IRIS. In total, 135 lesions in 110 patients were analyzed, including 43 cases (31.9%) of pathologically proven RN and 92 cases (68.1%) of TP. The top-performing connectomics features were three centrality measures of degree, betweenness, and eigenvector centralities. Combining these with the 10 top-performing mpRad features, an optimized IsoSVM model was able to produce a sensitivity of 0.87, specificity of 0.84, AUC-ROC of 0.89 (95% CI: 0.82–0.94), and AUC-PR of 0.94 (95% CI: 0.87–0.97).

Abstract Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and promising biomarkers and therapeutics in the central nervous system (CNS). Human brain‐derived EVs (BDEVs) provide a comprehensive snapshot of physiological changes in the brain's environment, however, the isolation of BDEVs and the comparison of different methods for this purpose have not been fully investigated. In this study, we compared the yield, morphology, subtypes and protein cargo composition of EVs isolated from the temporal cortex of aged human brains using three established separation methods: size‐exclusion chromatography (SEC), phosphatidylserine affinity capture (MagE) and sucrose gradient ultracentrifugation (SG‐UC). Our results showed that SG‐UC method provided the highest yield and collected larger EVs compared to SEC and MagE methods as assessed by transmission electron microscopy and nanoparticle tracking analysis (NTA). Quantitative tandem mass‐tag (TMT) mass spectrometry analysis of EV samples from three different isolation methods identified a total of 1158 proteins, with SG‐UC showing the best enrichment of common EV proteins with less contamination of non‐EV proteins. In addition, SG‐UC samples were enriched in proteins associated with ATP activity and CNS maintenance, and were abundant in neuronal and oligodendrocytic molecules. In contrast, MagE samples were more enriched in molecules related to lipoproteins, cell‐substrate junction and microglia, whereas SEC samples were highly enriched in molecules related to extracellular matrix, Alzheimer's disease and astrocytes. Finally, we validated the proteomic results by performing single‐particle analysis using the super‐resolution microscopy and flow cytometry. Overall, our findings demonstrate the differences in yield, size, enrichment of EV cargo molecules and single EV assay by different isolation methods, suggesting that the choice of isolation method will have significant impact on the downstream analysis and protein discovery.

Abstract Sensing of extracellular metabolites controls CD8+ T cell function. Their accumulation can occur through export by specialized molecules, such as the release channel Pannexin-1 (Panx1). Here, we report that T cell-specific Panx1 is needed for efficient CD8+ T cell responses to viral infection and cancer. Panx1 favors both the expansion of effector CD8+ T cells and the survival of memory CD8+ T cells. Our data suggests that, while memory CD8+ T cells require Panx1 for mitochondrial function, Panx1 promotes the glycolytic pathway in effector CD8+ T cells. Panx1 promotes memory CD8+ T cell survival together with the eATP sensor P2RX7. However, Panx1 induces effector CD8+ T cells independently of eATP. Rather, we found an unexpected link between Panx1, lactate export and the activation of effector CD8+ T cells. Therefore, Panx1 regulates effector and memory CD8+ T cells through export of distinct metabolites and by engaging different intracellular pathways.